Minigene cassettes, recombinant polynucleotides, and methods of their use

Abstract

The present disclosure relates to a minigene cassette comprising, from 5' to 3': a first intron, an alternatively-spliced exon, and a second intron comprising a U1 binding sequence, where (i) a portion of the alternatively-spliced exon is complementary to a portion of the second intron; the portion of the alternatively-spliced exon that is complementary to the portion of the second intron forms a hairpin loop structure comprising a hairpin stem and a hairpin loop; the hairpin stem comprises at least a portion of the U1 binding sequence; and the hairpin loop comprises an aptamer or (ii) a portion of the second intron forms a hairpin loop structure comprising a hairpin stem and a hairpin loop; the hairpin stem comprises at least a portion of the U1 binding sequence; and the hairpin loop comprises an aptamer. Also disclosed are polynucleotides encoding such minigene cassettes and methods of use thereof.

Description

MINIGENE CASSETTES, RECOMBINANT POLYNUCLEOTIDES, AND METHODS OF THEIR USE

[0001] This application claims the priority benefit of U.S. Provisional Patent Application Serial No.63 / 580,632, filed September 5, 2023, which is hereby incorporated by reference in its entirety.

[0002] This invention was made with government support under NS111631 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD

[0003] The present disclosure relates to minigene cassettes, polynucleotides encoding such minigene cassettes, and methods of use thereof. BACKGROUND

[0004] Numerous systems have been developed for small molecule control of gene expression. These systems are primarily designed to control the expression of transgenes, such as the widely used Tet-On / Off system (Gossen and Bujard, “Tight Control of Gene Expression in Mammalian Cells by Tetracycline-Responsive Promoters,” Proceedings of the National Academy of Sciences 89:5547-5551 (1992) and Gossen et al., “Transcriptional Activation by Tetracyclines in Mammalian Cells,” Science 268:1766-1769 (1995)). In the Tet-On / Off system, heterologous genes are expressed using a promoter that is engineered to bind to bacterial tetracycline-controlled DNA-binding proteins (Gossen and Bujard, “Tight Control of Gene Expression in Mammalian Cells by Tetracycline-Responsive Promoters,” Proceedings of the National Academy of Sciences 89:5547-5551 (1992) and Gossen et al., “Transcriptional Activation by Tetracyclines in Mammalian Cells,” Science 268:1766-1769 (1995)). These, and related systems, can be used to induce the expression of diverse transgenes.

[0005] In contrast to transgenes, it remains challenging to control the expression of endogenous genes with small molecules. In contrast to the promoters used by transgenes, endogenous promoter elements are controlled by diverse aspects of chromatin organization, which ensure tissue-specific, developmental, and regulated patterns of gene expression. Therefore, it is desirable to use small molecules to control endogenous genes, and thus determine the magnitude and duration of endogenous patterns of gene expression.

[0006] The main system to control endogenous gene expression is the CRISPRi / a system in which dCas9 fusion proteins are recruited to endogenous genomic loci using a guideRNA (Gilbert et al., “CRISPR-Mediated Modular RNA-Guided Regulation of Transcription inEukaryotes,” Cell 154:442-451 (2013); Qi et al., “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression,” Cell 152:1173-1183 (2013); Mali et al., “CAS9 Transcriptional Activators for Target Specificity Screening and Paired Nickases for Cooperative Genome Engineering,” Nat Biotechnol 31:833-838 (2013); Perez-Pinera et al., “RNA-Guided Gene Activation by CRISPR-Cas9-Based Transcription Factors,” Nat. Methods 10:973-976 (2013); and Maeder et al., “CRISPR RNA-Guided Activation of Endogenous Human Genes,” Nat. Methods 10:977-979 (2013)). The dCas9 protein is modified to contain transcription-regulatory elements that either increase or decrease gene expression. This system is cumbersome because it requires expression of protein components and is not readily controlled by small molecules. An ideal system would involve no additional foreign proteins, and be regulated solely by a highly selective small molecule.

[0007] The present invention is directed to overcoming these and other deficiencies in the art. SUMMARY

[0008] One aspect of the present disclosure relates to a minigene cassette comprising, from 5’ to 3’: a first intron, an alternatively-spliced exon, and a second intron comprising a U1 binding sequence, where (i) a portion of the alternatively-spliced exon is complementary to a portion of the second intron; the portion of the alternatively-spliced exon that is complementary to the portion of the second intron forms a hairpin loop structure comprising a hairpin stem and a hairpin loop; the hairpin stem comprises at least a portion of the U1 binding sequence; and the hairpin loop comprises an aptamer or (ii) a portion of the second intron forms a hairpin loop structure comprising a hairpin stem and a hairpin loop; the hairpin stem comprises at least a portion of the U1 binding sequence; and the hairpin loop comprises an aptamer.

[0009] Another aspect of the present disclosure relates to a recombinant polynucleotide comprising: a gene of interest and a minigene cassette according to the present disclosure for regulating expression of the gene of interest, where the minigene cassette is located within the protein coding sequence of the gene of interest.

[0010] Another aspect of the present disclosure relates to a recombinant polynucleotide comprising: a gene of interest and a minigene cassette according to the present disclosure for regulating expression of the gene of interest, where the minigene cassette is located upstream of the gene of interest.

[0011] Also disclosed are vectors comprising a minigene cassette or recombinant polynucleotide according to the present disclosure.

[0012] Also disclosed are cells comprising a recombinant polynucleotide of the present disclosure.

[0013] Another aspect of the present disclosure is directed to a method of modulating the expression of a gene of interest. This method involves introducing a recombinant polynucleotide or vector of the present disclosure into a cell and culturing the cell, where: (i) in the absence of a ligand specific for the aptamer located in the hairpin loop of the minigene cassette, transcription of the recombinant polynucleotide results in the production of a spliced transcript which includes the alternatively-spliced exon of the minigene cassette and allows for the translation of a truncated gene of interest or prevents translation of the gene of interest and (ii) in the presence of the ligand specific for the aptamer located in the hairpin loop of the minigene cassette, transcription of the recombinant polynucleotide results in the production of a spliced transcript which excludes the alternatively-spliced exon of the minigene cassette and allows for the translation of a functional gene of interest. The method further involves contacting the cell with the ligand that specifically binds said hairpin loop in an amount effective to induce dose- dependent transcription and translation of the functional gene of interest.

[0014] Another aspect of the present disclosure relates to a method of treating or preventing a disease or condition in a subject in need thereof. This method involves administering to the subject a recombinant polynucleotide or vector according to the present disclosure and administering to the subject a ligand that specifically binds said aptamer in the hairpin loop in an amount effective to induce dose-dependent transcription and translation of the functional gene of interest, where: (i) in the absence of the ligand specific for the aptamer in the hairpin loop of the minigene cassette, transcription of the recombinant polynucleotide results in the production of a spliced transcript which includes the alternatively-spliced exon of the minigene cassette and allows for the translation of a truncated gene of interest or prevents translation of the gene of interest; (ii) in the presence of the ligand specific for the aptamer in the hairpin loop of the minigene cassette, transcription of the recombinant polynucleotide results in the production of a spliced transcript which excludes the alternatively-spliced exon of the minigene cassette and which allows for the translation of a functional gene of interest; and (iii) administering the ligand that specifically binds said aptamer in the hairpin loop is effective to treat the disease or condition.

[0015] The Examples of the present disclosure describe a “portable” poison exon that can be inserted into genes to allow small molecule control of endogenous gene expression, without the need for any foreign protein. Poison exons are exons that contain a premature stop codon. Poison exons are normally regulated by splicing factors to enable their alternative splicing, thuscontrolling expression of the intended protein. The naturally occurring alternative splicing of exon 7 in SMN1 (survival of motor neuron 1) was adapted into a highly efficient small molecule- controlled poison exon. Exon 7 contains a hairpin structure that blocks access to the U1 component of the spliceosome. This hairpin structure was modified to contain an aptamer that binds acyclovir, a non-toxic FDA-approved small molecule. The modified exon 7, along with engineered intronic sequences derived from SMN1, together act as the Cyclone (acyclovir- controlled poison exon) cassette that can be inserted into diverse genes. The Examples of the present disclosure demonstrate that Cyclone enables tunable, reversible, and dynamic expression of transgenes and endogenous genes with acyclovir. Overall, the Examples demonstrate small molecule control of an endogenous gene regulatory splicing event by modifying a natural RNA structure with an RNA aptamer, thus achieving simple and efficient small molecule control of gene expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIGS.1A–1I illustrate the conversion of SMN1 exon 7 into a portable poison exon. FIG.1A is a schematic of the alternative splicing of an endogenous poison exon. The poison exon (rectangle marked “PE”) contains a premature stop codon (hexagon). Alternative splicing (dashed lines) is controlled by splicing factors (indicated above arrows), such as serine / arginine-rich splicing factors. The splicing isoform that retains the poison exon produces truncated proteins due to premature termination. The splicing isoform in which the poison exon is skipped produces full-length proteins. FIG.1B is a schematic of a poison exon controlled by the small molecule acyclovir. Acyclovir binds to the acyclovir aptamer (hairpin loop) and controls a sequence (hairpin stem) that is essential for poison exon splicing. FIG.1C is a schematic of the chemical structure of acyclovir. FIG.1D is a schematic showing the sequence and secondary structure of the acyclovir aptamer (5’-ACCGGAAUAA CCGAAGUAGU UUAUGGGCUA CCGAAAUCCG GU-3’; SEQ ID NO: 1). FIG.1E is a schematic of the splicing pattern of SMN1 exon 7. The SMN1 exon 7 is skipped ~6% of the time due to the partial sequestration of the U1-binding sequence (in the hairpins step, indicated by open arrowhead above intron 7). A stem-loop structure, primarily within exon 7, overlapping the exon 7 and intron 7 junction sequesters two nucleotides in intron 7, which comprise the first two nucleotides of the U1-binding sequence. FIG.1F is a schematic showing the sequence and secondary structure of the terminal stem-loop 2 (TSL2), which contains the 5’ splice site (5’ ss) and the U1- binding sequence (5’-guaagu-3’). The exon sequence is in uppercase, and the intron sequence is in lowercase (5’-UCACAUUCCU UAAAUUAAGG Aguaaguc-3’; SEQ ID NO:2). FIG.1G is a schematic showing the repurposing SMN1 exon 7 as a poison exon by inserting the intron 6trunc-exon 7-intron 7 cassette into Renilla luciferase. Two splicing isoforms are expected due to TSL2. Luminescence measurements showed inhibition of luciferase expression due to exon 7 retention. Renilla luciferase (RLuc) was normalized to Firefly luciferase (FLuc). TSL2 measurement was normalized to the no-poison-exon control (n = 3, ****P < 0.0001). RT- PCR using primers annealing adjacent to the cassette confirmed the two splicing isoforms. FIG. 1H is a schematic showing that modified TSL2 showed disruption of base pairing by six point mutations (bold) in modified TSL1 (5’-UCACAGCCAA GCAAUUAAGG Aguaaguc-3’; SEQ ID NO: 3). Luminescence measurements showed greater inhibition of luciferase expression (n = 3, ****P < 0.0001). RT-PCR showed a single isoform corresponding to exon 7 retention. Data in FIG.1G and FIG.1H are presented as mean ± s.d. n = number of biologically independent replicates. Statistics were analyzed by an unpaired t test. FIG.1I is a schematic showing the relative positions of Hairpin 1 (5’-AGACUUAUUC CUUAAAUUAA GGAguaaguc u-3’; SEQ ID NO: 4) and Hairpin 2 (5’-GGAguaaguc ugcgcgaaag cgcagacuua c-3’; SEQ ID NO: 5) to exon 7. Hairpin 1 (gray) is at the exon 7-intron 7 junction. Hairpin 2 (black) is in intron 7. The sequences and secondary structures of Hairpins 1 and 2 show complete sequestration of the U1-binding sequence (identified in bracketed text). RT-PCR showed one splicing isoform corresponding to exon 7 skipping. Luminescence measurements showed restoration of luciferase expression (n = 3, **P = 0.0032, ****P < 0.0001). Data are presented as mean ± s.d. n = number of biologically independent replicates. Statistics were analyzed by one-way ANOVA.

[0017] FIGS.2A–2H show control of SMN1 exon 7 skipping with an acyclovir aptamer. FIG.2A is a schematic of hairpin-aptamer fusions. Terminal loops and base pairs were removed from Hairpins 1 and 2. The stem of the acyclovir aptamer (5’-ACCGGAAUAA CCGAAGUAGU UUAUGGGCUA CCGAAAUCCG GU-3’; SEQ ID NO: 1) was shortened. Ac-Hairpin 1 (5’- AGACUUAUAA UAACCGAAGU AGUUUAUGGG CUACCGAAAU guaagucu-3’; SEQ ID NO: 6) has the aptamer in the exon (uppercase). Ac-Hairpin 25’-GGAguaaguc uaauaaccga aguaguuuau gggcuaccga aauagacuua c-3’; SEQ ID NO: 7) has the aptamer in the intron (lowercase). Acyclovir is expected to bind to the aptamer and enhance hairpin stability, leading to exon 7 skipping. FIG.2B is graph showing luminescence measurements in the presence and absence of acyclovir (500 µM). Poison exons containing Ac-Hairpin 1 and Ac- Hairpin 2 were inserted into Renilla luciferase to test for acyclovir-dependent gene activation in HEK293T cells. Dual-luciferase assay was used for normalization. Each measurement was then normalized to the no-poison-exon control (n = 3, ***P = 0.0003, ns = not significant). FIG.2Cshows a panel of transducer (shaded box) designs. Rationally designed transducer sequences were inserted between the aptamer and the hairpin to finely adjust the ligand-induced change in stem stability. Transducers 1-6 contained additional base pairs (purple). The base pair composition was altered in Transducers 7-12 through point mutations indicated by shaded circles. FIG.2D are bar graphs of luminescence measurements showing the gene regulatory performances of the transducers in HEK293T cells (n = 3, Transducer 1, ***P = 0.0002; Transducer 7, ***P = 0.0001). FIG.2E is a schematic showing the secondary structure of the winning candidate Ac-Hairpin 2 Transducer 7 (Ac-H2Tr7) (5’-GGAguaagug uaauaaccga aguaguuuau gggcuaccga aauacacuua c-3’; SEQ ID NO: 8) predicted by UNAfold. Data are presented as mean ± s.d. n = number of biologically independent replicates. Statistics were analyzed by multiple unpaired t tests. FIG.2F shows the secondary structure of the acyclovir aptamer (5’-AGGUGCUCAC AGCCAAGCAA UUAAGGAGUA AGUGUAAUAA CCGAAGUAGU UUAUGGGCUA CCGAAAUACA CUUACGCCAG CAUUAUGAAA G-3’; SEQ ID NO: 9) and the stem containing the U1-binding sequence (P1) overlaid with acyclovir- induced changes in DMS reactivity. DMS reactivities and M2 heatmaps (see FIGS.2G–2H) were used to generate the secondary structure model. Acyclovir-induced changes in DMS reactivity are depicted on a continuous gradient from blue (representing a large decrease in DMS reactivity) to grey (no change in DMS reactivity) to red (representing a large increase in DMS reactivity). FIGS.2G–2H show M2 DMS MaP heatmaps of mutagenized Ac-H2Tr7, both without acyclovir (FIG.2G) and with acyclovir (FIG.2H). The "Mutation Position" (row) represents the chemical reactivities at each nucleotide in the cassette when a mutation is introduced at the corresponding position. The "Mapped Position" (column) represents the Z- score normalized chemical reactivities at the specified position. Z-score normalization allows for the visualization of the mutation positions that caused the largest changes in chemical reactivity at the mapped nucleotides, indicating potential interactions between these nucleotides. Only positive Z-scores are plotted, using a gradient from 1 (white) to 10 (black).

[0018] FIGS.3A–3G show the design of a poison exon cassette that functions across different cell lines. FIG.3A is a schematic showing the mechanism of acyclovir-induced poison exon skipping and the performance of Ac-H2Tr7 in HeLa, Huh-7, and HT-1080 cells (n = 3, HeLa ***P = 0.0002, Huh-7 ***P = 0.0005, HT-1080 ****P < 0.0001). Data are presented as mean ± s.d. n = number of biologically independent replicates. Statistics were analyzed by multiple unpaired t tests. FIG.3B is a list of introns reported to be highly efficient. Introns longer than 300 nt were shortened by fusing the first 100 nt and the last 150 nt of each intron. FIG.3C is a schematic of an intron screening assay similar to the one in FIG.8B. Introns wereinserted into Renilla luciferase at positions that recapitulate native splice sites. Efficient splicing removes the intron to produce full-length proteins. FIG.3D is a bar graph showing luciferase expression normalized to the intronless control as a percentage of expression (n = 3, No intron vs. HBB intron 2, ****P < 0.0001, No intron vs. β-globin / IgG, ****P < 0.0001). Data are presented as mean ± s.d. n = number of biologically independent replicates. Statistics were analyzed by one-way ANOVA. FIG.3E is a schematic of replacing SMN1 introns with intron fragments from the HBB intron 2 or β-globin / IgG intron. A panel of six constructs was generated. “In” stands for intron. FIG.3F shows the results of a screen for lower background and improved ligand response in Huh-7 and HT-1080 cells (n = 3, Ac-H2Tr7 (Huh-7) **P = 0.0003, Ac-H2Tr7In4 (Huh-7) **P = 0.0005, Ac-H2Tr7 (HT-1080) ***P < 0.0001, Ac- H2Tr7In4 (HT-1080) ***P < 0.0001). Data are presented as mean ± s.d. n = number of biologically independent replicates. Statistics were analyzed by multiple unpaired t tests. FIG. 3G is a bar graph showing luciferase expression controlled by Ac-H2Tr7In4 in the absence and presence of acyclovir (500 µM) in six cell lines (n = 3, U-2 OS ***P = 0.0003, HCT 116 ***P = 0.0001, ****P < 0.0001). Data are presented as mean ± s.d. n = number of biologically independent replicates. Statistics were analyzed by multiple unpaired t tests.

[0019] FIGS.4A – 4G show the characterization of tunable and reversible expression. FIG.4A is a bar graphs showing the results of splicing analysis of Ac-H2Tr7-regulated Renilla luciferase. The upper panel shows a schematic representation of acyclovir-induced alternative splicing. The bottom panel shows a representative RT-PCR splicing assay, demonstrating the skipping of the poison exon in response to increasing acyclovir concentrations. cDNAs were amplified from HEK293T cells treated with acyclovir for 48 hours after transfection. Alternative splicing isoforms were detected using primers flanking Ac-H2Tr7. FIG.4B is a bar graphs showing dose-dependent luciferase expression controlled by Ac-H2Tr7 or Ac-H2Tr7In4. HEK293T cells were treated with acyclovir for 48 hours after transfection. The upper panel shows representative luminescence measurements. Renilla luciferase expression was normalized to Firefly luciferase and then expressed as a percentage relative to the control without any poison exon. Data are presented as the mean ± s.d. of three biological replicates. The bottom panel shows a representative western blot for Ac-H2Tr7-regulated Renilla luciferase tagged with N- terminal 3xFLAG. β-tubulin was used as the loading control. FIG.4C is a bar graph showing dose-dependent luciferase expression by Ac-H2Tr7 under different promoters. Data are presented as individual replicates with means connected. FIG.4D is a schematic of EGFP splitting. EGFP was split into two exons between G462 and G463. Ac-H2Tr7 was inserted between the two EGFP exons. Expression of the entire gene cassette was driven by a CMVpromoter. FIG.4E is a fluorescence microscopy image showing dose-dependent EGFP expression monitored by fluorescence microscopy. HEK293T cells were treated with acyclovir for 24 hours after transfection and collected fluorescence images. At the same exposure time, increasing acyclovir concentrations activated higher levels of green fluorescence. Scale bar, 50 μm. FIG.4F shows a time course for acyclovir-induced poison exon skipping. Cells were cultured for 24 hours after transfection before treatment with acyclovir. Cells were collected at 2-hour intervals after drug treatment. cDNAs were generated from total RNAs and amplified using primers flanking Ac-H2Tr7. The splicing analysis shows full induction at 6 hours after adding acyclovir. FIG.4G shows a time course for reversing poison exon skipping. Cells were cultured in acyclovir for 24 hours after transfection. Drug was removed by changing the media, then collected cells at 2-hour intervals for splicing analysis. RT-PCR was performed as in FIG. 4F. The representative gel shows that exon skipping is reversed 6 hours after washing out the drug.

[0020] FIGS.5A–5I demonstrate regulation of diverse genes. FIG.5A is a schematic of Ac-H2Tr7In4-regulated mouse thrombopoietin (Thpo). Thpo is expressed under the control of a CMV promoter. Thpo was split at one of two locations (between Q82 and D83, or between Q117 and V118) to produce two versions of regulated Thpo (FIG.13). FIG.5B is a bar graph showing dose-dependent Thpo expression was detected by ELISA. HEK293T cells were transiently transfected with plasmids encoding either of the two constructs. After treating the cells with acyclovir for 24 hours, the culture media containing the secreted Thpo protein was collected and the expression level was measured by ELISA. Data are presented as the mean ± s.d. of three biological replicates. FIG.5C is a schematic of Ac-H2Tr7-controlled Cre recombinase and a loxP-GFP Cre reporter N2a cell line. Ac-H2Tr7 was inserted into Cre between nucleotides G900 and G901 (FIG.13). Cre is C-terminally tagged with 3xFLAG and is expressed under the control of a CMV promoter. Cre activates the expression of GFP. FIG.5D is a representative western blot for Cre tagged with a C-terminal 3xFLAG using an anti-FLAG antibody. β-tubulin was used as the loading control. FIG.5E is an image showing acyclovir (62.5 µM)-induced GFP expression. N2a cells were treated with acyclovir for 48 hours. With the same exposure time, acyclovir activates GFP expression via regulation of Cre recombinase. Cell nuclei were labeled with Hoechst stain. Scale bar, 50 μm. FIG.5F is a bar graph showing quantification of N2a cells expressing GFP. Images from FIG.5E were collected and the green cells were manually counted to calculate the percentage of GFP-expressing cells over Hoechst- stained cells. Data are presented as the mean ± s.d. of three fields of view. FIG.5G is a schematic of a bicistronic cassette containing Ac-H2Tr7In4-controlled puromycin N-acetyltransferase (pac), P2A, and EGFP. Ac-H2Tr7In4 was inserted into pac between nucleotides G405 and G406 (FIG.13). The bicistronic cassette is expressed under the control of an EF‐1α promoter. FIG.5H is a representative western blot for puromycin N-acetyltransferase protein using an anti-puromycin N-acetyltransferase antibody. β-tubulin was used as the loading control. FIG.5I is an image showing regulation of EGFP by Ac-H2Tr7In4 in pac. HEK293T cells were treated with acyclovir (250 µM) for 48 hours. With the same exposure time, acyclovir activated EGFP expression. Scale bar, 50 μm.

[0021] FIGS.6A–6G show endogenous gene regulation by Pac-Cyclone. FIG.6A is a schematic of the Pac-Cyclone homology-directed repair (HDR) donor. This HDR donor contains an acyclovir-regulated puromycin selection marker, a P2A site, and a 3xFLAG epitope tag, all located between the left and right homology arms (300 bp). It is knocked into the N- terminus of the gene of interest to produce the protein of interest with a 3xFLAG tag and a puromycin N-acetyltransferase protein. The puromycin N-acetyltransferase protein is used to enrich edited cells. FIG.6B is a schematic of the knock-in design at the WTAP (WT1-associated protein) locus. Pac-Cyclone is inserted after the start codon (ATG, green) in exon 2. The sgRNA cuts adjacent to the insertion site (see FIG.15). The left homology arm comprises 300 bp before the start codon, overlapping with part of WTAP intron 1 and part of exon 2. The right homology arm comprises 300 bp after the start codon, overlapping with part of WTAP exon 2 and part of intron 2. The edited WTAP locus will have an acyclovir-controlled poison exon incorporated into the mRNA transcript. FIG.6C is a schematic of the knock-in design at the PCIF1 (phosphorylated CTD interacting factor 1) locus. This design is similar to the WTAP design, except for the specific homology arms. The PCIF1 start codon is located in exon 3. Therefore, the left homology arm overlaps with PCIF1 intron 2 and exon 3, while the right homology arm overlaps with PCIF1 exon 3, intron 3, and exon 4. FIG.6D is a schematic showing the workflow of Pac-Cyclone CRISPR-Cas9 knock-in. HCT 116 cells were seeded two days before transfection. Cas9 nuclease, sgRNA, and the ssDNA HDR donor were transfected in the presence of 250 µM acyclovir to maintain expression once Pac-Cyclone was inserted into the targeted genomic locus. Three days after transfection, puromycin selection (0.4 µg / ml) was initiated in the presence of 250 µM acyclovir. Puromycin selection was maintained for five days until all cells in the negative control had died. Cells were then recovered for two days in the presence of acyclovir (250 µM). This cell mixture contained both heterozygous and homozygous clones. Western blot experiments were performed (FIGS.6E–6F) to confirm Cyclone incorporation. Simultaneously, the cell mixture was subjected to clonal isolation (250 µM acyclovir) and genotyping by genomic PCR (FIGS.9A-9B). Homozygous clones wereexpanded (250 µM acyclovir), validated by western blot (FIG.6G), and frozen as aliquots. FIG. 6D was created with BioRender.com. FIG.6E is a western blot of the WTAP global cell mixture after puromycin selection, using anti-FLAG and anti-WTAP antibodies. GAPDH was used as the loading control. 3xFLAG-WTAP was expressed in a dose-dependent manner from the tagged allele. Cells were cultured at varying concentrations of acyclovir for two days. FIG.6F is a western blot of the PCIF1 global cell mixture after puromycin selection, using anti-FLAG and anti-PCIF1 antibodies. GAPDH was used as the loading control. 3xFLAG-PCIF1 was expressed in a dose-dependent manner from the tagged allele. Cells were cultured at varying concentrations of acyclovir for two days. FIG.6G is a representative western blot and splicing assay of the Cyclone-WTAP homozygous clone. 3xFLAG-WTAP is expressed in a dose- dependent manner. The expression level can be tuned to approach physiological levels, comparable to wild-type HCT 116 cells. Poison exon skipping was observed with increasing concentrations of acyclovir. Cells were cultured at varying concentrations of acyclovir for five days.

[0022] FIG.7 show isothermal titration calorimetry (ITC) analysis of the acyclovir aptamer.

[0023] FIGS.8A–8C show truncation of SMN1 introns for a portable poison exon cassette. FIG.8Ais a schematic of the splice sites and branch points of SMN1 intron 6 and intron 7. The internal sequence of intron 6 was removed when we designed the portable cassette. The lengths of the introns are marked using red letters. The schematic shows the insertion of SMN1 intron 6 and intron 7 into Renilla luciferase. Renilla luciferase was split into two exons in a way to provide the exonic sequences for forming the splice sites. Intron 6 was shortened to 200 nt by connecting the first 62 nt and the last 138 nt. FIG.8B is a schematic of an intron screening assay. If the intron can splice, the two exons will connect and produce luciferase proteins. The Renilla (RLuc) signal was normalized to the Firefly (FLuc) signal and then to the no-intron control as a percentage of expression (n = 3, No intron vs. Intron 6trunc, **P = 0.0083, No intron vs. Intron 7, **P = 0.0080). Data are presented as mean ± s.d. n = number of biologically independent replicates. Statistics were analyzed by one-way ANOVA. FIG.8C, is a schematic of inserting the SMN1 intron6trunc-exon 7-intron 7 cassette into Renilla luciferase. Renilla luciferase was split into two exons between methionine and valine to recapitulate the SMN1 splice sites. The cassette was inserted in-frame with the stop codon in exon 7.

[0024] FIGS.9A–9B are genomic PCR gel images for identifying clones with successful knock-ins. WTAP genomic PCR was performed using primers that anneal adjacent to the knock- in insertion site. Successful incorporation of the HDR donor sequence results in an upper band,with the shift in molecular weight matching the size of the HDR donor. For comparison, each gel image contains the amplicons of wild-type HCT 116 cells and the global pool after puromycin selection. 28 clones were isolated and tested for WTAP knock-in. Clones 1, 19, and 24 appear to be homozygous knock-ins (FIG.9A). Clone 19 was used for further characterization. PCIF1 genomic PCR was performed using a protocol similar to WTAP genotyping. 23 clones were isolated and tested for PCIF1 knock-in (FIG.9B). Nearly half of the tested clones are heterozygous, and no homozygous clones were identified.

[0025] FIG.10 is a table providing sequences related to the SMN1 intron 6-exon 7-intron 7 poison exon cassette.

[0026] FIG.11 is a table providing sequences of highly efficient introns (see FIGS.3B– 3D).

[0027] FIG.12 is a table providing acyclovir-controlled poison exons.

[0028] FIG.13 is a table showing the sites within the coding sequences of several example genes for the insertion of poison exons.

[0029] FIG.14 is a table providing the sequences of Pac-Cyclone for endogenous gene tagging.

[0030] FIG.15 is a table providing the sequences of sgRNA, HDR donor, and primers for WTAP knock-in experiment.

[0031] FIG.16 is a table providing the sequences of sgRNAs, HDR donor, and primers for PCIF1 knock-in experiment. DETAILED DESCRIPTION

[0032] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present application herein described for which they are suitable as would be understood by a person skilled in the art.

[0033] Singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure.

[0034] The term “about” includes being within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the term “about” depends onthe particular system under study, and can be readily appreciated by one of ordinary skill in the art.

[0035] The term “and / or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of” or “one or more” of the listed items is used or present.

[0036] In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “involving”, “having”, and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The term “consisting essentially of”, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and / or steps. In embodiments or claims where the term comprising (or the like) is used as the transition phrase, such embodiments can also be envisioned with replacement of the term “comprising” with the terms “consisting of” or “consisting essentially of.” The compositions and / or methods of the present disclosure can comprise, consist essentially of, or consist of, the components disclosed.

[0037] Certain terms employed in the specification, examples, and claims are collected herein. Unless defined otherwise, all technical and scientific terms used in this disclosure have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0038] Preferences and options for a given aspect, feature, embodiment, or parameter of the disclosure should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features, embodiments, and parameters of the disclosure. Minigene Cassettes

[0039] One aspect of the present disclosure relates to a minigene cassette comprising, from 5’ to 3’: a first intron, an alternatively-spliced exon, and a second intron comprising a U1 binding sequence, where (i) a portion of the alternatively-spliced exon is complementary to a portion of the second intron; the portion of the alternatively-spliced exon that is complementaryto the portion of the second intron forms a hairpin loop structure comprising a hairpin stem and a hairpin loop; the hairpin stem comprises at least a portion of the U1 binding sequence; and the hairpin loop comprises an aptamer or (ii) a portion of the second intron forms a hairpin loop structure comprising a hairpin stem and a hairpin loop; the hairpin stem comprises at least a portion of the U1 binding sequence; and the hairpin loop comprises an aptamer.

[0040] As used herein, “stem-loop structure” refers to a nucleic acid having a secondary structure that includes a region of nucleotides which are known or predicted to form a double strand (stem portion) that is linked on one side by a region of predominantly single-stranded nucleotides (loop portion). Such structures are well known in the art and these terms are used consistently with their known meanings in the art. As is known in the art, a stem-loop structure does not require exact base-pairing. Thus, the stem may include one or more base mismatches. In some embodiments, the hairpin stem is an imperfect hairpin stem. Alternatively, the base- pairing may be “exact” or “perfect”, i.e. not include any mismatches. The hairpin stem may be a perfect hairpin stem or an imperfect hairpin stem.

[0041] As used herein, the term “exon” refers to a portion of a gene that is present in the mature form of mRNA. The exons include sequences that encodes a protein as well as 5’ and 3’ untranslated regions (UTRs). A “protein-coding exon” refers to an exon that encodes (or at least partially encodes) a protein (or part of a protein).

[0042] As used herein, the term “intron” refers to any nucleic acid sequence that is transcribed but not translated, as encompassed by a gene (or an expressed polynucleotide sequence of interest). Introns include the expression sequences of DNA and the non-translated nucleic acid sequences within the corresponding sequences of the RNA molecules transcribed therefrom. Introns are removed from primary transcripts by cleavage at 5’ and 3’ splice sites, with recognition at the 5’ splice site mediated by the U1 small nuclear ribonucleoprotein (U1 snRNP) complex. In some embodiments of the minigene cassette according to the present disclosure, the second intron comprises a 5’ splice site (5’ ss) and the hairpin loop structure comprises the 5’ ss.

[0043] As used herein, the term “U1 binding sequence” refers to a nucleic acid sequence that binds to (i.e., is recognized by) the U1 spliceosomal RNA component of the snRNP complex. In some embodiments of the minigene cassette according to the present disclosure, the hairpin stem comprises at least 1 to 6 nucleic acids of the U1 binding sequence. The U1 binding sequence may comprise the nucleic acid sequence of:5’-GURAGU-3’, where R is A or G.

[0044] As used herein, the term “aptamer” refers to a nucleic acid molecule that binds specifically to a target. Nucleic acid aptamers are single-stranded, partially single-stranded,partially double-stranded, or double-stranded nucleotide sequences, advantageously a replicatable nucleotide sequence, capable of specifically recognizing a selected non- oligonucleotide molecule or group of molecules by a mechanism other than Watson-Crick base pairing or triplex formation. Aptamers include, without limitation, defined sequence segments and sequences comprising nucleotides, ribonucleotides, deoxyribonucleotides, nucleotide analogs, modified nucleotides, and nucleotides comprising backbone modifications, branchpoints, and non-nucleotide residues, groups, or bridges. Nucleic acid aptamers include partially and fully single-stranded and double-stranded nucleotide molecules and sequences; synthetic RNA, DNA, and chimeric nucleotides; hybrids; duplexes; heteroduplexes; and any ribonucleotide, deoxyribonucleotide, or chimeric counterpart thereof and / or corresponding complementary sequence, promoter, or primer-annealing sequence needed to amplify, transcribe, or replicate all or part of the aptamer molecule or sequence.

[0045] In some embodiments of the minigene cassette according to the present disclosure, the aptamer binds a ligand selected from the group consisting of a small-molecule, a protein, a peptide, a cell, a virus, a bacterium, and a nucleic acid.

[0046] The aptamer may bind a ligand selected from the group consisting of acyclovir, valacyclovir, famciclovir, penciclovir, and ganciclovir. In some embodiments, the aptamer binds acyclovir. In some embodiments, the acyclovir aptamer comprises the nucleic acid sequence of SEQ ID NO: 49 (5’- NATAACCGAA GTAGNNNNNN NNCTACCGAA AN-3’). Suitable exemplary acyclovir aptamers include, without limitation, those shown in Table 1 below. Table 1. Exemplary Acyclovir Aptamer Sequences

[0047] In some embodiments, the acyclovir aptamer comprises SEQ ID NO: 50, SEQ ID NO: 51, or SEQ ID NO: 52.

[0048] Inclusion of an alternatively-spliced exon comprising a stop codon into a RNA transcript may result in the production of truncated or nonfunctional proteins (see, e.g., Ni et al., “Ultraconserved Elements are Associated with Homeostatic Control of Splicing Regulators by Alternative Splicing and Nonsense-Mediated Decay,” Genes Dev 21:708-718 (2007) and Lareau et al., “Unproductive Splicing of SR Genes Associated with Highly Conserved and Ultraconserved DNA Elements,” Nature 446: 926-929 (2007), which are hereby incorporated by reference in their entirety). In some embodiments of the minigene cassette according to thepresent disclosure, the alternatively-spliced exon encodes a stop codon, a start codon, a polyadenylation signal sequence, one or more domains that encode a protein sequence of interest, a 5’ untranslated region sequence element, and / or a 3’ untranslated region sequence element.

[0049] When the alternatively-spliced exon encodes a stop codon, the stop codon may be an in-frame stop codon. Suitable “stop codons” are well known in the art and include, but are not limited to, UAA known as ochre, UAG known as amber, and UGA known as opal, which do not code for an amino acid but act as signals for the termination of protein synthesis. In some embodiments, the alternatively-spliced exon comprises multiple stop codons. For example, the alternatively-spliced exon may comprise one, two, three, or more stop codons.

[0050] In some embodiments of the minigene cassette according to the present disclosure, the alternatively-spliced exon does not encode a stop codon. In accordance with such embodiments, inclusion of the alternatively-spliced exon into a RNA transcript may result in the production of a frameshift mutation. As used herein, the term “frameshift mutation” refers to a type of genetic mutation in which the addition or deletion of one or more polynucleotides results in a shift in the reading frame of a codon in an mRNA. The shift in reading frame can result in an alteration of the amino acid sequence in protein translation, such as a missense mutation or a premature stop codon. In some embodiments, the alternatively-spliced exon may result in a frame shift mutation in a downstream exon.

[0051] In some embodiments of the minigene cassette according to the present disclosure, the alternatively-spliced exon may contain a start codon.

[0052] In some embodiments of the minigene cassette according to the present disclosure, the first intron contains an in / out of frame stop codon. In accordance with such embodiments, when the alternatively-spiced exon contains a start codon, inclusion of the alternatively-spliced exon in an mRNA transcript may result in expression of a protein of interest.

[0053] As used herein, the term “transducer” or “transducer region” or “transducer domain” refers to a portion of a nucleic acid molecule that connects a first portion of a nucleic acid molecule to a second portion of a nucleic acid molecule. The transducer region may comprise a plurality of nucleic acid molecules that form a secondary structure such as a hairpin stem. For example, the transducer region may form a hairpin stem comprising a plurality of base pairs. The transducer region may comprise 1–20 base pairs. In some embodiments, the transducer region may comprise 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more base pairs.

[0054] In some embodiments of the minigene cassette according to the present disclosure, the hairpin stem comprises a transducer region. In accordance with such embodiments, the transducer region may comprise 1–20 base pairs. In some embodiments, the transducer region comprises 6–20 base pairs. For example, the transducer region may comprise a first portion comprising any one of the transducer sequences shown in Table 2 and a second portion which is the reverse complement of the first portion. Suitable exemplary transducer region sequences and their reverse complement sequences are shown in Table 2 below and FIG. 2C. Table 2. Suitable Exemplary Transducer Sequences

[0055] The transducer region may comprise a first portion comprising a transducer region sequence from Table 2 and a second portion which is the reverse complement of the first portion. Thus, in some embodiments, the minigene cassette comprises, from the 5’ to 3’ direction, a 5’ transducer region sequence (e.g., a transducer region sequence of Table 2), an aptamer sequence (e.g., an acyclovir aptamer sequence), and a 3’ transducer region sequence which is the reverse complement of the 5’ transducer region sequence. Exemplary transducer- acyclovir aptamer-transducer reverse complement sequences include, without limitation, SEQ ID NOs: 53-64, as shown in Table 2 supra.

[0056] The first intron may be between 50 to 300 nucleotides in length. In some embodiments, the first intron is between 50 to 75 nucleotides in length, 50 to 100 nucleotides in length, 50 to 150 nucleotides in length, 50 to 200 nucleotides in length, 50 to 250 nucleotides inlength, 100 to 200 nucleotides in length, 100 to 250 nucleotides in length, 100 to 300 nucleotides in length, 150 to 200 nucleotides in length, 200 to 250 nucleotides in length, 250 to 300 nucleotides in length, 230 to 260 nucleotides in length, or any amount therebetween.

[0057] Suitable exemplary first intron sequences are shown in Table 3 below. Table 3. Suitable Exemplary First Intron Sequences

[0058] In some embodiments, the first intron comprises SEQ ID NO: 10, SEQ ID NO: 68, or SEQ ID NO: 65.

[0059] The alternatively-spliced exon may be between 20 to 100 nucleotides in length. In some embodiments, the alternatively-spliced exon is about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 nucleotides in length. In some embodiments, the alternatively-spliced exon is between 50 to 54 nucleotides in length.

[0060] Suitable exemplary alternatively-spliced exon sequences are shown in Table 4 below. Table 4. Suitable Exemplary Alternatively-Spliced Exon Sequences

[0061] In some embodiments, the alternatively-spliced exon comprises SEQ ID NOs: 66 or 67.

[0062] The second intron may be between 250 to 500 nucleotides in length. In some embodiments, the second intron is between 250 to 450 nucleotides in length, 250 to 400 nucleotides in length, 250 to 300 nucleotides in length, 300 to 450 nucleotides in length, 300 to 400 nucleotides in length, 350 to 400 nucleotides in length, or any amount therebetween.

[0063] Suitable exemplary second intron sequences are shown in Table 5 below. Table 5. Suitable Exemplary Second Intron Sequences

[0064] In some embodiments, the second intron comprises the nucleic acid sequence of SEQ ID NO: 72, SEQ ID NO: 11, SEQ ID NO: 69, SEQ ID NO: 70, or SEQ ID NO: 71.

[0065] One embodiment of the second intron of the minigene cassette according to the present disclosure is illustrated in FIG.2E. FIG 2E shows an exemplary second intron (i.e., Ac- Hairpin 2 Transducer 7 (Ac-H2Tr7); SEQ ID NO: 8) comprising a 5’ splice site, a U1 binding sequence (5’-GUAAGU-3’), and an acyclovir aptamer. As illustrated in FIG.2E, a portion of the second intron forms a hairpin loop structure comprising a hairpin stem and a hairpin loop; the hairpin stem comprises at least a portion of the U1 binding sequence; and the hairpin loop comprises an aptamer (e.g., an acyclovir aptamer).

[0066] The minigene cassette may comprise the nucleic acid sequence of any one of SEQ ID NOs: 73 to 85 (as shown in Table 6).Table 6. Suitable Exemplary SMN1 Intron 6trunc-Exon 7-Intron 7 Cassette Sequences

[0067] In some embodiments of the minigene cassette according to the present disclosure, the first intron, alternatively-spliced exon, and / or second intron may be derived from one or more genes selected from the group consisting of survival motor neuron 1 (SMN1), SMN2, HGH, PS2, Tau, Hnrnpa1, Ncam, IgM, β-TM, and β-globin.

[0068] In some embodiments, the first intron, alternatively-spliced exon, and / or second intron are derived from the same gene. In accordance with such embodiments, the first intron and the second intron may be derived from a human β-globin gene. For example, the first intron and the second intron may be derived from intron 2 of the human β-globin gene.

[0069] In some embodiments of the minigene cassette according to the present disclosure, wherein the first intron and the second intron are derived from a human β-globin gene, the alternatively-spliced exon may comprise at least a portion of the group consisting of: exon 2 of the human dihydrofolate reductase gene, mutant human Wilms tumor 1 exon 5, mouse calcium / calmodulin - dependent protein kinase II delta exon 16, and SIRT1 exon 6.

[0070] In some embodiments of the minigene cassette according to the present disclosure, the first intron, alternatively-spliced exon, and / or second intron are synthetic, recombinant, or comprise heterologous sequences.

[0071] In some embodiments, the alternatively-spliced exon contains a synthetic, recombinant, or heterologous sequence encoding an epitope tag. For example, the alternatively- spliced exon may comprise an epitope tag sequence. Suitable epitope tag sequences are well known in the art (see, e.g., Kimple et al., “Overview of Affinity Tags for Protein Purification,” Curr. Protoc. Protein Sci.73:Unit 9.9 (2013), which is hereby incorporated by reference in its entirety) and include, without limitation, a FLAG tag, a 6xHIS tag, a V5 tag, a c-Myc tag, an HA tag, a GST tag, a Strep II tag, an E2 epitope tag, an HSV epitope tag, a KT3 epitope tag, a polyaspartate tag, a polycysteine tag, a polyphenylalanine tag, an S1 tag, an S tag, a Strep tag, a VSV-G tag, an AU1 epitope tag, and an AU5 epitope tag (see, e.g., Table 7).Table 7. Suitable Exemplary Epitope Tag Sequences

[0072] In some embodiments of the minigene cassette according to the present disclosure, the first intron, alternatively-spliced exon, and / or second intron are derived from a human gene.

[0073] In some embodiments of the minigene cassette according to the present disclosure, (i) the first intron comprises at least a portion of SMN1 intron 6 (SEQ ID NO: 10) or at least about 5, 6, 7, 8, 9, or 10 continuous nucleic acids of SMN1 intron 6 (SEQ ID NO: 10); (ii) the alternatively-spliced exon comprises at least a portion of SMN1 exon 7 (SEQ ID NO: 66) or at least about 5, 6, 7, 8, 9, or 10 continuous nucleic acids of SMN1 exon 7 (see SEQ ID NO: 66); and / or (iii) the second intron comprises at least a portion of SMN1 intron 7 (SEQ ID NO: 94) or at least about 5, 6, 7, 8, 9, or 10 continuous nucleic acids of SMN1 intron 7 (see SEQ ID NO: 94).

[0074] The minigene cassette according to the present disclosure may further include an exon comprising a start codon at its 3’ end, wherein the exon comprising the start codon is immediately upstream of the first intron. In some embodiments, the exon comprising the startcodon comprises SEQ ID NO: 95 (5’- ATAATTCCCC CACCACCTCC CATATGTCCA GATTCTCTTG ATGATGCTGA TGCTTTGGGA AGTATGTTAA TTTCATGGTA CATGAGTGGC TATCATACTG GCTATTATAT G -3’).

[0075] In some embodiments of the minigene cassette according to the present disclosure, the exon comprising the start codon comprises a nucleotide sequence from a gene selected from the group consisting of survival motor neuron 1 (SMN1), SMN2, HGH, PS2, Tau, Hnrnpa1, Ncam, IgM, β-TM, and β-globin. In accordance with such embodiments, the exon comprising the start codon may comprise a nucleotide sequence from the same gene as the first intron sequence, the alternatively-spliced exon sequence, and / or the second intron sequence.

[0076] In some embodiments, the exon comprising the start codon comprises a synthetic sequence, recombinant sequence, or sequence heterologous to the first intron sequence, the alternatively-spliced exon sequence, and / or the second intron sequence.

[0077] The exon comprising the start codon may comprises a nucleotide sequence from a mammalian gene. For example, the mammalian gene may be a human gene. In some embodiments, the exon comprising the start codon may comprise at least a portion of SMN1 exon 6 (SEQ ID NO: 95) or at least 5, 6, 7, 8, 9, or 10 continuous nucleic acids of SMN1 exon 6 (SEQ ID NO: 95).

[0078] One embodiment of the minigene cassette according to the present disclosure is illustrated in FIG.2A. The top panel of FIG 2A (labeled “Hairpin 1 in exon”) shows a minigene cassette comprising, from 5’ to 3’: a first intron (shown as a black line labeled “6”), an alternatively-spliced exon (shown as a grey box labelled “7”) comprising a stop codon (shown as a hexagon labeled “stop”), and a second intron (shown as a black lined labeled “7”) comprising a U1 binding sequence, where (i) a portion of the alternatively-spliced exon is complementary to a portion of the second intron; the portion of the alternatively-spliced exon that is complementary to the portion of the second intron forms a hairpin loop structure comprising a hairpin stem and a hairpin loop; the hairpin stem comprises at least a portion of the U1 binding sequence (shown below the dashed lined marked “Truncate”); and the hairpin loop comprises an aptamer (e.g., an acyclovir aptamer comprising Acyclovir-Hairpin 1 (“Ac-Hairpin 1”; SEQ ID NO: 6).

[0079] Another embodiment of the minigene cassette according to the present disclosure is illustrated in FIG.2A. The top panel of FIG 2A (labeled “Hairpin 2 in intron”) shows a minigene cassette comprising, from 5’ to 3’: a first intron (shown as a black line labeled “6”), an alternatively-spliced exon (shown as a grey box labelled “7”) comprising a stop codon (shown as a hexagon labeled “stop”), and a second intron (shown as a black lined labeled “7”) comprising a U1 binding sequence, where (ii) a portion of the second intron forms a hairpin loop structurecomprising a hairpin stem and a hairpin loop; the hairpin stem comprises at least a portion of the U1 binding sequence; and the hairpin loop comprises an aptamer (e.g., an acyclovir aptamer such as Acyclovir-Hairpin 2 (“Ac-Hairpin 2”; SEQ ID NO: 7). Recombinant Polynucleotides

[0080] Another aspect of the present disclosure relates to a recombinant polynucleotide comprising: a gene of interest and a minigene cassette according to the present disclosure for regulating expression of the gene of interest, where the minigene cassette is located within the protein coding sequence of the gene of interest. In some embodiments, the alternatively-spliced exon does not comprise a stop codon in-frame with the gene of interest. In some embodiments of the recombinant polynucleotides according to the present disclosure, the alternatively-spliced exon does not comprise a stop codon in-frame with the target gene.

[0081] Another aspect of the present disclosure relates to a recombinant polynucleotide comprising: a gene of interest and a minigene cassette according to the present disclosure for regulating expression of the gene of interest, where the minigene cassette is located upstream of the gene of interest.

[0082] The terms “polynucleotide” and “nucleic acid molecule” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A nucleic acid molecule is represented by a nucleic acid sequence, which is primarily characterized by its base sequence. Polynucleotides may have any three-dimensional structure, and mya perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. “Recombinant polynucleotides” according to the present disclosure are nucleic acid molecules containing a combination of two or more genetic elements not naturally occurring together.

[0083] In some embodiments of the recombinant polynucleotides according to the present disclosure, the minigene cassette is positioned between the 5’ homology arm sequence and the 3’ homology arm sequence.

[0084] As used herein, the term “gene of interest” refers to any polynucleotide which encodes a polypeptide or protein of interest. In some embodiments of the recombinant polynucleotides according to the present disclosure, the gene of interest may be selected from the group consisting of a therapeutic protein, a reporter protein, an enzyme, a transcription factor, a cytokine, a chemokine, an antibody, a hormone, a transport protein, a growth factor, anintracellular protein, an extracellular protein, a cytoplasmic protein, a transmembrane protein, a secreted protein, a receptor molecule, an RNA binding protein, a DNA binding protein, a channel protein, a cell adhesion molecule, a kinase, a phosphatase, and a chaperone protein.

[0085] In some embodiments, the gene of interest is a therapeutic protein or peptide sequence.

[0086] Another aspect of the present disclosure relates to a recombinant polynucleotide comprising: a 5’ homology arm; a minigene cassette according to any one of claims 1 to 43; and a 3’ homology arm.

[0087] The term “homology arm” refers to a nucleic acid sequence which may direct insertion of a minigene cassette according to the present disclosure into a gene of interest (i.e., a target gene) by, for example, homologous recombination. Thus, as described in more detail infra, the recombinant polynucleotide may comprise a 5’ homology arm located 5’ to a minigene cassette according to the present disclosure, and a 3’ homology arm located 3’ to the minigene cassette.

[0088] The “homology arms” of the recombinant polynucleotides described herein are nucleotide sequences that are the same as or closely homologous (i.e., sharing significant sequence identity) to the nucleotide sequence of particular regions of the target gene, i.e., the gene in which the minigene cassette will be inserted into.

[0089] In some embodiments, the 5’ homology arm sequence and / or the 3’ homology arm sequence may be homologous to a gene of interest. In some embodiments, the 5’ homology arm and the 3’ homology arm targets a protein coding region of the gene of interest. In some embodiments, the gene of interest comprises a start codon.

[0090] The 5’ homology arm sequence and the 3’ homology arm sequence of the recombinant polynucleotide may be the same as or similar to the target gene sequence (e.g., the same as the sense strand of the target gene) immediately upstream and downstream of a start codon within the target gene.

[0091] In some embodiments, the 5’ homology arm sequence is homologous with a first portion of the gene of interest and the 3’ homology arm sequence is homologous with a second portion of the gene of interest. In some embodiments, the first portion of the gene of interest is adjacent to the second portion of the gene of interest.

[0092] In some embodiments, the 5’ homology arm sequence is homologous with a portion of the gene of interest 5’ to the start codon. In some embodiments, the 3’ homology arm sequence is homologous with a portion of the gene of interest 3’ to the start codon.

[0093] In some embodiments, the 5’ homology arm sequence is located at the 5’ end of the recombinant polynucleotide sequence. The percent identity between the 5’ homology arm sequence located at the 5’ end of the recombinant polynucleotide and the corresponding sequence of target gene (e.g., sense strand) may be at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%.

[0094] In some embodiments, the 3’ homology arm sequence is located at the 3’ end of the recombinant polynucleotide sequence. The percent identity between the 3’ homology arm sequence located at the 3’ end of the recombinant polynucleotide and the corresponding sequence of the target gene (e.g., sense strand) may be at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%.

[0095] In some embodiments, the 5’ homology arm sequence and the 3’ homology arm sequence (e.g., the 5’ and 3’ homology arms) are more than about 30 nucleotide residues in length, for example more than about any of 50 nucleotide residues, 100 nucleotide residues, 200 nucleotide residues, 300 nucleotide residues, 500 nucleotide residues, 800 nucleotide residues, 1,000 nucleotide residues, 1,500 nucleotide residues, 2,000 nucleotide residues, and 5,000 nucleotide residues in length. In some embodiments, the 3’ homology arm sequence and / or the 3’ homology arm sequence are approximately 300 nucleotides in length.

[0096] In some embodiments, the recombinant polynucleotide further comprises a nucleic acid sequence encoding a selection marker.

[0097] Suitable selection markers are known in the art, and include for example, antibiotic resistance genes (e.g., the puromycin resistance gene or the neomycin resistance gene), thymidine kinase, dihydrofolate reductase (together with methotrexate as a DHFR amplifier), aminoglycoside phosphotransferase, hygromycin B phosphotransferase, asparagine synthetase, adenosine deaminase, and metallothionein. In some embodiments, the antibiotic resistance gene is a puromycin resistance gene.

[0098] When the recombinant polynucleotide comprises a selection marker, the minigene cassette may be positioned within the nucleic acid sequence encoding the selection marker.

[0099] The recombinant polynucleotide according to the present disclosure may further comprise a nucleic acid sequence encoding a self-cleaving peptide. A “self-cleaving peptide” is a 18-22 amino-acid long viral oligopeptide sequence that mediates ribosome skipping during translation in eukaryotic cells (Liu et al., “Systemic Comparison of 2A peptides for Cloning Multi-Genes in a Polycistronic Vector,” Scientific Reports 7: Article Number 2193 (2017), which is hereby incorporated by reference in its entirety).

[0100] Exemplary self-cleaving peptides include, without limitation, porcine teschovirus- 12A (P2A), Foot and mouth disease virus 2A (F2A), thosea asigna virus 2A (T2A), equine rhinitis A virus 2A (E2A), cytoplasmic polyhedrosis virus (BmCPV 2A), and flacherie virus (BmIFV 2A). The nucleotide sequences encoding these self-cleaving peptides that are suitable for inclusion in the recombinant genetic construct described herein are provided in Table 8 below. Table 8. Suitable Exemplary Self-Cleaving Peptide Coding Nucleotide Sequences

[0101] When the recombinant polynucleotide comprises a nucleic acid sequence encoding a self-cleaving peptide, the nucleic acid sequence encoding the self-cleaving peptide may be positioned in a manner effective to mediate expression of an epitope tag.

[0102] In some embodiments, the recombinant polynucleotide further comprises a nucleic acid sequence encoding an epitope tag. Suitable exemplary epitope tags are described supra and include, without limitation, FLAG tag (e.g., a 3xFLAG tag), a 6xHIS tag, a V5 tag, a c-Myc tag, an HA tag, a GST tag, a Strep II tag, an E2 epitope tag, an HSV epitope tag, a KT3 epitope tag, a polyaspartate tag, a polycysteine tag, a polyphenylalanine tag, an S1 tag, an S tag, a Strep tag, a VSV-G tag, an AU1 epitope tag, and an AU5 epitope tag.

[0103] In some embodiments, the recombinant polynucleotide according to the present disclosure comprises: a 5’ homology arm sequence according to the present disclosure; a nucleicacid sequence encoding a selection marker according to the present disclosure positioned 3’ to the 5’ homology arm sequence; a minigene cassette according to the present disclosure positioned within the nucleic acid sequence encoding the selection marker; a nucleic acid sequences encoding a self-cleaving peptide according to the present disclosure positioned 3’ to the nucleic acid sequence encoding the selection marker; a nucleic acid sequence encoding an epitope tag according to the present disclosure positioned 3’ to the nucleic acid sequence encoding the self-cleaving peptide; and a 3’ homology arm sequence according to the present disclosure.

[0104] In some embodiments of the recombinant polynucleotides according to the present disclosure, the first and second introns are exogenous to the gene of interest.

[0105] FIGS.6A–6B are schematic illustrations of exemplary recombinant polynucleotides according to the present disclosure. As shown in FIG.6A, an exemplary recombinant polynucleotide may comprise, from the 5’ to the 3’ direction, a 5’ homology arm sequence (e.g., a homology arm sequence comprising 300 bp); a nucleic acid sequence encoding a selection marker (e.g., a puromycin N-acetyltransferase protein); a minigene cassette according to the present disclosure (e.g., an acyclovir-regulated minigene cassette) positioned within the nucleic acid sequence encoding the selection marker; a nucleic acid sequences encoding a self- cleaving peptide (e.g., P2A); a nucleic acid sequence encoding an epitope tag (e.g., a 3xFLAG tag); and a 3’ homology arm sequence (e.g., a homology arm sequence comprising 300 bp).

[0106] As shown in FIG.6B, an exemplary recombinant polynucleotide for insertion into the WTAP (WT1 associated protein) locus may comprise, from the 5’ to the 3’ direction, a 5’ homology arm sequence (e.g., a homology arm sequence comprising 300 bp before the start codon, overlapping with part of WTAP intron 1 and part of exon 2); a nucleic acid sequence encoding a selection marker (e.g., a puromycin N-acetyltransferase protein); a minigene cassette according to the present disclosure (e.g., an acyclovir-regulated minigene cassette) positioned within the nucleic acid sequence encoding the selection marker; a nucleic acid sequences encoding a self-cleaving peptide (e.g., P2A); a nucleic acid sequence encoding an epitope tag (e.g., a 3xFLAG tag); and a 3’ homology arm sequence (e.g., a homology arm sequence comprising 300 bp after the start codon, overlapping with part of WTAP exon 2 and part of intron 2).

[0107] As shown in FIG.6C, an exemplary recombinant polynucleotide for insertion into the PCIF1 (phosphorylated CTD interacting factor 1) locus may comprise, from the 5’ to the 3’ direction, a 5’ homology arm sequence (e.g., a homology arm sequence comprising 300 bp before the start codon, overlapping with part of PCIF1 intron 2 and exon 3); a nucleic acidsequence encoding a selection marker (e.g., a puromycin N-acetyltransferase protein); a minigene cassette according to the present disclosure (e.g., an acyclovir-regulated minigene cassette) positioned within the nucleic acid sequence encoding the selection marker; a nucleic acid sequences encoding a self-cleaving peptide (e.g., P2A); a nucleic acid sequence encoding an epitope tag (e.g., a 3xFLAG tag); and a 3’ homology arm sequence (e.g., a homology arm sequence comprising 300 bp after the start codon, overlapping with part of PCIF1 exon 3, intron 3, and exon 4). Vectors

[0108] In some embodiments, a minigene cassette according to the present disclosure or a recombinant polynucleotide according to the present disclosure is incorporated into a vector. Accordingly, vectors comprising a minigene cassette and / or recombinant polynucleotide of the present disclosure are also disclosed.

[0109] In some embodiments, the vector is an expression vector. For example, the expression vector may be a mammalian expression vector such as a pcDNA3.1 vector, a pCMV6 vector, or a pCAGGS expression vector (Niwa et al., “Efficient Selection for High-Expression Transfectants with a Novel Eukaryotic Vector,” Gene 108:193-200 (1991), which is hereby incorporated by reference in its entirety).

[0110] The vector may be a viral vector. Suitable viral vectors include, without limitation, adenoviral vectors, adeno-associated virus vectors, and lentiviral vectors.

[0111] As described herein, the minigene cassettes, recombinant polynucleotides, and / or vectors according to the present disclosure may be utilized to mediate site-specific integration into a gene of interest or an integration site. Methods of mediating site-specific integration into an integration site or gene of interest are well known in the art (see, e.g., Thyagarajan et al., “Site-Specific Genomic Integration in Mammalian Cells Mediated by Phage φC31 Integrase,” Mol. Cell. Biol.21(12):3926-3934 (2001) and Pandey et al., “Efficient Site-Specific Integration of Large Genes in Mammalian Cells via Continuously Evolved Recombinases and Prime Editing,” Nature Biomed. Eng.2024, which are hereby incorporated by reference in their entirety). Cells

[0112] Also disclosed are cells comprising a recombinant polynucleotide or vector according to the present disclosure. In some embodiments, the host cell is a eukaryotic host cell. Exemplary eukaryotic cells include a yeast cell, an insect cell, a fungal cell, a plant cell, and an animal cell (e.g., a mammalian cell). In some embodiments, the host cell is a mammalian cell.Suitable mammalian cells include, for example without limitation, human, non-human primate, cat, dog, sheep, goat, cow, horse, pig, rabbit, and rodent cells.

[0113] In some embodiments, the mammalian cell is a primary cell.

[0114] In some embodiments, the mammalian cell is a cell line. Suitable mammalian cell line cells include, but are not limited to, HeLa cells, U-2 OS cells, HCT 116 cells, Huh-7 cells, HT-1080 cells, human embryonic kidney (HEK) cells (e.g., HEK293 cells, HEK293T cells, HEK 293F cells), A549 cells, Chinese hamster ovary (CHO) cells (e.g., CHO Toronto / CHO pro 3, CHO K1, DG 44, DXB11, CHO K1 SV, CHOK1SV GS-KO), COS cells, PC3 cells, African Green Monkey kidney cells (e.g., Vero cells), baby hamster kidney (BHK) cells (e.g., BHK21), PER.C6®cells, and Madin-Darby canine kidney (MDCK) cells (e.g., MDCK cells, MDCK-HA cells, MDCK / London cells, MDCK.2 cells, MDCK SIAT-1 cells).

[0115] The above-described expression vectors may be introduced into appropriate host cells using conventional techniques in the art, e.g., transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection.

[0116] Once the vectors are introduced into cells, the cells are cultured in conventional nutrient media modified as appropriate for culturing the selected cells, selecting transfected cells, and / or monitoring expression of a gene of interest. Methods for expression of a gene of interest are known in the art, see, e.g., Paulina Balbas, Argelia Lorence (eds.) Recombinant Gene Expression: Reviews and Protocols (Methods in Molecular Biology), Humana Press; 2nd ed. 2004 (July 20, 2004) and Vladimir Voynov and Justin A. Caravella (eds.) Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology) Humana Press; 2nd ed.2012 (June 28, 2012), which are hereby incorporated by reference in their entirety. Methods of Modulating a Gene of Interest

[0117] Another aspect of the present disclosure is directed to a method of modulating the expression of a gene of interest. This method involves introducing a recombinant polynucleotide or vector of the present disclosure into a cell and culturing the cell, where: (i) in the absence of a ligand specific for the aptamer located in the hairpin loop of the minigene cassette, transcription of the recombinant polynucleotide results in the production of a spliced transcript which includes the alternatively-spliced exon of the minigene cassette and allows for the translation of a truncated gene of interest or prevents translation of the gene of interest and (ii) in the presence of the ligand specific for the aptamer located in the hairpin loop of the minigene cassette, transcription of the recombinant polynucleotide results in the production of a spliced transcript which excludes the alternatively-spliced exon of the minigene cassette and allows for the translation of a functional gene of interest. The method further involves contacting the cell withthe ligand that specifically binds said hairpin loop in an amount effective to induce dose- dependent transcription and translation of the functional gene of interest.

[0118] Suitable cells for carrying out the methods of the present disclosure include eukaryotic cells and are described in more detail supra. In some embodiments, the cell is a mammalian cell. Suitable mammalian cells include, for example without limitation, human, non-human primate, cat, dog, sheep, goat, cow, horse, pig, rabbit, and rodent cells. In some embodiments, the mammalian cell is a human cell.

[0119] In some embodiments of carrying out this method according to the present disclosure, the gene of interest is endogenous to the cell. The gene of interest may be down- regulated in a disease state, a stress state, or during a pathogen infection in a host cell. In some embodiments, the gene of interest is a chimeric or heterologous protein effective to treat the cell.

[0120] As described above, hairpin loop of the minigene cassette comprises an aptamer which may be bound by a ligand. Upon binding of the aptamer to its ligand, the U1-binding sequence in the second intron of the minigene cassette is sequestered by hybridization with a complementary strand, resulting in the exclusion of the alternatively-spliced exon from the transcript, which allows for the translation of a functional gene of interest.

[0121] In some embodiments of the methods according to the present disclosure, the ligand specific for the aptamer in the hairpin loop of the minigene cassette is selected from the group consisting of acyclovir, valacyclovir, famciclovir, penciclovir, and / or ganciclovir.

[0122] In some embodiments, the aptamer binds acyclovir. In accordance with such embodiments, the acyclovir aptamer may comprise the sequence of SEQ ID NO: 49 (NATAACCGAA GTAGNNNNNN NNCTACCGAA AN, where “N” can be any nucleic acid). For example, the acyclovir aptamer may comprise the sequence of SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52 (see, e.g., Table 1).

[0123] In some embodiments, expression of the gene of interest is at least about 2-fold to 1,000-fold higher when the ligand specific for the aptamer in the hairpin loop of the minigene cassette is present. In some embodiments, expression of the gene of interest is at least 10-fold higher, 20-fold higher, 30-fold higher, 40-fold higher, 50-fold higher, 60-fold higher, 70-fold higher, 80-fold higher, 90-fold higher, 100-fold higher, 200-fold higher, 300-fold higher, 400- fold higher, 500-fold higher, 600-fold higher, 700-fold higher, 800-fold higher, 900-fold higher, 1000-fold higher, or more when the ligand specific for the aptamer in the hairpin loop of the minigene cassette is present.

[0124] In some embodiments, expression of the gene of interest is at least about 10-fold higher, 50-fold higher, or 100-fold higher when the ligand specific for the aptamer in the hairpinloop of the minigene cassette is present. In some embodiments, expression of the gene of interest is at least about 300-fold higher when the ligand specific for the aptamer in the hairpin loop of the minigene cassette is present. In some embodiments, expression of the gene of interest is at least about 500-fold higher when the ligand specific for the aptamer in the hairpin loop of the minigene cassette is present.

[0125] As described above, the method involves contacting the cell with the ligand that specifically binds said hairpin loop in an amount effective to induce dose-dependent transcription and translation of the functional gene of interest. Contacting the cell with the ligand that specifically binds the hairpin loop may be carried out in vitro, in vivo, or in situ.

[0126] In some embodiments, the cell is contacted with about 1nM to about 2mM of a ligand according to the present disclosure. In some embodiments, the cell is contacted with about 10nM to about 2mM, 100nM to about 2mM, 1mM to about 2mM, 1nM to about 1mM, 1nM to about 100nM, 1nM to about 10nM, or any amount therebetween of a ligand according to the present disclosure.

[0127] In some embodiments, the cell is contacted with about 1μM to about 100μM of a ligand according to the present disclosure. In some embodiments, the cell is contacted with about 1μM to about 75μM, about 1μM to about 50μM, about 1μM to about 25μM, 10μM to about 90μM, 10μM to about 80μM, 10μM to about 70μM, 10μM to about 60μM, 10μM to about 50μM, 10μM to about 40μM, 10μM to about 30μM, 10μM to about 20μM, 15μM to about 90μM, 15μM to about 80μM, 15μM to about 70μM, 15μM to about 60μM, 15μM to about 50μM, 15μM to about 40μM, 15μM to about 30μM, 10μM to about 20μM, 20μM to about 40μM, or any amount therebetween of the ligand according to the present disclosure. In some embodiments, the cell is contacted with about 18.4 µM and 36.0 µM of the ligand. Methods of Treating or Preventing a Disease or Condition

[0128] Another aspect of the present disclosure relates to a method of treating or preventing a disease or condition in a subject in need thereof. This method involves administering to the subject a recombinant polynucleotide or vector according to the present disclosure and administering to the subject a ligand that specifically binds said aptamer in the hairpin loop in an amount effective to induce dose-dependent transcription and translation of the functional gene of interest, where: (i) in the absence of the ligand specific for the aptamer in the hairpin loop of the minigene cassette, transcription of the recombinant polynucleotide results in the production of a spliced transcript which includes the alternatively-spliced exon of the minigene cassette and allows for the translation of a truncated gene of interest or prevents translation of the gene of interest; (ii) in the presence of the ligand specific for the aptamer in thehairpin loop of the minigene cassette, transcription of the recombinant polynucleotide results in the production of a spliced transcript which excludes the alternatively-spliced exon of the minigene cassette and which allows for the translation of a functional gene of interest; and (iii) administering the ligand that specifically binds said aptamer in the hairpin loop is effective to treat the disease or condition.

[0129] The terms “treatment” or “treating” refer to therapy, prevention, and / or prophylaxis to reduce the extent of or likelihood of occurrence of a disease or condition. In some embodiments, the disease or condition is selected from the group consisting of a genetic disease or disorder, a neurological disease or disorder, a cardiovascular disease or disorder, an oncological disease or disorder, a lymphatic disease or disorder, a glandular disease or disorder, a viral infection, a bacterial infection, a fungal infection, a metabolic disorder such as diabetes, and obesity.

[0130] Suitable subjects include, without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. In some embodiments, the subject is a mammal (e.g., a human). The term does not denote a particular age. Thus, suitable subjects include newborn, infant, adolescent, adult, and geriatric individuals.

[0131] The recombinant polynucleotide, vector, or ligand according to the present disclosure may be administered to a subject by any mode of delivery including, without limitation, by parenteral administration, intravenous administration, intraperitoneal administration, oral administration, systemic administration, intramuscular administration, intravenous administration, subcutaneous administration, and intrathecal administration. In some embodiments, the ligand is administered by oral administration or by intravenous administration.

[0132] The compositions of the present disclosure may be administered as a single dose or in multiple doses (e.g., two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, nine doses, or more).

[0133] In some embodiments, the compositions of the present disclosure are administered in one, two, or three doses. In accordance with such examples, the timing between the doses can be at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 12 weeks, at least 2 months, at least 3 months, at least 4 months,at least 5 months, at least 6 months, at least 1 year, at least 2 years, or at least 5 years, such as 1-4 weeks, 2-3 weeks, 1-6 months, 2-4 months, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 12 weeks, 1 month, 2 months, 3, months, 4, months, 5 months, 6 months, 1 year, 2 years, 5 years, or 10 years, or combinations thereof (such as where there are at least three administrations, wherein the timing between the first and second, and second and third doses, can be the same or different).

[0134] The dose administered to the subject can depend on the species, age, weight, and general condition of the subject, as well as the particular composition being administered and its mode of administration. An appropriate dose can be determined by one of ordinary skill in the art. In some embodiments, the subject is administered about 1μg to about 1mg of a recombinant polynucleotide or vector according to the present disclosure. In some embodiments, the subject is administered about 1μg to about 100μg, about 1μg to about 50μg, or about 1μg to about 10μg of the recombinant polynucleotide or vector according to the present disclosure.

[0135] In some embodiments, the subject is administered a dose of about 100 mg to about 1000 mg of the ligand to the subject. In some embodiments, the dose administered to the subject is about 0.01–100 mg / kg, 0.01–90 mg / kg, 0.01–80 mg / kg, 0.01–70 mg / kg, 0.01–60 mg / kg, 0.01–50 mg / kg, 0.01–40 mg / kg, 0.01–30 mg / kg, 0.01–20 mg / kg, 0.01–10 mg / kg, 0.01–9 mg / kg, 0.01–8 mg / kg, 0.01–7 mg / kg, 0.01–6 mg / kg, 0.01–5 mg / kg, 0.01–4 mg / kg, 0.01–3 mg / kg, 0.01–2 mg / kg, 0.01–1 mg / kg, 0.05–10 mg / kg, 0.05–9 mg / kg, 0.05–8 mg / kg, 0.05–7 mg / kg, 0.05–6 mg / kg, 0.05–5 mg / kg, 0.05–4 mg / kg, 0.05–3 mg / kg, 0.05–2 mg / kg, 0.05–1 mg / kg, 0.1–10 mg / kg, 0.1–9 mg / kg, 0.1–8 mg / kg, 0.1–7 mg / kg, 0.1–6 mg / kg, 0.1–5 mg / kg, 0.1– 4 mg / kg, 0.1–3 mg / kg, 0.1–2 mg / kg, 0.1–1 mg / kg, 1–10 mg / kg, 2–10 mg / kg, 3–10 mg / kg, 4–10 mg / kg, 5–10 mg / kg, 6–10 mg / kg, 7–10 mg / kg, 8–10 mg / kg, 1–9 mg / kg, 1–9 mg / kg, 2–9 mg / kg, 3–9 mg / kg, 4–9 mg / kg, 5–9 mg / kg, 6–9 mg / kg, 7–9 mg / kg, 8–9 mg / kg, 1–8 mg / kg, 2–8 mg / kg, 3–8 mg / kg, 4–8 mg / kg, 5–8 mg / kg, 6–8 mg / kg, 7–8 mg / kg, 1–7 mg / kg, 2–7 mg / kg, 3–7 mg / kg, 4–7 mg / kg, 5–7 mg / kg, 6–7 mg / kg, 1–6 mg / kg, 2–6 mg / kg, 3–6 mg / kg, 4–6 mg / kg, 5–6 mg / kg, 1–5 mg / kg, 2–5 mg / kg, 3–5 mg / kg, 4–5 mg / kg, 1–4 mg / kg, 2–4 mg / kg, 3–4 mg / kg, 1–3 mg / kg, 2–3 mg / kg, 1–2 mg / kg, 0.05–2 mg / kg, or any amount therebetween. In some embodiments, the dose administered to the subject is about 5–10 mg / kg mass of the subject.

[0136] The gene of interest is selected from the group consisting of a therapeutic protein, a reporter protein, an enzyme, a transcription factor, a cytokine, a chemokine, an antibody, a hormone, a transport protein, a growth factor, an intracellular protein, an extracellular protein, a cytoplasmic protein, a transmembrane protein, a secreted protein, a receptor molecule, an RNAbinding protein, a DNA binding protein, a channel protein, a cell adhesion molecule, a kinase, a phosphatase, and a chaperone protein.

[0137] In some embodiments of the methods according to the present disclosure, the ligand specific for the aptamer in the hairpin loop of the minigene cassette is selected from the group consisting of acyclovir, valacyclovir, famciclovir, penciclovir, and / or ganciclovir.

[0138] In some embodiments, the aptamer bind acyclovir. In accordance with such embodiments, the acyclovir aptamer may comprise the sequence of SEQ ID NO: 49 (NATAACCGAA GTAGNNNNNN NNCTACCGAA AN, where “N” can be any nucleic acid). For example, the acyclovir aptamer may comprises the sequence of SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52 (see, e.g., Table 1).

[0139] In some embodiments, expression of the gene of interest is at least about 2-fold to 1,000-fold higher when the ligand specific for the aptamer in the hairpin loop of the minigene cassette is present. In some embodiments, expression of the gene of interest is at least 10-fold higher, 20-fold higher, 30-fold higher, 40-fold higher, 50-fold higher, 60-fold higher, 70-fold higher, 80-fold higher, 90-fold higher, 100-fold higher, 200-fold higher, 300-fold higher, 400- fold higher, 500-fold higher, 600-fold higher, 700-fold higher, 800-fold higher, 900-fold higher, 1000-fold higher, or more when the ligand specific for the aptamer in the hairpin loop of the minigene cassette is present.

[0140] In some embodiments, expression of the gene of interest is at least about 10-fold higher, 50-fold higher, or 100-fold higher when the ligand specific for the aptamer in the hairpin loop of the minigene cassette is present. In some embodiments, expression of the gene of interest is at least about 300-fold higher when the ligand specific for the aptamer in the hairpin loop of the minigene cassette is present. In some embodiments, expression of the gene of interest is at least about 500-fold higher when the ligand specific for the aptamer in the hairpin loop of the minigene cassette is present.

[0141] The method according to the present disclosure may further involve monitoring the subject for a predetermined period of time after administering the recombinant polynucleotide or the vector and determining the therapeutic effect of said administering the ligand that specifically binds said aptamer in the hairpin loop.

[0142] In carrying out the methods of the present disclosure, monitoring the subject may be carried out for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 12 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year, at least 2 years, or at least 5 years, such as 1-4 weeks, 2-3 weeks, 1-6 months, 2-4 months, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 12 weeks, 1 month, 2 months, 3, months, 4, months, 5 months, 6 months, 1 year, 2 years, 5 years, or 10 years, or more.

[0143] As used herein, the terms “modulation” or “modulating” refer to change, e.g., an increase or decrease in the dose and / or frequency of administration a recombinant polynucleotide, vector, or ligand according to the present disclosure.

[0144] In some embodiments, the method further involves modulating, based on said determining, said administering the recombinant polynucleotide or vector.

[0145] In some embodiments, the method further involves modulating, based on said determining, said administering the ligand.

[0146] The above disclosure generally describes the present disclosure. A more specific description is provided below in the following examples. The examples are described solely for the purpose of illustration and are not intended to limit the scope of the present disclosure. Changes in form and substitution of equivalents are contemplated as circumstances suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation. EXAMPLES

[0147] The examples below are intended to exemplify the practice of embodiments of the disclosure but are by no means intended to limit the scope thereof. Materials and Methods Plasmid Cloning, Characterization, and Purification

[0148] Introns and poison exon cassettes were ordered as gBlocks from IDT (Coralville, IA). Alternatively, they were ordered as double-stranded DNA gene fragments from Twist Bioscience (South San Francisco, CA). Each gene fragment contains 30-40 bp of vector-specific homologous regions on the tails of the fragment to accommodate Gibson Assembly with a linearized vector backbone.

[0149] Plasmid backbones were linearized by double digestion using restriction enzymes purchased from New England Biolabs (NEB) (Ipswich, MA). Appropriate experimental conditions and protocols for double digestion were generated using the NEBcloner web tool. The digestion mixture was separated on a 1% agarose gel to isolate the linearized fragment. Gel purification was carried out using the QIAquick Gel Extraction Kit (catalog number 28706). The psiCHECK-2 vector was purchased from Promega (catalog number C8021). pcDNA3-EGFP(Plasmid #13031), pCMV-Cre (Plasmid #123133), and LentiGuide Puro-P2A-EGFP (Plasmid #137729) were purchased from Addgene (Watertown, MA). The Thpo Mus musculus cDNA ORF clone in a pcDNA3.1+ / C-(K)-DYK backbone was ordered from GenScript (Piscataway, NJ).

[0150] Gene fragments were assembled with linearized backbones using the Gibson Assembly Master Mix (NEB E2611L) following the manufacturer’s protocol. The assembly mixture was transformed into NEB® 5-alpha Competent E. coli (High Efficiency) (NEB C2987I) following standard cloning procedures. All assembled plasmids were verified via Sanger sequencing by GENEWIZ (Azenta Life Sciences). Drug Preparation

[0151] Acyclovir (catalog number A1915-5G) was purchased from TCI AMERICA. It was dissolved in DMSO to create a 200 mM concentrated stock solution and kept frozen at - 20°C for long-term storage. Further dilutions of the stock solution into aqueous buffers or cell culture media should be made prior to performing biological experiments. No drug precipitation was observed as long as the final concentration did not exceed 2 mM. Cell Culture Conditions and Transfection Reagents

[0152] All cell lines were obtained directly from the American Type Culture Collection (ATCC) except for a loxP-GFP Cre reporter N2a cell line, which was a gift from the Zhang Lab at Broad Institute. The base media was supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS), 100 units / mL penicillin, and 100 μg / mL streptomycin (Gibco 15140122). HEK293T and Huh-7 were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (catalog number 11995065 or ATCC 30-2002), while HeLa, HT-1080, and N2a were cultured in Eagle's Minimum Essential Medium (EMEM) (ATCC 30-2003). U-2 OS and HCT 116 were cultured in McCoy's 5a Medium Modified (ATCC 30-2007). All cell lines were cultured at 37°C in a 5% CO2-humidified incubator under standard tissue culture conditions.

[0153] Transient transfections of HEK293T, Huh-7, HeLa, and U-2 OS were carried out using FuGENE HD transfection reagent (Promega E2311) following the manufacturer’s protocol. Transient transfections of HT-1080 and HCT 116 were done using Lipofectamine LTX reagent with PLUS reagent (Invitrogen A12621), and transient transfection of N2a was done using FuGENE 4K transfection reagent (Promega E5911). Transfection for CRISPR / Cas9 knock-ins in HCT 116 cells was carried out using Lipofectamine CRISPRMAX Cas9 transfection reagent (catalog number CMAX00008). Luciferase Assay

[0154] One day before transfection, cells were seeded into a clear-bottom, white, TC- treated 96-well microplate (Corning 3610). Each well holds 15,000 cells in 100 µl of growth medium. The edge wells of the plate were not seeded with cells; instead, they were filled with 1x PBS (phosphate-buffered saline) to minimize evaporation.

[0155] One hour before performing the transfection, the 200 mM acyclovir stock solution was diluted in the growth medium to achieve a concentration that was three times (3x) the final desired concentration. Then, 50 µl of this diluted acyclovir solution was added to the 100 µl of growth medium already present in each well without disturbing the cells. This process resulted in the final desired concentration of acyclovir (1x) in the wells.

[0156] Transfection was performed using Opti-MEM (Gibco 31985062) and transfection reagents appropriate for the cell line used in the experiment. Each plate contained blank wells for background subtraction and wells transfected with psiCHECK-2 control vectors for expression normalization, facilitating comparison among plates. Cells in each well were transfected with 100 ng of DNA plasmids. Transfection was performed according to the manufacturer's protocol. After transfection, the cells were returned to the incubator.

[0157] Two days after transfection, the acyclovir-containing medium was replaced with 75 µl of fresh medium per well. Luminescence was measured using the Dual-Glo Luciferase Assay System (Promega E2920). To measure Firefly luminescence, 75 µl of Dual-Glo Luciferase reaction mix was added to each well. After a 10-minute incubation at room temperature, the luminescence signal was measured using the SpectraMax iD3 Multi-Mode Microplate Reader. Renilla luminescence was measured by adding 75 µl of Dual-Glo Stop & Glo reaction mix to each well and following the same steps as for the Firefly luminescence measurements.

[0158] Luminescence data analysis was performed using Excel (Microsoft) and Prism (GraphPad). The ratio between Renilla and Firefly luciferase activity was calculated for each well to normalize for transfection efficiency and cell number. This ratio was then normalized to the ratio generated by the psiCHECK-2 control vector, expressed as a percentage of expression. RNA Extraction and RT–PCR

[0159] Total RNA was extracted using TRIzol Reagent (Invitrogen 15596018) according to the manufacturer’s protocol. RNA concentration was determined using a NanoDrop spectrophotometer (Thermo Scientific), and 1 μg of total RNA was treated with DNase I (NEB M0303S). cDNA was generated from the RNA using gene-specific primers and SuperScript IV Reverse Transcriptase (Invitrogen 18090010) according to the manufacturer’s protocol. ThecDNA was amplified by PCR using Phusion High-Fidelity PCR Master Mix (NEB M0531S) and then separated on a 2% agarose gel for imaging with a Bio-Rad Gel Doc XR imager. Western Blot

[0160] Cells expressing transgenes were treated with acyclovir for 48 hours after transfection before being harvested for western blot analysis. Cells expressing acyclovir- controlled WTAP or PCIF1 were treated with acyclovir for 5 days before being harvested for western blot analysis. Total protein was extracted from the cells using RIPA buffer with protease inhibitor (Thermo Scientific #78440) and quantified using the Quick Start Bradford Protein Assay (Bio-Rad 5000201). Equal amounts of total protein for each condition were separated on 4–12% NuPAGE Bis-Tris Mini Protein Gels (Invitrogen NP0321BOX) and transferred to Immobilon-P PVDF Membrane (Millipore IPVH00010). The membranes were blocked with 5% milk in PBS-T and incubated with the corresponding primary antibodies (anti- PCIF1 antibody (abcam ab300487); anti-WTAP antibody (Cell Signaling Technology 56501s); anti-puromycin N-acetyltransferase antibody (Invitrogen #702389); anti-FLAG antibody (Sigma- Aldrich F1804); anti-β-tubulin antibody (proteintech 10068-1-AP); anti-GAPDH antibody (GeneTex GT239)) at the recommended dilution factors at 4°C overnight, followed by incubation with secondary antibodies at room temperature for 1 hour. The membranes were treated with Pierce ECL Western Blotting Substrate (Thermo Scientific 32106) and imaged using a Bio-Rad Gel Doc XR imager. Fluorescence Imaging

[0161] Cells were plated in 24-well glass bottom tissue culture plates (MatTek) treated with Poly-D-Lysine (Cultrex 3439-100-01) and mouse Laminin (Cultrex 3400-010-02) and cultured for 24 hours before transfection. After transient transfection and acyclovir treatment, the cells were incubated for another 48 hours before being subjected to imaging experiments. To reduce background fluorescence, the culture media were replaced with complete FluoroBrite DMEM (Gibco A1896701) 1 hour before imaging. Hoechst 33342 was added to a final concentration of 0.5 μg / ml. The 24-well plate was transferred to a Tokai Hit stage top incubator (37°C and 5% CO2). Live cell fluorescence images were obtained with a CoolSnap HQ2 CCD camera (Photometrics) through a ×20 air objective mounted on a Nikon Eclipse Ti-E microscope and analyzed with NIS-Elements software to detect EGFP (470 ± 20 nm excitation, 495 nm dichroic, 525 ± 25 nm emission) and Hoechst (350 ± 25 nm excitation, 400 nm dichroic, 460 ± 25 nm emission). Image analysis was conducted in ImageJ software.Isothermal Titration Calorimetry (ITC)

[0162] ITC binding experiments were performed using an Affinity ITC (TA Instruments). Titrations were carried out with the acyclovir aptamer RNA in the sample cell and acyclovir ligand as the titrant in the syringe. Acyclovir aptamer RNA (70 µM) was folded by heating for 10 minutes at 75°C in ITC running buffer (100 mM HEPES pH 7.4, 500 mM NaCl, 0.5 mM MgCl2, and 0.25% (v / v) DMSO) and then cooled to room temperature over 1 hour on the benchtop. The acyclovir stock solution (200 mM) was diluted to a final concentration of 495.6 µM in ITC running buffer to serve as the titrant solution. The sample cell was loaded with acyclovir aptamer RNA, and the injection syringe was loaded with the acyclovir titrant solution. At the start of the ITC run, the instrument was equilibrated at 25°C, and the acyclovir titrant solution was injected into the sample cell with an initial injection of 1 µl followed by 16 injections of 3 µl. Data analysis was performed using NanoAnalyze (TA Instruments). After baseline correction, the area under each peak was integrated and fitted to a one-site independent binding model. The heat of dilution was subtracted from the integrated peak area. The first data point was removed from all data sets due to the different injection volume. ELISA

[0163] HEK293T cells were plated in a 96-well plate as described in the luciferase assay section. After transient transfection and acyclovir treatment, cells were incubated for 24 hours before removing 2.5 µl of media to measure Thpo protein concentration using the Thrombopoietin (TPO / THPO) Mouse ELISA Kit (Invitrogen #EMTHPO) according to the manufacturer’s protocol. ELISA samples were measured with a SpectraMax iD3 Multi-Mode Microplate Reader. M2 DMS MaP reagents and equipment

[0164] Dimethyl sulfate (CAS 77-78-1) was purchased from Sigma Aldrich (St. Louis, MO). DNA primers were ordered from IDT. DNA Clean & Concentrator-5 kit, RNA Clean & Concentrator-5 kit, and RNA Clean & Concentrator-25 kit were purchased from Zymo Research. AMPure XP Bead-Based Reagents were purchased from Beckman Coulter. Phusion High- Fidelity PCR Master Mix with HF Buffer, Induro Reverse Transcriptase, 5X Induro RT Reaction Buffer, and Barcoded sequencing oligos were purchased from NEB. AmpliScribe T7 High Yield Transcription Kit was purchased from Biosearch Technologies. RNaseOUT Recombinant Ribonuclease Inhibitor was purchased from Invitrogen.Error-Prone PCR and in vitro Transcription for M2 DMS MaP

[0165] The DNA template for mutate-and-map experiments was prepared as previously described Cheng et al., “RNA Structure Inference through Chemical Mapping after Accidental or Intentional Mutations,” Proc. Natl. Acad. Sci. U S A 114:9876-9881 (2017) and Wilson and Keefe, “Random Mutagenesis by PCR,” Curr. Protoc. Mol. Biol. Chapter 8, Unit8.3 (2001), which are hereby incorporated by reference in their entirety), using error-prone PCR with Taq polymerase. The DNA amplicons were evaluated on an agarose gel and purified with the DNA Clean & Concentrator-5 kit. 200 ng of mutated DNA was used as a template to perform in vitro transcription with the AmpliScribe T7 High Yield Transcription Kit. The reaction was purified with the RNA Clean & Concentrator-25 kit. RNA quality was assessed by visualization on 10% Novex TBE-Urea Gels (Invitrogen EC6875BOX) stained with SYBR Gold. DMS Modification of in vitro-Transcribed RNA

[0166] In vitro-transcribed RNA was probed as previously described, with some minor changes to the protocol (Mustoe et al., “RNA Base-Pairing Complexity in Living Cells Visualized by Correlated Chemical Probing,” Proc. Natl. Acad. Sci. U S A 116:24574-24582 (2019), which is hereby incorporated by reference in its entirety). Briefly, 1 µg of non- mutagenized or mutagenized RNA was refolded by heating to 95°C for 3 minutes, then snap- cooled on ice for 1 minute in a buffer that promotes the modification of m1G and m3U (200 mM Bicine, 100 mM KCl, pH 8.37 at room temperature). MgCl2(5 mM) and acyclovir (250 µM) or DMSO were then added, followed by incubation of the RNA at 37°C for 10 minutes. DMS probing was performed by adding neat DMS (1.7 M in anhydrous ethanol) to the folded RNA and incubating at 37°C for 6 minutes. The reaction was quenched by adding 4 volumes of 20% β-mercaptoethanol in water while incubating on ice. The RNA was purified from the quenched DMS reaction using the RNA Clean & Concentrator-5 kit and eluted in 10 µl water. The RNA was either processed directly or stored at -80°C. Reverse Transcription and Sequencing Library Preparation

[0167] Reverse transcription was performed using Induro Reverse Transcriptase (NEB M0681S) according to the manufacturer's protocol, with the following changes. RNA (500 ng) was mixed with 10 mM dNTP (2 µl), 2 µM RT primer (1 µl), and water to a final volume of 13 µl during the annealing step. A custom RT master mix was prepared by combining 5X Induro RT Reaction Buffer (4 µl), 100 mM DTT (1 µl), RNaseOUT Recombinant Ribonuclease Inhibitor (0.5 µl), Induro Reverse Transcriptase (200 U / µl, 1 µl), and water (0.5 µl). Reverse transcription reactions were incubated at 25°C for 10 minutes, followed by 57°C for 4 hours. To hydrolyze the RNA, 1 N NaOH (5 µl) and 500 mM EDTA (5 µl) were added to the reaction,which was then incubated at 95°C for 3 minutes. The cDNA was purified using the DNA Clean & Concentrator-5 kit and eluted in 15 µl water.

[0168] Sequencing libraries were generated by a 2-step PCR protocol using Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB M0531S). Step 1 PCR amplifies cDNA to install the Illumina oligo binding site. cDNA (2 µl) was amplified for 20 cycles using the primers below. The reaction mix was purified using the DNA Clean & Concentrator-5 kit and eluted in 15 µl water. Step 1 PCR forward primer (5' ^ 3') ACCACGACGCTCTTCCGATCTNNNNNTTTTCCTTACAGGGTTTCAGACAAAATCAAAAAGA (SEQ ID NO: 96) Step 1 PCR reverse primer (5' ^ 3') GACGTGTGCTCTTCCGATCTNNNNNACATTTGTTTTCCACAAACCATAAAGTTTTACAAA (SEQ ID NO: 97)

[0169] Step 2 PCR was performed with NEBNext Multiplex Oligos for Illumina. The Step 2 PCR reaction mix contained purified DNA from Step 1 (2-10 ng in 12 µl water), i5 index primer (4 µl), i7 index primer (4 µl), and Phusion High-Fidelity PCR Master Mix with HF Buffer (20 µl). The PCR cycle number was optimized for each experiment. The reaction mix was size-selected with 0.9X AMPure XP Beads, eluted in 21 µl water, and quantified using Qubit 1X dsDNA High Sensitivity (HS) and Broad Range (BR) Assay Kits (Invitrogen). Libraries were pooled, cleaned up again with 0.9X AMPure XP Beads, and eluted in 28 µl water. The final library was quantified using Qubit 1X dsDNA High Sensitivity (HS) and Broad Range (BR) Assay Kits (Invitrogen) and sequenced on the NovaSeq X Plus PE 2 x 100 (~10 million reads per sample in the library). Data Processing

[0170] DMS MaP sequencing data were processed using the ShapeMapper pipeline (Busan and Weeks, “Accurate Detection of Chemical Modifications in RNA by Mutational Profiling (MaP) with ShapeMapper 2,” Rna 24:143-148 (2018) and Mitchell et al., Mutation Signature Filtering Enables High-Fidelity RNA Structure Probing at all Four Nucleobases with DMS,” Nucleic Acids Res.51:8744-8757 (2023), which are hereby incorporated by reference in their entirety). Sequencing data were aligned to a custom FASTA sequence for the Ac-H2Tr7 amplicon. Sequencing data alignment was performed with ShapeMapper v2.2 using the following settings: --amplicon --output-parsed --output-aligned-reads --output-counted-mutations --min-mutation-separation 2 (Oleynikov and Jaffrey, “RNA Tertiary Structure andConformational Dynamics Revealed by BASH MaP,” bioRxiv (2024), which is hereby incorporated by reference in its entirety).

[0171] For M2 DMS MaP, sequencing data were aligned to a single FASTA sequence with ShapeMapper v2.2 using the following settings: --amplicon --dms --output-parsed --min- mutation-separation 2. M2 heatmaps were generated as previously described (Oleynikov and Jaffrey, “RNA Tertiary Structure and Conformational Dynamics Revealed by BASH MaP,” bioRxiv (2024), which is hereby incorporated by reference in their entirety). Briefly, the output parsed mutation file was processed with the script mut_to_simple.py, which retains only sequencing reads that cover the entire length of the RNA. The resulting .simple file was then used to calculate a unique mutational profile for each point mutant with the script simple_to_M2_map.py. Z-score normalization was performed using the script M2_Map_to_Zscores.py. The normalized matrix was then visualized in Prism GraphPad as a heatmap. Design of sgRNA and HDR Donor Constructs

[0172] The overall workflow of our knock-in experiment is built upon the widely used endogenous tagging protocol based on the 2A peptide homology-directed repair (HDR) donor cassette (Nabet et al., “The dTAG System for Immediate and Target-Specific Protein Degradation,” Nat. Chem. Biol.14:431-441 (2018) and Philip et al., “qTAG: An Adaptable CRISPR-Based Endogenous Tagging Protocol using Optimized Repair Cassettes,” bioRxiv, 2023.2011.2001.565029 (2023), which are hereby incorporated by reference in their entirety). The Pac-Cyclone HDR donor is inserted after the start codon and therefore tags the endogenous gene at the N-terminus.

[0173] Before making any designs, it is recommended to research the gene of interest using the NCBI (National Center for Biotechnology Information) Gene database or the Ensembl genome database. Besides identifying the location of the start codon, it is also helpful to know which chromosome the gene is located on to determine the copy number in the cell line to be used. Learning the orientation of the gene is informative during the design step of the HDR donor. Additionally, it is important to ensure that the knock-in design can target all transcripts and isoforms of the gene of interest. Next, it is recommended to check the expression level of this gene in the cell line to be used for the knock-in experiment using The Human Protein Atlas database to confirm that the gene will be expressed.

[0174] The sgRNAs and HDR donors targeting the WTAP and PCIF1 genes were generated using a web-based design tool from GenScript (Homology-Directed Repair (HDR) Knock-in Templates). This tool provides a graphic display and interactive interface of the targetgenomic locus for convenient design of the sgRNAs, HDR donors, and custom primers for assessing editing efficiency. To design the gene-specific HDR donor containing the poison exon, search for the gene ID and transcript ID, or provide the genome coordinates of the start codon. After placing the cursor behind the start codon, input the sequence of Pac-Cyclone (FIG. 14). The design tool will by default add 300 bp long homology arms on both ends to ensure that the donor overlaps with the locus for efficient homology-directed repair. Choose the “add silent mutations” option to disrupt the PAM sequence and avoid sgRNA binding and re-editing events. Design one HDR donor with mutations for testing multiple sgRNAs. The HDR donor was ordered directly from GenScript in single-stranded DNA format (3 µg). The web tool recommends the top five sgRNAs in this region of the genome. Choose 2-3 sgRNAs with cut sites within 30 bp of the insertion site for better editing efficiency. It is also advised to consider the “on-target score” to avoid off-target editing events. Finally, custom primers for assessing editing efficiency were generated automatically by the design tool. Delivery of CRISPR-Cas9 Editing Components and Cell Culture

[0175] Before delivering the editing components into cells, the efficiency of the sgRNAs was tested using the GenCrispr sgRNA Screening Kit (GenScript L00689). HCT 116 cells were chosen due to their predominantly diploid nature, fast growth rate, and susceptibility to transfection. The knock-in experiment was started at the third passage of the HCT 116 cells, which were obtained directly from ATCC.

[0176] Two days before transfection, cells were plated into a 6-well TC-treated plate (0.4 million cells per well in 2 ml medium). Seed additional wells as transfection control and puromycin selection control. One day before transfection, cells were treated with nocodazole (5 ng / ml) (catalog number SML1665-1ML) for 15 hours. Nocodazole-containing medium was replaced with acyclovir-containing medium (250 µM acyclovir, 3 ml per well) 30 minutes before transfection. GenCRISPR™ Ultra NLS-Cas9 protein (6.25 µg) (GenScript Z03621), sgRNA (1.2 µg), and single-stranded HDR donor (2.5 µg) were delivered by chemical transfection using Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent (Invitrogen CMAX00008). Replace the medium with fresh medium containing 250 µM acyclovir 24 hours after transfection. Incubate for another 48 hours before starting puromycin selection.

[0177] Three days after transfection, cells were passaged from the 6-well plate into a 10- cm dish using medium containing acyclovir and puromycin (250 µM acyclovir, 0.4 µg / ml puromycin) (Sigma-Aldrich P4512). The puromycin concentration was determined by establishing an antibiotic kill curve to find the minimum concentration required to kill unedited cells. Wash cells daily to remove dead cells and debris. Passage cells to a 15-cm dish if neededto avoid contact inhibition. Puromycin selection was maintained for 5 days as the control cells all died. Cells were then recovered in acyclovir-only medium for two days before starting clonal expansion. Limited Dilution and Clonal Expansion

[0178] After puromycin selection, limiting dilution was performed to isolate monoclonal cell populations for genotyping. During this process, cells were cultured in complete medium containing 250 µM acyclovir. Cells were washed with 1x PBS (Gibco 10010023) and then trypsinized using TrypLE™ Express Enzyme (Gibco 12604013) to obtain a homogenized cell solution. Cell concentration was quantified using the Countess 3 Automated Cell Counter (Invitrogen AMQAX2000). Prepare 10 ml of cell solution with a concentration of 5 cells / ml for each 96-well plate to be used. Transfer 100 µL of this cell solution into each well of a 96-well plate to obtain an average density of 0.5 cells / well. Seed one well at the corner with ~1000 cells to help find the focal plane. Cells were left undisturbed in an incubator for 7–14 days. After ~7 days, scan the plate for cell growth. Wells containing monoclonal populations were expanded into 24-well plates. One monoclonal population was split into two wells: one for genotyping and one for further expansion. After genotyping, populations with undesired genotypes were discarded. Edited clones with both alleles tagged were expanded into larger culture dishes, screened for acyclovir-controlled expression, and then frozen into aliquots and stored in liquid nitrogen. Genotyping by Genomic PCR

[0179] Monoclonal cells for genotyping were grown in 24-well plates as described in the previous section. Extract genomic DNA from wild-type and tagged cells by adding 200 µl of QuickExtract™ DNA Extraction Solution (catalog number QE09050) to cells trypsinized and collected from each well. Genomic DNA (ranging from 0.5 µl to 5 µl, depending on cell density) was used for PCR amplification with Phusion high-fidelity polymerase (NEB M0531L) using primers that anneal outside the donor sequence to check for both successful editing and homozygosity. Amplicons were analyzed on 1% agarose gels. Wild-type cells show a single band corresponding to two unedited alleles. Heterozygous editing shows the appearance of an upper band corresponding to integration of the donor sequence. A single band of higher molecular weight indicates homozygous editing of both alleles. Amplicons from homozygous clones were sent for Sanger sequencing using the amplicon sequencing service provided by GENEWIZ.Example 1 – Selection of SMN1 Exon 7 and Acyclovir for Creation of a Portable Poison Exon

[0180] Poison exons are alternatively-spliced exons that introduce premature stop codons into mRNA transcripts (Ni et al., “Ultraconserved Elements are Associated with Homeostatic Control of Splicing Regulators by Alternative Splicing and Nonsense-Mediated Decay,” Genes Dev 21:708-718 (2007) and Lareau et al., “Unproductive Splicing of SR Genes Associated with Highly Conserved and Ultraconserved DNA Elements,” Nature 446: 926-929 (2007), which are hereby incorporated by reference in their entirety) (FIG.1A). Inclusion of poison exons often leads to the production of truncated and nonfunctional proteins (Ni et al., “Ultraconserved Elements are Associated with Homeostatic Control of Splicing Regulators by Alternative Splicing and Nonsense-Mediated Decay,” Genes Dev 21:708-718 (2007) and Lareau et al., “Unproductive Splicing of SR Genes Associated with Highly Conserved and Ultraconserved DNA Elements,” Nature 446: 926-929 (2007), which are hereby incorporated by reference in their entirety). By selectively including or skipping poison exons, cells can regulate the expression levels of specific proteins. Poison exons are found in several genes, including serine / arginine-rich splicing factors (SRSFs), that regulate each other by controlling poison exon splicing (Leclair et al., “Poison Exon Splicing Regulates a Coordinated Network of SR Protein Expression during Differentiation and Tumorigenesis,” Mol Cell 80:648-665.e649 (2020) and Thomas et al., “RNA Isoform Screens uncover the Essentiality and Tumor-Suppressor Activity of Ultraconserved Poison Exons. Nat Genet 52:84-94 (2020), which are hereby incorporated by reference in their entirety).

[0181] Although poison exon skipping is regulated by splicing factors, it was sought to develop a poison exon whose skipping was induced by a small molecule (FIG.1B). In this way, the gene would normally produce a nonfunctional truncated protein due to the poison exon, but switch to the full-length protein when the small molecule induces skipping of the poison exon during pre-mRNA splicing. Importantly, the ability to insert this small molecule-regulated poison exon into endogenous genes in a way that preserves its promoter, enhancer, and chromatin-based regulatory pathways was desired. Only the magnitude of expression would be controlled by the small molecule, and this would occur in a manner that is rapid and reversible, as well as tunable based on the concentration of the small molecule.

[0182] The objective was therefore to modify a poison exon with a small molecule- binding RNA aptamer (FIG.1B). The modified poison exon would therefore be skipped based on the presence of the small molecule. A small molecule was first selected to control the poison exon. In the past, theophylline and tetracycline-controlled aptamers have been used to controlribozymes and other RNAs, as these small molecules are readily available and the aptamers remain functional in the mammalian cellular environment (Weigand and Suess, “Tetracycline Aptamer-Controlled Regulation of pre-mRNA Splicing in Yeast,” Nucleic Acids Res.35:4179- 4185 (2007); Vogel et al., “A Small, Portable RNA Device for the Control of Exon Skipping in Mammalian Cells,” Nucleic Acids Res.46:e48 (2018); Finke et al., “Efficient Splicing-Based RNA Regulators for Tetracycline-Inducible Gene Expression in Human Cell Culture and C. elegans,” Nucleic Acids Res.49:e71 (2021); Spöring et al., “Aptamer-Mediated Control of Polyadenylation for Gene Expression Regulation in Mammalian Cells,” ACS Synth. Biol. 9:3008-3018 (2020); Xiang et al., “Massively Parallel RNA Device Engineering in Mammalian Cells with RNA-Seq,” Nature Communications 10:4327 (2019); and Beilstein et al. “Conditional Control of Mammalian Gene Expression by Tetracycline-Dependent Hammerhead Ribozymes,” ACS Synth. Biol.4:526-534 (2015), which are hereby incorporated by reference in their entirety). However, antibiotics are problematic since they can promote antibiotic resistance (Grossman, T.H., “Tetracycline Antibiotics and Resistance,” Cold Spring Harbor Perspectives in Medicine 6:a025387 (2016), which is hereby incorporated by reference in its entirety) and tetracycline induces mitochondrial dysfunction, even at low doses (Chatzispyrou et al., “Tetracycline Antibiotics Impair Mitochondrial Function and its Experimental use Confounds Research,” Cancer Res.75:4446-4449 (2015) and Moullan et al., “Tetracyclines Disturb Mitochondrial Function across Eukaryotic Models: A Call for Caution in Biomedical Research,” Cell Rep 10:1681-1691 (2015), which are hereby incorporated by reference in their entirety). Theophylline inhibits phosphodiesterases, leading to increased cyclic AMP levels and alterations in cell signaling (Polson et al., “Inhibition of Human Pulmonary Phosphodiesterase Activity by Therapeutic Levels of Theophylline,” Clin. Exp. Pharmacol. Physiol.5:535-539 (1978) and Banner and Page, “Theophylline and Selective Phosphodiesterase Inhibitors as Anti- Inflammatory Drugs in the Treatment of Bronchial Asthma,” Eur. Respir. J.8:996-1000 (1995), which are hereby incorproated by reference in their entirety). Therefore, the objective was to select a molecule that has minimal or no effects on cellular physiology .

[0183] Acyclovir (FIG.1C), an antiviral medication used for treating herpes infections (Lycke et al., “Acyclovir Levels in Serum and Cerebrospinal Fluid After Oral Administration of Valacyclovir,” Antimicrob Agents Chemother.47:2438-2441 (2003); Elion, G.B., “The Biochemistry and Mechanism of Action of Acyclovir,” J. Antimicrob Chemother.12 Suppl B:9- 17 (1983); Mindel et al., “Dosage and Safety of Long-Term Suppressive Acyclovir Therapy for Recurrent Genital Herpes,” Lancet 1:926-928 (1988); Fletcher and Bean, “Evaluation of Oral Acyclovir Therapy,” Drug Intell. Clin. Pharm.19:518-524 (1985); and Elion, G.B., “Mechanismof Action and Selectivity of Acyclovir,” Am. J. Med.73:7-13 (1982), which are hereby incorporated by reference in their entirety), was chosen. Acyclovir appears to have minimal effects on cell function, as evidenced by over 40 years of routine use in humans for treatment and prophylaxis of herpes infections. Acyclovir can be administered to cells at concentrations exceeding 1 mM with no apparent cytotoxicity (de Jalón et al., “Increased Efficacy of Acyclovir- Loaded Microparticles Against Herpes Simplex Virus Type 1 in Cell Culture,” Eur. J. Pharm. Biopharm.56:183-187 (2003); Yao et al., “Turning an Antiviral into an Anticancer Drug: Nanoparticle Delivery of Acyclovir Monophosphate,” J. Control Release 170:414-420 (2013); and Tanabe et al., “Cytotoxic Effect of Acyclovir on Cultured Mammalian Cells to which Herpesvirus Thymidine Kinase Gene was Introduced,” Jpn J. Vet. Res.37:181-185 (1989), which are hereby incorporated by reference in their entirety), although it is used therapeutically in humans with typical serum concentrations between 18.4 µM and 36.0 µM (Lycke et al., “Acyclovir Levels in Serum and Cerebrospinal Fluid After Oral Administration of Valacyclovir,” Antimicrob Agents Chemother.47:2438-2441 (2003), which is hereby incorproated by reference in its entirety), with therapeutic serum concentrations reaching up to ~114 µM (Kacirova et al., “Therapeutic Monitoring of Serum Concentrations of Acyclovir and its Metabolite 9-(Carboxymethoxymethyl) Guanine in Routine Clinical Practice,” Biomed. Pharmacother.156:113852 (2022), which is hereby incorporated by reference in its entreity). Acyclovir is generally nontoxic to mammalian cells since it is phosphorylated in cells by a herpes-encoded viral thymidine kinase, which produces a nucleotide analog that inhibits herpes- encoded viral polymerase (Elion, G.B., “The Biochemistry and Mechanism of Action of Acyclovir,” J. Antimicrob Chemother.12 Suppl B:9-17 (1983) and and Elion, G.B., “Mechanism of Action and Selectivity of Acyclovir,” Am. J. Med.73:7-13 (1982), which are hereby incorporated by reference in their entirety), but has minimal effect on human polymerases (Elion, G.B., “The Biochemistry and Mechanism of Action of Acyclovir,” J. Antimicrob Chemother.12 Suppl B:9-17 (1983) and and Elion, G.B., “Mechanism of Action and Selectivity of Acyclovir,” Am. J. Med.73:7-13 (1982), which are hereby incorporated by reference in their entirety). Although acyclovir may have effects in cells, these do not appear to be clinically relevant based on the safety of this drug in humans. Thus, acyclovir has multiple features that make it desirable for controlling synthetic RNA devices.

[0184] A recent automated SELEX approach described the development of a series of small molecule-binding aptamers, including an acyclovir-binding aptamer (Townshend et al., “A Multiplexed, Automated Evolution Pipeline Enables Scalable Discovery and Characterization of Biosensors,” Nat. Commun.12:1437 (2021), which is hereby incorporated by reference in itsentirety) (FIG.1D). The acyclovir aptamer contains a helical stem region that was incorporated into a functionally important helical stem region in a ribozyme and enabled a ~2.0-fold increase in ribozyme activity in yeast upon addition of acyclovir (Townshend et al., “A Multiplexed, Automated Evolution Pipeline Enables Scalable Discovery and Characterization of Biosensors,” Nat. Commun.12:1437 (2021), which is hereby incorporated by reference in its entirety). It was confirmed that the acyclovir aptamer binds acyclovir, with an affinity of ~1.3 µM (FIG.7). Although this aptamer did not initially seem promising due to its relatively weak ability to control ribozyme-mediated cleavage compared to tetracycline and theophylline-controlled ribozymes (Xiang et al., “Massively Parallel RNA Device Engineering in Mammalian Cells with RNA-Seq,” Nature Communications 10:4327 (2019) and Beilstein et al. “Conditional Control of Mammalian Gene Expression by Tetracycline-Dependent Hammerhead Ribozymes,” ACS Synth. Biol.4:526-534 (2015), which are hereby incorporated by reference in their entirety), this aptamer was nevertheless tested for controlling poison exon skipping.

[0185] Next, the aim was to identify a naturally occurring poison exon that could be readily modified with the acyclovir aptamer. The acyclovir aptamer is predicted to contain a helical stem (Townshend et al., “A Multiplexed, Automated Evolution Pipeline Enables Scalable Discovery and Characterization of Biosensors,” Nat. Commun.12:1437 (2021), which is hereby incorporated by reference in its entirety) (FIG.1D), which would therefore allow it to be fused to a functionally important helical stem in a poison exon. However, the well-known poison exons appear to contain linear SRSF-binding motifs (Leclair et al., “Poison Exon Splicing Regulates a Coordinated Network of SR Protein Expression during Differentiation and Tumorigenesis,” Mol Cell 80:648-665.e649 (2020) and Thomas et al., “RNA Isoform Screens uncover the Essentiality and Tumor-Suppressor Activity of Ultraconserved Poison Exons. Nat Genet 52:84-94 (2020), which are hereby incorporated by reference in their entirety), which confer poison exon inclusion / skipping upon SRSF binding. Therefore, it was not clear how to incorporate an acyclovir aptamer into these exons.

[0186] Instead, inspiration was taken from the highly studied exon 7 in the SMN1 gene, which contains a functionally important stem (FIG.1E). Exon 7 is the last coding exon and contains the stop codon of the gene; therefore, it is not naturally a poison exon. Notably, ~6% of SMN1 transcripts exhibit skipping of exon 7 (Singh et al., “Modulating Role of RNA Structure in Alternative Splicing of a Critical Exon in the Spinal Muscular Atrophy Genes,” Nucleic Acids Res.35:371-389 (2007), which is hereby incorporated by reference in its entirety), resulting in mRNAs containing exon 6 directly connected to exon 8. Exon 7 seems to undergo skipping because the 5’ splice site, which is located at the junction of exon 7 and intron 7, is partiallyinaccessible due to a helical stem (FIGS.1E–1F). The 5’ splice site comprises the U1-binding sequence. The U1-binding sequence of this particular 5' splice site is unusual because it forms base pairs with a sequence at the 3’ end of exon 7. As a result, the 5’ splice site is obscured in a stem-loop structure, termed terminal stem-loop 2 (TSL2) (Singh et al., “Modulating Role of RNA Structure in Alternative Splicing of a Critical Exon in the Spinal Muscular Atrophy Genes,” Nucleic Acids Res.35:371-389 (2007), which is hereby incorporated by reference in its entirety) (FIGS.1E–1F). Previous studies have demonstrated that TSL2 accounts for the occasional skipping of exon 7. Adding more base pairs to further stabilize TSL2 more efficiently sequesters, or “hides”, the U1-binding sequence, leading to more skipping of exon 7 (Singh et al., “Modulating Role of RNA Structure in Alternative Splicing of a Critical Exon in the Spinal Muscular Atrophy Genes,” Nucleic Acids Res.35:371-389 (2007), which is hereby incorporated by reference in its entirety). Conversely, decreasing the number of base pairs to destabilize TSL2 results in greater accessibility of the U1-binding sequence, leading to less skipping of exon 7 (Singh et al., “Modulating Role of RNA Structure in Alternative Splicing of a Critical Exon in the Spinal Muscular Atrophy Genes,” Nucleic Acids Res.35:371-389 (2007), which is hereby incorporated by reference in its entirety). Thus, the TSL2 structure in exon 7 of SMN1 appeared to be a useful entry point for creating an acyclovir-regulated poison exon. Example 2 – Conversion of SMN1 Exon 7 into a Portable Poison Exon

[0187] To create a poison exon cassette from SMN1 intron 6-exon 7-intron 7, confirmation that this exon along with the two flanking introns can function outside of the SMN1 gene was needed. Intron 6 is problematic due its long length (5764 nt). However, previous studies showed that intron 6 can be truncated to 200 nt, comprising the first 62 nt and the last 138 nt of the original intron 6 (FIG.8A), and can still be efficiently spliced as measured in a splicing minigene cassette (Cartegni and Krainer, “Disruption of an SF2 / ASF-Dependent Exonic Splicing Enhancer in SMN2 causes Spinal Muscular Atrophy in the Absence of SMN1,” Nat. Genet.30:377-384 (2002), which is hereby incorporated by reference in its entirety). Indeed, it was confirmed that inserting the truncated intron 6 (termed intron 6trunc), or inserting the entire 444-nt intron 7, into a Renilla luciferase gene did not impair luciferase expression (FIGS.8A– 8B). This indicates that both introns are efficiently spliced out of the luciferase transcript. Intron 7 was not further truncated since previous studies showed that truncation impairs its splicing (Cartegni and Krainer, “Disruption of an SF2 / ASF-Dependent Exonic Splicing Enhancer in SMN2 causes Spinal Muscular Atrophy in the Absence of SMN1,” Nat. Genet.30:377-384 (2002), which is hereby incorporated by reference in its entirety). Overall, both intron 6truncand intron 7 can splice efficiently in a foreign gene context.

[0188] It was next asked if the contiguous intron 6trunc-exon 7-intron 7 cassette can be inserted into the luciferase gene, and if this would allow exon 7 to be efficiently inserted into the mRNA (FIG.1G). In mammalian splicing, the 5’ and 3’ splice sites are primarily located at the termini of introns. However, the 5’ splice site also includes 2-3 nt (typically AG or UG) from the 3’ end of the previous exon. The 3’ splice site includes the first nucleotide (typically G) of the following exon (Sibley et al., “Lessons from Non-Canonical Splicing,” Nat. Rev. Genet. 17:407-421 (2016) and Zhang et al., “Sequence Information for the Splicing of Human pre- mRNA Identified by Support Vector Machine Classification,” Genome Res.13:2637-2650 (2003), which are hereby incorporated by reference in their entirety). In order to preserve the SMN1 splice sites, the intron 6trunc-exon 7-intron 7 cassette was inserted into the middle of the Renilla luciferase gene between UG521-522 and G523 (FIG.8C). This location also maintains the reading frame with the stop codon in exon 7.

[0189] After insertion of the intron 6trunc-exon 7-intron 7 cassette, it was found that luciferase expression was reduced by ~92% (FIG.1G). This indicates that exon 7 is spliced into the luciferase transcript, and exon 7 acts as a poison exon. This was confirmed by RT-PCR using primers flanking the insertion site, which demonstrated that the majority (~83%) of the luciferase transcript contained an additional ~50 nt sequence, which reflects the size of exon 7 (FIG.1G). Thus, insertion of the intron 6trunc-exon 7-intron 7 cassette can cause exon 7 to be spliced into a heterologous transcript as a poison exon.

[0190] It was reasoned that the occasional exon 7 skipping (~17%) that led to functional luciferase was due to occasional inaccessibility, i.e., hiding, of the U1-binding sequence. The RNA stem-loop structure exists in a folded:unfolded equilibrium, with the hairpin predominantly being in the unfolded form, which could cause exon 7 inclusion. However, transient hybridization of the stem would sequester the U1-binding sequence, leading to exon 7 skipping. To test this, base pairs in TSL2 (FIG.1H) were removed to increase the accessibility of the U1- binding sequence. This led to essentially complete loss of luciferase activity (FIG.1H) and no skipping of exon 7 (FIG.1H). Thus, the hybridization state of TSL2 is a determinant of exon 7 skipping. Example 3 – Design of Alternative Stem-Loop Structures that Control Exon 7 Skipping

[0191] The next objective was to control the degree of exon 7 skipping with acyclovir. To achieve this, the first step was to identify a highly stable stem that efficiently sequesters theU1-binding sequence and thus exhibits complete skipping of exon 7. Subsequently, the goal was to insert the acyclovir aptamer into the stem in a way that the structure of the stem is destabilized, and the U1-binding sequence is exposed. This would cause all transcripts to contain exon 7 and ensure low background expression in the absence of acyclovir. This construct has to be designed so that addition of acyclovir causes the aptamer to undergo a conformational change that leads to efficient sequestration of the U1-binding sequence and skipping of exon 7. This would enable acyclovir-dependent control of gene expression.

[0192] To improve chances of finding an optimal site for insertion of the acyclovir aptamer, the objective was to develop multiple stem-loop structures that sequester the U1- binding sequence and efficiently induce exon 7 skipping.

[0193] The initial focus was on TSL2. To increase skipping of exon 7, a modified TSL2 described by Singh and colleagues (Singh et al., “Modulating Role of RNA Structure in Alternative Splicing of a Critical Exon in the Spinal Muscular Atrophy Genes,” Nucleic Acids Res.35:371-389 (2007), which is hereby incorporated by reference in its entirety) where the 6 nt of the U1-binding sequence are fully base paired with TSL2 was used (FIG.1I). Incorporation of intron 6trunc-exon 7-intron 7 cassette with this modified TSL2 rescued Renilla luciferase expression (FIG.1I), demonstrating that exon 7 was efficiently skipped (FIG.1I). Interestingly, the level of protein expression was higher than the transcript lacking the cassette. This likely reflects the stimulatory effect of splicing on mRNA nuclear export (Le Hir et al., The Exon-Exon Junction Complex Provides a Binding Platform for Factors Involved in mRNA Export and Nonsense-Mediated mRNA Decay,” Embo J.20:4987-4997 (2001), which is hereby incorporated by reference in its entirety), which is mediated by exon junction complexes deposited during splicing. Splicing is also associated with more efficient capping and polyadenylation of mRNAs (Le Hir et al., The Exon-Exon Junction Complex Provides a Binding Platform for Factors Involved in mRNA Export and Nonsense-Mediated mRNA Decay,” Embo J.20:4987-4997 (2001); Wiegand et al., “Exon Junction Complexes Mediate the Enhancing Effect of Splicing on mRNA Expression,” Proc. Natl. Acad. Sci. U S A 100:11327-11332 (2003); and Dahan et al., “Regulatory Mechanisms and Networks Couple the Different Phases of Gene Expression,” Trends Genet.27:316-322 (2011), which are hereby incorproated by reference in their entirety). This stabilized TSL2 was designated as “Hairpin 1” (FIG.1I) for further modification with the acyclovir aptamer.

[0194] The next aim was to create a second hairpin structure that can be tested as an entry point for the acyclovir aptamer. TSL2 forms a hairpin that traverses the exon-intron junction. An “intron-only” hairpin involving the U1-binding sequence on the 5’ side of thestem-loop structure followed by a loop and a newly inserted 6 nt-long U1-complementary sequence was therefore considered (FIG.1I). Since exon 7 already contains nucleotides that are complementary to the U1-binding sequence (i.e. one strand of TSL2) (FIG.1F), these nucleotides were mutated to ensure that they would not compete with the folding of this new hairpin. Incorporation of the intron 6trunc-exon 7-intron 7 cassette with this new hairpin led to highly efficient luciferase expression (FIG.1I) and efficient skipping of exon 7 (FIG.1I). This hairpin was designated as “Hairpin 2”. Thus, Hairpin 1 and Hairpin 2 can serve as entry points for an acyclovir aptamer. Example 4 – Control of SMN1 Exon 7 Skipping using an Acyclovir Aptamer

[0195] Given that both Hairpins 1 and 2 efficiently sequester the U1-binding sequence and thus cause complete exon 7 skipping, it was next sought to make these hairpins sequester the U1-binding sequence only upon the application of acyclovir. To do this, the acyclovir aptamer needs to be inserted into the hairpins so that the acyclovir-binding pocket is in close proximity to the U1-binding sequence, enabling ligand-dependent sequestration of the U1-binding sequence (FIG.2A).

[0196] To fuse the acyclovir aptamer with Hairpins 1 and 2, extraneous base pairs were first removed from each of these hairpins (FIG.2A). Above the base paired region that sequesters the U1-binding sequence, Hairpins 1 and 2 have additional base pairs and loop sequences. The acyclovir aptamer also has an extended stem below the putative acyclovir- binding site (Townshend et al., “A Multiplexed, Automated Evolution Pipeline Enables Scalable Discovery and Characterization of Biosensors,” Nat. Commun.12:1437 (2021), which is hereby incorporated by reference in its entirety). This stem was completely removed except for one A•U base pair. Base pairs above the U1-binding sequence of Hairpins 1 and 2 were removed, and fused this to the remaining part of the acyclovir aptamer to create the initial acyclovir- regulated splicing cassettes, which were termed Ac-Hairpin 1 and Ac-Hairpin 2 (FIG.2A).

[0197] Both cassettes were inserted into Renilla luciferase to test for acyclovir-induced expression of luciferase. The cassette that used Ac-Hairpin 1 did not generate any acyclovir- induced increase in luminescence: the baseline luciferase expression was 5.3% of the expression of the control luciferase lacking the poison exon, and it did not increase with acyclovir (500 µM, FIG.2B). In contrast, Ac-Hairpin 2 led to a 29-fold (0.4% to 12.7%) increase in luminescence upon addition of acyclovir (500 µM, FIG.2B).

[0198] The next objective was to increase the magnitude of acyclovir-induced luciferase expression from Ac-Hairpin 2. To obtain more acyclovir-induced poison exon skipping thestrategy was to induce more sequestration of the U1-binding sequence upon acyclovir binding. Adding one (Transducer 1 and 2), two (Transducer 3 and 4), and three base pairs (Transducer 5 and 6) of different compositions between the acyclovir aptamer and Hairpin 2 (FIG.2C) were tested. Among these, Transducer 1 showed the greatest improvement with gene activation (35.4%) while maintaining low background expression (1.0%) (FIG.2D).

[0199] It was next asked whether the activation of Transducer 1 could be further increased, while maintaining low background, by changing the base composition. Transducers 1-6 demonstrated increasing background with increasing transducer length (FIG.2D). Therefore, the transducer was kept at zero or one base pair to avoid background while testing an array of base pair compositions for increased activation (FIG.2C). It was found that these different sequence variants, termed Transducers 7-12, resulted in large changes in the performance (FIG.2D). In the case of Transducer 7 (Ac-H2Tr7), the signal ranged from 0.3% luciferase expression (no acyclovir) to 90.5% expression (500 µM acyclovir), which reflected a 295-fold gene induction by acyclovir. Therefore Ac-H2Tr7 was used for further analysis (FIG. 2E and FIG.12). Example 5 – M2 DMS MaP Reveals Acyclovir-Induced Conformational Change

[0200] To understand the structure of Ac-H2Tr7 and the conformational changes induced by acyclovir, a multidimensional chemical-mapping method called “mutate-and-map” dimethyl sulfate mutational profiling (M2 DMS MaP) was used. M2 DMS MaP is a powerful method for identifying pairs of interacting nucleotides (Cheng et al., “RNA Structure Inference through Chemical Mapping after Accidental or Intentional Mutations,” Proc. Natl. Acad. Sci. U S A 114:9876-9881 (2017) and Oleynikov and Jaffrey, “RNA Tertiary Structure and Conformational Dynamics Revealed by BASH MaP,” bioRxiv (2024), which are hereby incorporated by reference in their entirety). It relies on sparsely mutated nucleotides to induce structural perturbations in partner nucleotides, which are then detected through DMS probing and mutational profiling. Heatmaps generated by M2 DMS MaP reveal helix signatures as diagonal lines.

[0201] M2 DMS MaP was performed on in vitro transcribed 800-nt long Ac-H2Tr7 RNA sequence, including the adjacent intronic sequences. Mutations were introduced using error- prone PCR. To identify structural features, DMS probing was performed in the absence or presence of acyclovir (250 μM) on the full-length Ac-H2Tr7 RNA sequence. Targeted RT-PCR and amplicon sequencing were then performed on the region surrounding the acyclovir aptamer and the stem containing the U1-binding sequence (P1) (FIG.2F).

[0202] First, the one-dimensional DMS reactivity was plotted onto the secondary structure of this region (FIG.2F). Acyclovir lowers the overall one-dimensional DMS reactivity in the aptamer domain and the P1 stem, suggesting ligand-induced structural folding (FIG.2F). The residues at the putative acyclovir-binding pocket undergo the most significant change in DMS reactivity, and the large conformational change in the aptamer strengthens the folding of the P1 stem due to their close proximity, supporting the proposed regulatory mechanism.

[0203] Next, the amplicon sequencing data was analyzed to generate M2 heatmaps for the acyclovir-free and acyclovir-bound states (FIGS.2G–2H). The M2 heatmap of the acyclovir-bound state displays two large helical domains, as well as intervening box-like patterns (FIG.2H), which are notably absent in the ligand-free heatmap (FIG.2G). Box-like patterns in M2 heatmaps often indicate the presence of a complex tertiary conformation (Oleynikov and Jaffrey, “RNA Tertiary Structure and Conformational Dynamics Revealed by BASH MaP,” bioRxiv (2024), which is hereby incorporated by reference in its entirety), and thus likely represent the ligand-binding pocket of the acyclovir aptamer. Thus, the multidimensional M2 heatmaps collectively show a large conformational change at the ligand-binding pocket.

[0204] The M2 heatmaps display a diagonal pattern in P1 both with and without acyclovir (FIGS.2G–2H), indicating that the U1-binding sequence base pairs with its complementary sequence in both conditions. Pre-folding in the stem is likely promoted by the high Mg2+concentration (5 mM) used in the mapping procedure. Nevertheless, further strengthening of the stem by acyclovir was observed, as shown by the increased intensity in the heatmap (FIG.2H). Notably, the acyclovir-induced box-like network of interactions includes the immediately adjacent base pairs in the P1 stem, suggesting that these residues are directly regulated by acyclovir binding. This base pairing likely stabilizes the remainder of the P1 stem, thus facilitating the overall sequestration of the U1-binding sequence.

[0205] Overall, M2 DMS MaP data reveals marked acyclovir-induced formation of complex tertiary interactions at the ligand-binding pocket of the aptamer, which suggests that the acyclovir aptamer is especially suitable for ligand-induced regulation of RNA-based devices. Example 6 – Design of a Poison Exon that Functions in Multiple Cell Lines

[0206] To determine if Ac-H2Tr7 exhibits the same low background and high activation in different cell types, Renilla luciferase containing Ac-H2Tr7 was expressed in three additional cell lines of diverse origins: HeLa, Huh-7 (human hepatocellular carcinoma cell line), and HT- 1080 (human fibrosarcoma). In Huh-7 and HT-1080 cells, elevated background expression was observed, suggesting that the poison exon was not efficiently spliced (FIG.3A). It was thereforesought to modify the SMN1 introns using more efficient intron junctional sequences to enhance poison exon splicing.

[0207] Several well-studied introns that splice with high efficiencies were tested (FIG. 11): MAP3K7 intron 4 (North et al., “Synthetic Introns Enable Splicing Factor Mutation- Dependent Targeting of Cancer Cells,” Nat. Biotechnol.40:1103-1113 (2022), which is hereby incorporated by reference in its entirety), ORAI2 intron 1 (North et al., “Synthetic Introns Enable Splicing Factor Mutation-Dependent Targeting of Cancer Cells,” Nat. Biotechnol.40:1103-1113 (2022), which is hereby incorporated by reference in its entirety), TMEM14C intron 1 (North et al., “Synthetic Introns Enable Splicing Factor Mutation-Dependent Targeting of Cancer Cells,” Nat. Biotechnol.40:1103-1113 (2022), which is hereby incorporated by reference in its entirety), HBB intron 2 (Zhang et al., “Intron Function in the Nonsense-Mediated Decay of Beta-Globin mRNA: Indications that pre-mRNA Splicing in the Nucleus can Influence mRNA Translation in the Cytoplasm,” Rna 4:801-815 (1998), which is hereby incorporated by reference in its entirety), a β-globin / IgG chimeric intron (Bothwell et al., “Heavy Chain Variable Region Contribution to the NPb Family of Antibodies: Somatic Mutation Evident in a Gamma 2a Variable Region,” Cell 24:625-637 (1981) and Brinster et al., “Introns Increase Transcriptional Efficiency in Transgenic Mice,” Proc. Natl. Acad. Sci.U S A 85:836-840 (1988), which is hereby incorporated by reference in its entirety), the MINX intron (Zapp et al., “Evidence for Nuclear Factors Involved in Recognition of 5' Splice Sites,” Nucleic Acids Res.17:2655-2674 (1989), which is hereby incorporated by reference in its entriety), and TPI1 introns 4 and 5 (Cheng and Maquat, “Nonsense Codons can Reduce the Abundance of Nuclear mRNA without Affecting the Abundance of pre-mRNA or the Half-Life of Cytoplasmic mRNA,” Mol. Cell Biol.13:1892- 1902 (1993), which is hereby incorporated by reference in its entirety). The long introns were first truncated to 250 nt by removing internal sequence while preserving splice junctions and branch points (FIG.3B). Then, each intron was inserted into Renilla luciferase to test their ability to undergo splicing (FIG.3C). Among these introns, HBB intron 2 and β-globin / IgG chimeric intron showed better performance than the SMN1 introns (FIG.8B and FIG.3D). Besides restoring luciferase expression by removing themselves from the coding sequence, HBB intron 2 and chimeric intron were able to enhance overall protein expression by 2-fold and 3-fold respectively (FIG.3D).

[0208] The next objective was to confer the improved splicing properties to the Ac- H2Tr7-containing cassette by partially replacing the SMN1 introns with the stronger introns. For these experiments, the intron 6 and intron 7 sequence were presented immediately flanking exon 7. The remaining parts of intron 6 and 7 were replaced, alone or in combination, with strongerintrons to create six chimeric introns (FIG.3E) and then tested for performance in Huh-7 and HT-1080 cells (FIG.3F). Among these, a modified version of intron 6 that exhibited almost no background expression and high levels of acyclovir-induced activation in both cell lines was identified (FIG.3F). This construct comprised the first 100 nt of β-globin / IgG chimeric intron and the terminal 138 nt of intron 6, and maintained the SMN1 intron 7. This construct was termed Ac-H2Tr7In4 (FIG.12).

[0209] It was next investigated whether Ac-H2Tr7In4 enabled acyclovir-induced poison exon skipping in other cell types. Four additional cell lines, HeLa, U-2 OS, HEK293T, and HCT 116, were tested and low background and high acyclovir-induced luciferase expression to at least 50% the level seen with the control luciferase lacking the poison exon in all cell types was found (FIG.3G).

[0210] Overall, these experiments demonstrate that Ac-H2Tr7In4 converts exon 7 into a portable poison exon with high performance in multiple different cell types. The Ac-H2Tr7In4- containing cassette was designated Cyclone (acyclovir-controlled poison exon). Example 7 – Tunable and Reversible Protein Expression by Acyclovir-Dependent Skipping of the Poison Exon

[0211] The next aim was to confirm that the mechanism of acyclovir-induced luciferase expression is due to acyclovir-induced exon 7 skipping. To test this, Cyclone-Renilla luciferase was expressed in HEK293T cells treated with acyclovir ranging from 0 µM to 2 mM for 48 hours. In the absence of acyclovir, a single RT-PCR product (251 nt) corresponding to poison exon inclusion was observed (FIG.4A). As the acyclovir concentration was increased, a 197-nt RT-PCR product appeared, reflecting poison exon skipping (FIG.4A). The poison exon-skipped splicing isoform continued to increase as the acyclovir concentration increased. Similarly, protein expression increased with acyclovir concentration, with 250 µM acyclovir leading to ~100% gene activation relative to the control lacking the poison exon (FIG.4B). Acyclovir- dependent regulation of luciferase expression was also seen when the construct was expressed using promoters of different strengths (FIG.4C). Overall, these data demonstrate that acyclovir dose can control poison exon skipping and protein expression levels.

[0212] Notably, Cyclone enhanced luciferase expression by ~2-3-fold compared to Ac- H2Tr7 at every acyclovir dose tested (FIG.4B). Besides tuning expression between 0% and 100% relative to the control lacking the poison exon, doses of acyclovir higher than 250 µM resulted in higher expression than the no-intron control. The maximal acyclovir-induced activation increased from 101.4% (2 mM acyclovir, Ac-H2Tr7) to 349.5% (2 mM acyclovir, Ac-H2Tr7In4 / Cyclone). Thus, Cyclone may allow subphysiological to supraphysiological levels of gene expression.

[0213] Next, the time course of acyclovir-induced poison exon skipping was examined. For these experiments, Cyclone was inserted into enhanced green fluorescent protein (EGFP) (FIG.4D and FIG.13). Similar to the regulation of Renilla luciferase, a dose-dependent change in green fluorescence was observed by live cell imaging (FIG.4E). Poison exon skipping from the EGFP transcript was detected ~2 hours after treating cells with acyclovir (500 µM). Maximal poison exon skipping was seen after 6 h (FIG.4F).

[0214] The reversibility of acyclovir-induced exon skipping was next assessed. HEK293T cells were cultured in 500 µM acyclovir for 24 hours before washing out the drug by exchanging fresh media without acyclovir. At the beginning of the washout, it was observed that most EGFP transcript lacked the poison exon, indicating high exon skipping (FIG.4G). After 2 hours, a clear increase in EGFP transcript containing the poison exon was observed. And at 6 hours, nearly all EGFP transcript contained the poison exon (FIG.4G). Thus, acyclovir induces poison exon skipping in a dose-dependent manner, and acyclovir withdrawal results in rapid switching from poison exon skipping to poison exon inclusion. Example 8 – Cyclone Enables Acyclovir-Dependent Expression of Diverse Genes

[0215] One feature of the Cyclone cassette is that it can be used for traceless protein expression, which is the ability to completely remove itself without affecting the sequence of the expressed protein. This requires the Cyclone cassette to be directly inserted into the gene of interest (Sibley et al., “Lessons from Non-Canonical Splicing,” Nat. Rev. Genet.17:407-421 (2016) and Zhang et al., “Sequence Information for the Splicing of Human pre-mRNA Identified by Support Vector Machine Classification,” Genome Res.13:2637-2650 (2003), which are hereby incorporated by reference in their entirety). Based on the splice site consensus motifs, the Cyclone cassette can be inserted in the coding portion of a gene between an NAG and GNN codon. In this way, the stop codon of Cyclone will be in-frame. The coding sequence provides the exonic portion of the splice sites that are contiguous with the 5’ end of the β-globin / IgG chimeric intron and the 3’ end of SMN1 intron 7. Since many codons can undergo synonymous mutations that maintain the identity of the encoded amino acid, Cyclone can be inserted in-frame after any glutamine, glutamate or lysine that is followed by a valine, alanine, aspartate, glutamate or glycine. The versatility ensures that multiple potential insertion sites will likely exist for any single gene of interest.

[0216] To test whether insertion location affects the performance of Cyclone, Cyclone was inserted into the mouse thrombopoietin (Thpo) gene at two positions (FIG.5A): between Q82 and D83 or between Q117 and V118 (FIG.13). Each Cyclone-Thpo construct was expressed in HEK293T cells. Thrombopoietin levels were measured in the culture media by ELISA (FIG.5B). At both insertion sites, thrombopoietin was expressed in an acyclovir dose- dependent manner with minimal background and similar degrees of maximal activation.

[0217] Thus, poison exon insertion can lead to traceless control of protein expression and appears to be relatively versatile with respect to the insertion site.

[0218] To further test Cyclone, a Cre recombinase construct containing Cyclone was created (FIG.13) and transfected into a loxP-GFP Cre reporter N2a cell line (Segel et al., “Mammalian Retrovirus-Like Protein PEG10 Packages its own mRNA and can be Pseudotyped for mRNA Delivery,” Science 373:882-889 (2021), which is hereby incorporated by reference in its entirety) to measure Cre activity (FIG.5C). In the absence of acyclovir, no Cre expression was detected by western blot (FIG.5D), and minimal GFP expression was observed (FIGS.5E– 5F). Cre expression increased with higher doses of acyclovir (FIG.5D), with approximately 40% of cells exhibiting green fluorescence (FIGS.5E–5F). Overall, these data demonstrate that acyclovir can control Cre expression and Cre function.

[0219] An expression system using Cyclone was developed that enables the efficient selection of cells exhibiting acyclovir-induced expression of any transgene. A puromycin N- acetyltransferase (pac)-P2A-EGFP fusion protein was generated. The P2A sequence is a self- cleaving peptide (Liu et al., “Systematic Comparison of 2A Peptides for Cloning Multi-Genes in a Polycistronic Vector,” Scientific Reports 7:2193 (2017), which is hereby incorporated by reference in its entirety) that causes puromycin N-acetyltransferase and EGFP to be produced as separate proteins. Cyclone was inserted into the pac gene (FIG.13) and expressed the construct in HEK293T cells cultured in the presence of acyclovir. Puromycin-selected HEK293T cells exhibited acyclovir dose-dependent EGFP expression, as determined by fluorescence microscopy (FIGS.5G–5I). Overall, this approach provides a general method for creating cell lines with acyclovir-induced transgene expression. Example 9 – Cyclone Enables Control of Endogenous Gene Expression with Acyclovir

[0220] Because the Cyclone cassette regulates gene expression without interfering with promoter elements, it was asked whether Cyclone could control endogenous gene expression. To create a general and simple approach, a Pac-Cyclone (FIG.14), a cassette that can be knocked into almost any gene at the start codon using standard CRISPR methods, was designed(FIG.6A). Pac-Cyclone contains Cyclone inserted within the pac selection marker, followed by a P2A site. A 3xFLAG sequence was also included after the P2A site to simplify detection of the endogenous protein. After insertion of Pac-Cyclone immediately after the start codon, the endogenous promoter induces translation of puromycin N-acetyltransferase and the protein encoded by the endogenous gene. The intervening P2A site ensures that these are produced as separate proteins. Pac-Cyclone is simpler than Cyclone, as Cyclone needs to be inserted at sites compatible with the exonic splice site consensus sequences. In contrast, the 5' and 3' ends of Pac-Cyclone are coding sequences, and the Cyclone sequence is localized internally. Thus, Pac- Cyclone can be inserted at any location within a coding sequence (FIGS.6B–6C), and knocked- in cell lines can be obtained with puromycin selection.

[0221] Pac-Cyclone was knocked into two endogenous genes, WTAP (WT1 associated protein) and PCIF1 (phosphorylated CTD interacting factor 1). WTAP and PCIF1 have different exon-intron structures and produce proteins of different sizes, which can demonstrate the versatility of Pac-Cyclone. WTAP was chosen since this is an essential gene and therefore stable knockout cell lines cannot be generated (Poh et al., “Alternative Splicing of METTL3 Explains Apparently METTL3-Independent m6A Modifications in mRNA,” PLoS Biol 20:e3001683 (2022), which is herby incorporated by reference in its entirety). Thus, Cyclone can allow transient depletion of WTAP protein using acyclovir withdrawal. In both cases, homology arms from each gene for CRISPR / Cas9 knock-in were used (FIGS.15–16). After knock-in into HCT 116 cells, cells were cultured with acyclovir (250 µM) to maintain expression of the endogenous gene and with puromycin to select for stable integration, and in the case of WTAP, clonal cell lines (FIG.6D).

[0222] Prior to the selection of clonal cell lines, acyclovir-induced expression from the tagged allele was readily detected by anti-FLAG western blotting (FIG.6E). Anti-WTAP western blotting revealed the endogenous WTAP and a slightly larger band corresponding to 3xFLAG-WTAP (FIG.6E). Similar results were obtained with PCIF1 (FIG.6F). The media was replaced with media containing 0, 62.5, and 250 µM acyclovir for 2 days, which demonstrated dose-dependent WTAP and PCIF1 expression.

[0223] Since these results showed both the endogenous protein and the 3xFLAG-tagged protein, the cells likely comprise a mix of heterozygous and homozygous knock-in. Clonal selection was therefore performed to obtain homozygous Cyclone-controlled WTAP cell lines (FIG.9A). In these clonal cell lines, only a single WTAP band was observed, reflecting the 3xFLAG-WTAP (FIG.6G). The cells were then cultured in varying concentrations of acyclovir for 5 days and found dose-dependent WTAP expression with no detectable WTAP at 0 µMacyclovir (FIG.6G). Notably, doses of acyclovir above 62.5 µM resulted in WTAP levels that exceeded wild-type levels, demonstrating that both depletion and overexpression can be obtained depending on the acyclovir dose. Acyclovir-induced poison exon skipping was also detected using a splicing assay with primers annealing adjacent to the targeted WTAP genomic locus (FIG.6G). Discussion of Examples 1 – 9

[0224] Poison exons are common regulatory elements that modulate gene expression through alternative splicing. In this study, a natural alternative splicing event was transformed into a drug-regulated poison exon, creating a highly portable intron-exon-intron cassette. This cassette can be seamlessly inserted into various genes, including both transgenes and endogenous genes. Notably, Cyclone does not require exogenous proteins, which makes Cyclone a straightforward system for small molecule control of splicing and gene expression in various contexts.

[0225] The Cyclone cassette, featuring optimized introns flanking the poison exon, can be easily inserted into endogenous genes using standard CRISPR knock-in techniques. Although a cassette containing an N-terminal puromycin selection marker, which is removed from the protein of interest using a P2A sequence, was used, Cyclone can also be used without puromycin selection. In this case, Cyclone can be inserted within a gene of interest, thus preserving the N- terminus of the target gene if necessary. While Cyclone can be used to control transgene expression, it can be inserted into diverse gene loci, thus enabling gene expression regulation at physiologically relevant expression levels.

[0226] Creating artificial splicing is challenging due to the complex regulation of splicing, which is influenced by splice site strengths (Yeo and Burge, “Maximum Entropy Modeling of Short Sequence Motifs with Applications to RNA Splicing Signals,” J. Comput. Biol.11:377-394 (2004), which is hereby incorporated by reference in its entirety), exon-intron architecture (Fox-Walsh et al., “The Architecture of pre-mRNAs Affects Mechanisms of Splice- Site Pairing,” Proc. Natl. Acad. Sci. U S A 102:16176-16181 (2005), which is hereby incorporated by reference in its entirety), splicing enhancers and silencers (Black, D.L., “Mechanisms of Alternative Pre-Messenger RNA Splicing,” Annu. Rev. Biochem.72:291-336 (2003), which is hereby incorporated by reference in its entirety), and the role of RNA polymerase II during transcription (Kornblihtt, A.R., “Promoter Usage and Alternative Splicing,” Curr. Opin. Cell Biol.17:262-268 (2005), which is hereby incorporated by reference in its entirety). This prompted us to identify natural alternative splicing events that rely on secondarystructures at splice junctions. Stem-loop structures near splice junctions, including TSL2 in SMN1, are considered important for maintaining splicing isoform ratios in the cell (Warf and Berglund, “Role of RNA Structure in Regulating pre-mRNA Splicing,” Trends Biochem. Sci. 35:169-178 (2010) and Bose et al., “Causes, Functions, and Therapeutic Possibilities of RNA Secondary Structure Ensembles and Alternative States,” Cell Chem. Biol.31:17-35 (2024), which are hereby incorporated by reference in their entirety). By incorporating an acyclovir aptamer into TSL2, this natural structure was successfully modified to achieve small molecule- controlled gene regulation. This approach demonstrates robust control of alternative splicing and protein expression using a naturally derived gene cassette, which might account for the highly improved performance compared to earlier synthetic drug-controlled artificial splicing systems (Vogel et al., “A Small, Portable RNA Device for the Control of Exon Skipping in Mammalian Cells,” Nucleic Acids Res.46:e48 (2018) and Finke et al., “Efficient Splicing-Based RNA Regulators for Tetracycline-Inducible Gene Expression in Human Cell Culture and C. elegans,” Nucleic Acids Res.49:e71 (2021), which are hereby incorporated by reference in their entirety).

[0227] RNA-binding small molecules have previously been used to regulate splicing. The most notable is branaplam, which binds to the 3’ end of exon 7 of SMN2. SMN2 is similar to SMN1 except for a C-to-T mutation in exon 7, which causes exon 7 skipping. Branaplam acts as molecular glue that stabilizes the transient interaction between SMN2 pre-mRNA and the U1 small nuclear ribonucleoprotein (U1 snRNP) complex (Palacino et al., “SMN2 Splice Modulators Enhance U1-pre-mRNA Association and Rescue SMA Mice,” Nat. Chem. Biol. 11:511-517 (2015), which is hereby incorporated by reference in its entirety). The enhanced recruitment of U1 snRNP increases SMN2 exon 7 inclusion. Rather than binding U1 snRNP, acyclovir acts to alter the accessibility of U1 snRNP to SMN1 pre-mRNA. Acyclovir directly binds to RNA and stabilizes a helical stem, which leads to more efficient sequestration of the U1-binding site. Thus, the design of Cyclone results in a fundamentally different mechanism for splicing control, ultimately leading to an opposite outcome compared to branaplam, i.e., drug- induced skipping of exon 7.

[0228] Many RNA-based devices have been generated, but they typically exhibit relatively poor activation by small molecules. For example, theophylline- and tetracycline- regulated ribozymes (Xiang et al., “Massively Parallel RNA Device Engineering in Mammalian Cells with RNA-Seq,” Nature Communications 10:4327 (2019); and Beilstein et al. “Conditional Control of Mammalian Gene Expression by Tetracycline-Dependent Hammerhead Ribozymes,” ACS Synth. Biol.4:526-534 (2015), which are hereby incorporated by reference in their entirety) only show 8-9-fold regulation in cells. Metabolite-binding aptamers that induce folding andfluorescence of fluorogenic aptamers, such as Broccoli, typically exhibit less than 20-fold increases in fluorescence (You et al., “Imaging Metabolite Dynamics in Living Cells Using a Spinach-Based Riboswitch,” Proceedings of the National Academy of Sciences 112:E2756- E2765 (2015) and Moon et al., “Naturally Occurring Three-Way Junctions can be Repurposed as Genetically Encoded RNA-Based Sensors,” Cell Chemical Biology 28:1569-1580. e1564 (2021), which are hereby incorporated by reference in their entirety). In contrast, the acyclovir-regulated stem-loop leads to an unusually large effect on splicing, with approximately a 300-fold increase in transgene expression induced by acyclovir. In this case, the high performance is likely due to the coupling of RNA conformational changes to spliceosome binding and subsequent splicing (Panchapakesan and Breaker, “The Case of the Missing Allosteric Ribozymes,” Nat. Chem. Biol. 17:375-382 (2021), which is hereby incorporated by reference in its entirety). In particular, the no-acyclovir state leads to sufficient exposure of the U1-binding site to ensure U1 snRNP binding, splicing, and inclusion of the poison exon, resulting in low background. However, acyclovir binding sequesters the U1-binding site, which is sufficient to block U1 snRNP binding and prevent splicing. This may account for the remarkably effective control of splicing by an RNA-binding small molecule.

[0229] Another key feature of Cyclone is the acyclovir-binding aptamer. Structural analysis reveals significant structural reorganization of the acyclovir-binding aptamer, especially within the ligand-binding pocket, upon acyclovir binding. Additionally, the acyclovir-binding pocket includes base pairs directly integrated into the helical stem that sequesters the U1-binding sequence. In this way, acyclovir binding efficiently regulates the hybridization of the helical stem containing the U1-binding sequence. This makes the acyclovir aptamer highly promising for the design of RNA-based devices.

[0230] An important consideration in small-molecule control of gene expression is ensuring the molecule has well-documented low toxicity. Acyclovir, a prodrug converted into a nucleotide analog by a viral kinase, specifically inhibits viral polymerase without affecting mammalian proteins (Elion, G.B., “The Biochemistry and Mechanism of Action of Acyclovir,” J. Antimicrob Chemother.12 Suppl B:9-17 (1983) and Elion, G.B., “Mechanism of Action and Selectivity of Acyclovir,” Am. J. Med.73:7-13 (1982), which are hereby incorporated by reference in their entirety). Its well-documented safety in humans indicates that acyclovir does not negatively impact cellular function, making it more appealing than commonly used alternatives like tetracycline, which can have adverse effects on cells. Therefore, the acyclovir aptamer is likely to be widely adopted in RNA-based devices for cellular applications.

[0231] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Claims

WHAT IS CLAIMED IS:

1. A minigene cassette comprising, from 5’ to 3’: a first intron; an alternatively-spliced exon; a second intron comprising a U1 binding sequence, wherein: (i) a portion of the alternatively-spliced exon is complementary to a portion of the second intron; the portion of the alternatively-spliced exon that is complementary to the portion of the second intron forms a hairpin loop structure comprising a hairpin stem and a hairpin loop; the hairpin stem comprises at least a portion of the U1 binding sequence; and the hairpin loop comprises an aptamer; or (ii) a portion of the second intron forms a hairpin loop structure comprising a hairpin stem and a hairpin loop; the hairpin stem comprises at least a portion of the U1 binding sequence; and the hairpin loop comprises an aptamer.

2. The minigene cassette according to claim 1, wherein the hairpin stem is a perfect hairpin stem.

3. The minigene cassette according to claim 1, wherein the hairpin stem is an imperfect hairpin stem.

4. The minigene cassette according to any one of claims 1 to 3, wherein the second intron comprises a 5’ splice site (5’ ss) and the hairpin loop structure comprises the 5’ ss.

5. The minigene cassette according to any one of claims 1 to 4, wherein the hairpin stem comprises at least 1 to 6 nucleic acids of the U1 binding sequence.

6. The minigene cassette according to any one of claims 1 to 5, wherein the aptamer binds a ligand selected from the group consisting of a small-molecule, a protein, a peptide, a cell, a virus, a bacterium, and a nucleic acid.

7. The minigene cassette according to any one of claims 1 to 6, wherein the aptamer binds a ligand selected from the group consisting of acyclovir, valacyclovir, famciclovir, penciclovir, and ganciclovir.

8. The minigene cassette according to claim 7, wherein the aptamer binds acyclovir.

9. The minigene cassette according to claim 8, wherein the aptamer comprises SEQ ID NO:

49.

10. The minigene cassette according to claim 9, wherein the aptamer comprises SEQ ID NO: 50, SEQ ID NO: 51, or SEQ ID NO:

52.

11. The minigene cassette according to any one of claims 1 to 10, wherein the alternatively-spliced exon encodes a stop codon, a start codon, a polyadenylation signal sequence, one or more domains that encode a protein sequence of interest, a 5’ untranslated region sequence element, and / or a 3’ untranslated region sequence element.

12. The minigene cassette according to any one of claims 1 to 10, wherein the alternatively-spliced exon does not encode a stop codon.

13. The minigene cassette according to claim 12, wherein the alternatively- spliced exon results in a frame shift mutation in a downstream exon.

14. The minigene cassette according to any one of claims 1 to 13, wherein the alternatively-spliced exon contains a start codon.

15. The minigene cassette according to any one of claims 1 to 14, wherein the first intron contains an in / out of frame stop codon.

16. The minigene cassette according to any one of claims 1 to 15, wherein the hairpin stem comprises a transducer region.

17. The minigene cassette according to claim 16, wherein the transducer region comprises 6–20 base pairs.

18. The minigene cassette according to claim 16 or claim 17, wherein the transducer region comprises a first portion comprising a transducer region sequence from Table 2 and a second portion which is the reverse complement of the first portion.

19. The minigene cassette according to any one of claims 1 to 18, wherein the first intron is between 50 to 300 nucleotides in length.

20. The minigene cassette according to any one of claims 1 to 19, wherein the first intron comprises SEQ ID NO: 10 , SEQ ID NO: 68, or SEQ ID NO:

65.

21. The minigene cassette according to any one of claims 1 to 20, wherein the alternatively-spliced exon is between 50 to 54 nucleotides in length.

22. The minigene cassette according to any one of claims 1 to 21, wherein the alternatively-spliced exon comprises SEQ ID NOs: 66 or 67.

23. The minigene cassette according to any one of claims 1 to 24, wherein the second intron is between 250 to 500 nucleotides in length.

24. The minigene cassette according to any one of claims 1 to 23, wherein the second intron comprises the nucleic acid sequence of SEQ ID NO: 72, SEQ ID NO: 11, SEQ ID NO: 69, SEQ ID NO: 70, or SEQ ID NO:

71.

25. The minigene cassette according to any one of claims 1 to 24, wherein the minigene cassette comprises the nucleic acid sequence of any one of SEQ ID NOs: 73 to 85.

26. The minigene cassette according to any one of claims 1 to 25, wherein the first intron, alternatively-spliced exon, and / or second intron are derived from one or more genes selected from the group consisting of survival motor neuron 1 (SMN1), SMN2, HGH, PS2, Tau, Hnrnpa1, Ncam, IgM, β-TM, and β-globin.

27. The minigene cassette according to claim 26, wherein the first intron, alternatively-spliced exon, and / or second intron are derived from the same gene.

28. The minigene cassette according to claim 27, wherein the first intron and the second intron are derived from a human β-globin gene.

29. The minigene cassette according to claim 28, wherein the first intron and the second intron are derived from intron 2 of the human β-globin gene.

30. The minigene cassette according to claim 28 or claim 29, wherein the alternatively-spliced exon comprises at least a portion of the group consisting of: exon 2 of the human dihydrofolate reductase gene, mutant human Wilms tumor 1 exon 5, mouse calcium / calmodulin - dependent protein kinase II delta exon 16, and SIRT1 exon 6 31. The minigene cassette according to any one of claims 1 to 30, wherein the first intron, alternatively-spliced exon, and / or second intron are synthetic, recombinant, or comprise heterologous sequences.

32. The minigene cassette according to any one of claims 1 to 31, wherein the alternatively-spliced exon contains a synthetic, recombinant, or heterologous sequence encoding an epitope tag.

33. The minigene cassette according to claim 32, wherein the tag sequence is selected from the group consisting of FLAG tag, a 6xHIS tag, a V5 tag, a c-Myc tag, an HA tag, a GST tag, a Strep II tag, an E2 epitope tag, an HSV epitope tag, a KT3 epitope tag, a polyaspartate tag, a polycysteine tag, a polyphenylalanine tag, an S1 tag, an S tag, a Strep tag, a VSV-G tag, an AU1 epitope tag, and an AU5 epitope tag.

34. The minigene cassette according to any one of claims 1 to 33, wherein the first intron, alternatively-spliced exon, and / or second intron are derived from a human gene.

35. The minigene cassette according to any one of claims 1 to 34, wherein: (i) the first intron comprises at least a portion of SMN1 intron 6 (SEQ ID NO: 10) or at least about 5, 6, 7, 8, 9, or 10 continuous nucleic acids of SMN1 intron 6 (SEQ ID NO: 10);(ii) the alternatively-spliced exon comprises at least a portion of SMN1 exon 7 (SEQ ID NO: 66) or at least about 5, 6, 7, 8, 9, or 10 continuous nucleic acids of SMN1 exon 7 (see SEQ ID NO: 66); and / or (iii) the second intron comprises at least a portion of SMN1 intron 7 (SEQ ID NO: 94) or at least about 5, 6, 7, 8, 9, or 10 continuous nucleic acids of SMN1 intron 7 (see SEQ ID NO: 94).

36. The minigene cassette according to any one of claims 1 to 35 further comprising: an exon comprising a start codon at its 3’ end, wherein the exon comprising the start codon is immediately upstream of the first intron.

37. The minigene cassette according to claim 36, wherein the exon comprising the start codon comprises SEQ ID NO:

95.

38. The minigene cassette according to claim 36 or claim 37, wherein the exon comprising the start codon comprises a nucleotide sequence from a gene selected from the group consisting of survival motor neuron 1 (SMN1), SMN2, HGH, PS2, Tau, Hnrnpa1, Ncam, IgM, β-TM, and β-globin.

39. The minigene cassette according to claim 38, wherein the exon comprising the start codon comprises a nucleotide sequence from the same gene as the first intron sequence, the alternatively-spliced exon sequence, and / or the second intron sequence.

40. The minigene cassette according to claim 38, wherein the exon comprising the start codon comprises a synthetic sequence, recombinant sequence, or sequence heterologous to the first intron sequence, the alternatively-spliced exon sequence, and / or the second intron sequence.

41. The minigene cassette according to any one of claims 36-39, wherein the exon comprising the start codon comprises a nucleotide sequence from a mammalian gene.

42. The minigene cassette according to claim 41, wherein the exon comprising the start codon comprises a nucleotide sequence from a human gene.

43. The minigene cassette according to claim 42, wherein the exon comprising the start codon comprises at least a portion of SMN1 exon 6 (SEQ ID NO: 95) or at least 5, 6, 7, 8, 9, or 10 continuous nucleic acids of SMN1 exon 6 (SEQ ID NO: 95).

44. A recombinant polynucleotide comprising: a gene of interest and the minigene cassette according to any one of claims 1 to 35 for regulating expression of the gene of interest, wherein the minigene cassette is located within the protein coding sequence of the gene of interest.

45. The recombinant polynucleotide according to claim 44 or claim 45, wherein the alternatively-spliced exon does not comprise a stop codon in-frame with the gene of interest.

46. A recombinant polynucleotide comprising: a gene of interest and the minigene cassette according to any one of claims 36 to 43 for regulating expression of the gene of interest, wherein the minigene cassette is located upstream of the gene of interest.

47. A recombinant polynucleotide comprising: a 5’ homology arm sequence; the minigene cassette according to any one of claims 1 to 43; and a 3’ homology arm sequence.

48. The recombinant polynucleotide according to claim 47, wherein the minigene cassette is positioned between the 5’ homology arm sequence and the 3’ homology arm sequence.

49. The recombinant polynucleotide according to claim 47 or claim 48, wherein the 5’ homology arm sequence and / or the 3’ homology arm sequence is homologous toa gene of interest, optionally wherein the 5’ homology arm and the 3’ homology arm targets a protein coding region of the gene of interest.

50. The recombinant polynucleotide according to claim 49, wherein the gene of interest comprise a start codon, wherein the 5’ homology arm sequence is homologous with a portion of the gene of interest 5’ to the start codon; and wherein the 3’ homology arm sequence is homologous with a portion of the gene of interest 3’ to the start codon.

51. The recombinant polynucleotide according to any one of claims 47 to 50, wherein the 3’ homology arm sequence and / or the 3’ homology arm sequence are approximately 300 nucleotides in length.

52. The recombinant polynucleotide according to any one of claims 44 to 51, wherein the gene of interest is selected from the group consisting of a therapeutic protein, a reporter protein, an enzyme, a transcription factor, a cytokine, a chemokine, an antibody, a hormone, a transport protein, a growth factor, an intracellular protein, an extracellular protein, a cytoplasmic protein, a transmembrane protein, a secreted protein, a receptor molecule, an RNA binding protein, a DNA binding protein, a channel protein, a cell adhesion molecule, a kinase, a phosphatase, and a chaperone protein.

53. The recombinant polynucleotide according to any one of claims 44 to 52, wherein the first and second introns are exogenous to the gene of interest.

54. The recombinant polynucleotide according to any one of claims 44 to 53, wherein the recombinant polynucleotide further comprises a nucleic acid sequence encoding a selection marker.

55. The recombinant polynucleotide according to claim 54, wherein the minigene cassette is positioned within the nucleic acid sequence encoding the selection marker.

56. The recombinant polynucleotide according to any one of claims 54 to 56, wherein the selection marker is an antibiotic resistance gene.

57. The recombinant polynucleotide according to claim 56, wherein the antibiotic resistance gene is a puromycin resistance gene.

58. The recombinant polynucleotide according to any one of claims 44 to 57 further comprising a nucleic acid sequence encoding a self-cleaving peptide.

59. The recombinant polynucleotide according to claim 58, wherein the nucleic acid sequence encoding the self-cleaving peptide is selected from the group consisting of porcine teschovirus-12A (P2A), thosea asigna virus 2A (T2A), equine rhinitis A virus 2A (E2A), cytoplasmic polyhedrosis virus (BmCPV 2A), and flacherie virus (BmIFV 2A).

60. The recombinant polynucleotide according to claim 60 or claim 61, wherein the nucleic acid sequence encoding the self-cleaving peptide is positioned in a manner effective to mediate expression of an epitope tag.

61. The recombinant polynucleotide according to any one of claims 44 to 59 further comprising a nucleic acid sequence encoding an epitope tag.

62. The recombinant polynucleotide according to claim 60 or claim 61, wherein the epitope tag is selected from the group consisting of FLAG tag, a 6xHIS tag, a V5 tag, a c-Myc tag, an HA tag, a GST tag, a Strep II tag, an E2 epitope tag, an HSV epitope tag, a KT3 epitope tag, a polyaspartate tag, a polycysteine tag, a polyphenylalanine tag, an S1 tag, an S tag, a Strep tag, a VSV-G tag, an AU1 epitope tag, and an AU5 epitope tag.

63. The recombinant polynucleotide according to any one of claims 47 to 62, wherein the polynucleotide sequence comprises: the 5’ homology arm; a nucleic acid sequence encoding a selection marker positioned 3’ to the 5’ homology arm; the minigene cassette according to any one of claims 1 to 43 positioned within the nucleic acid sequence encoding the selection marker; the nucleic acid sequences encoding the self-cleaving peptide positioned 3’ to the nucleic acid sequence encoding the selection marker;the nucleic acid sequence encoding an epitope tag positioned 3’ to the nucleic acid sequence encoding the self-cleaving peptide; and the 3’ homology arm.

64. The recombinant polynucleotide according to any one of claims 44 or 46 to 63, wherein the alternatively-spliced exon comprises a stop codon in-frame with the target gene.

65. A vector encoding the minigene cassette according to any one of claims 1 to 43 or comprising the recombinant polynucleotide of any one of claims 44 to 64.

66. The vector according to claim 65, wherein the vector is an expression vector.

67. The vector according to claim 65 or claim 6, wherein the vector is a viral vector.

68. The vector according to claim 67, wherein the viral vector is selected from the group consisting of adenoviral vector, adeno-associated virus vector, and lentiviral vector.

69. A cell comprising the recombinant polynucleotide of any one of claims 44 to 64 or the vector according to any one of claims 65 to 68.

70. The cell according to claim 69, wherein the cell is a mammalian cell.

71. The cell according to claim 70, wherein the mammalian cell is a human cell.

72. A method of modulating the expression of a gene of interest, said method comprising: introducing the recombinant polynucleotide of any one of claims 44 to 64 or the vector according to any one of claims 65 to 68 into a cell and culturing the cell, wherein:(i) in the absence of a ligand specific for the aptamer located in the hairpin loop of the minigene cassette, transcription of the recombinant polynucleotide results in the production of a spliced transcript which includes the alternatively-spliced exon of the minigene cassette and allows for the translation of a truncated gene of interest or prevents translation of the gene of interest and (ii) in the presence of the ligand specific for the aptamer located in the hairpin loop of the minigene cassette, transcription of the recombinant polynucleotide results in the production of a spliced transcript which excludes the alternatively-spliced exon of the minigene cassette and allows for the translation of a functional gene of interest; and contacting the cell with the ligand that specifically binds said hairpin loop in an amount effective to induce dose-dependent transcription and translation of the functional gene of interest.

73. The method according to claim 73, wherein the ligand specific for the aptamer in the hairpin loop of the minigene cassette is selected from the group consisting of acyclovir, valacyclovir, famciclovir, penciclovir, and ganciclovir.

74. The method according to claim 72, wherein the aptamer binds acyclovir.

75. The method according to claim 74, wherein the aptamer comprises SEQ ID NO:

49.

76. The method according to claim 75, wherein the aptamer comprises SEQ ID NO: 50, SEQ ID NO: 51, or SEQ ID NO:

52.

77. The method according to any one of claims 72 to 76, wherein expression of the gene of interest is at least about 2-fold to 1,000-fold higher when the ligand specific for the aptamer in the hairpin loop of the minigene cassette is present.

78. The method according to any one of claims 72 to 76, wherein expression of the gene of interest is at least about 10-fold higher, 50-fold higher, or 100-fold higher when the ligand specific for the aptamer in the hairpin loop of the minigene cassette is present.

79. The method according to any one of claims 72 to 76, wherein expression of the gene of interest is at least about 300-fold higher when the ligand specific for the aptamer in the hairpin loop of the minigene cassette is present.

80. The method according to any one of claims 72 to 76, wherein expression of the gene of interest is at least about 500-fold higher when the ligand specific for the aptamer in the hairpin loop of the minigene cassette is present.

81. The method according to any one of claim 72 to 80, wherein the cell is a mammalian cell.

85. The method according to claim 81, wherein the mammalian cell is a human cell.

83. The method according to any one of claims 72 to 85, wherein said contacting is carried out in vitro.

84. The method according to any one of claims 72 to 85, wherein said contacting is carried out in vivo.

85. A method of treating or preventing a disease or condition in a subject in need thereof, said method comprising: administering to the subject the recombinant polynucleotide of any one of claims 44 to 4 or the vector according to any one of claims 65 to 68; and administering to the subject a ligand that specifically binds said aptamer in the hairpin loop in an amount effective to induce dose-dependent transcription and translation of the functional gene of interest, wherein: (i) in the absence of the ligand specific for the aptamer in the hairpin loop of the minigene cassette, transcription of the recombinant polynucleotide results in the production of a spliced transcript which includes the alternatively-spliced exon of the minigene cassette and allows for the translation of a truncated gene of interest or prevents translation of the gene of interest; (ii) in the presence of the ligand specific for the aptamer in the hairpin loop of the minigene cassette, transcription of the recombinant polynucleotide results in the productionof a spliced transcript which excludes the alternatively-spliced exon of the minigene cassette and which allows for the translation of a functional gene of interest; and (iii) said administering the ligand that specifically binds said aptamer in the hairpin loop is effective to treat the disease or condition.

86. The method according to claim 85, wherein said administering steps are carried out by parenteral administration, intravenous administration, intraperitoneal administration, oral administration, systemic administration, intramuscular administration, intravenous administration, subcutaneous administration, and intrathecal administration.

87. The method according to claim 85 or claim 86, wherein the gene of interest is selected from the group consisting of a therapeutic protein, a reporter protein, an enzyme, a transcription factor, a cytokine, a chemokine, an antibody, a hormone, a transport protein, a growth factor, an intracellular protein, an extracellular protein, a cytoplasmic protein, a transmembrane protein, a secreted protein, a receptor molecule, an RNA binding protein, a DNA binding protein, a channel protein, a cell adhesion molecule, a kinase, a phosphatase, and a chaperone protein.

88. The method according to any one of claims 85 to 87, wherein the subject is a mammalian subject.

89. The method according to claim 88, wherein the subject is a human subject.

90. The method according to any one of claims 85 to 89, wherein the ligand specific for the aptamer in the hairpin loop of the minigene cassette is selected from the group consisting of acyclovir, valacyclovir, famciclovir, penciclovir, and / or ganciclovir.

91. The method according to claim 90, wherein the aptamer binds acyclovir.

92. The method according to claim 91, wherein the acyclovir aptamer comprises SEQ ID NO: 49.

93. The method according to claim 92, wherein the acyclovir aptamer comprises SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO:

52.

94. The method according to any one of claims 85 to 93, wherein expression of the gene of interest is at least about 2-fold to 1,000-fold higher when the ligand specific for the aptamer in the hairpin loop of the minigene cassette is present.

95. The method according to any one of claims 85 to 93, wherein expression of the gene of interest is at least about 10-fold higher, 50-fold higher, or 100-fold higher when the ligand specific for the aptamer in the hairpin loop of the minigene cassette is present.

96. The method according to any one of claims 85 to 93, wherein expression of the gene of interest is at least about 300-fold higher when the ligand specific for the aptamer in the hairpin loop of the minigene cassette is present.

97. The method according to any one of claims 85 to 93, wherein expression of the gene of interest is at least about 500-fold higher when the ligand specific for the aptamer in the hairpin loop of the minigene cassette is present.

98. The method according to any one of claims 85 to 97, wherein the disease or condition is selected from the group consisting of a genetic disease or disorder, a neurological disease or disorder, a cardiovascular disease or disorder, an oncological disease or disorder, a lymphatic disease or disorder, a glandular disease or disorder, a viral infection, a bacterial infection, a fungal infection, a metabolic disorder such as diabetes, and obesity.

99. The method according to any one of claims 85 to 98, further comprising: monitoring the subject for a predetermined period of time after administering the recombinant polynucleotide or the vector and determining the therapeutic effect of said administering the ligand that specifically binds said aptamer in the hairpin loop.

100. The method according to claim 99 further comprising: modulating, based on said determining, said administering the recombinant polynucleotide or vector.

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