Compositions for and methods of engineering the transcriptome

Abstract

Disclosed herein are CRISPR-free compositions for the production of chimeric RNA molecules via trans-splicing and methods of producing the chimeric RNA molecules, and methods of using chimeric RNA molecules produced via trans-splicing to treat and / or prevent genetic diseases or conditions.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Publication Number (43) Publication Date (21) Application Number 202480035887.X (22) Application Date 2024.03.29 (30) Priority Data 63 / 493,696 2023.03.31 US (85) PCT International Application Entering National Phase Date 2025.11.27 (86) PCT International Application Application Data PCT / US2024 / 022327 2024.03.29 (87) PCT International Application Publication Data WO2024 / 206891 EN 2024.10.03 (71) Applicant Duke University Address North Carolina, USA (72) Inventors A. Asokan D. Pfeifferis (74) Patent Agency King & Wood Mallesons, Beijing 11256 Patent Attorneys Chen Wenping and Yuan Quan (51) Int.Cl. C12N 15 / 113 (2006.01) C12N 15 / 86 (2006.01) A61K 31 / 7105 (2006.01) (54) Invention Title: Compositions and Methods for Engineered Transcriptomes (57) Abstract: This document discloses CRISPR-free compositions for generating chimeric RNA molecules via trans-splicing and methods for generating said chimeric RNA molecules, as well as methods for treating and / or preventing genetic diseases or conditions using chimeric RNA molecules generated via trans-splicing. Claims (3 pages), Description (109 pages), Sequence Listing (electronic publication), Drawings (20 pages), CN 121195069 A, 2025.12.23, CN 1 21 19 50 69 A. 1. A nucleic acid molecule comprising: one or more RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' hemiintron; and exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA. 2. A nucleic acid molecule comprising: exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; a 5' hemiintron; one or more RNA targeting motifs; and one or more RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09. 3. The nucleic acid molecule of claim 1 or claim 2, wherein the targeted endogenous pre-mRNA contains one or more mutations in one or more exons. 4. The nucleic acid molecule of claim 3, wherein one or more mutations are located in the 3' portion of the targeted endogenous pre-mRNA. 5. The nucleic acid molecule of claim 3, wherein one or more mutations are located in the 5' portion of the targeted endogenous pre-mRNA.6. The nucleic acid molecule of claim 1, wherein the targeted endogenous pre-mRNA encodes a protein-encoding gene. 7. The nucleic acid molecule of claim 6, wherein the protein-coding gene comprises one or more coding regions of the following: ABCA1, ABCA12, ABCA13, ABCA2, ABCA3, ABCA4, ABCA5, ABCC1, ABCC2, ABCC6, ABCC8, ABCC9, ACAN, ADAMTS13, ADCY10, ADGRV1, AGL, AGRN, AHDC1, ALK, ALMS1, ALPK3, ALS2, ANAPC1, ANK1, ANK2, ANK3, ANKRD11, ANKRD26, APC, APC2, APOB, ARFGEF2, ARHGAP31, ARHGEF10, ARHGEF18, ARID1A, ARID1B, ARID2, ASH1L, ASPM, ASXL1, ASXL2, ASXL3, ATM, ATP7A, ATP7B, ATR, ATRX, BAZ1A, BAZ2B, BCOR, BCORL1, BDP1, BLM, BPTF, BRCA1, BRCA2, BRD4, BRWD3, C2CD3, C3, C5, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, CACNA1H, CACNA1S, CAD, CAMTA1, CARMIL2, CC2D2A, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2, CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP152, CEP164, CEP250, CEP290, CFAP43, CFAP44, CFAP65, CFTR / ABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT, CLIP1, CLTC, CNOT1, CNTNAP1, COL11A1, COL11A2, COL12A1, COL17A1, COL18A1, COL1A1, COL1A2, COL27A1, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A3, COL7A1, CPAMD8, CPLANE1, CPS1, CPSF1, CRB1, CREBBP, CUBN, CUL7, CUX1, DCC, DCHS1, DEPDC5, DICER1, DIP2B,DLC1、DMD、DMXL2、DNAH1、DNAH11、DNAH17、DNAH2、DNAH5、DNAH7、DNAH8、DNAH9、DNMBP、DNMT1 DOCK2, DOCK3, DOCK6, DOCK7, DOCK8, DSCAM, DSP, DST, DUOX2, DYNC1H1, DYNC2H1, DYSF. EIF2AK4, EP300, EPG5, ERCC6, ERCC6L2, EXPH5, ​​EYS, F5, F8, FANCA, FANCD2, FANCM, FAT1 FAT4, FBN1, FBN2, FLG, FLG2, FLNA, FLNB, FLNC, FLT4, FMN2, FN1, FRAS1, FREM1, FREM2, FSIP2. FXN, FYCO1, GLI2, GLI3, GPR179, GREB1L, GRIN2A, GRIN2B, GRIN2D, HCFC1, HECW2, HERC1 HERC2, HFM1, HIVEP1, HIVEP2, HMCN1, HSPG2, HTT, HUWE1, HYDIN, IFT140, IFT172, IGF1R. IGF2R, IGSF1, INSR, INTS1, IQSEC2, ITGB4, ITPR1, ITPR2, JMJD1C, KALRN, KANK1, KAT6A. KAT6B, KDM3B, KDM5B, KDM5C, KDM6A, KDM6B, KDR, KIAA0586, KIAA1109, KIAA1549, KIDINS220. KIF14, KIF1A, KIF1B, KIF21A, KIF26B, KIF7, KMT2A, KMT2B, KMT2C, KMT2D, KMT2E, KNL1 LAMA1, LAMA2, LAMA3, LAMA4, LAMA5, LAMB1, LAMB2, LAMC3, LCT, LMNA, LOXHD1, LPA, LRBA. LRP1, LRP2, LRP4, LRP5, LRP6, LRPPRC, LRRK1, LRRK2, LTBP2, LTBP4, LYST, MACF1, MADD A MAGI2, MAP1B, MAP3K1, MAPK8IP3, MAPKBP1, MAST1, MBD5, MCM3AP, MED12, MED12L, MED13.MED13L、MED23、MEGF8、MET、MLH3、MPDZ、MSH6、MTOR、MYH10、MYH11、MYH14、MYH2、MYH3、MYH6、 MYH7、MYH7B、MYH8、MYH9、MYLK、MYO15A、MYO18B、MYO3A、MYO5A、MYO5B、MYO7A、MYO9A、NALCN、 NBAS、NBEA、NBEAL2、NCAPD2、NCAPD3、NEB、NEXMIF、NEXMIF、NF1、NFASC、NHS、NIN、NIPBL、 NLRP1、NOTCH1、NOTCH2、NOTCH3、NPHP4、NRXN1、NRXN3、NSD1、NSD2、NUP155、NUP188、NUP205、 OBSCN、OBSL1、OTOF、OTOG、OTOGL、PARD3、PBRM1、PCDH15、PCLO、PCNT、PHIP、PI4KA、PIEZO1、 PIEZO2、PIK3C2A、PIKFYVE、PKD1、PKD1L1、PKHD1、PLCE1、PLEC、PLEKHG2、PNPLA6、POGZ、 POLA1、POLE、POLR1A、POLR2A、POLR3A、PRG4、PRKDC、PRPF8、PRR12、PRX、PTCH1、PTPN23、 PTPRF、PTPRJ、PTPRQ、PXDN、QRICH2、RAB3GAP2、RAI1、RALGAPA1、RANBP2、RB1CC1、RELN、RERE、 REV3L、RIC1、RIMS1、RIMS2、RNF213、ROBO1、ROBO2、ROBO3、ROS1、RP1、RP1L1、RTTN、RUSC2、 RYR1、RYR2、SACS、SAMD9、SAMD9L、SBF2、SCAPER、SCN10A、SCN11A、SCN1A、SCN2A、SCN3A、 SCN4A、SCN5A、SCN8A、SCN9A、SETBP1、SETD1A、SETD1B、SETD2、SETD5、SETX、SHANK2、SHANK3、 SHROOM4、SI、SIPA1L3、SLIT2、SLX4、SMARCA2、SMARCA4、SMCHD1、SNRNP200、SON、SPEF2、SPEG、 SPG11、SPTA1、SPTAN1、SPTB、SPTBN2、SPTBN4、SRCAP、STRC、SVIL、SYNE1、SYNGAP1、SYNJ1、SZT2, TAF1, TANC2, TCF20, TCOF1, TDRD9, TECPR2, TECTA, TENM3, TENM4, TET3, TEX14, TEX15, TG, THOC2, TMEM94, TNC, TNIK, TNR, TNRC6B, TNXB, TOGARAM1, TONSL, TRIO, TRIOBP, TRIP11, TRIP12, TRPM1, TRPM6, TRPM7, TRRAP, TSC2, TTC37, TTN, TUBGCP6, UBR1, UNC80, USH2A, USP9X, VCAN, VPS13A, VPS13B, VPS13C, VPS13D, VWF, WDFY3, WDR19, WDR62, WDR81, WNK1, WRN, ZFHX2, ZFYVE26, ZNF142, ZNF292, ZNF335, ZNF407, ZNF462, or ZNF469. 8. The nucleic acid molecule of any one of claims 3-7, wherein one or more mutations in one or more exons contribute to the pathogenesis in one or more cells. 9. The nucleic acid molecule of claim 8, wherein one or more cells are in a subject. 10. The nucleic acid molecule of claim 1, wherein the 5' portion of the targeted endogenous pre-mRNA is trans-spliced ​​with exogenous RNA. 11. The nucleic acid molecule of claim 2, wherein the 3' portion of the targeted endogenous pre-mRNA is trans-spliced ​​with exogenous RNA. 12. The nucleic acid molecule of claim 1 or claim 2, wherein the RNA targeting motif binds to the targeted endogenous pre-mRNA. 13. The nucleic acid molecule of claim 12, wherein the RNA targeting motif comprises an antisense oligonucleotide. 14. The nucleic acid molecule of claim 13, wherein the antisense oligonucleotide comprises about 15 nucleotides to about 50 nucleotides. 15. The nucleic acid molecule of claim 1, wherein the RNA targeting motif binds to the 3' end of the targeted endogenous pre-mRNA. 16. The nucleic acid molecule of claim 2, wherein the RNA targeting motif binds to the 5' end of the targeted endogenous pre-mRNA. 17. The nucleic acid molecule of claim 3, wherein the RNA targeting motif is specific for endogenous pre-mRNA having one or more mutations. 18. The nucleic acid molecule of claim 1, wherein the RNA targeting motif is directed to the 3' intron of an exon of the targeted endogenous pre-mRNA to be spliced ​​therewith. 19. The nucleic acid molecule of claim 2, wherein the RNA targeting motif is directed to the 5' intron of an exon of the targeted endogenous pre-mRNA to be spliced ​​therewith. 20. The nucleic acid molecule of claim 1, wherein the 3' half-intron is linked to exogenous RNA to be trans-spliced ​​with endogenous mRNA.21. The nucleic acid molecule of claim 1, wherein the 3' hemiintron is recognized by the nuclear splicing component in the host cell. 22. The nucleic acid molecule of claim 1, wherein the 3' hemiintron comprises (i) a 3' splice region including a branch point, (ii) a polypyrimidine string, and (iii) a 3' splice acceptor site. 23. The nucleic acid molecule of claim 2, wherein the 5' hemiintron is linked to exogenous RNA to be trans-spliced ​​with endogenous mRNA. 24. The nucleic acid molecule of claim 2, wherein the 5' hemiintron is recognized by the nuclear splicing component in the host cell. 25. The nucleic acid molecule of claim 2, wherein the 5' hemiintron comprises a 5' splice site. 26. The nucleic acid molecule according to any of the preceding claims, wherein one or more RNA structures bind to one or more RNA-binding proteins. 27. A nucleic acid molecule according to any one of the preceding claims, wherein one or more RNA structures (i) improve and / or enhance trans-splicing efficiency, (ii) stabilize the resulting chimeric RNA transcript, (iii) localize the RNA to the cell nucleus, (iv) stabilize the interaction between the targeted endogenous pre-mRNA molecule and the exogenous RNA to be trans-spliced, or (v) any combination thereof. 28. A viral vector comprising: a nucleic acid molecule according to any one of claims 1-27. 29. A pharmaceutical formulation comprising: the vector of claim 28; and one or more pharmaceutically acceptable carriers. 30. A method of producing a chimeric RNA molecule, the method comprising: contacting a targeted endogenous pre-mRNA in one or more cells with a nucleic acid molecule according to any one of claims 1-27 or the vector of claim 28, wherein the resulting chimeric RNA molecule comprises a trans-spliced ​​nucleic acid sequence. 31. The method of claim 30, wherein one or more cells are in a subject. 32. The method of claim 31, wherein the subject has been diagnosed with or is suspected of having a genetic disease or genetic condition. 33. A method for treating a genetic disease or hereditary condition, the method comprising: generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of the vector of claim 28 or the pharmaceutical preparation of claim 29 to a subject in need; wherein the resulting chimeric RNA molecule comprises a trans-spliced ​​nucleic acid sequence; and wherein the resulting chimeric RNA molecule restores one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic disorders. 34. The method of claim 33, wherein restoring one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic disorders comprises restoring the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes. 35. The method of claim 33, wherein the therapeutically effective amount of the vector comprises about 1 × 10¹⁰ vg to about 2 × 10¹⁴ vg.36. The method of claim 33, further comprising administering to the subject a therapeutically effective amount of one or more therapeutic agents. 37. The method of claim 33, further comprising administering to the subject a therapeutically effective amount of one or more immune modulators. 38. The method of any one of claims 33-37, further comprising repeating the administration step. 39. The method of any one of claims 33-38, further comprising monitoring the subject for adverse effects. 40. The method of any one of claims 33-39, wherein one or more aspects of restoring cellular homeostasis and / or cellular functional and / or metabolic dysfunction include (i) correcting cellular starvation in one or more cells; (ii) correcting, preventing, reducing and / or alleviating autophagy; (iii) improving, enhancing, restoring and / or preserving mitochondrial functional and / or structural integrity; (iv) improving, enhancing, restoring and / or preserving organelle functional and / or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing and / or slowing the rate of progression of a multisystemic manifestation of a genetic disease or condition; (vii) reversing, inhibiting, preventing, stabilizing and / or slowing the rate of progression of a genetic disease or condition, or (viii) any combination thereof. Claims 3 / 3 Page 4 CN 121195069 A Compositions and methods for engineered transcriptomes I. Cross-reference to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 493,696, filed March 31, 2023, the entire contents of which are incorporated herein by reference.

[0002] II. Statement regarding federal funding This invention was made with government support under federal grant No. NS099371 granted by the National Institute of Neurological Disorders and Stroke (NIH / NINDS). The federal government has certain rights to this invention.

[0003] III. Reference to the sequence listing The sequence listing, filed March 29, 2024, as an XML file entitled “23-2078-WO_Sequence Listing”, created on March 29, 2024, and measuring 95 kilobytes, is incorporated herein by reference in accordance with 37 CFR §1.52(e)(5).

[0004] IV. Background Art In eukaryotes, chromosomal DNA is transcribed into precursor RNA information (pre-mRNA), which contains protein-coding regions (exons) and intercalated non-protein-coding regions (introns). Prior to processing, these pre-mRNA molecules do not possess the sequences for ribosome-initiated translation due to the preservation of non-coding intron sequences. Therefore, before nuclear export, the exons of the pre-mRNA transcript are joined by a cellular mechanism called splicing. This mechanism is characterized by a large number of spliceosomes called spliceosomes.Ribonucleoprotein structure-mediated double transesterification. In the first transesterification, the branching point sequence of the intercalated intron attacks the 5' splice site, forming a lasso structure. This reaction releases the 5' splice site to attack the 3' splice site, thereby removing the intercalated intron and connecting the adjacent exon. After all intron sequences are removed, the precursor information matures into a mature RNA transcript capable of translation, which is transported to the ribosome, where it is decoded to produce cellular proteins.

[0005] In mammalian cells, mutations in transcriptionally active regions of chromosomal DNA produce pre-mRNA with the same mutation. If the mutation is located in a non-coding region, it can alter or eliminate the processing of the pre-mRNA. If the mutation is located in an exon region of the pre-mRNA, the mutation will be passed on to the mature mRNA sequence. These mutations can help suppress the complete translation of the encoded protein (speechless mutation) or modify the primary structure of the encoded protein in a counter-productive manner (missense mutation). In summary, these genetically encoded mutations can play a role in promoting pathogenesis in eukaryotes.

[0006] The aim of gene therapy is to correct such genetic abnormalities through adoptive gene transfer of recombinant nucleic acids carrying sequences of protein products capable of producing mutated genes. This strategy, often referred to as "classical gene therapy," has proven to be a safe and effective phenotypic correction strategy for genetic disorders, and several gene therapy products are available on the market.

[0007] However, current gene therapy approaches are limited by: toxicity caused by transcript overexpression, inability to deliver large transcripts exceeding the packaging capacity of AAV vectors (i.e., the standard vectors considered for gene therapy and having a packaging capacity of ~4.7 KB), inability to correct and replace large segments of pathogenic mRNA, and inability to correct and replace autosomal dominant inheritance with a single transcript and / or method.

[0008] Therefore, there is an urgent need for a minimally invasive, definitive therapy to address the root causes and sequelae of symptoms associated with these various genetic diseases and conditions. Therefore, this disclosure provides compositions and methods for generating chimeric RNA molecules via trans-splicing (and without CRISPR) and for treating and / or preventing genetic diseases and / or conditions, which can be used alone or in combination with other treatments. Specification 1 / 109 pages 5 CN 121195069 A

[0009] V. Brief Description of the Drawings Figure 1 (left) shows the mechanism of 5' trans-splicing, while Figure 1 (right) shows the mechanism of 3' trans-splicing. The schematic diagram on the left first shows the construct trans-splicing for 5' substitution after trans-splicing confirmed by trans-splicing. If the RNA species is in cis...If the RNA species is spliced ​​in cis, the stop codon in the first half of the open reading frame blocks translation. If the trans-spliced ​​RNA successfully edits the RNA, the open reading frame of EGFP is restored and fluorescence expression is restored. The schematic diagram on the right first shows the construct trans-splicing used for 3' substitution after trans-splicing. If the RNA species is spliced ​​in cis, the stop codon in the second half of the open reading frame blocks translation. If the trans-spliced ​​RNA successfully edits the RNA, the open reading frame of EGFP is restored and fluorescence expression is restored. GRAFT is a guide RNA helper fragment for trans-splicing.

[0010] Figures 2A–2B show a set of 5'-splicing motifs. These are flow cytometry data from co-transfection experiments of 5'-trans-splicing candidate molecules. In Figure 2A, the percentage of cells expressing green fluorescence when the trans-spliced ​​RNA candidate was delivered to cells was measured. RNA structures 1–11 are plotted on the x-axis and the percentage of GFP-positive cells is plotted on the y-axis. In Figure 2B, the mean fluorescence intensity of cells was measured when the trans-spliced ​​RNA candidate was delivered. RNA structures 1-11 are plotted on the x-axis, and the mean fluorescence intensity is plotted on the y-axis.

[0011] Figures 3A–3D show a set of 3' trans-spliced ​​motifs. Flow cytometry data from co-transfection experiments of 3' trans-spliced ​​RNA candidate molecules. In Figure 3A, the percentage of cells expressing green fluorescence was measured when the trans-spliced ​​RNA candidate was delivered to cells. RNA structures 1-11 are plotted on the x-axis, and the percentage of GFP-positive cells is plotted on the y-axis. In Figure 3B, the mean fluorescence intensity of cells was measured when the trans-spliced ​​RNA candidate was delivered. RNA structures 1-11 are plotted on the x-axis, and the mean fluorescence intensity is plotted on the y-axis. The data in Figures 3C and 3D were measured in the same manner for different target introns.

[0012] Figures 4A–4B show the replication validation of the top-ranked 3' trans-spliced ​​RNA candidates. Flow cytometry data from co-transfection experiments of 3' trans-splicing candidate molecules. In Figure 4A, the percentage of cells expressing green fluorescence was measured when the trans-splicing RNA candidate was delivered to the cells. The RNA structures are plotted on the x-axis, and the percentage of GFP-positive cells is plotted on the y-axis. In Figure 4B, the mean fluorescence intensity of the cells was measured when the trans-splicing RNA candidate was delivered. The RNA structures are plotted on the x-axis, and the mean fluorescence intensity is plotted on the y-axis.

[0013] Figures 5A–5B show the replication validation of the top-ranked 3' trans-splicing RNA candidates for the novel target. Flow cytometry data from co-transfection experiments of 3' trans-splicing candidate molecules were measured. In Figure 5A, the percentage of cells expressing green fluorescence was measured when the trans-splicing RNA candidate was delivered to the cells. RNA structures 1–11 are plotted on the x-axis, andThe percentage of GFP-positive cells is plotted on the y-axis. In Figure 5B, the average fluorescence intensity of cells is measured when the trans-spliced ​​RNA candidate is delivered. RNA structures 1-11 are plotted on the x-axis, and the average fluorescence intensity is plotted on the y-axis.

[0014] Figures 6A–6G provide plasmid maps of the constructs used in Example 1 and / or disclosed herein. Figure 6A shows an exemplary 3' replacement construct (empty). Figure 6B shows a 3' replacement construct (3-GRAFT-RYR2) for RYR2, while Figure 6C shows a 5' replacement construct (5-GRAFT-RYR2) for RYR2. Figure 6D shows a 3' replacement construct (3-GRAFT-LMNA) for LMNA, while Figure 6E shows a 3' replacement construct (3-GRAFT-FXN) for FXN. Figure 6F shows a Split GFP reporter construct for LMNA, while Figure 6G shows a split GFP reporter construct for RYR2.

[0015] Figure 7A shows the 5' substitution construct for DMD in Example 2. Figure 7B shows the 3' substitution construct for DMD in Example 2.

[0016] VI. Summary of the Invention This document discloses a nucleic acid molecule comprising a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' hemi-intron; one or more RNA targeting motifs; and one or more RNA structures. Specification 2 / 109 pages 6 CN 121195069 A

[0017] This document discloses a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' hemi-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA.

[0018] This document discloses a non-viral or viral vector comprising a nucleic acid molecule comprising a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' hemi-intron; one or more RNA targeting motifs; and one or more RNA structures.

[0019] This document discloses a non-viral or viral vector comprising a nucleic acid molecule, said nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' hemi-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA.

[0020] This document discloses a pharmaceutical formulation comprising a non-viral or viral vector, said non-viral or viral vector comprising a nucleic acid molecule, said nucleic acid molecule comprising a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; a 5' hemi-intron; one or more RNA targeting motifs; and one or more RNA structures.

[0021] This document discloses a pharmaceutical preparation comprising a non-viral vector or a viral vector, said non-viral vector or viral vector comprising a nucleic acid molecule, said nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA.

[0022] This document discloses a method for treating a genetic disease or condition, said method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a disclosed non-viral vector or a disclosed viral vector or a pharmaceutical preparation thereof to a subject in need of the condition, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic disorders.

[0023] This document discloses a method for treating a genetic disease or condition, said method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a disclosed non-viral vector or a disclosed viral vector or a pharmaceutical preparation thereof to a subject in need of the condition, wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of a missing, defective, and / or mutated protein or enzyme.

[0024] This document discloses a method for treating a genetic disease or condition, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a nonviral vector, a viral vector, an AAV particle, or a pharmaceutical preparation thereof to a subject in need of the molecule, said nonviral vector, viral vector, AAV particle, or pharmaceutical preparation thereof comprising (i) one or more 5' substitution constructs, (ii) one or more 3' substitution constructs, or (iii) one or more 5' substitution constructs and / or one or more 3' substitution constructs, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic disorders, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of a missing, defective, and / or mutated protein or enzyme.

[0025] This document discloses a method for inhibiting and / or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a disclosed nonviral vector or a disclosed viral vector, or a pharmaceutical preparation thereof, to a subject in need of the molecule, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic disorders.

[0026] This document discloses a method for inhibiting and / or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a disclosed nonviral vector or a disclosed viral vector or a pharmaceutical preparation thereof to a subject in need, wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of a missing, defective, and / or mutated protein or enzyme. (Specification)Page 3 / 109, CN 121195069 A

[0027] This document discloses a method for inhibiting and / or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a nonviral vector, viral vector, AAV particle, or pharmaceutical preparation thereof to a subject in need thereof, said nonviral vector, viral vector, AAV particle, or pharmaceutical preparation thereof comprising (i) one or more 5' substitution constructs, (ii) one or more 3' substitution constructs, or (iii) one or more 5' substitution constructs and / or one or more 3' substitution constructs, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes.

[0028] VII. Detailed Description This disclosure describes formulations, composite compositions, kits, capsules, containers, and / or methods thereof. It should be understood that various aspects of the invention are not limited to specific synthetic methods unless otherwise stated, or are not limited to specific reagents unless otherwise stated, and therefore variations are of course possible. It should also be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the invention, exemplary methods and materials are now described.

[0029] All publications mentioned herein are incorporated herein by reference to disclose and describe methods and / or materials in relation to the cited publications. The publications discussed herein are provided only because their disclosures predate the filing date of this application. Nothing herein should be construed as an admission that the invention is not entitled to precede such publications by virtue of prior invention.

[0030] A. Definitions Before disclosing and describing the compounds, compositions, articles, systems, devices and / or methods of the invention, it should be understood that they are not limited to specific synthetic methods unless otherwise stated, or to specific reagents unless otherwise stated, and therefore can certainly vary. It should also be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the invention, exemplary methods and materials are now described.

[0031] This disclosure describes the inventive concept with reference to specific embodiments. However, it is intended to cover all modifications, equivalents, and alternatives to the inventive concept consistent with this disclosure.

[0032] The singular forms “a,” “an,” and “the” as used in the specification and appended claims include plural indicators unless the context clearly indicates otherwise.

[0033] The phrase “consisting substantially of…” limits the scope of the claims to the components or methods described in the composition.The steps described herein, and those that do not substantially affect the basic and novel features of the claimed composition or the claimed method. The phrase “composes of” excludes any component, step, or element not recited in the claims. The phrase “comprising” is synonymous with “including,” “containing,” or “characterized in,” and is inclusive or open-ended. “Comprising” does not exclude additional, unlisted components or steps.

[0034] As used herein, when referring to any numerical value, the term “about” means a value falling within ±10% of the stated value.

[0035] A range may be expressed herein as from “about” one particular value and / or to “about” another particular value. When such a range is expressed, a further aspect includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation by using the antecedent “about,” it should be understood that a particular value forms a further aspect. It should be further understood that the endpoints of each range are significant relative to and independent of the other endpoint. It should also be understood that many values ​​are disclosed herein, and each value is also disclosed herein as “about” that particular value, in addition to the value itself. For example, specification 4 / 109 page 8 CN 121195069 A. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It should also be understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0036] The reference to the parts by weight of a particular element or component in the composition in the specification and the final claims indicates the weight relationship between that element or component and any other element or component in the composition or article expressed in parts by weight. Thus, in a complex containing 2 parts by weight of component X and 5 parts by weight of component Y, X and Y are present in a weight ratio of 2:5, and are present in this ratio regardless of whether the complex contains other components.

[0037] As used herein, the terms “optional” or “optionally” mean that the events or conditions described below may or may not occur, and the specification includes cases in which the events or conditions occur and cases in which they do not occur. In one aspect, the disclosed method may optionally include one or more additional steps, such as repeating the application step or changing the application step.

[0038] As used herein, “isolated” means that the nucleic acid molecule or nucleic acid sequence has been substantially isolated from, produced separately from, or purified from other biological components in the cells or tissues of an organism in which the component is present, such as other cells, chromosomes, and extrachromosomal DNA and RNA and proteins. “Isolated” nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. The term also covers nucleic acids and proteins prepared by recombinant expression in host cells and chemically synthesized nucleic acids and proteins.

[0039] As used herein, the term "subject" refers to the target of the application, such as a human. The term "subject" also includes domestic animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mice, rabbits, rats, guinea pigs, fruit flies, etc.). Therefore, the subject of the methods disclosed herein can be a vertebrate, such as a mammal, fish, bird, reptile, or amphibian. Alternatively, the subject of the methods disclosed herein can be a human, a non-human primate, a horse, pig, rabbit, dog, sheep, goat, cattle, cat, guinea pig, or rodent. This term does not indicate a particular age or sex and is therefore intended to cover adult and child subjects, as well as fetuses, whether male or female. In one aspect, the subject can be a human patient. In another aspect, the subject can have a disease or condition, be suspected of having a disease or condition, or be at risk of developing a disease or condition (e.g., a genetic disease or condition). In another aspect, the subject can be untreated.

[0040] As used herein, “regulatory element” can refer to promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Regulatory elements can include those guide nucleotide sequences constitutively expressed in many types of host cells and those guide nucleotide sequences expressed only in certain host cells (e.g., tissue-specific regulatory sequences).

[0041] As used herein, the term “diagnostic” means that a person has been examined by a technician, such as a physician, and has been found to have a condition that can be diagnosed or treated by one or more publicly available nucleic acid molecules, publicly available vectors, publicly available pharmaceutical preparations, or combinations thereof, or by one or more publicly available methods. For example, “diagnosed with a disease or condition” means that a person has been examined by a technician, such as a physician, and has been found to have a condition (e.g., a genetic disease or condition) that can be treated by one or more publicly available nucleic acid molecules, publicly available vectors, publicly available pharmaceutical preparations, or combinations thereof, or by one or more publicly available methods. For example, “suspected to have a disease or condition” can mean that an examination has been conducted by a technician, such as a physician, and a condition has been found that can be treated with one or more publicly disclosed nucleic acid molecules, publicly disclosed vectors, publicly disclosed pharmaceutical preparations, or combinations thereof, or with one or more publicly disclosed methods (e.g., genetic diseases or conditions). In one aspect, the examination can be physical and can involve various tests (e.g., blood tests, genotyping, biopsies, etc.) and assays (e.g., enzyme assays), or combinations thereof.

[0042] “Patient” refers to a subject suffering from a disease or condition (e.g., a genetic disease or condition). In one aspect, a patient can refer to a subject who has been diagnosed with or is suspected of having a disease or condition. In another aspect, a patient can refer to a subject who has been diagnosed with or is suspected of having a disease or condition and is seeking or receiving treatment for a disease or condition.Instructions 5 / 109 Page 9 CN 121195069 A

[0043] The phrase “identified as requiring treatment of a disease or condition” as used herein refers to selecting a subject based on the requirement of treatment of a disease or condition. For example, a subject may be identified as requiring treatment of a disease or condition (e.g., a genetic disease or condition) based on an early diagnosis made by someone skilled in the art, and then treatment of the genetic disease or condition may be administered. In one aspect, the identification may be performed by a person different from the person making the diagnosis. In one aspect, the administration may be performed by the person making the diagnosis.

[0044] The terms “inhibition,” “inhibition,” and “prevention” as used herein refer to attenuation or reduction of activity, level, response, condition, severity, disease, or other biological parameters. This may include, but is not limited to, complete elimination of activity, level, response, condition, severity, disease, or other biological parameters. This may also include, for example, 10% inhibition or reduction of activity, level, response, condition, severity, disease, or other biological parameters compared to natural or control levels (e.g., subjects without a disease or condition such as a genetic disease or condition). Therefore, in one aspect, the inhibition or reduction compared to the natural or control level can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any reduction between these values. In one aspect, the inhibition or reduction compared to the natural or control level can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100%. In one aspect, the inhibition or reduction compared to the natural or control level can be 0-25%, 25-50%, 50-75%, or 75-100%. In one aspect, the natural or control level can be the pre-disease or pre-symptom level.

[0045] The terms “treatment” or “conducting treatment” or “management” include palliative treatment, i.e., treatment designed to eliminate symptoms rather than cure a disease, pathological condition, or symptom; preventive treatment, i.e., treatment aimed at minimizing or partially or completely suppressing the development of a related disease, pathological condition, or symptom; and supportive treatment, i.e., treatment used to complement another specific therapy aimed at improving a related disease, pathological condition, or symptom. In one aspect, the term covers any treatment of a subject (including mammals, e.g., humans) and includes: (i) preventing the occurrence of undesirable physiological changes, diseases, pathological conditions, or symptoms in subjects who may be susceptible to the disease but have not yet been diagnosed with it; (ii) suppressing physiological changes, diseases, pathological conditions, or symptoms, i.e., halting their development; or (iii) eliminating physiological changes, diseases, pathological conditions, or symptoms, i.e., causing the remission of the disease. For example, in one aspect, treatment of a disease or symptom may, compared to a control (e.g., an individual without a genetic disease or symptom), result in the treatment of the disease or symptom.Treatment reduces the severity of an identified disease or condition in a subject by 1%–100%. In one aspect, treatment can refer to a reduction in the severity of a disease or condition (e.g., a genetic disease or condition) by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. For example, treatment of a disease or condition can reduce one or more symptoms of the disease or condition in a subject by 1%–100% compared to a control (e.g., an individual without a genetic disease or condition). In one aspect, treatment can refer to a reduction in one or more symptoms of an identified disease or condition by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. It should be understood that treatment does not necessarily mean a cure or complete elimination or eradication of the disease or condition. However, in one aspect, treatment can mean a cure or complete elimination or eradication of the disease or condition.

[0046] As used herein, the terms “prevention” or “preventing” or “preventing” refer to stopping, avoiding, excluding, preemptively preventing, terminating, or hindering the occurrence of something, particularly by taking early action. It should be understood that in the context of the use of “reduce,” “inhibit,” or “prevent” herein, the use of the other two terms is also explicitly disclosed unless otherwise specifically stated. In one aspect, it aims to prevent a disease or condition having chromatin dysregulation and / or abnormalities in chromatin regulation. The terms “prevention,” “preventing,” and “preventing” also refer to preventive or preventive measures for protecting or preventing a subject (e.g., an individual) who does not have a given disease or condition (e.g., a genetic disease or condition) or related complications from progressing to that complication.

[0047] As used herein, the terms “administer” and “administer” refer to any method of providing a subject with one or more disclosed nucleic acid molecules, disclosed vectors, disclosed pharmaceutical preparations, or combinations thereof. Such methods are well known to those skilled in the art and include, but are not limited to, the following: oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, intrauterine administration, intrahepatic administration, intravaginal administration, ocular administration, intraauricular administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable methods such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration may also include intrahepatic artery administration or administration via the portal vein (HPV). Disclosed nucleic acid molecules, disclosed carriers, disclosed pharmaceutical compositions, disclosed therapeutic agents, disclosed immunomodulators, disclosed proteasome inhibitors, disclosed small molecules, disclosed endonucleases, disclosed oligonucleotides, and / or disclosed RNA therapeutics.The administration of a therapeutic agent may include direct application to the CNS or PNS. Administration may be continuous or intermittent. Administration may include a combination of one or more pathways.

[0048] In one aspect, a person skilled in the art may determine an effective dose, effective regimen, and effective route of administration for one or more of a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical preparation, or a combination thereof to treat or prevent a disease or condition (e.g., a genetic disease or condition). In one aspect, a person skilled in the art may also modify, alter, or modify one aspect of the administration procedure to improve the efficacy of one or more of a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical preparation, or a combination thereof.

[0049] “Measured amount” refers to the absolute quantification of a particular analyte (e.g., an mRNA sequence containing a specific tag) or the determination of the relative abundance of a particular analyte (e.g., the amount compared to mRNA sequences containing different tags). This phrase includes direct or indirect measurements of abundance (e.g., the amount of a single mRNA transcript may be quantified, or the amount of an mRNA sequence amplified under certain conditions for a certain period of time may be used as an alternative to quantifying a single transcript) or both.

[0050] As used herein, “modifying the method” can include modifying or altering one or more features or aspects of one or more steps of the disclosed method. For example, in one aspect, the method can be modified by changing the amount of one or more of the disclosed nucleic acid molecule, disclosed carrier, disclosed pharmaceutical preparation or combination thereof administered to a subject, or by changing the frequency of administration of one or more of the disclosed nucleic acid molecule, disclosed carrier, disclosed pharmaceutical preparation or combination thereof to a subject, by changing the duration of administration of one or more of the disclosed nucleic acid molecule, disclosed carrier, disclosed pharmaceutical preparation or combination thereof to a subject, or by replacing one or more of the disclosed components and / or reagents with similar or equivalent components and / or reagents. This also applies to all disclosed therapeutic agents, immunomodulators, immunosuppressants, proteasome inhibitors, etc.

[0051] As used herein, the term “pharmaceutical-grade carrier” refers to a sterile aqueous or non-aqueous solution, dispersion, suspension or emulsion, and a sterile powder intended for recombination into a sterile injectable solution or dispersion prior to use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or media include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethyl cellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. In one aspect, the drug carrier used can be solid, liquid, or gaseous. In one aspect, examples of solid carriers may include lactose, kaolin, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, and stearic acid. In one aspect, examples of liquid carriers may include syrup, peanut oil, olive oil, and water. In one aspect, examples of gaseous carriers may include…Carbon dioxide and nitrogen. Any convenient pharmaceutical medium can be used in preparing the disclosed compositions for oral dosage forms. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, etc., can be used to form oral liquid preparations, such as suspensions, elixirs, and solutions; while carriers, such as starch, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrants, etc., can be used to form oral solid preparations, such as powders, capsules, and tablets. Tablets and capsules are preferred oral dosage units where solid pharmaceutical carriers are used due to their ease of administration. Optionally, tablets can be coated using standard aqueous or non-aqueous techniques. For example, by using coating materials such as lecithin, appropriate flowability can be maintained in the case of dispersions by maintaining the desired particle size and by using surfactants. These compositions may also contain excipients such as preservatives, wetting agents, emulsifiers, and dispersants. Prevention of microbial action can be ensured by including various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. Isotonic agents, such as sugars and sodium chloride, may also be desired. Prolonged absorption of injectable drug forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin. Injectable reservoir forms are prepared by forming microcapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolic acid, poly(orthoester), and poly(anhydride). The drug release rate can be controlled depending on the ratio of drug to polymer and the properties of the specific polymer used. Reservoir injectable formulations are also prepared by encapsulating the drug in liposomes or microemulsions compatible with body tissues. Injectable formulations can be sterilized, for example, by filtration through a bacterial trap or by incorporating a sterilizing agent that can be dissolved or dispersed in sterile water or other sterile injectable media in the form of a sterile solid composition before use. Suitable inert carriers may include sugars, such as lactose. Ideally, at least 95% by weight of the active ingredient particles have an effective particle size in the range of 0.01 to 10 micrometers.

[0052] As used herein, the term “excipient” refers to an inert substance that is commonly used as a diluent, medium, preservative, binder, or stabilizer, and includes, but is not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkyl sulfonates, caprylates, etc.), surfactants (e.g., SDS, polysorbates, nonionic surfactants, etc.), sugars (e.g., sucrose, maltose, trehalose, etc.), and polyols (e.g., mannitol, sorbitol, etc.). For reference, also see Remington's Pharmaceutical Sciences, (1990) Mack Publishing Co., Easton,Pa., the entire contents of which are incorporated herein by reference.

[0053] As used herein, “in parallel” means (1) simultaneously in time, or (2) at different times during a co-treatment regimen.

[0054] As used herein, the term “contact” means to bring together one or more disclosed nucleic acid molecules, disclosed carriers, disclosed pharmaceutical preparations or combinations thereof with a target region or intended target region in such a manner that one or more of the disclosed nucleic acid molecules, disclosed carriers, disclosed pharmaceutical preparations or combinations thereof directly or indirectly act on the intended target or target region. The target region may include one or more cells, and in one aspect, the one or more cells may be in a subject. The target region or intended target region may be one or more organs of the subject (e.g., lungs, heart, liver, kidneys, brain, etc.). In one aspect, the target region or intended target region may be any cell or any organ infected by a disease or condition (e.g., a genetic disease or condition). In one aspect, the target region or intended target region may be any organ, tissue or cell affected by a disease or condition (e.g., a genetic disease or condition).

[0055] As used herein, “determine” can refer to measuring or ascertaining the presence and severity of a disease or condition, such as a genetic disease or condition. Methods and techniques for determining the presence and / or severity of a disease or condition are generally known in the medical field. For example, ways of identifying and / or diagnosing the presence, severity, or both of a disease or condition (e.g., a genetic disease or condition) are well-known in the art.

[0056] As used herein, “effective amount” and “effective quantity” can refer to an amount sufficient to achieve a desired outcome, such as treatment and / or prevention of a disease or condition (e.g., a genetic disease or condition) or suspected disease or condition. As used herein, the terms “effective amount” and “effective quantity” can refer to an amount sufficient to achieve a desired effect on an undesirable condition (e.g., a disease or condition). For example, a “therapeutic effective amount” refers to an amount sufficient to achieve a desired therapeutic outcome or have an effect on undesirable symptoms, but generally insufficient to cause adverse side effects. In one respect, a “therapeutic effective dose” means the amount of a disclosed nucleic acid molecule, a disclosed carrier, or a disclosed pharmaceutical preparation that (i) treats a specific disease, condition, or symptom (e.g., a genetic disease or symptom), (ii) reduces, alleviates, or eliminates one or more symptoms of a specific disease, condition, or symptom (e.g., a genetic disease or symptom), or (iii) delays the onset of one or more symptoms of a specific disease, condition, or symptom (e.g., a genetic disease or symptom). The specific therapeutic effective dose level for any particular patient will depend on a number of factors, including the condition being treated and its severity; the disclosed nucleic acid molecule, disclosed carrier, or disclosed pharmaceutical preparation used; the disclosed method used; the patient’s age, weight, general health condition, sex, and diet; the time of administration; the route of administration; and the amount of the disclosed nucleic acid molecule, disclosed carrier, or disclosed pharmaceutical preparation used.The excretion rate of the disclosed carrier or the disclosed pharmaceutical preparation; the duration of treatment; the drugs used in combination with or concurrently with the disclosed nucleic acid molecule, the disclosed carrier, or the disclosed pharmaceutical preparation (page 8 / 109, 12 CN 121195069 A), and other similar factors well known in the medical field. For example, starting the dose of the disclosed nucleic acid molecule, the disclosed carrier, or the disclosed pharmaceutical preparation at a level below that required to achieve the desired therapeutic effect and gradually increasing the dose until the desired effect is achieved is within the scope of the art. If necessary, the effective daily dose can be divided into multiple doses for the purpose of administration. Thus, a single dose of the disclosed nucleic acid molecule, the disclosed carrier, or the disclosed pharmaceutical preparation may contain such an amount or an approximation thereof to constitute the daily dose. If there are any contraindications, the dose may be adjusted by an individual physician. The dose may vary and may be administered daily in one or more doses for one or more days. Guidance on the appropriate dose for a given class of pharmaceutical products can be found in the literature. In further aspects, the preparation can be administered in a “preventively effective amount”; that is, an amount that effectively prevents disease or condition, such as disease or symptom caused by a missing, defective, and / or mutated protein or enzyme.

[0057] As used herein, “RNA therapeutic agent” can refer to the use of oligonucleotides to target RNA. RNA therapeutic agents can offer the prospect of uniquely targeting precise nucleic acids involved in a specific disease with greater specificity, improved efficacy, and reduced toxicity. This may be particularly effective for genetic diseases, where targeting RNA rather than protein is most advantageous. In one aspect, therapeutic RNA can include one or more expression sequences. As known in the art, expression sequences can include RNAi, shRNA, mRNA, non-coding RNA (ncRNA), antisense such as antisense RNA, miRNA, morpholino oligonucleotides, peptide-nucleic acid (PNA), or ssDNA (having natural and modified nucleotides, including but not limited to LNA, BNA, 2'-O-Me-RNA, 2'-MEO-RNA, 2'-F-RNA), or analogs or conjugates thereof. In one aspect, the disclosed therapeutic RNA may include one or more long non-coding RNAs (lncRNAs), such as long intergenic non-coding RNAs (lincRNAs), pre-transcripts, pre-miRNAs, pre-mRNAs, competitive endogenous RNAs (ceRNAs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), pseudogenes, rRNAs, or tRNAs. In one aspect, the ncRNA may be a piwi-interacting RNA (piRNA), a primary miRNA (pri-miRNA), or a pre-mature miRNA (pre-miRNA). In one aspect, the disclosed therapeutic RNA or RNA therapeutic agent may include an antisense oligonucleotide that inhibits mRNA translation.Antisense oligonucleotides (ASOs), oligonucleotides that function via the RNA interference (RNAi) pathway, RNA molecules that behave like enzymes (ribosomal enzymes), RNA oligonucleotides that bind to proteins and other cellular molecules, and ASOs that bind to mRNA and form structures recognized by RNase H that lead to the cleavage of the mRNA target. In one aspect, RNA therapeutics may include ASOs that inhibit mRNA translation and RNAi. Generally, as is known in the art, RNAi specifically and posttranscribedly manipulates sequences by activating ribonucleases that, together with other enzymes and complexes, co-degrade RNA after the original RNA target has been cleaved into smaller fragments, while antisense oligonucleotides bind to their target nucleic acids via Watson-Crick base pairing and inhibit or alter gene expression via steric hindrance, splicing alteration, initiation of target degradation, or other events.

[0058] As used herein, “small molecule” can refer to any organic or inorganic material that is not a polymer. Small molecules do not include large macromolecules, such as large proteins (e.g., proteins with a molecular weight exceeding 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000), large nucleic acids (e.g., nucleic acids with a molecular weight exceeding 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000), or large polysaccharides (e.g., polysaccharides with a molecular weight exceeding 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000). In one aspect, "small molecule" can be, for example, a drug that can readily enter cells due to its low molecular weight. In one aspect, small molecules can be used in combination with disclosed compositions in the disclosed methods.

[0059] In one aspect, the term "ex vivo" can generally refer to activities occurring outside an organism or subject, such as experiments, modifications, differentiations, manipulations, and / or measurements performed in or on living tissue in an artificial environment outside an organism. In one aspect, ex vivo experiments, ex vivo modifications, ex vivo differentiations, ex vivo manipulations, and / or ex vivo measurements can occur with minimal alteration to natural conditions. In one aspect, "ex vivo" can include living cells, tissues, or organs (e.g., cells requiring trans-splicing of one or more protein-coding genes) taken from a subject or donor subject and cultured and / or maintained and / or perfused in a laboratory setting, typically under sterile conditions and typically for a limited duration (e.g., a few hours or up to about 24 hours, up to about 48 hours, up to about 72 hours, up to about 96 hours, up to about 120 hours).The time may be as long as approximately 144 hours, approximately 168 hours or longer, depending on the environment and / or desired characteristics. In one aspect, tissues, cells or organs may be collected, frozen and subsequently thawed for in vitro processing.

[0060] As used herein, “operably linked” means that the expression of a gene or transgene is under the control of a promoter spatially linked to it. The promoter may be located at the 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter originates. As is known in the art, variations in this distance can be adapted without loss of promoter function.

[0061] As used herein, “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may constitute a protein sequence. The term “peptide” may refer to a short chain of amino acids, including, for example, natural peptides, recombinant peptides, synthetic peptides, or any combination thereof. Proteins and peptides may include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins.

[0062] As used herein, “nucleic acid” or “oligonucleotide” or “polynucleotide” means at least two nucleotides covalently linked together. The description of a single strand may also define the sequence of the complementary strand. Thus, nucleic acid can encompass the complementary strand of the single strand depicted. Many variants of nucleic acids can be used for the same purpose as a given nucleic acid. Thus, nucleic acid can encompass substantially the same nucleic acid and its complement. A single strand can provide a probe that can hybridize with a target sequence under strict hybridization conditions. Thus, nucleic acid can encompass probes that hybridize under strict hybridization conditions. Nucleic acids can be single-stranded or double-stranded, or may contain portions of both double-stranded and single-stranded sequences. Nucleic acids can be DNA, genome and cDNA, RNA or hybrids, wherein the nucleic acids may contain combinations of deoxyribonucleotides and ribonucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis or by recombinant methods. Similarly, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid construct,” “nucleotide sequence,” and “polynucleotide” as used herein can refer to linear or branched, single-stranded or double-stranded RNA or DNA, or hybrids thereof. The term can encompass RNA / DNA hybrids. When dsRNA is synthesized, less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine, etc., can also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides containing C-5 propyne analogs of uridine and cytidine have been shown to bind RNA with high affinity and are gene expression inhibitors.Antisense inhibitors. Other modifications may also be made, such as modifications to the 2'-hydroxyl group in the ribose group of the phosphodiester backbone or RNA. As used herein, "synthetic" nucleic acid or polynucleotide refers to a nucleic acid or polynucleotide that is not found in nature but is artificially constructed and is therefore not a product of nature.

[0063] "Polynucleotide" is a sequence of nucleotide bases and can be RNA, DNA, or DNA-RNA hybrid sequences (including both naturally occurring and non-naturally occurring nucleotides).

[0064] A “fragment” or “part” of a nucleotide sequence may be understood to mean a nucleotide sequence that is reduced in length (e.g., reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides) relative to a reference nucleic acid or nucleotide sequence, and comprises, is substantially composed of or is composed of the following nucleotide sequences: identical or nearly identical (e.g. 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) of a reference nucleic acid or nucleotide sequence. According to this disclosure, such nucleic acid fragments or portions may (where appropriate) be included in larger polynucleotides in which the nucleic acid fragment or portion is a component. In one aspect, fragments or portions of a nucleotide sequence or nucleic acid sequence may include sequences encoding exons having one or more mutations, as per specification page 10 / 109, column 14, CN 121195069 A.

[0065] A “fragment” or “part” of an amino acid sequence can be understood as representing an amino acid sequence that is reduced in length relative to a reference amino acid sequence (e.g., reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more amino acids), and comprises, is substantially composed of or is composed of the following amino acid sequences: identical or nearly identical to the reference amino acid sequence (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%). A series of amino acids (88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical). According to this disclosure...Such amino acid fragments or portions may be included, where appropriate, in the larger amino acid sequence in which they constitute.

[0066] The terms “heterologous” or “recombinant” nucleotide or amino acid sequences are used interchangeably herein and may refer to nucleotide or amino acid sequences that are not naturally associated with the host cell to which they are introduced, including multiple non-natural copies of naturally occurring nucleotide or amino acid sequences.

[0067] The term “endogenous” as used herein may refer to genes, proteins, compounds, or activities (e.g., pre-mRNA) that are normally present in the host cell. The term “exogenous” nucleic acid molecule, construct, or sequence (e.g., RNA sequence to be trans-spliced) as used herein may refer to a nucleic acid molecule or portion of a nucleic acid molecule that is not natural to the host cell but may be homologous to a nucleic acid molecule or portion of a nucleic acid molecule derived from the host cell.

[0068] Different nucleic acids or proteins that are homologous may be referred to as “homologs.” The term homologs include homologous sequences from the same and other species and orthologous sequences from the same and other species. "Homology" refers to the level of similarity between two or more nucleic acid and / or amino acid sequences, expressed as a percentage of positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of similar functional properties between different nucleic acids or proteins. Therefore, the disclosed compositions and methods may include homologs of the disclosed nucleotide sequences and / or disclosed polypeptide sequences.

[0069] As used herein, "orthologous homologs" may refer to homologous nucleotide and / or amino acid sequences in different species arising from a common ancestral gene during speciation. Homologous forms of a disclosed nucleotide sequence or a disclosed polypeptide may have substantially the same sequence identity as the disclosed nucleotide sequence or the disclosed polypeptide (e.g., at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and / or 100%).

[0070] As used herein, “complementary” or “complementary” means that nucleic acids may be paired in Watson-Crick (e.g., A-T / U and C-G) or Hoogsteen base pairs between nucleotides or nucleotide analogs of a nucleic acid molecule. “Complementarity” refers to a property shared between two nucleic acid sequences such that when they are antiparallel aligned to each other, the nucleotide bases at each position will be complementary.

[0071] The terms "promoter" or "multiple promoters" as used herein are known in the art. A variety of promoter elements can be used depending on the desired level and tissue-specific expression. Promoters can be tissue-specific or ubiquitous, and can be...Constitutive or inducible, depending on the desired gene expression pattern. Promoters can be natural (endogenous) or exogenous (exogenous) and can be natural or synthetic sequences. Exogenous or exogenous means that the transcription start region is absent in the wild-type host where it is introduced.

[0072] “Tissue-specific promoters” are known in the art, including but not limited to neuron-specific promoters, muscle-specific promoters, liver-specific promoters, skeletal muscle-specific promoters, and heart-specific promoters.

[0073] “Liver-specific promoters” are known in the art, including but not limited to thyroxine-binding globulin (TBG) promoters, α1-microglobulin / bikuin enhancer / thyroxine-binding globulin promoters, human albumin (hALB) promoters, thyroxine-binding globulin promoters, α-1-antitrypsin promoters, bovine albumin (bAlb) promoters, and mouse albumin promoters. (Instructions for use 11 / 109 pages, 15 CN 121195069 A) The liver-specific promoter may include a human α1-antitrypsin (hAAT) promoter, an ApoEhAAT promoter containing an ApoE enhancer and an hAAT promoter, a transthyretin (TTR) promoter, a liver fatty acid-binding protein promoter, a hepatitis B virus (HBV) promoter, a DC172 promoter containing an hAAT promoter and an α1-microglobulin enhancer, a DC190 promoter containing a human albumin promoter and a prothrombin enhancer, or any other natural or synthetic liver-specific promoter. In one aspect, the liver-specific promoter may comprise approximately 845 bp and includes a thyroid hormone-binding globulin promoter sequence (2382 to 13), two copies of an α1-microglobulin / bicubin enhancer sequence (22,804 to 22,704), and a 71 bp leader sequence, as described by Ill CR et al. (1997).

[0074] Ubiquitous / constitutive promoters are known in the art and include, but are not limited to, the CMV major immediate early enhancer / chicken β-actin promoter, the cytomegalovirus (CMV) major immediate early promoter, the elongation factor 1-a (EF1-a) promoter, the simian vacuolating virus 40 (SV40) promoter, the AmpR promoter, the PyK promoter, the human ubiquitin C gene (Ubc) promoter, the MFG promoter, the human β-actin promoter, the CAG promoter, the EGR1 promoter, the FerH promoter, the FerL promoter, the GRP78 promoter, the GRP94 promoter, the HSP70 promoter, the β-kin promoter, the mouse phosphoglycerate kinase (mPGK) or human PGK (hPGK) promoter, the ROSA promoter, the human ubiquitin B promoter, the Rous sarcoma virus promoter, or any other natural or synthetic ubiquitous / constitutive promoter.

[0075] As used herein, “inducible promoter” refers to a promoter that can be modulated by positive or negative controls. Factors that can modulate inducible promoters include, but are not limited to, chemical agents (e.g., metallothionein promoters or hormone-inducible promoters), temperature, and light.

[0076] As used herein, the term “serotype” is used to refer to an AAV having a capsid that is serologically different from other AAV serotypes. Serological uniqueness can be determined by the lack of cross-reactivity between antibodies against an AAV compared to another AAV. This difference in cross-reactivity is typically due to differences in capsid protein sequence / antigenic determinants (e.g., due to differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes).

[0077] As used herein, “tropism” refers to the specificity of AAV capsid proteins present in AAV viral particles for infecting a specific type of cell or tissue. The tropism of the AAV capsid for a specific type of cell or tissue can be determined by measuring the ability of an AAV vector particle containing a hybrid AAV capsid protein to infect or transduce a specific type of cell or tissue using standard assays well known in the art (e.g., those disclosed in the embodiments of this application). As used herein, the term "liver tropism" or "liver tropism" refers to tropism toward the liver or liver tissue and cells (including hepatocytes).

[0078] "Sequence identity" and "sequence similarity" can be determined by comparing two peptide or two nucleotide sequences using a global or local alignment algorithm. When the sequences are optimally aligned, they can then be described as "substantially identical" or "substantially similar." For example, sequence similarity or identity can be determined by searching databases such as FASTA, BLAST, etc., but hits should be retrieved and compared side-by-side to compare sequence identity. Two proteins or two protein domains or two nucleic acid sequences may have "substantially sequence identity" if the percentage of sequence identity is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or higher, preferably 90%, 95%, 98%, 99%, or higher. Such sequences are also referred to herein as "variants," for example, other variants of deleted, defective, and / or mutated proteins or enzymes. It should be understood that sequences having substantially the same sequence identity do not necessarily have the same length and can differ in length. For example, sequences having the same nucleotide sequence but one having additional nucleotides on the 3'- and / or 5'- sides are 100% identical.

[0079] "Codon optimization" as used herein can refer to the process of modifying a nucleic acid sequence to enhance expression in a target host cell by replacing one or more codons of a natural sequence with codons that are more frequently or most frequently used in the gene of the host cell, while maintaining the natural amino acid sequence. Various species exhibit specific amino acid responses to different amino acids.Special preference for certain codons in acids. As considered herein, genes can be tailored based on codon optimization to determine optimal gene expression in an organism. Codon usage tables are readily available, for example, in “codon usage databases.” Many methods and software tools for codon optimization have been previously reported (see, for example, genomes.urv.es / OPTIMIZER / ).

[0080] In one aspect, “RNA editing” can be posttranscriptional modification, in which the nucleotide sequence of the precursor mRNA (pre-mRNA) is altered by base insertion, deletion, or modification. The extent of RNA editing varies, from hundreds of bases in trypanosome mitochondrial DNA to a single base in mammalian nuclear genes. Even a single base change in pre-mRNA can convert a codon of one amino acid to a codon of another amino acid or a stop codon. This type of recoding can significantly affect the structure and function of proteins and can result in multiple variants of proteins from a single gene.

[0081] In one aspect, insertional and deletional RNA editing can involve adding and deleting specific nucleotides or sequences of nucleotides from pre-mRNA. In one aspect, substitutional RNA editing via base modification has been observed in higher eukaryotes, wherein bases are modified without altering the length of the pre-mRNA.

[0082] As used herein, “immune tolerance,” “immunological tolerance,” and “immune tolerance” refer to a state of unresponsive or blunted response of the immune system to a substance capable of evoking an immune response in a subject (e.g., a publicly disclosed nucleic acid molecule, a publicly disclosed vector, a publicly disclosed transgenic product, a publicly disclosed pharmaceutical preparation, a publicly disclosed therapeutic agent, etc.). Immune tolerance is induced by prior exposure to a specific antigen. Immune tolerance in a subject can be determined by measuring antibodies against a specific antigen or by liver-restricted transgenic expression using a viral vector (e.g., AAV). Low antibody titers or the absence of antibody titers over time are indicators of immune tolerance. For example, in some embodiments, immune tolerance can be determined by having an IgG antibody titer less than or equal to about 12,000, 11,500, 11,000, 10,500, 10,000, 9,500, 9,000, 8,500, 8,000, 7,500, 7,000, 6,500, or 6,000 after gene therapy (e.g., administration of a transgenic protein or enzyme encoded by a gene, such as a deleted, defective, or mutated protein or enzyme).

[0083] As is known in the art, antibodies (Abs) can alleviate AAV infection through a variety of mechanisms, including by binding to the AAV capsid.And by blocking key steps in transduction, such as cell surface attachment and uptake, endosome escape, productive transport to the nucleus, or uncoating, and promoting AAV opsonization by phagocytes, they mediate rapid clearance from circulation. For example, in humans, serological studies have revealed a high prevalence of NAbs in the global population, with approximately 67% of people having antibodies against AAV1, 72% against AAV2, and approximately 40% against AAV serotypes 5 to 9. Vector immunogenicity represents a major challenge in the reapplication of AAV vectors.

[0084] In one aspect, this document also discloses partially self-complementary parvovirus (e.g., disclosed AAV) genomes, plasmid vectors encoding said parvovirus genomes, and parvovirus (e.g., disclosed AAV) particles comprising these genomes. In one aspect, this document provides a plasmid vector comprising a nucleotide sequence encoding a disclosed parvovirus genome (e.g., disclosed AAV). In one aspect, this document provides a partially self-complementary parvovirus genome comprising a payload construct, a parvovirus ITR flanking the payload construct, and a self-complementary region flanking one ITR. The self-complementary region may contain a nucleotide sequence complementary to the payload construct. A disclosed self-complementary region may have a length less than the entire length of the payload construct.

[0085] In one aspect, the disclosed self-complementary region of the disclosed parvovirus genome may contain a minimum length while still having a length less than the entire length of the payload construct. In one aspect, the disclosed self-complementary region may contain a length of at least 50 bases, at least 100 bases, at least 200 bases, at least 300 bases, at least 400 bases, at least 500 bases, at least 600 bases, at least 700 bases, at least 800 bases, at least 900 bases, or at least 1,000 bases.

[0086] In one aspect, a “self-complementary parvovirus genome” may be a single-stranded polynucleotide having a first parvovirus ITR sequence, a heterologous sequence (e.g., a payload construct containing, for example, the desired gene), a second parvovirus ITR sequence, a second heterologous sequence, and a third parvovirus ITR sequence in the 5' to 3' direction, wherein the second heterologous sequence is complementary to the first heterologous sequence. In contrast to a self-complementary genome, a “partially self-complementary genome” does not contain three parvovirus ITRs, and the second heterologous sequence complementary to the first heterologous sequence has a length less than the entire length of the first heterologous sequence (e.g., the payload construct). Thus, a partially self-complementary genome is a single-stranded polynucleotide having a first parvovirus ITR sequence in the 5' to 3' direction or a second parvovirus ITR sequence in the 3' to 5' direction.The device comprises a first parvovirus ITR sequence, a heterologous sequence (e.g., a payload construct), a second parvovirus ITR sequence, and a self-complementary region of a length complementary to a portion of the heterologous sequence and having a length less than the entire length of the heterologous sequence.

[0087] As used herein, “immunomodulatory” refers to the ability of a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical preparation, or a disclosed reagent to alter (modulate) one or more aspects of the immune system. The immune system protects an organism from infection and foreign antigens through cellular and humoral mechanisms involving lymphocytes, macrophages, and other antigen-presenting cells, which are mutually regulated through a variety of cell-cell interactions and through the elaboration of soluble factors (including lymphokines and antibodies) that have autocrine, paracrine, and endocrine effects on immune cells.

[0088] As used herein, “immunomodulator” refers to a reagent capable of modulating a given immune response to a desired level (e.g., as in immune enhancement, immunosuppression, or induction of immune tolerance). Examples of immunomodulators include, but are not limited to, disclosed immunomodulators that may include aspirin, azathioprine, belimumab, betamethasone dipropionate, betamethasone valerate, bortezomib, brebrutinib, cyazathioprine, cyclophosphamide, cyclosporine, deoxyguanidine, didemnin B, fluocinolone acetonide, leucovorin, ibuprofen, IL-6 inhibitors (e.g., sarilumab), indomethacin, inebilizumab, intravenous gamma globulin (IVIG), methotrexate, methylprednisolone, and mycophenolate mofetil. (mofetil), naproxen, prednisolone, prednisolone, prednisolone, indomethacin, rapamycin, rituximab, sirolimus, sulindac, synthetic vaccine particles containing rapamycin (SVP-rapamycin or ImmTOR), thalidomide, tocilizumab, tometidine, triamcinolone acetonide, anti-CD3 antibody, anti-CD4 antibody, anti-CD19 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD40 antibody, anti-FcRN antibody, anti-IL6 antibody, anti-IGFIR antibody, IL2 mutant protein, BTK inhibitor, or combinations thereof. In one aspect, the disclosed immunomodulatory agent may include one or more Treg (regulatory T cell) infusions (e.g., antigen-specific Treg cells against AAV). In one aspect, the disclosed immunomodulatory agent may be bortezomib or SVP-rapamycin. In one aspect, the immunomodulatory agent may be administered via any suitable route of administration, including but not limited to sub-administration.Intrauterine, intra-CSF, intrathecal, intravenous, subcutaneous, transdermal, intradermal, intramuscular, oral, percutaneous, intraperitoneal (IP), or vaginal administration. In one aspect, a combination of routes may be used to administer the disclosed immunomodulatory agent. Administration may also include administration via the hepatic artery or via the portal vein (HPV). Administration of the immunomodulatory agent may be continuous or intermittent, and administration may include a combination of one or more routes.

[0089] The term “immune-tolerant” as used herein refers to unresponsiveness to antigens (e.g., vectors, therapeutic proteins, transgenic products, etc.). Immune-tolerant promoters can reduce, mitigate, or prevent transgenic-induced immune responses that may be associated with gene therapy. Assays known in the art for measuring immune responses, such as immunohistochemical assays of cytotoxic T-cell responses, may be used to determine whether one or more promoters can confer immune-tolerant properties.

[0090] The term “packaging insert” as used herein refers to instructions for use typically included in the commercial packaging of a therapeutic product, which contains information about indications, usage, dosage, administration, contraindications, and / or warnings about the use of such therapeutic products.

[0091] As used herein, in the context of administering other therapies (e.g., other agents), the term “combination” includes using more than one therapy (e.g., pharmaceutical therapy). Administration in combination with one or more other therapeutic agents includes simultaneous (e.g., concurrent) and sequential administration in any order. The use of the term “combination” does not limit the order in which the therapies are administered to the subject. As a non-limiting example, a first therapy (e.g., a disclosed nucleic acid molecule, a disclosed vector, a disclosed pharmaceutical preparation, or a combination thereof) may be administered to a subject who has or is diagnosed with a disease or condition (e.g., a hereditary disease or condition) before (e.g., at 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks) administering a second therapy (e.g., an agent). The drug can be administered at 1 week, 5 weeks, 6 weeks, 8 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks), in parallel, or after (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or longer).

[0092] Components for preparing the disclosed nucleic acid molecules, disclosed carriers, or disclosed pharmaceutical formulations are disclosed, as well as the disclosed nucleic acid molecules, disclosed carriers, or disclosed pharmaceutical formulations used in the methods disclosed herein. This document discloses...These and other materials, and it should be understood that when combinations, subsets, interactions, groups, etc., of these materials are disclosed, while specific mentions of every different individual and collective combination and arrangement of these compounds are not explicitly disclosed, each is specifically considered and described herein. For example, if a particular compound is disclosed and discussed, and many modifications that can be made to a number of molecules including the compound are discussed, then the compound and possible modifications, as well as every combination and arrangement thereof, are specifically considered, unless otherwise expressly indicated to the contrary. Thus, if a class of molecules A, B, and C and a class of molecules D, E, and F are disclosed, and examples of combinations of molecules A-D are disclosed, then each is considered individually and collectively, even if each is not individually enumerated, meaning that combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Similarly, any subsets or combinations of these are also disclosed. Thus, for example, subgroups of A-E, B-F, and C-E should be considered disclosed. This concept applies to all aspects of this application, including but not limited to steps in methods of preparing and using the compositions of the invention. Therefore, if various additional steps are available, it should be understood that each of these additional steps can be performed using any particular embodiment or combination of embodiments of the method of the present invention.

[0093] B. Compositions for Transcriptome Engineering 1. Nucleic Acid Molecule 5' Replacement Construct This document discloses a nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures.

[0094] This document discloses a nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures comprising the sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09.

[0095] This document discloses a nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures, wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA.

[0096] This document discloses a nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures comprising the sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09, wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA.5' substitution of the targeted endogenous pre-mRNA. Specification 15 / 109 pages 19 CN 121195069 A

[0097] In one aspect, the disclosed targeted endogenous pre-mRNA may contain one or more mutations. In one aspect of the disclosed targeted endogenous pre-mRNA, one or more disclosed mutations may be in the 5' portion of the pre-mRNA. In one aspect, one or more disclosed mutations in one or more exons may contribute to the pathogenesis of one or more cells.

[0098] In one aspect, the disclosed cells may be in a subject. In one aspect, the subject may be a human patient and may be male or female. In one aspect, the subject may have a genetic disease or condition. In one aspect, the subject may be untreated.

[0099] In one aspect, one or more disclosed mutations may inhibit the translation of the encoded protein. In one aspect, one or more disclosed mutations may modify the translation of the encoded protein. In one aspect, one or more disclosed mutations may produce the encoded protein with nonsense or missense mutations.

[0100] In one aspect, the disclosed targeted endogenous pre-mRNA may contain one or more mutations in one or more exons. In one aspect, the disclosed targeted endogenous pre-mRNA may contain one or more mutations in one or more introns. In one aspect, one or more disclosed exon mutations may contribute to pathogenesis in one or more cells. In one aspect, one or more disclosed intron mutations may contribute to pathogenesis in one or more cells.

[0101] In one aspect, the disclosed targeted endogenous pre-mRNA may be a primary transcript of a protein-coding gene. In one aspect of the publicly disclosed targeted endogenous pre-mRNA, the disclosed protein-coding gene may contain one or more of the following coding regions: ABCA1, ABCA12, ABCA13, ABCA2, ABCA3, ABCA4, ABCA5, ABCC1, ABCC2, ABCC6, ABCC8, ABCC9, ACAN, ADAMTS13, ADCY10, ADGRV1, AGL, AGRN, AHDC1, ALK, ALMS1, ALPK3, ALS2, ANAPC1, ANK1, ANK2, ANK3, ANKRD11, ANKRD26, APC, APC2, APOB, ARFGEF2, ARHGAP31, ARHGEF10, ARHGEF18, ARID1A, ARID1B, ARID2, ASH1L, ASPM, ASXL1, ASXL2, ASXL3, ATM, ATP7A.ATP7B, ATR, ATRX, BAZ1A, BAZ2B, BCOR, BCORL1, BDP1, BLM, BPTF, BRCA1, BRCA2, BRD4, BRWD3 C2CD3, C3, C5, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, CACNA1H CACNA1S, CAD, CAMTA1, CARMIL2, CC2D2A, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2 CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP152, CEP164, CEP250, CEP290, CFAP43, CFAP44 CFAP65, CFTR / ABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT, CLIP1, CLTC, CNOT1 CNTNAP1、COL11A1、COL11A2、COL12A1、COL17A1、COL18A1、COL1A1、COL1A2、COL27A1、COL2A1、 COL3A1、COL4A1、COL4A2、COL4A3、COL4A4、COL4A5、COL4A6、COL5A1、COL5A2、COL6A3、COL7A1、 CPAMD8, CPLANE1, CPS1, CPSF1, CRB1, CREBBP, CUBN, CUL7, CUX1, DCC, DCHS1, DEPDC5, DICER1 DIP2B, DLC1, DMD, DMXL2, DNAH1, DNAH11,DNAH17,DNAH2,DNAH5,DNAH7,DNAH8,DNAH9,DNAMBP.” DNMT1、DOCK2、DOCK3、DOCK6、DOCK7、DOCK8、DSCAM、DSP、DST、DUOX2、DYNC1H1、DYNC2H1、DYSF、 EIF2AK4, EP300, EPG5, ERCC6, ERCC6L2, EXPH5, ​​EYS, F5, F8, FANCA, FANCD2, FANCM, FAT1 FAT4、FBN1、FBN2、FLG、FLG2、FLNA、FLNB、FLNC、FLT4、FMN2、FN1、FRAS1、FREM1、FREM2、FSIP2、 FXN、FYCO1、GLI2、GLI3、GPR179、GREB1L、GRIN2A、GRIN2B、GRIN2D、HCFC1、HECW2、HERC1、HERC2, HFM1, HIVEP1, HIVEP2, HMCN1, HSPG2, HTT, HUWE1, HYDIN, IFT140, IFT172, IGF1R. IGF2R, IGSF1, INSR, INTS1, IQSEC2, ITGB4, ITPR1, ITPR2, JMJD1C, KALRN, KANK1, KAT6A. KAT6B, KDM3B, KDM5B, KDM5C, KDM6A, KDM6B, KDR, KIAA0586, KIAA1109, KIAA1549, KIDINS220. KIF14, KIF1A, KIF1B, KIF21A, KIF26B, KIF7, KMT2A, KMT2B, KMT2C, KMT2D, KMT2E, KNL1 LAMA1, LAMA2, LAMA3, LAMA4, LAMA5, LAMB1, LAMB2, LAMC3, LCT, LMNA, LOXHD1, LPA, LRBA LRP1, LRP2, LRP4, LRP5, LRP6, LRPPRC, LRRK1, LRRK2, LTBP2, LTBP4, LYST, MACF1, MADD. MAGI2, MAP1B, MAP3K1, MAPK8IP3, MAPKBP1, MAST1, MBD5, MCM3AP, MED12, MED12L, MED13. MED13L, MED23, MEGF8, MET, MLH3, MPDZ, MSH6, MTOR, MYH10, MYH11, MYH14, MYH2, MYH3, MYH6. MYH7, MYH7B, MYH8, MYH9, MYLK, MYO15A, MYO18B, MYO3A, MYO5A, MYO5B, MYO7A, MYO9A, NALCN. NBAS, NBEA, NBEAL2, NCAPD2, NCAPD3, NEB, NEXMIF, NEXMIF, NF1, NFASC, NHS, NIN, NIPBL. NLRP1, NOTCH1, NOTCH2, NOTCH3, NPHP4, NRXN1, NRXN3, NSD1, NSD2, NUP155, NUP188, NUP205. OBSCN, OBSL1, OTOF, OTOG, OTOGL, PARD3, PBRM1, PCDH15, PCLO, PCNT, PHIP, PI4KA, PIEZO1.PIEZO2, PIK3C2A, PIKFYVE, PKD1, PKD1L1, PKHD1, PLCE1, PLEC, PLEKHG2, PNPLA6, POGZ, POLA1, POLE, POLR1A, POLR2A, POLR3A, PRG4, PRKDC, PRPF8, PRR12, PRX, PTCH1, PTPN23, PTPRF, PTPRJ, PTPRQ, PXDN, QRICH2, RAB3GAP2, RAI1, RALGAPA1, RANBP2, RB1CC1, RELN, RERE, REV3L, RIC1, RIMS1, RIMS2, RNF213, ROBO1, ROBO2, ROBO3, ROS1, RP1, RP1L1, RTTN, RUSC2, RYR1, RYR2, SACS, SAMD9, SAMD9L, SBF2, SCAPER, SCN10A, SCN11A, SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SETBP1, SETD1A, SETD1B, SETD2, SETD5, SETX, SHANK2, SHANK3, SHROOM4, SI, SIPA1L3, SLIT2, SLX4, SMARCA2, SMARCA4, SMCHD1, SNRNP200, SON, SPEF2, SPEG, SPG11, SPTA1, SPTAN1, SPTB, SPTBN2, SPTBN4, SRCAP, STRC, SVIL, SYNE1, SYNGAP1, SYNJ1, SZT2, TAF1, TANC2, TCF20, TCOF1, TDRD9, TECPR2, TECTA, TENM3, TENM4, TET3, TEX14, TEX15, TG, THOC2, TMEM94, TNC, TNIK, TNR, TNRC6B, TNXB, TOGARAM1, TONSL, TRIO, TRIOBP, TRIP11, TRIP12, TRPM1, TRPM6, TRPM7, TRRAP, TSC2, TTC37, TTN, TUBGCP6, UBR1, UNC80, USH2A, USP9X, VCAN, VPS13A, VPS13B, VPS13C, VPS13D, VWF, WDFY3, WDR19, WDR62, WDR81, WNK1, WRN, ZFHX2, ZFYVE26, ZNF142, ZNF292, ZNF335, ZNF407, ZNF462, or NF469. In one aspect, publicly disclosed protein-coding bases...It may contain one or more coding regions of CFTR, MDX, DYSF / TTN, DMPK, COL7A1, K14, MAPT, FVIII, HTT, RHO, DNA-PKcs, SMN2, or CD40L. In one aspect, the disclosed protein-coding gene may contain one or more coding regions of FXN, LMNA, or RYR2. In one aspect, the disclosed protein-coding gene may contain a portion of the disclosed protein-coding gene (e.g., exon 1 or exon 4, etc.).

[0102] In one aspect, the 3' portion of the disclosed targeted endogenous pre-mRNA can be trans-spliced ​​with exogenous RNA. In one aspect, the disclosed RNA targeting motif can bind to the targeted endogenous pre-mRNA. In one aspect, the disclosed RNA targeting motif can bind to the 5' end of the targeted endogenous pre-mRNA. In one aspect, the disclosed RNA targeting motif can be specific to endogenous pre-mRNAs with one or more mutations. In one aspect, the disclosed RNA targeting motif may be specific to endogenous pre-mRNA having one or more exon mutations. In one aspect, the disclosed RNA targeting motif may be specific to endogenous pre-mRNA having one or more intron mutations.

[0103] In one aspect, the disclosed RNA targeting motif may comprise an antisense oligonucleotide. In one aspect, the disclosed antisense oligonucleotide may comprise about 15 nucleotides to about 50 nucleotides. In one aspect, the disclosed antisense oligonucleotide may comprise about 30 nucleotides. In one aspect, the disclosed RNA targeting motif may be directed to an intron adjacent to the 5' of the exon of the targeted endogenous pre-mRNA to be spliced.

[0104] In one aspect, the disclosed 5' hemi-intron may comprise a 5' splice site. In one aspect, the disclosed 5' splice site may comprise a shared 5' splice site. In one aspect, the disclosed 5' splice site may comprise MAG|GURAGU (SEQ ID NO: 13), where | denotes exon-intron connection, where M=A or C, and where R=A or G. In one aspect, the disclosed 5' hemi-intron may be recognized by nuclear splicing components in host cells. In one aspect, the disclosed 5' hemi-intron may be recognized by spliceosomes in host cells. In one aspect, the disclosed 5' hemi-intron may facilitate trans-splicing of exogenous RNA to the 3' exon of an intron immediately adjacent to the target intron in the endogenous pre-mRNA.

[0105] In one aspect, the disclosed exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA may comprise proteinOne or more exons of the white-coding gene. In one aspect, the disclosed exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA may comprise a primary sequence of the coding sequence of one or more exons having one or more mutations. In another aspect, the disclosed exogenous RNA may be trans-spliced ​​to the 3' end of the targeted endogenous pre-mRNA.

[0106] In one aspect of the disclosed exogenous RNA, the disclosed protein-coding gene may comprise one or more of the following coding regions: ABCA1, ABCA12, ABCA13, ABCA2, ABCA3, ABCA4, ABCA5, ABCC1, ABCC2, ABCC6, ABCC8, ABCC9, ACAN, ADAMTS13, ADCY10, ADGRV1, AGL, AGRN, AHDC1, ALK, ALMS1, ALPK3, ALS2, ANAPC1, ANK1, ANK2, ANK3, ANKRD11, ANKRD26, APC, APC2, APOB, ARFGEF2, ARHGAP31, ARHGEF10, ARHGEF18, ARID1A, ARID1B, ARID2, ASH1L, ASPM, ASXL1, ASXL2, ASXL3, ATM, ATP7A, ATP7B. ATR, ATRX, BAZ1A, BAZ2B, BCOR, BCORL1, BDP1, BLM, BPTF, BRCA1, BRCA2, BRD4, BRWD3, C2CD3, C3, C5, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, CACNA1H, CACNA1S, CAD, CAMTA1, CARMIL2, CC2D2A, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2, CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP152, CEP164, CEP250, CEP290, CFAP43, CFAP44, CFAP65, CFTR / ABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT, CLIP1, CLTC, CNOT1, CNTNAP1, COL11A1, COL11A2, COL12A1, COL17A1, COL18A1, COL1A1, COL1A2, COL27A1, COL2A1, COL3A1, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A3, COL7A1,CPAMD8, CPLANE1, CPS1, CPSF1, CRB1, CREBBP, CUBN, CUL7, CUX1, DCC, DCHS1, DEPDC5, DICER1. DIP2B, DLC1, DMD, DMXL2, DNAH1, DNAH11, DNAH17, DNAH2, DNAH5, DNAH7, DNAH8, DNAH9, and DNMBP. DNMT1, DOCK2, DOCK3, DOCK6, DOCK7, DOCK8, DSCAM, DSP, DST, DUOX2, DYNC1H1, DYNC2H1, DYSF. EIF2AK4, EP300, EPG5, ERCC6, ERCC6L2, EXPH5, ​​EYS, F5, F8, FANCA, FANCD2, FANCM, FAT1 FAT4, FBN1, FBN2, FLG, FLG2, FLNA, FLNB, FLNC, FLT4, FMN2, FN1, FRAS1, FREM1, FREM2, FSIP2. FXN, FYCO1, GLI2, GLI3, GPR179, GREB1L, GRIN2A, GRIN2B, GRIN2D, HCFC1, HECW2, HERC1 HERC2, HFM1, HIVEP1, HIVEP2, HMCN1, HSPG2, HTT, HUWE1, HYDIN, IFT140, IFT172, IGF1R. IGF2R, IGSF1, INSR, INTS1, IQSEC2, ITGB4, ITPR1, ITPR2, JMJD1C, KALRN, KANK1, KAT6A. KAT6B, KDM3B, KDM5B, KDM5C, KDM6A, KDM6B, KDR, KIAA0586, KIAA1109, KIAA1549, KIDINS220. KIF14, KIF1A, KIF1B, KIF21A, KIF26B, KIF7, KMT2A, KMT2B, KMT2C, KMT2D, KMT2E, KNL1 LAMA1, LAMA2, LAMA3, LAMA4, LAMA5, LAMB1, LAMB2, LAMC3, LCT, LMNA, LOXHD1, LPA, LRBA. LRP1, LRP2, LRP4, LRP5, LRP6, LRPPRC, LRRK1, LRRK2, LTBP2, LTBP4, LYST, MACF1, MADD. MAGI2, MAP1B, MAP3K1, MAPK8IP3, MAPKBP1, MAST1, MBD5, MCM3AP, MED12, MED12L, MED13.MED13L、MED23、MEGF8、MET、MLH3、MPDZ、MSH6、MTOR、MYH10、MYH11、MYH14、MYH2、MYH3、MYH6、 MYH7、MYH7B、MYH8、MYH9、MYLK、MYO15A、MYO18B、MYO3A、MYO5A、MYO5B、MYO7A、MYO9A、NALCN、 NBAS、NBEA、NBEAL2、NCAPD2、NCAPD3、NEB、NEXMIF、NEXMIF、NF1、NFASC、NHS、NIN、NIPBL、 NLRP1、NOTCH1、NOTCH2、NOTCH3、NPHP4、NRXN1、NRXN3、NSD1、NSD2、NUP155、NUP188、NUP205、 OBSCN、OBSL1、OTOF、OTOG、OTOGL、PARD3、PBRM1、PCDH15、PCLO、PCNT、PHIP、PI4KA、PIEZO1、 PIEZO2、PIK3C2A、PIKFYVE、PKD1、PKD1L1、PKHD1、PLCE1、PLEC、PLEKHG2、PNPLA6、POGZ、 POLA1、POLE、POLR1A、POLR2A、POLR3A、PRG4、PRKDC、PRPF8、PRR12、PRX、PTCH1、PTPN23、 PTPRF、PTPRJ、PTPRQ、PXDN、QRICH2、RAB3GAP2、RAI1、RALGAPA1、RANBP2、RB1CC1、RELN、RERE、 22 CN 121195069 SCN4A、SCN5A、SCN8A、SCN9A、SETBP1、SETD1A、SETD1B、SETD2、SETD5、SETX、SHANK2、SHANK3、 SHROOM4、SI、SIPA1L3、SLIT2、SLX4、SMARCA2、SMARCA4、SMCHD1、SNRNP200、SON、SPEF2、SPEG、SPG11, SPTA1, SPTAN1, SPTB, SPTBN2, SPTBN4, SRCAP, STRC, SVIL, SYNE1, SYNGAP1, SYNJ1, SZT2, TAF1, TANC2, TCF20, TCOF1, TDRD9, TECPR2, TECTA, TENM3, TENM4, TET3, TEX14, TEX15, TG, THOC2, TMEM94, TNC, TNIK, TNR, TNRC6B, TNXB, TOGARAM1, TONSL, TRIO, TRIOBP, TRIP11, TRIP12, TRPM1, TRPM6, TRPM7, TRRAP, TSC2, TTC37, TTN, TUBGCP6, UBR1, UNC80, USH2A, USP9X, VCAN, VPS13A, VPS13B, VPS13C, VPS13D, VWF, WDFY3, WDR19, WDR62, WDR81, WNK1, WRN, ZFHX2, ZFYVE26, ZNF142, ZNF292, ZNF335, ZNF407, ZNF462, or ZNF469. In one aspect, the disclosed protein-coding gene may contain one or more coding regions of CFTR, MDX, DYSF / TTN, DMPK, COL7A1, K14, MAPT, FVIII, HTT, RHO, DNA-PKcs, SMN2, or CD40L. In one aspect, the disclosed protein-coding gene may contain one or more coding regions of FXN, LMNA, or RYR2.

[0107] In one aspect, the disclosed nucleic acid sequence to be trans-spliced ​​may encode LMNA / C (SEQ ID NO: 20) or a portion thereof. LMNA / C is known in the art (e.g., Gene ID 4000), and the nucleotide sequence may contain nucleotides 4974-62517 in accession number NG008692.2. The nuclear layer consists of a two-dimensional matrix of proteins located adjacent to the nuclear membrane. Proteins of the lamin family constitute the matrix and are highly conserved in evolution. During mitosis, the nuclear matrix is ​​reversibly broken down as lamins are phosphorylated. Lamins are involved in nuclear stability, chromatin structure, and gene expression. Vertebrate lamins consist of two types, A and B. Alternative splicing produces a variety of transcript variants. Mutations in this gene lead to several diseases: Emery-Dreifuss muscular dystrophy, familial partial lipodystrophy, limb-girdle muscular dystrophy, dilated cardiomyopathy, Charcot-Marie-Tooth disease, and Hutchinson-Gilford progeria.

[0108] In one aspect, the disclosed nucleic acid sequence to be trans-spliced ​​may encode DP71 or a portion thereof. DP71 is known in the art (e.g., Gene ID 13405).

[0109] In one aspect, the disclosed nucleic acid sequence to be trans-spliced ​​may encode CFTR (SEQ ID NO: 19) or a portion thereof. CFTR is known in the art (e.g., Gene ID 1080), and the nucleotide sequence may contain nucleotides 19180-207882 in accession number NG016465.4. This gene encodes a member of the ATP-binding cassette (ABC) transporter superfamily. The encoded protein acts as a chloride ion channel, making it unique among members of this protein family, and controls the secretion and uptake of ions and water in epithelial tissues. Channel activation is mediated by a cycle of phosphorylation of the regulatory domain, ATP binding of the nucleotide-binding domain, and ATP hydrolysis. Mutations in this gene lead to cystic fibrosis, the most common fatal genetic disease in people of Nordic descent. The most common mutation in cystic fibrosis, DeltaF508, results in impaired folding and transport of the encoded protein. Several pseudogenes have been identified in the human genome.

[0110] In one aspect, the disclosed nucleic acid sequence to be trans-spliced ​​may encode DMPK (SEQ ID NO: 21) or a portion thereof. DMPK is known in the art (e.g., Gene ID 1760), and the nucleotide sequence may contain nucleotides 5068-17841 of accession number NG009784.1. DMPK is a serine-threonine kinase, closely associated with other kinases that interact with members of the Rho family of small GTPases. The substrates of this enzyme include myopoietin (the β-subunit of an L-type calcium channel) and phosphatases. The 3' untranslated region of this gene contains 5-38 copies of a CTG trinucleotide repeat sequence. Amplifying this unstable motif to 50-5,000 copies causes type I myotonic dystrophy, the severity of which increases with increasing copy number of the repeat element. Repeated amplification is associated with condensation that disrupts the local chromatin structure that expresses the gene in this region. Several alternative splicing transcript variants of this gene have been described, but the full-length nature of some of these variants has not yet been determined. Specification 19 / 109 pages 23 CN 121195069 A

[0111] In one aspect, the disclosed gene may be DMD (dystrophin) (SEQ ID NO: 17). DMD is known in the art (e.g., Gene ID 1756), and the nucleotide sequence may contain nucleotides 5001-2225382 in accession number NG012232.1. DMD spans a genome range greater than 2 Mb and encodes N-terminal...A large protein containing an actin-binding domain and multiple spectrin repeats. The encoded protein forms a component of the dystrophic glycoprotein complex (DGC), which bridges the internal cytoskeleton and extracellular matrix. Deletions, duplications, and point mutations at this gene locus can cause Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), or cardiomyopathy. The use of alternative promoters and alternative splicing result in many different transcript variants and protein isotypes for this gene.

[0112] In one aspect, the disclosed nucleic acid sequence to be trans-spliced ​​may encode LRRK2 (SEQ ID NO: 18) or a portion thereof. LRRK2 is known in the art (e.g., Gene ID 120892), and the nucleotide sequence may comprise nucleotides 5001-149275 of accession number NG011709.1. LRRK2 is a member of the leucine-rich repeat kinase family and encodes a protein having a repeat region, a leucine-rich repeat (LRR) domain, a kinase domain, a DFG-like motif, a RAS domain, a GTPase domain, an MLK-like domain, and a WD40 domain. This protein is primarily located in the cytoplasm but is also associated with the outer mitochondrial membrane. Mutations in this gene are associated with Parkinson's disease.

[0113] In one aspect, the disclosed exogenous RNA to be trans-spliced ​​may further include a UTR.

[0114] In one aspect, one or more disclosed RNA structures may bind to one or more RNA-binding proteins. In one aspect, one or more disclosed RNA structures may bind to one or more double-stranded RNA-binding proteins (dsRBPs). In one aspect, dsRBPs are known to those skilled in the art and include, but are not limited to, ADAR1, ADAR2, DICER, NFAR, PACT, PKR, RHA RNase III, Stauffen, TRBP, TSEN, or any combination thereof.

[0115] In one aspect, one or more disclosed RNA structures may comprise any one of the sequences of SEQ ID NO: 01-SEQ ID NO: 09. In one aspect, one or more disclosed RNA structures may improve and / or enhance trans-splicing efficiency. In one aspect, one or more disclosed RNA structures may stabilize pre-mRNA. In one aspect, one or more disclosed RNA structures may localize RNA to the cell nucleus. In one aspect, one or more disclosed RNA structures may stabilize the interaction between the targeted endogenous pre-mRNA molecule and the exogenous RNA to be trans-spliced. In one aspect, the disclosed nucleic acid molecule may lack CRISPR-related proteins.

[0116] In one aspect, the disclosed resulting chimeric RNA transcript may comprise the 5' endogenous pre-mRNA of the targeted endogenous pre-mRNA.The disclosed chimeric RNA transcript may contain the 3' portion of the targeted endogenous pre-mRNA and the 5' portion of the exogenous RNA.

[0117] In one aspect, the disclosed targeted endogenous pre-mRNA and the disclosed exogenous RNA may encode the same protein-coding gene. In one aspect, the disclosed targeted endogenous pre-mRNA and the disclosed exogenous RNA may contain one or more exons of the same protein-coding gene.

[0118] In one aspect, the disclosed nucleic acid molecule may be packaged into a viral vector. In one aspect, the disclosed viral vector may contain an AAV vector. In one aspect, the disclosed nucleic acid molecule may be packaged into a non-viral vector. In one aspect, the disclosed nucleic acid molecule may be incorporated into a plasmid. In one aspect, the disclosed nucleic acid molecule may be incorporated into lipid nanoparticles.

[0119] In one aspect, the disclosed nucleic acid molecule may further contain a polyadenylated sequence. In one aspect, the disclosed nucleic acid molecule may further contain a sequence for a promoter. In one aspect, the disclosed nucleic acid molecule may further contain a spacer region. In one aspect, the disclosed spacer region may separate the 5' splice region from one or more RNA structures. In one aspect, the disclosed spacer region may contain any known spacer. In one aspect, the disclosed spacer region may contain a common splicing motif (e.g., U1 or U2). In one aspect, the disclosed spacer region may contain a limited number of common splicing motifs (e.g., U1 or U2). Specification 20 / 109 pages 24 CN 121195069 A

[0120] In one aspect, the disclosed nucleic acid molecule may further contain one or more nuclear localization signals. NLS are known to those skilled in the art. In one aspect, the disclosed NLS may include any NLS known in the art. As known in the art (see, for example, Lu J et al. (2021) Cell Commun Signal. 19:60, whose teachings on NLS are incorporated herein by reference), nuclear localization signals (NLS) are typically short peptides that act as signal fragments mediating the transport of proteins from the cytoplasm to the nucleus.

[0121] In one aspect, the disclosed nucleic acid molecule may further contain one or more nuclear retention elements (NREs). NREs are known to those skilled in the art. In one aspect, the disclosed NRE may comprise SIRLOIN (SEQ ID NO:15) or BORG (SEQ ID NO:16).

[0122] In one aspect, the disclosed nucleic acid molecule may further comprise one or more flavivirus genetic elements. In one aspect, the flavivirus genetic element may comprise one or more flavivirus 3' untranslated regions (3'UTRs) or one or more subunits.The elements are: flavivirus RNA (sfRNA) elements, one or more flavivirus XRN1 resistance RNA (xrRNA) elements, one or more flavivirus dumbbell (DB) RNA elements, one or more flavivirus 3' stem-loop (3'SL) elements, or any combination thereof. (For a description of flavivirus gene elements, see WO 2022 / 182835).

[0123] In one aspect, the disclosed exogenous RNA can induce splicing events. In one aspect, the disclosed 5' hemiintron can be recognized by nuclear splicing components within the host cell.

[0124] In one aspect, the disclosed promoter for the 5' substitution construct can be tissue-specific or ubiquitous, and can be constitutive or inducible, depending on the desired expression pattern. The promoter can be natural or exogenous, and can be a natural or synthetic sequence. Exogenous means that the transcription start region is not present in the wild-type host in which the transcription start region is introduced. In one aspect, the disclosed promoter can be a promoter / enhancer. In one aspect, the disclosed promoter for the disclosed nucleic acid molecule can be an endogenous promoter. In one aspect, the disclosed endogenous promoter may be an endogenous promoter / enhancer. In one aspect, the disclosed endogenous promoter or disclosed endogenous promoter / enhancer may generally be obtained from a non-coding region upstream of the transcription start site of the target gene. In one aspect, the disclosed endogenous promoter or disclosed endogenous promoter / enhancer may be used for constitutive and efficient expression of a disclosed protein-coding gene. In one aspect, the disclosed promoter for one or more disclosed guide RNA sequences may be a CMV promoter or a CMV promoter / enhancer. CMV promoters and CMV promoters / enhancers are well known in the art. In one aspect, the disclosed promoter for one or more disclosed guide RNA sequences may be any eukaryotic RNA polymerase II promoter.

[0125] This document discloses an expression cassette comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures. This document discloses an expression cassette comprising: exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures comprising the sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09.5' substitution of pre-mRNA. This document discloses an expression cassette comprising: exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising sequences of any one of SEQ ID NO: 01-SEQ ID NO: 09, wherein the nucleic acid molecules enable 5' substitution of the targeted endogenous pre-mRNA.

[0126] In one aspect, expression of the disclosed protein-coding gene can be restored and / or returned to wild-type, normal, or control expression levels. In one aspect, the disclosed nucleic acid molecules can restore one or more aspects of cellular homeostasis and / or cellular function and / or metabolic disorders. In one aspect, the disclosed nucleic acid molecules can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above). In one aspect, one or more aspects of restoring cellular homeostasis and / or cellular function may include one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (e.g., correcting, preventing, reducing, and / or mitigating autophagy); (iii) improving, enhancing, restoring, and / or preserving mitochondrial function and / or structural integrity; (iv) improving, enhancing, restoring, and / or preserving organelle function and / or structural integrity; (v) correcting enzymatic dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of a multisystemic manifestation of a genetic disease or condition; (vii) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of a genetic disease or condition, or (viii) any combination thereof. In one aspect, one or more aspects of restoring cellular homeostasis may include one or more aspects of improving, enhancing, restoring, and / or preserving cellular structural and / or functional integrity.

[0127] In one aspect, restoring the activity and / or functionality of a missing, defective, and / or mutated protein or enzyme may include 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of restoration compared to a pre-existing level, such as a pre-treatment level. In one aspect, the amount of restoration may be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% higher than a pre-existing level (e.g., a pre-treatment level). In one aspect, restoration may be measured against a control or reference level (e.g., using a protein or enzyme without missing, defective, and / or mutated proteins or enzymes).(The altered protein or enzyme is determined by one or more subjects). In one aspect, recovery may be partial or incomplete. In another aspect, recovery may be complete or near-complete, such that the levels of expression, activity, and / or function are similar to those of wild-type or control levels.

[0128] In one aspect, the disclosed 5' substitution construct can be used specifically in methods for treating subjects with autosomal dominant genetic diseases or conditions (e.g., progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert's disease, hereditary hemorrhagic telangiectasia, hereditary elliptosis, hereditary spherocytosis, Huntington's disease, idiopathic hypoparathyroidism, intestinal polyposis, marble bone disease, Marfan syndrome, neurofibromatosis, polycystic kidney disease (adults), protein C deficiency, osteogenesis imperfecta, Treacher Collins syndrome, tuberous sclerosis, Von Willebrand's disease, etc.).

[0129] 3' Replacement Construct This document discloses a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA. This document discloses a nucleic acid molecule comprising one or more RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA. This document discloses a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables 3' replacement of the targeted endogenous pre-mRNA. This document discloses a nucleic acid molecule comprising one or more RNA structures, said RNA structures comprising the sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a 3' half-intron to be trans-spliced ​​into the targeted RNA.The exogenous RNA of the endogenous pre-mRNA, wherein the nucleic acid molecule enables the 3' substitution of the targeted endogenous pre-mRNA.

[0130] In one aspect, the disclosed targeted endogenous pre-mRNA may contain one or more mutations in one or more exons. In one aspect, one or more disclosed mutations may be in the 3' portion of one or more exons of the pre-mRNA. In one aspect, the disclosed targeted endogenous pre-mRNA may contain one or more mutations in one or more introns. In one aspect, one or more disclosed mutations in one or more exons may contribute to the pathogenesis of one or more cells. In one aspect, the disclosed cells may be in a subject. In one aspect, the subject may be a human patient and may be male or female. In one aspect, the subject may have a genetic disease or condition. In one aspect, the subject may be untreated. In one aspect, one or more disclosed mutations may inhibit the translation of the encoded protein. In one aspect, one or more disclosed mutations may modify the translation of the encoded protein. In one aspect, during and / or after translation, one or more publicly disclosed mutations can produce proteins with nonsense or missense mutations. In one aspect, one or more publicly disclosed exon mutations can contribute to pathogenesis in one or more cells. In one aspect, one or more publicly disclosed intron mutations can contribute to pathogenesis in one or more cells. In one aspect, a publicly disclosed targeted endogenous pre-mRNA can encode a protein-coding gene.

[0131] In one aspect of the disclosed targeted endogenous pre-mRNA, the disclosed protein-coding gene may comprise one or more of the following coding regions: ABCA1, ABCA12, ABCA13, ABCA2, ABCA3, ABCA4, ABCA5, ABCC1, ABCC2, ABCC6, ABCC8, ABCC9, ACAN, ADAMTS13, ADCY10, ADGRV1, AGL, AGRN, AHDC1, ALK, ALMS1, ALPK3, ALS2, ANAPC1, ANK1, ANK2, ANK3, ANKRD11, ANKRD26, APC, APC2, APOB, ARFGEF2, ARHGAP31, ARHGEF10, ARHGEF18, ARID1A, ARID1B, ARID2, ASH1L, ASPM, ASXL1, ASXL2, ASXL3, ATM, ATP7A.ATP7B, ATR, ATRX, BAZ1A, BAZ2B, BCOR, BCORL1, BDP1, BLM, BPTF, BRCA1, BRCA2, BRD4, BRWD3 C2CD3, C3, C5, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, CACNA1H CACNA1S, CAD, CAMTA1, CARMIL2, CC2D2A, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2 CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP152, CEP164, CEP250, CEP290, CFAP43, CFAP44 CFAP65, CFTR / ABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT, CLIP1, CLTC, CNOT1 CNTNAP1、COL11A1、COL11A2、COL12A1、COL17A1、COL18A1、COL1A1、COL1A2、COL27A1、COL2A1、 COL3A1、COL4A1、COL4A2、COL4A3、COL4A4、COL4A5、COL4A6、COL5A1、COL5A2、COL6A3、COL7A1、 CPAMD8, CPLANE1, CPS1, CPSF1, CRB1, CREBBP, CUBN, CUL7, CUX1, DCC, DCHS1, DEPDC5, DICER1 DIP2B, DLC1, DMD, DMXL2, DNAH1, DNAH11,DNAH17,DNAH2,DNAH5,DNAH7,DNAH8,DNAH9,DNAMBP.” DNMT1、DOCK2、DOCK3、DOCK6、DOCK7、DOCK8、DSCAM、DSP、DST、DUOX2、DYNC1H1、DYNC2H1、DYSF、 EIF2AK4, EP300, EPG5, ERCC6, ERCC6L2, EXPH5, ​​EYS, F5, F8, FANCA, FANCD2, FANCM, FAT1 FAT4、FBN1、FBN2、FLG、FLG2、FLNA、FLNB、FLNC、FLT4、FMN2、FN1、FRAS1、FREM1、FREM2、FSIP2、 FXN、FYCO1、GLI2、GLI3、GPR179、GREB1L、GRIN2A、GRIN2B、GRIN2D、HCFC1、HECW2、HERC1、HERC2, HFM1, HIVEP1, HIVEP2, HMCN1, HSPG2, HTT, HUWE1, HYDIN, IFT140, IFT172, IGF1R. IGF2R, IGSF1, INSR, INTS1, IQSEC2, ITGB4, ITPR1, ITPR2, JMJD1C, KALRN, KANK1, KAT6A. KAT6B, KDM3B, KDM5B, KDM5C, KDM6A, KDM6B, KDR, KIAA0586, KIAA1109, KIAA1549, KIDINS220. KIF14, KIF1A, KIF1B, KIF21A, KIF26B, KIF7, KMT2A, KMT2B, KMT2C, KMT2D, KMT2E, KNL1 LAMA1, LAMA2, LAMA3, LAMA4, LAMA5, LAMB1, LAMB2, LAMC3, LCT, LMNA, LOXHD1, LPA, LRBA. LRP1, LRP2, LRP4, LRP5, LRP6, LRPPRC, LRRK1, LRRK2, LTBP2, LTBP4, LYST, MACF1, MADD. MAGI2, MAP1B, MAP3K1, MAPK8IP3, MAPKBP1, MAST1, MBD5, MCM3AP, MED12, MED12L, MED13. MED13L, MED23, MEGF8, MET, MLH3, MPDZ, MSH6, MTOR, MYH10, MYH11, MYH14, MYH2, MYH3, MYH6. MYH7, MYH7B, MYH8, MYH9, MYLK, MYO15A, MYO18B, MYO3A, MYO5A, MYO5B, MYO7A, MYO9A, NALCN. NBAS, NBEA, NBEAL2, NCAPD2, NCAPD3, NEB, NEXMIF, NEXMIF, NF1, NFASC, NHS, NIN, NIPBL NLRP1, NOTCH1, NOTCH2, NOTCH3, NPHP4, NRXN1, NRXN3, NSD1, NSD2, NUP155, NUP188, NUP205. OBSCN, OBSL1, OTOF, OTOG, OTOGL, PARD3, PBRM1, PCDH15, PCLO, PCNT, PHIP, PI4KA, PIEZO1.PIEZO2, PIK3C2A, PIKFYVE, PKD1, PKD1L1, PKHD1, PLCE1, PLEC, PLEKHG2, PNPLA6, POGZ, POLA1, POLE, POLR1A, POLR2A, POLR3A, PRG4, PRKDC, PRPF8, PRR12, PRX, PTCH1, PTPN23, PTPRF, PTPRJ, PTPRQ, PXDN, QRICH2, RAB3GAP2, RAI1, RALGAPA1, RANBP2, RB1CC1, RELN, RERE, REV3L, RIC1, RIMS1, RIMS2, RNF213, ROBO1, ROBO2, ROBO3, ROS1, RP1, RP1L1, RTTN, RUSC2, RYR1, RYR2, SACS, SAMD9, SAMD9L, SBF2, SCAPER, SCN10A, SCN11A, SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SETBP1, SETD1A, SETD1B, SETD2, SETD5, SETX, SHANK2, SHANK3, SHROOM4, SI, SIPA1L3, SLIT2, SLX4, SMARCA2, SMARCA4, SMCHD1, SNRNP200, SON, SPEF2, SPEG, SPG11, SPTA1, SPTAN1, SPTB, SPTBN2, SPTBN4, SRCAP, STRC, SVIL, SYNE1, SYNGAP1, SYNJ1, SZT2, TAF1, TANC2, TCF20, TCOF1, TDRD9, TECPR2, TECTA, TENM3, TENM4, TET3, TEX14, TEX15, TG, THOC2, TMEM94, TNC, TNIK, TNR, TNRC6B, TNXB, TOGARAM1, TONSL, TRIO, TRIOBP, TRIP11, TRIP12, TRPM1, TRPM6, TRPM7, TRRAP, TSC2, TTC37, TTN, TUBGCP6, UBR1, UNC80, USH2A, USP9X, VCAN, VPS13A, VPS13B, VPS13C, VPS13D, VWF, WDFY3, WDR19, WDR62, WDR81, WNK1, WRN, ZFHX2, ZFYVE26, ZNF142, ZNF292, ZNF335, ZNF407, ZNF462, or ZNF469. In one aspect, the publicly disclosed protein coding...The gene may contain one or more coding regions of CFTR, MDX, DYSF / TTN, DMPK, COL7A1, KI4, MAPT, FVIII HTT, RHO, DNA-PKcs, SMN2, or CD40L. In one aspect, the disclosed protein-coding gene may contain one or more coding regions of FXN, LMNA, or RYR2. In one aspect, the disclosed protein-coding gene may contain a portion of the disclosed protein-coding gene (e.g., exon 1 or exon 4, etc.).

[0132] In one aspect, the 5' portion of the disclosed targeted endogenous pre-mRNA may be trans-spliced ​​with exogenous RNA.

[0133] In one aspect, the disclosed RNA targeting motif may bind to the targeted endogenous pre-mRNA. In one aspect, the disclosed RNA targeting motif may bind to the 3' end of the targeted endogenous pre-mRNA. In one aspect, the disclosed RNA targeting motif may be specific to endogenous pre-mRNAs with one or more mutations. In one aspect, the disclosed RNA targeting motif may be specific to endogenous pre-mRNA having one or more exon mutations. In one aspect, the disclosed RNA targeting motif may be specific to endogenous pre-mRNA having one or more intron mutations. In one aspect, the disclosed RNA targeting motif may comprise an antisense oligonucleotide.

[0134] In one aspect, the disclosed antisense oligonucleotide may comprise about 15 to about 50 nucleotides. In one aspect, the disclosed antisense oligonucleotide may comprise about 30 nucleotides. In one aspect, the disclosed RNA targeting motif may be directed to an intron immediately adjacent to the 3' of the exon of the targeted endogenous pre-mRNA to be spliced.

[0135] In one aspect, the disclosed 3' hemiintron may comprise (i) a 3' splice region containing a branch point, (ii) a polypyrimidine tract, and (iii) a 3' splice acceptor site. In one aspect, the disclosed branch point may comprise the sequence of SEQ ID NO: 10. In one aspect, the disclosed 3' splice acceptor site may comprise the sequence YAG, where Y is a pyrimidine (SEQ ID NO: 11). In one aspect, the disclosed 3' hemiintron may be recognized by nuclear splicing components in host cells. In one aspect, the disclosed 3' hemiintron may be recognized by spliceosomes in host cells. In one aspect, the disclosed 3' hemiintron may facilitate trans-splicing of exogenous RNA to the 5' exon of the targeted intron immediately adjacent to the endogenous pre-mRNA.

[0136] In one aspect, the disclosed exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA may comprise a protein.One or more exons of the white-coding gene. In one aspect, the exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA may comprise a primary sequence of the coding sequence of one or more exons having one or more mutations. In one aspect, the exogenous RNA may be trans-spliced ​​to the 5' end of the targeted endogenous pre-mRNA.

[0137] In one aspect of the disclosed exogenous RNA, the disclosed protein-coding gene may comprise one or more of the following coding regions: ABCA1, ABCA12, ABCA13, ABCA2, ABCA3, ABCA4, ABCA5, ABCC1, ABCC2, ABCC6, ABCC8, ABCC9, ACAN, ADAMTS13, ADCY10, ADGRV1, AGL, AGRN, AHDC1, ALK, ALMS1, ALPK3, ALS2, ANAPC1, ANK1, ANK2, ANK3, ANKRD11, ANKRD26, APC, APC2, APOB, ARFGEF2, ARHGAP31, ARHGEF10, ARHGEF18, ARID1A, ARID1B, ARID2, ASH1L, ASPM, ASXL1, ASXL2, ASXL3, ATM, ATP7A, ATP7B. ATR, ATRX, BAZ1A, BAZ2B, BCOR, BCORL1, BDP1, BLM, BPTF, BRCA1, BRCA2, BRD4, BRWD3, C2CD3, C3, C5, CACNA1A, CACNA1B, CACNA1C, CACNA1D, CACNA1E, CACNA1F, CACNA1G, CACNA1H, CACNA1S, CAD, CAMTA1, CARMIL2, CC2D2A, CCDC88A, CCDC88C, CCNB3, CDH23, CDK13, CDK5RAP2, CELSR1, CEMIP2, CENPE, CENPF, CENPJ, CEP152, CEP164, CEP250, CEP290, CFAP43, CFAP44, CFAP65, CFTR / ABCC7, CHD1, CHD2, CHD3, CHD4, CHD7, CHD8, CIC, CIT, CLIP1, CLTC, CNOT1, CNTNAP1, COL11A1, COL11A2, COL12A1, COL17A1, COL18A1, COL1A1, COL1A2, COL27A1, COL2A1,COL3A1、COL4A1、COL4A2、COL4A3、COL4A4、COL4A5、COL4A6、COL5A1、COL5A2、COL6A3、COL7A1、 CPAMD8、CPLANE1、CPS1、CPSF1、CRB1、CREBBP、CUBN、CUL7、CUX1、DCC、DCHS1、DEPDC5、DICER1、 DIP2B、DLC1、DMD、DMXL2、DNAH1、DNAH11、DNAH17、DNAH2、DNAH5、DNAH7、DNAH8、DNAH9、DNMBP、 DNMT1、DOCK2、DOCK3、DOCK6、DOCK7、DOCK8、DSCAM、DSP、DST、DUOX2、DYNC1H1、DYNC2H1、DYSF、 EIF2AK4、EP300、EPG5、ERCC6、ERCC6L2、EXPH5、EYS、F5、F8、FANCA、FANCD2、FANCM、FAT1、 FAT4、FBN1、FBN2、FLG、FLG2、FLNA、FLNB、FLNC、FLT4、FMN2、FN1、FRAS1、FREM1、FREM2、FSIP2、 FXN、FYCO1、GLI2、GLI3、GPR179、GREB1L、GRIN2A、GRIN2B、GRIN2D、HCFC1、HECW2、HERC1、 HERC2、HFM1、HIVEP1、HIVEP2、HMCN1、HSPG2、HTT、HUWE1、HYDIN、IFT140、IFT172、IGF1R、 IGF2R、IGSF1、INSR、INTS1、IQSEC2、ITGB4、ITPR1、ITPR2、JMJD1C、KALRN、KANK1、KAT6A、 KAT6B、KDM3B、KDM5B、KDM5C、KDM6A、KDM6B、KDR、KIAA0586、KIAA1109、KIAA1549、KIDINS220、 KIF14、KIF1A、KIF1B、KIF21A、KIF26B、KIF7、KMT2A、KMT2B、KMT2C、KMT2D、KMT2E、KNL1、 LAMA1、LAMA2、LAMA3、LAMA4、LAMA5、LAMB1、LAMB2、LAMC3、LCT、LMNA、LOXHD1、LPA、LRBA、 LRP1、LRP2、LRP4、LRP5、LRP6、LRPPRC、LRRK1、LRRK2、LTBP2、LTBP4、LYST、MACF1、MADD、MAGI2, MAP1B, MAP3K1, MAPK8IP3, MAPKBP1, MAST1, MBD5, MCM3AP, MED12, MED12L, MED13, MED13L, MED23, MEGF8, MET, MLH3, MPDZ, MSH6, MTOR, MYH10, MYH11, MYH14, MYH2, MYH3, MYH6, MYH7, MYH7B, MYH8, MYH9, MYLK, MYO15A, MYO18B, MYO3A, MYO5A, MYO5B, MYO7A, MYO9A, NALCN, NBAS, NBEA, NBEAL2, NCAPD2, NCAPD3, NEB, NEXMIF, NEXMIF, NF1, NFASC, NHS, NIN, NIPBL, NLRP1, NOTCH1, NOTCH2, NOTCH3, NPHP4, NRXN1, NRXN3, NSD1, NSD2, NUP155, NUP188, NUP205, OBSCN, OBSL1, OTOF, OTOG, OTOGL, PARD3, PBRM1, PCDH15, PCLO, PCNT, PHIP, PI4KA, PIEZO1, PIEZO2, PIK3C2A, PIKFYVE, PKD1, PKD1L1, PKHD1, PLCE1, PLEC, PLEKHG2, PNPLA6, POGZ, POLA1, POLE, POLR1A, POLR2A, POLR3A, PRG4, PRKDC, PRPF8, PRR12, PRX, PTCH1, PTPN23, PTPRF, PTPRJ, PTPRQ, PXDN, QRICH2, RAB3GAP2, RAI1, RALGAPA1, RANBP2, RB1CC1, RELN, RERE, REV3L, RIC1, RIMS1, RIMS2, RNF213, ROBO1, ROBO2, ROBO3, ROS1, RP1, RP1L1, RTTN, RUSC2, RYR1, RYR2, SACS, SAMD9, SAMD9L, SBF2, SCAPER, SCN10A, SCN11A, SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SETBP1, SETD1A, SETD1B, SETD2, SETD5, SETX, SHANK2, SHANK3, SHROOM4, SI, SIPA1L3, SLIT2, SLX4, SMARCA2, SMARCA4, SMCHD1, SNRNP200, SON, SPEF2, SPEG, Description Page 25 / 109 Page 29CN 121195069 A SPG11, SPTA1, SPTAN1, SPTB, SPTBN2, SPTBN4, SRCAP, STRC, SVIL, SYNE1, SYNGAP1, SYNJ1, SZT2, TAF1, TANC2, TCF20, TCOF1, TDRD9, TECPR2, TECTA, TENM3, TENM4, TET3, TEX14, TEX15, TG, THOC2, TMEM94, TNC, TNIK, TNR, TNRC6B, TNXB, TOGARAM1, TONSL, TRIO, TRIOBP, TRIP11, TRIP12, TRPM1, TRPM6, TRPM7, TRRAP, TSC2, TTC37, TTN, TUBGCP6, UBR1, UNC80, USH2A, USP9X, VCAN, VPS13A, VPS13B, VPS13C, VPS13D, VWF, WDFY3, WDR19, WDR62, WDR81, WNK1, WRN, ZFHX2, ZFYVE26, ZNF142, ZNF292, ZNF335, ZNF407, ZNF462, or ZNF469. In one aspect, the disclosed protein-coding gene may contain one or more coding regions of CFTR, MDX, DYSF / TTN, DMPK, COL7A1, KI4, MAPT, FVIII HTT, RHO, DNA-PKcs, SMN2, or CD40L. In one aspect, the disclosed protein-coding gene may contain one or more coding regions of FXN, LMNA, or RYR2.

[0138] In one aspect, the disclosed nucleic acid sequence to be trans-spliced ​​may encode LMNA / C (SEQ ID NO: 20) or a portion thereof. LMNA / C is as described above. In one aspect, the disclosed nucleic acid sequence to be trans-spliced ​​may encode DP71 or a portion thereof. In one aspect, the disclosed nucleic acid sequence to be trans-spliced ​​may encode CFTR (SEQ ID NO: 19) or a portion thereof. CFTR is as described above. In one aspect, the disclosed nucleic acid sequence to be trans-spliced ​​may encode DMPK (SEQ ID NO: 21) or a portion thereof. DMPK is as described above. In one aspect, the disclosed gene may be DMD (dystrophin) (SEQ ID NO: 17). DMD is discussed above.

[0139] In one aspect, the disclosed exogenous RNA to be trans-spliced ​​may further include UTR.

[0140] In one aspect, one or more disclosed RNA structures may bind to one or more RNA-binding proteins. In one aspect, one or more disclosed RNA structures may bind to one or more double-stranded RNA-binding proteins (dsRBP).In one aspect, dsRBP is known to those skilled in the art and includes, but is not limited to, ADAR1, ADAR2, DICER, NFAR, PACT, PKR, RHA RNase III, Stauffen, TRBP, TSEN, or any combination thereof.

[0141] In one aspect, one or more disclosed RNA structures may bind to one or more RNA-binding proteins. In one aspect, one or more disclosed RNA structures may bind to one or more double-stranded RNA-binding proteins. In one aspect, one or more disclosed RNA structures may comprise a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09. In one aspect, one or more disclosed RNA structures may improve and / or enhance trans-splicing efficiency. In one aspect, one or more disclosed RNA structures may stabilize pre-mRNA. In one aspect, one or more disclosed RNA structures may localize RNA to the cell nucleus. In one aspect, one or more disclosed RNA structures may stabilize the interaction between the targeted internal pre-mRNA molecule and the exogenous RNA to be trans-spliced.

[0142] In one aspect, the disclosed nucleic acid molecule may lack CRISPR-related proteins.

[0143] In one aspect, the disclosed chimeric RNA transcript may comprise the 5' portion of the targeted endogenous pre-mRNA and the 3' portion of the exogenous RNA. In one aspect, the disclosed chimeric RNA transcript may comprise the 3' portion of the targeted endogenous pre-mRNA and the 5' portion of the exogenous RNA. In one aspect, the disclosed targeted endogenous pre-mRNA and the disclosed exogenous RNA may encode the same protein-coding gene. In one aspect, the disclosed targeted endogenous pre-mRNA and the disclosed exogenous RNA may comprise one or more exons of the same protein-coding gene.

[0144] In one aspect, the disclosed nucleic acid molecule may be packaged into a viral vector. In one aspect, the disclosed viral vector may comprise an AAV vector. In one aspect, the disclosed nucleic acid molecule may be packaged into a non-viral vector. In one aspect, the disclosed nucleic acid molecule may be incorporated into a plasmid. In one aspect, the disclosed nucleic acid molecule may be incorporated into lipid nanoparticles.

[0145] In one aspect, the disclosed nucleic acid molecule may further comprise a polyadenylated sequence. In one aspect, the disclosed nucleic acid molecule may further include sequences for promoters. In one aspect, the disclosed 3' half-intron may be recognized by intracellular nuclear splicing components. In one aspect, the disclosed exogenous RNA may induce splicing events. In one aspect, the disclosed nucleic acid molecule may further include a spacer region. In one aspect, the disclosed spacer region may connect the 5' splice region to...One or more RNA structures are separated. In one aspect, the disclosed spacer region may contain any known spacer. In one aspect, the disclosed spacer region may contain a shared splicing motif (e.g., U1 or U2). In one aspect, the disclosed spacer region may contain a limited number of shared splicing motifs (e.g., U1 or U2). In one aspect, the disclosed nucleic acid molecule may further contain a nuclear localization signal (NLS). In one aspect, the disclosed nucleic acid molecule may further contain one or more nuclear retention elements (NREs). NREs are known to those skilled in the art. In one aspect, the disclosed NRE may contain SIRLOIN (SEQ ID NO:15) or BORG (SEQ ID NO:16).

[0146] In one aspect, the disclosed nucleic acid molecule may further contain one or more flavivirus genetic elements. In one aspect, the flavivirus genetic element may contain one or more flavivirus 3' untranslated region (3'UTR), one or more subgenomic flavivirus RNA (sfRNA) elements, one or more flavivirus XRN1 resistance RNA (xrRNA) elements, one or more flavivirus dumbbell (DB) RNA elements, one or more flavivirus 3' stem-loop (3'SL) elements, or any combination thereof. (For a description of flavivirus gene elements, see WO 2022 / 182835).

[0147] In one aspect, the disclosed nucleic acid molecule may comprise the sequence of one or more regulatory elements (e.g., a post-transcriptional regulatory element (WPRE) of marmot hepatitis virus (WHV), a triple helix from MALAT1, a PRE (HPRE) of hepatitis B virus, and an iron response element). For example, the disclosed regulatory element may comprise a promoter operatively linked to the disclosed nucleic acid molecule, wherein the promoter drives the expression of a disclosed variant capsid protein, a disclosed encoded polypeptide, a disclosed encoded therapeutic agent, or both.

[0148] In one aspect, the disclosed promoter for the 3' substitution construct may be tissue-specific or ubiquitous, and may be constitutive or inducible, depending on the desired expression pattern. The promoter may be natural or exogenous, and may be a natural or synthetic sequence. Exogenous means that the transcription start region is not present in the wild-type host in which the transcription start region is introduced. In one aspect, the disclosed promoter may be a promoter / enhancer. In one aspect, the publicly disclosed promoter for a publicly disclosed nucleic acid molecule can be an endogenous promoter. In another aspect, the publicly disclosed endogenous promoter can be an endogenous promoter / enhancer. In another aspect, the publicly disclosed endogenous promoter or publicly disclosed endogenous promoter / enhancer can typically be obtained from a non-coding region upstream of the transcription start site of the target gene. In another aspect, the publicly disclosed endogenous promoter or publicly disclosed endogenous promoter / enhancer can be used for the constitutive and efficient expression of the publicly disclosed gene. In another aspect, for one or more...A publicly disclosed promoter for a publicly disclosed guide RNA sequence may be a CMV promoter or a CMV promoter / enhancer. CMV promoters and CMV promoters / enhancers are well known in the art. In one aspect, a publicly disclosed promoter for one or more publicly disclosed guide RNA sequences may be any eukaryotic RNA polymerase II promoter.

[0149] This document discloses an expression cassette comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA. This document discloses an expression cassette comprising one or more RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA. This document discloses an expression cassette comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA, wherein the nucleic acid molecules enable 3' substitution of the targeted endogenous pre-mRNA. This document discloses an expression cassette comprising one or more RNA structures comprising sequences of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA, wherein the nucleic acid molecules enable 3' substitution of the targeted endogenous pre-mRNA.

[0150] In one aspect, the expression of the disclosed protein-coding gene can be restored and / or returned to wild-type, normal, or control expression levels. In one aspect, the disclosed nucleic acid molecules can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation. In another aspect, the disclosed nucleic acid molecules can restore the functional and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above). In one aspect, restoring one or more aspects of cellular homeostasis and / or cellular functionality may include one or more of the following: (i) correcting cell starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (e.g., correcting, preventing, reducing, and / or alleviating autophagy); (iii) improving, enhancing, restoring, and / or preserving mitochondrial function.(iv) Improving, enhancing, restoring and / or preserving organelle functionality and / or structural integrity; (v) Correcting enzyme dysregulation; (vi) Reversing, inhibiting, preventing, stabilizing and / or slowing the rate of progression of a multisystemic manifestation of a genetic disease or condition; (vii) Reversing, inhibiting, preventing, stabilizing and / or slowing the rate of progression of a genetic disease or condition, or (viii) any combination thereof. In one aspect, restoring one or more aspects of cellular homeostasis may include improving, enhancing, restoring and / or preserving cellular structural and / or functional integrity. In one aspect, restoring the activity and / or functionality of missing, defective and / or mutated proteins or enzymes may include a restoration of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any amount compared to pre-existing levels, such as pre-treatment levels. In one aspect, the amount of recovery can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% higher than the pre-existing level, for example, the pre-treatment level. In another aspect, recovery can be measured against a control or reference level (e.g., determined using one or more subjects without the missing, defective, and / or mutated protein or enzyme). In one aspect, recovery can be partial or incomplete. In another aspect, recovery can be complete or near-complete, such that the levels of expression, activity, and / or functionality are similar to wild-type or control levels.

[0151] In one aspect, the disclosed 3' substitution construct can be used specifically in methods for treating subjects with autosomal dominant genetic diseases or conditions (e.g., progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert's disease, hereditary hemorrhagic telangiectasia, hereditary elliptic polycythemia, hereditary spherocytosis, Huntington's disease, idiopathic hypoparathyroidism, intestinal polyposis, marble osteodystrophy, Marfan syndrome, neurofibromatosis, polycystic kidney disease (adults), protein C deficiency, osteogenesis imperfecta, TracyCollins syndrome, tuberous sclerosis, Villebrand disease, etc.).

[0152] 2. Transcriptome Engineering System This document discloses a transcriptome engineering system comprising one or more of the disclosed 3' substitution constructs, one or more of the disclosed 5' substitution constructs, or any combination thereof. This article discloses a transcriptome engineering system comprising one or more of the following: (i) a nucleic acid molecule containing exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' hemiintron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA...(ii) a nucleic acid molecule comprising, (ii) a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the foreign RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising any one of the sequences in SEQ ID NO: 01-SEQ ID NO: 09; (iii) a nucleic acid molecule comprising, (iii) a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the foreign RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA; and (iv) a nucleic acid molecule comprising, (ii) a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the foreign RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising, (iii) a sequence in SEQ ID NO: 01-SEQ ID NO: 09; (iv) a sequence in SEQ ID NO: 09; (v ... The sequence of any one of 09, wherein the nucleic acid molecule enables the 5' substitution of the targeted endogenous pre-mRNA. (Page 32, CN 121195069 A, Instructions 28 / 109)

[0153] This document discloses a transcriptome engineering system comprising one or more of the following: (i) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; (ii) a nucleic acid molecule comprising one or more RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; (iii) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA; and (iv) a nucleic acid molecule, It comprises one or more RNA structures, said RNA structures comprising the sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA.

[0154] This article discloses a transcriptome engineering system comprising one or more of the following: (i) a nucleic acid molecule containing exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures; (ii) a nucleic acid molecule containing exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures comprising SEQ ID NO: 01-SEQ ID NO: The sequence of any one of SEQ ID NO: 09; (iii) a nucleic acid molecule comprising a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; a 5' half-intron linked to the foreign RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA; and (iv) a nucleic acid molecule comprising a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; a 5' half-intron linked to the foreign RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising SEQ ID NO: 01-SEQ ID NO: (v) A nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; (vi) A nucleic acid molecule comprising one or more RNA structures comprising SEQ ID NO: 01-SEQ ID NO: (vii) A nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; and (viii) A nucleic acid molecule comprising one or more RNA structures, said RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA; and (viii) A nucleic acid molecule comprising one or more RNA structures, said RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a 3' half-intron linked to a foreign RNA to be trans-spliced; and a 3' half-intron linked to the targeted endogenous pre-mRNA; ... and a 3' half-intron linked to the targeted endogenous pre-mRNA; and a 3' half-intron linked to the targeted endogenous pre-mRNA; and a 3' half-intron linked to the targeted endogenous pre-mRNA; and aThe exogenous RNA is attached to the targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA.

[0155] 3. Vector This document discloses a vector comprising the disclosed nucleic acid molecule. In one aspect, the disclosed vector may be a non-viral vector or a viral vector. This document discloses a non-viral vector comprising the disclosed nucleic acid molecule. This document discloses a non-viral vector comprising one or more disclosed nucleic acid molecules. This document discloses a viral vector comprising the disclosed nucleic acid molecule. 29 / 109 pages 33 CN 121195069 A This document discloses a viral vector comprising one or more disclosed nucleic acid molecules. This document discloses a non-viral or viral vector comprising a nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures. This document discloses a non-viral or viral vector comprising a nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09.

[0156] This document discloses a non-viral or viral vector comprising a nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures, wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA. This document discloses a non-viral or viral vector comprising a nucleic acid molecule containing a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the foreign RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures comprising sequences of any one of SEQ ID NO: 01-SEQ ID NO: 09, wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA. This document also discloses a non-viral or viral vector comprising a nucleic acid molecule containing one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to the foreign RNA to be trans-spliced; and the foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA. Finally, this document discloses a non-viral or viral vector comprising a nucleic acid molecule containing one or more RNA structures comprising sequences of SEQ ID NO: 01-SEQ ID NO: 09.The present invention discloses a non-viral or viral vector comprising a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA. This document discloses a nonviral or viral vector comprising a nucleic acid molecule comprising one or more RNA structures, the RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' hemiintron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA. This document also discloses a nonviral or viral vector comprising one or more 5' substitution constructs. This document further discloses a nonviral or viral vector comprising one or more 3' substitution constructs. This document also discloses a nonviral or viral vector comprising one or more 5' substitution constructs and / or one or more 3' substitution constructs. In one aspect, the disclosed 5' substitution construct to be used in combination with one or more other substitution constructs may comprise any disclosed 5' substitution construct. In one aspect, the disclosed 5' substitution construct may comprise (i) a nucleic acid molecule containing exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures; (ii) a nucleic acid molecule containing exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; and (iii ... and (iv) a 5' half-intron linked to the exogenous RNA to be trans-spliced; and (v) a 5' half-intron linked to the exogenous RNA to be trans-spliced; and (v) a 5' half-intron linked to the A 5' half-intron linked to the target RNA; one or more RNA targeting motifs; one or more RNA structures wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA, or (iv) a nucleic acid molecule containing exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs.Book 30 / 109, page 34, CN 121195069 A; one or more RNA structures comprising sequences of any one of SEQ ID NO: 01-SEQ ID NO: 09, wherein the nucleic acid molecule enables a 5' substitution of the targeted endogenous pre-mRNA, or (v) any combination thereof.

[0157] In one aspect, a disclosed 3' substitution construct to be used in combination with one or more other substitution constructs may comprise any disclosed 3' substitution construct. In one aspect, the disclosed 3' substitution construct may comprise (i) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; (ii) a nucleic acid molecule comprising one or more RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; (iii) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA; (iv) a nucleic acid molecule comprising one or more RNA structures comprising a sequence of SEQ ID NO: 01-SEQ ID NO: 09 sequence; one or more RNA targeting motifs; a 3' half-intron linked to the exogenous RNA to be trans-spliced; and the exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables a 3' substitution of the targeted endogenous pre-mRNA, or (v) any combination thereof.

[0158] In one aspect, the disclosed vector can be formulated for administration via one or more routes. These methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, intrauterine administration, intrahepatic administration, intravaginal administration, ocular administration, intraauricular administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable administration, such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration may also include intra-hepatic artery administration or administration via the portal vein (HPV). The administration of a disclosed therapeutic agent, a disclosed pharmaceutical composition, or a combination thereof may include direct application to...In the CNS (e.g., intraparenchymal, intravenous, intrasheathic cisterns, intrasheath (lumbar), deep gray matter delivery, convection-enhanced delivery to deep gray matter) or PNS. Administration may be continuous or intermittent. Administration may include administration of a viral vector and / or a generated optimized viral vector. Administration of the disclosed vector may be continuous or intermittent.

[0159] In one aspect, a therapeutically effective amount of the disclosed AAV particles or the disclosed AAV vector may include a range of about 1 × 10¹⁰ vg / kg to about 2 × 10¹⁴ vg / kg. In one aspect, for example, the disclosed vector may be administered at a dose of about 1 × 10¹¹ to about 8 × 10¹³ vg / kg or about 1 × 10¹² to about 8 × 10¹³ vg / kg. In one aspect, the disclosed vector may be administered at a dose of about 1 × 10¹³ to about 6 × 10¹³ vg / kg. In one aspect, the disclosed carrier can be administered at doses of at least about 1×10¹⁰, at least about 5×10¹⁰, at least about 1×10¹¹, at least about 5×10¹¹, at least about 1×10¹², at least about 5×10¹², at least about 1×10¹³, at least about 5×10¹³, or at least about 1×10¹⁴ vg / kg. In another aspect, the disclosed carrier can be administered at doses not exceeding about 1×10¹⁰, not exceeding about 5×10¹⁰, not exceeding about 1×10¹¹, not exceeding about 5×10¹¹, not exceeding about 1×10¹², not exceeding about 5×10¹², not exceeding about 1×10¹³, not exceeding about 5×10¹³, or not exceeding about 1×10¹⁴ vg / kg. In one aspect, the disclosed carrier can be administered at a dose of 1×10¹² vg / kg. In another aspect, the disclosed carrier can be administered at a dose of 1×10¹¹ vg / kg. In one aspect, the disclosed carrier may be administered in single or multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses) as needed for the desired therapeutic outcome.

[0160] In one aspect, the therapeutically effective amount of the disclosed AAV particles or the disclosed AAV carrier may include a range from about 1 × 10¹² vg to about 1 × 10¹⁷ vg per subject in total. In one aspect, the therapeutically effective amount of the disclosed AAV particles or the disclosed AAV carrier may include a range from about 1 × 10¹² vg, about 1 × 10¹³ vg, about 1 × 10¹⁴ vg, about 1 × 10¹⁵ vg, about 1 × 10¹⁶ vg, or about 1 × 10¹⁷ vg per subject in total. In one aspect, a therapeutically effective amount of the disclosed AAV particles or the disclosed AAV carrier can be delivered via retrograde ureteral infusion and / or renal artery administration, and may include each recipientThe total amount of the AAV particles may range from approximately 1 × 10¹² vg to approximately 1 × 10¹⁷ vg per subject.

[0161] In one aspect, the therapeutically effective amount of the disclosed AAV particles may include approximately 1 × 10⁶ DRP / mL to approximately 1 × 10¹⁴ DRP / mL. In one aspect, the disclosed pharmaceutical formulation may include approximately 1 × 10⁶ DRP / mL, 1 × 10⁷ DRP / mL, 1 × 10⁸ DRP / mL, 1 × 10⁹ DRP / mL, 1 × 10¹⁰ DRP / mL, 1 × 10¹¹ DRP / mL, 1 × 10¹² DRP / mL, 1 × 10¹³ DRP / mL, or 1 × 10¹⁴ DRP / mL. In one aspect, the therapeutically effective amount of the disclosed AAV particles or the disclosed AAV carrier may include a range determined by a person skilled in the art.

[0162] In one aspect, the disclosed non-viral carrier may be a polymer-based carrier, a peptide-based carrier, a lipid nanoparticle, a solid lipid nanoparticle, or a cationic lipid-based carrier. In one aspect, the disclosed vector may include exosomes, extracellular vesicles, and virus-like particles.

[0163] In one aspect, the disclosed viral vector may be an adenovirus vector, AAV vector, herpes simplex virus vector, retrovirus vector, lentivirus vector, alphavirus vector, flavivirus vector, rhabdovirus vector, measles virus vector, Newcastle disease virus vector, poxvirus vector, or piconemavirus vector. In one aspect, the disclosed nucleic acid sequence may have a coding sequence of less than about 4.5 kilobases.

[0164] In one aspect, the disclosed AAV vector may include naturally isolated serotypes, including but not limited to AAV1, AAV2, AAV3 (including 3a and 3b), AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAVrh39, AAVrh43, AAVcy.7, as well as bovine AAV, goat AAV, canine AAV, horse AAV, sheep AAV, bird AAV, primate AAV, non-primate AAV, and any other virus classified as AAV by the International Committee on Taxonomy of Viruses (ICTV). In one aspect, the AAV capsid may be a chimera generated by capsid evolution or by rational capsid engineering from naturally isolated AAV variants to capture desired serotype characteristics, such as enhanced or specific tissue tropism and / or host immune response escape. Naturally isolated AAV variants include, but are not limited to, AAV-DJ, AAV-HAE1, AAV-HAE2, AAVM41, AAV-1829, AAV2 Y / F, and AAV2.T / V, AAV2i8, AAV2.5, AAV9.45, AAV9.61, AAV-B1, AAV-AS, AAV9.45A-String (e.g., AAV9.45-AS), AAV9.45Angiopep, AAV9.47-Angiopep, and AAV9.47-AS, AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV-F, AAVcc.47, and AAVcc.81. In one aspect, the disclosed AAV carrier may be AAV-Rh74 or a related variant (e.g., a capsid variant such as RHM4-1). In one aspect, the disclosed AAV carrier may be AAV.cc47. In one aspect, the disclosed AAV carrier may be AAV.cc81. In one aspect, the disclosed AAV carrier may be a self-complementary AAV.

[0165] In one aspect, the disclosed vector may comprise one or more ITRs (e.g., ITRs from AAV2).

[0166] In one aspect, the disclosed vector may further comprise one or more nuclear localization signals (NLS). NLS are known to those skilled in the art. In one aspect, the disclosed NLS may comprise any NLS known in the art. As known in the art (see, for example, Lu J et al. (2021) Cell Commun Signal. 19:60, whose teachings on NLS are incorporated herein by reference), nuclear localization signals (NLS) are typically short peptides that act as signal fragments mediating the transport of proteins from the cytoplasm to the nucleus.

[0167] In one aspect, the disclosed vector may further comprise one or more nuclear retention elements (NREs). NREs are known to those skilled in the art. In one aspect, the disclosed NRE may comprise SIRLOIN (SEQ ID NO:15) or BORG (SEQ ID NO:16).

[0168] In one aspect, the disclosed vector may further comprise one or more flavivirus genetic elements. In one aspect, according to the flavivirus specification 32 / 109, page 36, CN 121195069 A, viral genetic elements may include one or more flavivirus 3' untranslated regions (3'UTR), one or more subgenomic flavivirus RNA (sfRNA) elements, one or more flavivirus XRN1 resistance RNA (xrRNA) elements, one or more flavivirus dumbbell (DB) RNA elements, one or more flavivirus 3' stem-loop (3'SL) elements, or any combination thereof. (For a description of flavivirus genetic elements, see WO 2022 / 182835).

[0169] In one aspect, the disclosed vector may further comprise encoding therapeutic proteins, therapeutic agents, and / or therapeutic agents.The disclosed therapeutic protein may include a polypeptide and / or glycopeptide. In one aspect, the disclosed therapeutic agent may include an oligonucleotide therapeutic agent. In one aspect, the disclosed oligonucleotide therapeutic agent may be single-stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA, non-coding RNA (ncRNA), antisense molecule, miRNA, morpholinonucleotide, peptide nucleic acid (PNA), or analogs or conjugates thereof. In one aspect, the disclosed therapeutic agent may be ASO or RNAi. In one aspect, the disclosed therapeutic agent may contain a CRISPR-based endonuclease (e.g., Cas9). In one aspect, the disclosed CRISPR-based endonuclease may be derived from a CRISPR / Cas Type I, II, or Type III system.

[0170] In one aspect, the disclosed therapeutic RNA may comprise ribosomal RNA (rRNA), transfer RNA (tRNA), heterogeneous nuclear RNA (hnRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), single-guide RNA (sgRNA), non-coding RNA (ncRNA), long non-coding RNA (lncRNA), 7SL, Xist, short enhancer RNA (eRNA), circular RNA, intergenic RNA, or any combination thereof. In one aspect, the disclosed RNA may include lncRNA, siRNA, shRNA, sgRNA, circular RNA, snoRNA, miRNA, or any combination thereof. In one aspect, the disclosed coding RNA may include functional non-coding RNA elements.

[0171] In one aspect, the disclosed vector may comprise one or more promoters operatively linked to a disclosed nucleic acid molecule (e.g., a 5' substitution construct and / or a 3' substitution construct), a disclosed transgene, a disclosed sequence to be trans-spliced, and / or a disclosed nucleic acid sequence. In one aspect of the disclosed vector, the disclosed nucleic acid molecule may be operatively linked to one or more transcriptional regulatory elements. In one aspect, one or more regulatory elements (e.g., a marmot hepatitis virus (WHV) posttranscriptional regulatory element (WPRE), a triple helix from MALAT1, a hepatitis B virus PRE (HPRE), and an iron response element) may increase the transcription and / or expression of the disclosed nucleic acid molecule (e.g., a 5' substitution construct and / or a 3' substitution construct), the disclosed transgene, the disclosed sequence to be trans-spliced, and / or the disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and / or a disclosed therapeutic RNA). In one aspect, the disclosed promoter may be located at a disclosed [unclear text - possibly a location] under its control.The promoter can be located at the 5' (upstream) or 3' (downstream) of a nucleic acid molecule (e.g., a 5' substitution construct and / or a 3' substitution construct), a disclosed transgene, a disclosed sequence to be trans-spliced, and / or a disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and / or a disclosed therapeutic RNA). The distance between the disclosed promoter and the disclosed nucleic acid molecule (e.g., a 5' substitution construct and / or a 3' substitution construct), the disclosed transgene, the disclosed sequence to be trans-spliced, and / or the disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and / or a disclosed therapeutic RNA) can be approximately the same as the distance between the promoter and the disclosed nucleic acid molecule (e.g., a 5' substitution construct and / or a 3' substitution construct), the disclosed transgene, the disclosed sequence to be trans-spliced, and / or the disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and / or a disclosed therapeutic RNA) under its control. As is known in the art, variations in this distance can be modulated without loss of promoter function.

[0172] In one aspect of the disclosed vector, the disclosed promoter may be tissue-specific or ubiquitous, and may be constitutive or inducible, depending on the desired expression pattern. The disclosed promoter may be natural or exogenous, and may be a natural or synthetic sequence. Exogenous means that the transcription start region is not present in the wild-type host in which the transcription start region is introduced. In one aspect, the disclosed promoter may be a promoter / enhancer. In one aspect, the promoter used for the disclosed nucleic acid molecule (e.g., 5' substitution construct and / or 3' substitution construct), the disclosed transgene, the disclosed sequence to be trans-spliced, and / or the disclosed nucleic acid sequence (e.g., encoding the disclosed therapeutic protein and / or the disclosed therapeutic RNA) may be an endogenous promoter. In one aspect, the disclosed endogenous promoter may be an endogenous promoter / enhancer. In one aspect, the disclosed endogenous promoter or the disclosed endogenous promoter / enhancer may generally be obtained from a non-coding region upstream of the transcription start site of the target gene. In one aspect, a disclosed endogenous promoter or a disclosed endogenous promoter / enhancer can be used for the constitutive and efficient expression of a disclosed protein-coding gene. In another aspect, the disclosed promoter used for a disclosed nucleic acid molecule (e.g., a 5' substitution construct and / or a 3' substitution construct), a disclosed transgene, a disclosed sequence to be trans-spliced, and / or a disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and / or a disclosed therapeutic RNA) can be a CMV promoter or a CMV promoter / enhancer. CMV promoters and CMV promoters / enhancers are well known in the art. In one aspect, the disclosed endogenous promoter or endogenous promoter / enhancer used for a disclosed nucleic acid molecule (e.g., a 5' substitution construct and / or a 3' substitution construct), a disclosed transgene, a disclosed trans-spliced ​​sequence, and / or a disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and / or a disclosed therapeutic RNA) can be a CMV promoter or a CMV promoter / enhancer. CMV promoters and CMV promoter / enhancers are well known in the art.The disclosed promoter of the open sequence to be trans-spliced ​​and / or the disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and / or a disclosed therapeutic RNA) can be any eukaryotic RNA polymerase II promoter. In one aspect, the disclosed AAV vector can be used to generate AAV particles. In one aspect, the disclosed AAV vector can be used to generate AAV particles containing, under its control, a disclosed nucleic acid molecule (e.g., a 5' substitution construct and / or a 3' substitution construct), a disclosed transgene, a disclosed sequence to be trans-spliced ​​and / or the disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and / or a disclosed therapeutic RNA).

[0173] AAV particles are disclosed herein containing, under its control, a disclosed nucleic acid molecule (e.g., a 5' substitution construct and / or a 3' substitution construct), a disclosed transgene, a disclosed sequence to be trans-spliced ​​and / or the disclosed nucleic acid sequence (e.g., encoding a disclosed therapeutic protein and / or a disclosed therapeutic RNA).

[0174] In one aspect, the disclosed carrier or the disclosed AAV particles can be used, particularly, in methods of treating subjects with autosomal dominant genetic diseases or conditions (e.g., progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert's disease, hereditary hemorrhagic telangiectasia, hereditary elliptic polycythemia, hereditary spherocytosis, Huntington's disease, idiopathic hypoparathyroidism, intestinal polyposis, marbled bone disease, Marfan syndrome, neurofibromatosis, polycystic kidney disease (adults), protein C deficiency, osteogenesis imperfecta, TracyCollins syndrome, tuberous sclerosis, von Willebrand disease, etc.).

[0175] 4. Pharmaceutical Formulation This article discloses a pharmaceutical formulation comprising the disclosed nucleic acid molecule. This article discloses a pharmaceutical formulation comprising the disclosed nucleic acid molecule and a pharmaceutically acceptable carrier. This article discloses a pharmaceutical formulation comprising the disclosed carrier. This article discloses a pharmaceutical formulation comprising the disclosed carrier and a pharmaceutically acceptable carrier. This article discloses a pharmaceutical formulation comprising the disclosed AAV particles. This article discloses a pharmaceutical formulation comprising the disclosed AAV particles and a pharmaceutically acceptable carrier.

[0176] This article discloses a pharmaceutical formulation comprising a non-viral vector or viral vector containing nucleic acid molecules, comprising exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures. This article discloses a pharmaceutical formulation comprising a non-viral vector or viral vector containing nucleic acid molecules, comprising exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures, said RNA structures comprising SEQ ID NO:This document discloses a pharmaceutical formulation comprising a non-viral or viral vector containing nucleic acid molecules, comprising exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures wherein the nucleic acid molecules enable 5' substitution of the targeted endogenous pre-mRNA. This document discloses a pharmaceutical formulation comprising a non-viral or viral vector containing nucleic acid molecules, comprising exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising sequences of any one of SEQ ID NO: 01-SEQ ID NO: 09, wherein the nucleic acid molecules enable 5' substitution of the targeted endogenous pre-mRNA. This article discloses a pharmaceutical formulation comprising a non-viral or viral vector containing a nucleic acid molecule, said nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA. This article also discloses a pharmaceutical formulation comprising a non-viral or viral vector containing a nucleic acid molecule, said nucleic acid molecule comprising one or more RNA structures, said RNA structures comprising sequences of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA. This article discloses a pharmaceutical formulation comprising a non-viral or viral vector containing a nucleic acid molecule, said nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA. This article also discloses a pharmaceutical formulation comprising a non-viral or viral vector containing a nucleic acid molecule, said nucleic acid molecule comprising one or more RNA structures, said RNA structures comprising sequences of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA, wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA.3' substitution of mRNA.

[0177] In one aspect, the disclosed pharmaceutical formulation may comprise (i) one or more active agents, (ii) bioactive agents, (iii) one or more pharmaceutically active agents, (iv) one or more immunotherapeutic agents, (v) one or more clinically approved pharmaceutical agents, or (vi) combinations thereof. In one aspect, the disclosed composition may comprise one or more immunomodulators. In one aspect, the disclosed composition may comprise one or more proteasome inhibitors. In one aspect, the disclosed composition may comprise one or more immunosuppressive agents or immunosuppressants. In one aspect, the immunosuppressant may be antithymocyte globulin (ATG), cyclosporine (CSP), mycophenolate mofetil (MMF), or a combination thereof. In one aspect, the disclosed formulation may comprise an anaplerotic agent (e.g., a C7 compound, such as triheptanylglycerol or MCT).

[0178] In one aspect, the disclosed formulation may comprise an RNA therapeutic agent. The RNA therapeutic agent may comprise RNA-mediated interference (RNAi) and / or antisense oligonucleotides (ASO). In one aspect, the disclosed RNA therapeutic agent may target any protein or enzyme that is overexpressed or overactive due to a missing, defective, and / or mutated protein or enzyme. In one aspect, the disclosed RNA therapeutic agent may include a therapy delivered via LNP. In one aspect, the disclosed formulation may contain an enzyme or enzyme precursor for enzyme replacement therapy (ERT).

[0179] In one aspect, the disclosed formulation may contain the disclosed small molecule. In one aspect, the disclosed small molecule may help restore the functionality and / or structural integrity of a missing, defective, and / or mutated protein or enzyme.

[0180] In one aspect, any disclosed pharmaceutical formulation may contain one or more excipients and / or pharmaceutically acceptable carriers. Excipients and / or pharmaceutically acceptable carriers are known in the art and discussed above.

[0181] In one aspect, a therapeutically effective amount of the disclosed pharmaceutical formulation may include the range of about 1 × 10¹⁰ vg / kg to about 2 × 10¹⁴ vg / kg of the disclosed carrier and / or disclosed AAV particles. In one aspect, for example, the dosage of the disclosed pharmaceutical preparation may include about 1×10¹¹ to about 8×10¹³ vg / kg or about 1×10¹² to about 8×10¹³ vg / kg. In one aspect, the dosage of the disclosed pharmaceutical preparation may include about 1×10¹³ to about 6×10¹³ vg / kg. In one aspect, the dosage of the disclosed pharmaceutical preparation may include at least about 1×10¹⁰, at least about 5×10¹⁰, at least about 1×10¹¹, at least about 5×10¹¹, at least about 1×10¹², at least about 5×10¹², at least about 1×10¹³, at least about 5×10¹³, or at least about 1×10¹⁴ vg / kg. In one aspect,The dosage of the disclosed pharmaceutical preparation may include no more than about 1×10¹⁰, no more than about 5×10¹⁰, no more than about 1×10¹¹, not exceeding about 5×10¹¹, no more than about 1×10¹², no more than about 5×10¹², no more than about 1×10¹³, or no more than about 1×10¹⁴ vg / kg. In one aspect, the dosage of the disclosed pharmaceutical preparation may include about 1×10¹² vg / kg. In another aspect, the dosage of the disclosed pharmaceutical preparation may include about 1×10¹¹ vg / kg. In another aspect, the dosage of the disclosed pharmaceutical preparation may include a single dose or multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses) as needed according to the desired therapeutic outcome.

[0182] In one aspect, the therapeutically effective amount of the disclosed pharmaceutical formulation may include a range from about 1 × 10¹² vg to about 1 × 10¹⁷ vg per subject in total. In one aspect, the therapeutically effective amount of the disclosed pharmaceutical formulation may include a range from about 1 × 10¹² vg, about 1 × 10¹³ vg, about 1 × 10¹⁴ vg, about 1 × 10¹⁵ vg, about 1 × 10¹⁶ vg, or about 1 × 10¹⁷ vg per subject in total. In one aspect, the therapeutically effective amount of the disclosed pharmaceutical formulation may include from about 1 × 10⁶ DRP / mL to about 1 × 10¹⁴ DRP / mL. In one aspect, the therapeutically effective amount of the disclosed pharmaceutical preparation may include about 1×10⁶ DRP / mL, 1×10⁷ DRP / mL, 1×10⁸ DRP / mL, 1×10⁹ DRP / mL, 1×10¹⁰ DRP / mL, 1×10¹¹ DRP / mL, 1×10¹² DRP / mL, 1×10¹³ DRP / mL, or 1×10¹⁴ DRP / mL.

[0183] In one aspect, the therapeutically effective amount of the disclosed pharmaceutical preparation may include a range determined by a person skilled in the art.

[0184] In one aspect, the disclosed pharmaceutical preparation can be used to restore and / or return a disclosed protein-coding gene to wild-type, normal, or control expression levels. In one aspect, the disclosed pharmaceutical preparation can restore one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic disorders. In one aspect, the disclosed nucleic acid molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above). In one aspect, restoring cellular homeostasis and / or cellular function may include one or more of the following: (i) correcting cellular starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway.(e.g., correcting, preventing, reducing, and / or mitigating autophagy); (iii) improving, enhancing, restoring, and / or preserving mitochondrial functionality and / or structural integrity; (iv) improving, enhancing, restoring, and / or preserving organelle functionality and / or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of a multisystemic manifestation of a genetic disease or condition; (vii) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of a genetic disease or condition, or (viii) any combination thereof. In one aspect, restoring one or more aspects of cellular homeostasis may include improving, enhancing, restoring, and / or preserving cellular structural and / or functional integrity. In one aspect, restoring the activity and / or function of missing, defective, and / or mutated proteins or enzymes may include a restoration of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount compared to pre-existing levels, such as pre-treatment levels. In one aspect, the amount of recovery can be higher than the pre-existing level, such as 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% of the pre-treatment level. In another aspect, recovery can be measured against a control or reference level (e.g., determined using one or more subjects without the missing, defective, and / or mutated protein or enzyme). In one aspect, recovery can be partial or incomplete. In another aspect, recovery can be complete or near-complete, such that the levels of expression, activity, and / or function are similar to wild-type or control levels.

[0185] In one aspect, the disclosed pharmaceutical formulation can be used specifically in methods for treating subjects suffering from autosomal dominant genetic diseases or conditions (e.g., progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert's disease, hereditary hemorrhagic telangiectasia, hereditary elliptic polycythemia, hereditary spherocytosis, Huntington's disease, idiopathic hypoparathyroidism, intestinal polyposis, marbled bone disease, Marfan syndrome, neurofibromatosis, polycystic kidney disease (adults), protein C deficiency, osteogenesis imperfecta, TracyCollins syndrome, tuberous sclerosis, von Willebrand disease, etc.). Specification 36 / 109 pages 40 CN 121195069 A

[0186] 5. Plasmids Plasmids comprising one or more disclosed nucleic acid molecules are disclosed herein. Plasmids comprising one or more disclosed vectors are disclosed herein. This document discloses plasmids used in methods for preparing disclosed compositions, such as disclosed nucleic acid molecules, disclosed vectors, or disclosed pharmaceutical formulations. Plasmids and the use of plasmids are known in the art. This document discloses a plasmid containing SEQ ID.This document discloses a plasmid comprising a sequence or fragment thereof having at least 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the sequences shown in ... Techniques for achieving transfection and transduction are known in the art, and the use of transfected or transduced cells is also known in the art. In one aspect, human cell lines transduced with one or more disclosed viral vectors or transfected with one or more disclosed nucleic acids, one or more disclosed nonviral vectors, or one or more disclosed plasmids are disclosed herein. In one aspect, human cell lines having one or more genetic diseases or conditions are disclosed herein, which are contacted with one or more nucleic acid molecules, one or more disclosed vectors, and / or one or more disclosed pharmaceutical preparations. This document discloses treatment of cells obtained from subjects with one or more disclosed nucleic acid molecules, one or more disclosed vectors, one or more disclosed plasmids, and / or one or more disclosed pharmaceutical preparations.

[0188] 7. Animals This document discloses animals treated with one or more disclosed nucleic acid molecules, one or more disclosed alternative constructs, one or more disclosed vectors, one or more disclosed AAV particles, one or more disclosed pharmaceutical preparations, and / or one or more disclosed plasmids. Transgenic animals are known in the art, and techniques for producing transgenic animals are also known in the art.

[0189] 8. Library This document discloses libraries of one or more disclosed nucleic acid molecules. This document discloses libraries of one or more disclosed 5' alternative constructs. This document discloses libraries of one or more publicly disclosed 3' substitution builder structures. This document discloses libraries of one or more publicly disclosed 5' substitution builder structures and / or publicly disclosed 3' substitution builder structures. This document discloses libraries of one or more publicly disclosed vectors. This document discloses libraries of one or more publicly disclosed vectors containing one or more publicly disclosed 5' substitution builder structures, one or more publicly disclosed 3' builder structures, or any combination thereof. This document discloses one or more publicly disclosed AAVs.A library of particles comprising one or more disclosed 5' substitution constructs, one or more disclosed 3' constructs, or any combination thereof. Libraries of one or more disclosed plasmids are disclosed herein.

[0190] 9. Kit This document discloses a kit comprising one or more disclosed nucleic acid molecules, one or more disclosed vectors or disclosed AAV particles, disclosed pharmaceutical formulations, or any combination thereof. This document discloses a kit comprising one or more disclosed nucleic acid molecules, one or more disclosed vectors, one or more disclosed pharmaceutical formulations, or any combination thereof. In one aspect, the kit may comprise disclosed nucleic acid molecules, disclosed vectors or disclosed AAV particles, disclosed pharmaceutical formulations, disclosed therapeutic agents, or combinations thereof, and one or more reagents. “Reagents” and “therapeutic agents” are known in the art and are described as on pages 37 / 109 of the specification above, 41 CN 121195069 A.

[0191] In one aspect, one or more reagents may treat, prevent, inhibit, and / or alleviate one or more comorbidities in a subject. In one aspect, one or more active agents can treat, inhibit, prevent, and / or alleviate cellular and / or metabolic complications associated with missing, defective, and / or mutated proteins or enzymes.

[0192] In one aspect, the disclosed kit may comprise at least two components constituting the kit. These components together constitute a functional unit for a given purpose (e.g., treating a subject diagnosed with or suspected of having a genetic disease or condition). The individual component components may be physically packaged together or separately. For example, a kit that includes instructions for use may or may not physically include instructions for use with other individual component components. Instead, the instructions for use may be provided as a separate component, in paper or electronic form, which may be available on a computer-readable storage device or downloaded from an Internet website, or presented as a recording. In one aspect, a kit for the disclosed method may comprise one or more containers containing the disclosed nucleic acid molecule, the disclosed vector, the disclosed pharmaceutical preparation, the disclosed RNA therapeutic agent, or a combination thereof, and a label or packaging insert with instructions for use. In one aspect, suitable containers include, for example, bottles, vials, syringes, blister packs, etc. Containers may be formed from various materials such as glass or plastic. The container may contain disclosed nucleic acid molecules, disclosed carriers, disclosed pharmaceutical preparations, or combinations thereof, and may have a sterile inlet (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). Labels or packaging inserts may indicate that the disclosed nucleic acid molecules, disclosed carriers, disclosed AAV particles, disclosed pharmaceutical preparations, disclosed RNA therapeutics, or combinations thereof are intended for the treatment, prevention, inhibition, and / or alleviation of a disease or condition or its complications and / or related to the disease or condition.Or symptoms. The disclosed kit may contain additional components required for administration, such as other buffers, diluents, filters, needles, and syringes. In one aspect, the disclosed kit can be used in any of the disclosed methods. In one aspect, the disclosed kit can be used to generate one or more chimeric RNA molecules. In one aspect, the disclosed kit can be used to treat genetic diseases or genetic conditions. In one aspect, the disclosed kit can be used to inhibit and / or minimize disease progression.

[0193] C. Method for generating chimeric RNA molecules This document discloses a method for generating chimeric RNA molecules in cells, the method comprising contacting endogenous pre-mRNA in cells with a disclosed 5' substitution construct, wherein the resulting chimeric RNA transcript comprises a 3' portion of the targeted endogenous pre-mRNA and a 5' portion of the exogenous RNA.

[0194] This document discloses a method for generating chimeric RNA molecules, the method comprising contacting one or more cells with a disclosed 5' substitution construct, wherein the resulting chimeric RNA transcript comprises a 3' portion of the targeted endogenous pre-mRNA and a 5' portion of the exogenous RNA.

[0195] This document discloses a method for generating chimeric RNA molecules, the method comprising contacting one or more cells with a nucleic acid molecule containing an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, wherein the resulting chimeric RNA transcript comprises a 3' portion of the targeted endogenous pre-mRNA and a 5' portion of the exogenous RNA.

[0196] This document discloses a method for generating chimeric RNA molecules, the method comprising contacting one or more cells with a nucleic acid molecule containing an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising a sequence comprising any one of SEQ ID NO: 01-SEQ ID NO: 09, wherein the resulting chimeric RNA transcript comprises a 3' portion of the targeted endogenous pre-mRNA and a 5' portion of the exogenous RNA.

[0197] This document discloses a method for generating chimeric RNA molecules, the method comprising contacting one or more cells with a nucleic acid molecule containing an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' hemiintron linked to the exogenous RNA to be trans-spliced ​​(see specification 38 / 109 pages 42 CN 121195069 A); one or more RNA targeting motifs; one or more RNA structures, wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA transcript contains the targeted endogenous pre-mRNA.

[0198] This document discloses a method for generating chimeric RNA molecules, the method comprising contacting one or more cells with a nucleic acid molecule containing an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures containing sequences of any one of SEQ ID NO: 01-SEQ ID NO: 09, wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA transcript contains the 3' portion of the targeted endogenous pre-mRNA and the 5' portion of the exogenous RNA.

[0199] This document discloses a method for generating chimeric RNA molecules in cells, the method comprising contacting endogenous pre-mRNA in cells with a disclosed 3' substitution construct, wherein the resulting chimeric RNA transcript contains the 5' portion of the targeted endogenous pre-mRNA and the 3' portion of the exogenous RNA.

[0200] This document discloses a method for generating chimeric RNA molecules, the method comprising contacting one or more cells with a disclosed 3' substitution construct, wherein the resulting chimeric RNA transcript comprises a 5' portion of a targeted endogenous pre-mRNA and a 3' portion of exogenous RNA.

[0201] This document discloses a method for generating chimeric RNA molecules, the method comprising contacting one or more cells with a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' hemiintron linked to exogenous RNA to be trans-spliced; and exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; wherein the resulting chimeric RNA transcript may comprise a 5' portion of the targeted endogenous pre-mRNA and a 3' portion of exogenous RNA.

[0202] This document discloses a method for generating chimeric RNA molecules, the method comprising contacting one or more cells with a nucleic acid molecule, the nucleic acid molecule comprising one or more RNA structures, the RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; wherein the resulting chimeric RNA transcript may comprise a 5' portion of the targeted endogenous pre-mRNA and a 3' portion of the foreign RNA.

[0203] This document discloses a method for generating chimeric RNA molecules, the method comprising contacting one or more cells with a nucleic acid molecule, the nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA.The exogenous RNA linked to a 3' half-intron; and the exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables a 3' substitution of the targeted endogenous pre-mRNA, and wherein the resulting chimeric RNA transcript may contain a 5' portion of the targeted endogenous pre-mRNA and a 3' portion of the exogenous RNA.

[0204] This document discloses a method for generating a chimeric RNA molecule, the method comprising contacting one or more cells with a nucleic acid molecule comprising one or more RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to the exogenous RNA to be trans-spliced; and the exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables a 3' substitution of the targeted endogenous pre-mRNA, and wherein the resulting chimeric RNA transcript may contain a 5' portion of the targeted endogenous pre-mRNA and a 3' portion of the exogenous RNA.

[0205] In one aspect, one or more cells may be in the subject. In one aspect, the subject may be diagnosed with or may be suspected of having a genetic disease or condition. In one aspect, the disease or condition may include any disease or condition caused by a disclosed gene or a deleted, defective, and / or mutated gene. In one aspect, the subject may be a subject who requires treatment for a disclosed disease or condition (e.g., a genetic disease or condition). Genetic diseases and conditions are those discussed extensively herein on page 39 / 109 of CN 121195069 A.

[0206] In one aspect, the disclosed method of generating a chimeric RNA molecule may further include identifying a subject who requires the generation of a chimeric RNA molecule.

[0207] In one aspect of the disclosed method of generating a chimeric RNA molecule, the disclosed vector or disclosed nucleic acid molecule may be formulated for administration via one or more routes. Such methods are well known to those skilled in the art and include, but are not limited to, the following routes: oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, intrauterine administration, intrahepatic administration, intravaginal administration, ocular administration, intraauricular administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable methods such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration may also include intra-hepatic artery administration or administration via the portal vein (HPV). Administration of the disclosed therapeutic agents, disclosed pharmaceutical compositions, or combinations thereof may include direct administration to the CNS (e.g., intraparenchymal, intravenous, intrasheathic cisterns, intrasheath (lumbar), deep gray matter delivery, convection-enhanced delivery to deep gray matter) or PNS. Administration may be continuous or intermittent.

[0208] In one aspect, administration can be performed via one or more ex vivo methods, such as an ex vivo perfusion protocol. In one aspect, the ex vivo perfusion protocol can be used with one or more cells, tissues, and / or organs affected by a genetic disease or condition obtained from a subject. In one aspect, one or more cells and / or one or more tissues and / or one or more organs can be obtained from a subject in need, can be used for ex vivo perfusion and / or treatment and / or contact protocols, and can be returned to the subject in need, wherein the one or more cells produce chimeric RNA transcripts.

[0209] In one aspect, the disclosed method for producing chimeric RNA molecules in cells can include verifying trans-splicing events and / or the production of chimeric RNA molecules. Verification of trans-splicing events and / or the production of chimeric RNA molecules can be performed using methods and techniques known in the art (e.g., sequencing, northern blotting, FISH, PCR, RNA-Seq, 3'RACE, 5'RACE, etc.).

[0210] In one aspect, the disclosed method for generating chimeric RNA molecules may include preparing a disclosed 5' substitution construct, a disclosed 3' substitution construct, a disclosed nonviral vector or a disclosed viral vector, a disclosed nucleic acid molecule, a disclosed pharmaceutical preparation, or any combination thereof.

[0211] In one aspect, the disclosed method can be performed in vitro. In one aspect, administration can be performed by one or more in vitro methods, such as an in vitro perfusion protocol. In one aspect, the in vitro perfusion protocol can be used with one or more cells, tissues, and / or organs obtained from a subject affected by a genetic disease or condition. In one aspect, one or more cells and / or one or more tissues and / or one or more organs can be obtained from a subject in need, an in vitro perfusion and / or treatment and / or contact protocol can be performed, and they can be returned to the subject in need, wherein one or more cells produce chimeric RNA transcripts.

[0212] In one aspect, the disclosed method can restore the activity and / or functionality of missing, defective, and / or mutated proteins or enzymes, and may include restoration of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount compared to a pre-existing level, such as a pre-treatment level. In one aspect, the amount of restoration may be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% higher than a pre-existing level, such as a pre-treatment level. In one aspect, restoration may be measured against a control or reference level (e.g., determined using one or more subjects who do not have missing, defective, and / or mutated proteins or enzymes). In one aspect, restoration may be partial.Partial or incomplete recovery. In one aspect, recovery can be complete or near-complete recovery, such that the levels of expression, activity, and / or functionality are similar to wild-type or control levels, and / or the expression levels of one or more protein-coding genes are restored to wild-type, normal, or control expression levels. Specification 40 / 109 pages 44 CN 121195069 A

[0213] In one aspect, the disclosed method can restore one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic disorders. In one aspect, the disclosed method can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0214] In one aspect, one or more aspects of restoring cellular homeostasis and / or cellular functionality may include one or more of the following: (i) correcting cellular starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (e.g., correcting, preventing, reducing, and / or alleviating autophagy); (iii) improving, enhancing, restoring, and / or preserving mitochondrial functionality and / or structural integrity; (iv) improving, enhancing, restoring, and / or preserving organelle functionality and / or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of a multisystemic manifestation of a genetic disease or condition; (vii) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of a genetic disease or condition, or (viii) any combination thereof. In one aspect, one or more aspects of restoring cellular homeostasis may include one or more aspects of improving, enhancing, restoring, and / or preserving cellular structural and / or functional integrity.

[0215] In one aspect, the disclosed chimeric RNA can be used specifically in methods for treating subjects with autosomal dominant genetic diseases or conditions (e.g., progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert's disease, hereditary hemorrhagic telangiectasia, hereditary elliptic polycythemia, hereditary spherocytosis, Huntington's disease, idiopathic hypoparathyroidism, intestinal polyposis, marble bone disease, Marfan syndrome, neurofibromatosis, polycystic kidney disease (adults), protein C deficiency, osteogenesis imperfecta, TracyCollins syndrome, tuberous sclerosis, Villebrand disease, etc.).

[0216] D. Method for Treating Genetic Diseases or Conditions This document discloses a method for treating genetic diseases or conditions, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a disclosed nonviral vector or a disclosed viral vector or a pharmaceutical preparation thereof to a subject in need, wherein the resulting chimeric RNA molecule can restore cellular homeostasis and / or one or more aspects of cellular functional and / or metabolic disorders.

[0217] This article discloses a method for treating a genetic disease or condition, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a disclosed nonviral vector or a disclosed viral vector or a pharmaceutical preparation thereof to a subject in need of the molecule, wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0218] This document discloses a method for treating a genetic disease or condition, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector containing a nucleic acid molecule to a subject in need of the molecule, the nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic disorders, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0219] This document discloses a method for treating a genetic disease or condition, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector containing a nucleic acid molecule to a subject in need of the molecule, the nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures comprising sequences of any one of SEQ ID NO: 01-SEQ ID NO: 09, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic disorders, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0220] This document discloses a method for treating a genetic disease or condition, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector containing a nucleic acid molecule to a subject in need of the molecule, said nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' hemiintron linked to the exogenous RNA to be trans-spliced; one or more RNA-targeting motifs; and one or more RNA structures wherein the nucleic acidThe molecules enable the 5' substitution of targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic dysregulation, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0221] This document discloses a method for treating a genetic disease or condition, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector containing a nucleic acid molecule to a subject in need of the molecule, the nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures comprising sequences of any one of SEQ ID NO: 01-SEQ ID NO: 09, wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic disorders, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0222] This document discloses a method for treating a genetic disease or condition, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector containing a nucleic acid molecule to a subject in need of the molecule, the nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' hemiintron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic disorders, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0223] This document discloses a method for treating a genetic disease or condition, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector containing a nucleic acid molecule to a subject in need of the molecule, the nucleic acid molecule comprising one or more RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; and a 3' hemisphere linked to a foreign RNA to be trans-spliced.Intron; and exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic dysregulation, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0224] This document discloses a method for treating a genetic disease or condition, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector containing a nucleic acid molecule to a subject in need of the molecule, the nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic disorders, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0225] This document discloses a method for treating a genetic disease or condition, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector containing a nucleic acid molecule to a subject in need of the molecule, the nucleic acid molecule comprising one or more RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic disorders, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0226] This document discloses a method for treating a genetic disease or condition, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a nonviral vector, a viral vector, AAV particles, or a pharmaceutical preparation thereof to a subject in need of the molecule, wherein the nonviral vector, viral vector, AAV particles, or pharmaceutical preparation thereof comprises (i) one or more(ii) one or more 3' replacement constructs, or (iii) one or more 5' replacement constructs and / or one or more 3' replacement constructs, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic dysregulation, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0227] In one aspect of the disclosed therapeutic method, the disclosed 5' replacement construct to be used in combination with one or more other replacement constructs may comprise any disclosed 5' replacement construct. In one aspect, the disclosed 5' substitution construct may comprise (i) a nucleic acid molecule containing exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures; (ii) a nucleic acid molecule containing exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures comprising SEQ ID NO: 01-SEQ ID NO: The sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09, (iii) a nucleic acid molecule comprising a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; a 5' half-intron linked to the foreign RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA, or (iv) a nucleic acid molecule comprising a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; a 5' half-intron linked to the foreign RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising the sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09 wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA, or (v) any combination thereof.

[0228] In one aspect of the disclosed treatment method, the disclosed 3' substitution construct to be used in combination with one or more other substitution constructs may comprise any disclosed 3' substitution construct. In one aspect, the disclosed 3' substitution construct may comprise (i) a nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' hemiintron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; (ii) a nucleic acid molecule comprising one or more RNA structures, the RNA structures comprising SEQ ID NO.(iii) A nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA; (iv) A nucleic acid molecule comprising one or more RNA structures, the RNA structures comprising SEQ ID NO: 01-SEQ ID NO: The sequence of any one of 09; one or more RNA targeting motifs; a 3' half-intron linked to the exogenous RNA to be trans-spliced; and the exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables a 3' substitution of the targeted endogenous pre-mRNA, or (v) any combination thereof.

[0229] In one aspect of the disclosed treatment method, the subject may suffer from a genetic disease or condition associated with and / or caused by one or more of the disclosed genes, including those disclosed in Section VII(B)(1). In one aspect of the disclosed treatment method, the subject may suffer from a genetic disease or condition associated with and / or caused by one or more of the disclosed genes, including those disclosed in Section VII(B)(1).

[0230] In one aspect of the disclosed treatment method, the expression of the disclosed protein-coding gene may be restored and / or returned to wild-type, normal, or control expression levels. In one aspect, the disclosed method for treating a genetic disease or condition may restore the functionality and / or structural integrity of a missing, defective, and / or mutated protein or enzyme. In one aspect, the disclosed treatment methods may include one or more aspects of restoring cellular homeostasis and / or cellular functional and / or metabolic dysregulation. In one aspect, restoring cellular homeostasis and / or cellular functional and / or metabolic dysregulation includes restoring the functional and / or structural integrity of missing, defective, and / or mutated proteins or enzymes. In one aspect, restoring cellular homeostasis and / or cellular functionalization may include one or more of the following: (i) correcting cellular starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (e.g., correcting, preventing, reducing, and / or alleviating autophagy); (iii) improving, enhancing, restoring, and / or preserving mitochondrial functional and / or structural integrity; (iv) improving, enhancing, restoring, and / or...Or, preserve organelle functionality and / or structural integrity; (v) correct enzyme dysregulation; (vi) reverse, inhibit, prevent, stabilize, and / or slow the rate of progression of a multisystemic manifestation of a genetic disease or condition; (vii) reverse, inhibit, prevent, stabilize, and / or slow the rate of progression of a genetic disease or condition, or (viii) any combination thereof. In one aspect, one or more aspects of restoring cellular homeostasis may include improving, enhancing, restoring, and / or preserving the structural and / or functional integrity of cells. In one aspect, restoring the activity and / or functionality of missing, defective, and / or mutated proteins or enzymes (e.g., encoded by protein-coding genes described above) may include a restoration of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount compared to a pre-existing level, such as a pre-treatment level. In one aspect, the amount of recovery may be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% higher than a pre-existing level, such as the pre-treatment level. In one aspect, recovery may be measured against a control or reference level (e.g., determined using one or more subjects without the missing, defective, and / or mutated protein or enzyme). In one aspect, recovery may be partial or incomplete. In one aspect, recovery may be complete or near-complete, such that the levels of expression, activity, and / or functionality are similar to wild-type or control levels.

[0231] In one aspect, the disclosed treatment method may further include monitoring the subject's metabolic and / or physiological improvements after one administration step and / or after multiple administration steps. In one aspect, a clinician may measure and / or determine the subject's metabolic and / or physiological status over time to identify one or more improvements and / or one or more attenuations. In one aspect of the disclosed method, clinicians may use the subject's metabolic and / or physiological state and / or trends in the subject's metabolic and / or physiological state to make treatment decisions and / or modify one aspect of the disclosed method and / or continue to treat the subject and / or continue to administer the disclosed AAV particles, the disclosed carrier, the disclosed nucleic acid molecule, the disclosed pharmaceutical preparation, the disclosed therapeutic agent and / or the disclosed immunomodulator, or any combination thereof. In one aspect, metabolic and / or physiological data may inform clinicians and treatment plans. Specification 44 / 109 pages 48 CN 121195069 A

[0232] In one aspect of the disclosed treatment method, techniques for monitoring, measuring and / or assessing the restoration of one or more aspects of cellular homeostasis and / or cellular function may include qualitative (or subjective) means as well as quantitative (or objective) means.These methods are known to those skilled in the art. For example, representative regulatory variables and sensors related to systemic homeostasis are provided below.

[0233] In one aspect, the disclosed treatment method may further include performing one or more invasive or non-invasive diagnostic assessments on the subject. Diagnostic assessments are known in the art. In one aspect, the disclosed non-invasive diagnostic assessment may include x-ray, computed tomography (CT) scan, magnetic resonance imaging (MRI) scan, ultrasound, positron emission tomography (PET) scan, or any combination thereof. In one aspect, the disclosed invasive diagnostic assessment may include tissue biopsy or exploratory surgery.

[0234] In one aspect, the disclosed treatment method may be used to repair one or more diseased and / or disordered cell types in cells, tissues, and / or organs.

[0235] In one aspect, the disclosed treatment method may be used to improve and / or enhance the subject's quality of life compared to pre-treatment levels. In one aspect, the disclosed treatment method may improve the subject's quality of life by at least 50% when compared to the subject's pre-treatment quality of life.

[0236] In one aspect, the disclosed treatment method can be used to improve and / or enhance the quality of life of a subject compared to a pre-treatment level. In one aspect, the disclosed treatment method can improve the quality of life of a subject by at least 50% compared to the subject's pre-treatment quality of life.

[0237] In one aspect, the disclosed treatment method can be used to diminish and / or reduce one or more symptoms associated with and / or related to a subject's genetic disease and / or genetic condition. In one aspect, the disclosed treatment method can be used to prevent the occurrence of undesirable physiological changes, diseases, pathological conditions, or symptoms in a subject. In one aspect, the disclosed treatment method can be used to inhibit physiological changes, diseases, pathological conditions, or symptoms in a subject, i.e., to halt their development. In one aspect, the disclosed treatment method can be used to eliminate physiological changes, diseases, pathological conditions, or symptoms in a subject, i.e., to induce disease regression.

[0238] In one aspect of the disclosed treatment method, administration to a subject may include contacting one or more cells with one or more disclosed nucleic acid molecules, disclosed carriers or disclosed AAV particles, disclosed pharmaceutical preparations, or any combination thereof.

[0239] In one aspect of the disclosed treatment method, administration of the disclosed nucleic acid molecule, the disclosed carrier, the disclosed AAV particles, the disclosed pharmaceutical formulation, or any combination thereof may include one or more routes. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, intrauterine administration, intrahepatic administration, intravaginal administration, ocular administration, intraauricular administration, intracerebral administration, rectal administration, etc.Sublingual, buccal, and parenteral administration, including injectable, such as intravenous, intra-CSF, intra-arterial, intramuscular, and subcutaneous administration. Administration may also include intrahepatic artery administration or administration via the portal vein (HPV). Administration of the disclosed nucleic acid molecule, disclosed carrier, disclosed AAV particles, disclosed pharmaceutical formulation, or any combination thereof may include direct administration to the CNS (e.g., intraparenchymal, intravenous, intrasheathic cisterns, intrasheath (lumbar), deep gray matter delivery, convection-enhanced delivery to deep gray matter) or PNS. Administration may be continuous or intermittent.

[0240] In one aspect, the disclosed treatment method may be administered to the subject via multiple routes of administration. In one aspect, the disclosed treatment method may be administered via a first route of administration, which may be the same as or different from a second and / or subsequent route of administration.

[0241] In one aspect of the disclosed method of treating a genetic disease or condition, the therapeutically effective amount of the disclosed carrier or the disclosed pharmaceutical preparation may include a range of about 1 × 10¹⁰ vg / kg to about 2 × 10¹⁴ vg / kg. In one aspect, for example, the therapeutically effective amount of the disclosed carrier or the disclosed pharmaceutical preparation may include a dose of about 1 × 10¹¹ vg / kg to about 8 × 10¹³ vg / kg or about 1 × 10¹² vg / kg to about 8 × 10¹³ vg / kg. In one aspect, for example, the therapeutically effective amount of the disclosed carrier or the disclosed pharmaceutical preparation may include a dose of about 1 × 10¹³ vg / kg to about 6 × 10¹³ vg / kg. In one aspect, the therapeutically effective amount of the disclosed carrier or the disclosed pharmaceutical preparation may include a dose of at least about 1×10¹⁰ vg / kg, at least about 5×10¹⁰ vg / kg, at least about 1×10¹¹ vg / kg, at least about 5×10¹¹ vg / kg, at least about 1×10¹² vg / kg, at least about 5×10¹² vg / kg, at least about 1×10¹³ vg / kg, at least about 5×10¹³ vg / kg, or at least about 1×10¹⁴ vg / kg. In one aspect, the therapeutically effective amount of the disclosed carrier or the disclosed pharmaceutical preparation may include doses not exceeding about 1 × 10¹⁰ vg / kg, not exceeding about 5 × 10¹⁰ vg / kg, not exceeding about 1 × 10¹¹ vg / kg, not exceeding about 5 × 10¹¹ vg / kg, not exceeding about 1 × 10¹² vg / kg, not exceeding about 5 × 10¹² vg / kg, not exceeding about 1 × 10¹³ vg / kg, not exceeding about 5 × 10¹³, or not exceeding about 1 × 10¹⁴ vg / kg. In another aspect, the therapeutically effective amount of the disclosed carrier or the disclosed pharmaceutical preparation may include about 1 × 10¹² vg / kg or about 1 × 10¹¹ vg / kg.A dose of vg / kg. In one aspect, the therapeutically effective amount of the disclosed carrier or the disclosed pharmaceutical formulation may include administration in single or multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses) as needed for the desired therapeutic outcome.

[0242] In one aspect, the therapeutically effective amount of the disclosed carrier or the disclosed pharmaceutical formulation may include a range from about 1 × 10¹² vg to about 1 × 10¹⁷ vg per subject in total, and in another aspect, the therapeutically effective amount of the disclosed carrier or the disclosed pharmaceutical formulation may include a range of about 1 × 10¹² vg, about 1 × 10¹³ vg, about 1 × 10¹⁴ vg, about 1 × 10¹⁵ vg, about 1 × 10¹⁶ vg, or about 1 × 10¹⁷ vg per subject in total.

[0243] In one aspect, the therapeutically effective amount of the disclosed carrier or the disclosed pharmaceutical preparation may include about 1 × 10⁶ DRP / mL to about 1 × 10¹⁴ DRP / mL. In one aspect, the therapeutically effective amount of the disclosed carrier or the disclosed pharmaceutical preparation may include about 1 × 10⁶ DRP / mL, 1 × 10⁷ DRP / mL, 1 × 10⁸ DRP / mL, 1 × 10⁹ DRP / mL, 1 × 10¹⁰ DRP / mL, 1 × 10¹¹ DRP / mL, 1 × 10¹² DRP / mL, 1 × 10¹³ DRP / mL, or 1 × 10¹⁴ DRP / mL. In one aspect, the therapeutically effective amount of the disclosed carrier or the disclosed pharmaceutical preparation may include a range determined by a person skilled in the art.

[0244] In one aspect, the disclosed method of treating a genetic disease or condition may further include administering a therapeutically effective amount of the therapeutic agent to a subject. The therapeutic agent may be any disclosed agent that achieves the desired clinical outcome.

[0245] In one aspect, the disclosed method of treating a genetic disease or condition may further include monitoring the subject for adverse reactions. In one aspect, in the absence of adverse reactions, the method may further include continuing treatment of the subject. In one aspect, in the presence of adverse reactions, the method may further include modifying the treatment steps. Methods for monitoring the health of the subject may include subjective and objective criteria (and as described above). Such methods are known to those skilled in the art.

[0246] In one aspect, the disclosed treatment method may further include administering a therapeutically effective amount of a reagent to the subject, the reagent being capable of correcting one or more aspects of a disordered metabolic or enzymatic pathway. In one aspect, such a reagent may include an enzyme for enzyme replacement therapy. In one aspect, the disclosed enzyme may replace a disordered or dysfunctional metabolic or enzymatic pathway.Any enzyme in the pathway. In one aspect, the disclosed treatment method may include replacing one or more enzymes in the dysregulated or dysfunctional metabolic pathway.

[0247] In one aspect, the disclosed method of treating a genetic disease or condition may further include administering one or more immunomodulators. In one aspect, the disclosed immunomodulator may be methotrexate, rituximab, intravenous gamma globulin, or bortezomib or a combination thereof. In one aspect, the disclosed immunomodulator may be bortezomib or SVP-rapamycin. In one aspect, the disclosed immunomodulator may be tacrolimus. In one aspect, the disclosed immunomodulator, such as methotrexate, may be administered in an instantaneous low to high dose. In one aspect, the disclosed immunomodulator may be administered at a dose of about 0.1 mg / kg body weight to about 0.6 mg / kg body weight. In one aspect, the disclosed immunomodulator may be administered at a dose of about 0.4 mg / kg body weight. In one aspect, the disclosed immunomodulator may be administered at a daily dose of about 0.4 mg / kg body weight for 3 to 5 or more cycles, each cycle lasting up to three days. In one aspect, the disclosed immunomodulatory agent can be administered at a daily dose of about 0.4 mg / kg body weight for at least 3 cycles, each cycle lasting 3 days. In one aspect, those skilled in the art can determine the appropriate number of cycles. In one aspect, the disclosed immunomodulatory agent can be administered multiple times as needed to achieve the desired clinical effect.

[0248] In one aspect, the disclosed immunomodulatory agent can be administered orally about 1 hour before the disclosed therapeutic agent. In one aspect, the disclosed immunomodulatory agent can be administered subcutaneously about 15 minutes before the disclosed therapeutic agent. In one aspect, the disclosed immunomodulatory agent can be administered concurrently with the disclosed therapeutic agent. In one aspect, the disclosed immunomodulatory agent can be administered orally about 1 hour or several days before the disclosed nucleic acid molecule, disclosed carrier, disclosed pharmaceutical preparation or combination thereof. In one aspect, the disclosed immunomodulatory agent can be administered subcutaneously about 15 minutes or several days before the disclosed nucleic acid molecule, disclosed carrier, disclosed pharmaceutical preparation or combination thereof. In one aspect, the disclosed immunomodulatory agent can be administered concurrently with the disclosed nucleic acid molecule, disclosed carrier, disclosed pharmaceutical preparation or combination thereof.

[0249] In one aspect, the disclosed method of treating a genetic disease or condition may further include administering one or more proteasome inhibitors (e.g., bortezomib, carfilzomib, marizomib, ixazomib, and oprozomib). In one aspect, the proteasome inhibitor may be an agent acting on plasma cells (e.g., daratumumab). In one aspect, acting on plasma cells...The reagents for cell preparation can be melphalan hydrochloride, melphalan, disodium pamidronate, carmustine, carfilzomib, cyclophosphamide, daratumumab, doxorubicin hydrochloride liposomes, doxorubicin hydrochloride liposomes, elotuzumab, melphalan hydrochloride, pambistat, ixazomib citrate, carfilzomib, lenalidomide, melphalan hydrochloride, prasaf, ixazomib citrate, disodium pamidronate, pambistat, prasaf, pomalidomide, lenalidomide, celiniso, thalidomide, bortezomib, celiniso, zoledronic acid, or zoledronic acid.

[0250] In one aspect, the disclosed method of therapeutic stability may further include administering one or more proteasome inhibitors or plasma cell-acting agents prior to the administration of the disclosed nucleic acid molecule, the disclosed carrier, the disclosed AAV particles, the disclosed pharmaceutical preparation, or any combination thereof. In one aspect, the disclosed treatment method may include administering one or more proteasome inhibitors or plasma cell-acting agents in parallel with the administration of the disclosed nucleic acid molecule, the disclosed carrier, the disclosed AAV particles, the disclosed pharmaceutical preparation, or any combination thereof. In one aspect, the disclosed treatment method may include administering one or more proteasome inhibitors or one or more plasma cell-acting agents after the administration of the disclosed nucleic acid molecule, the disclosed carrier, the disclosed AAV particles, the disclosed pharmaceutical preparation, or any combination thereof. In one aspect, the disclosed treatment method specification 47 / 109 pages 51 CN 121195069 A may further include administering one or more proteasome inhibitors more than once. In one aspect, the disclosed treatment method may include repeated administration of one or more proteasome inhibitors over time.

[0251] In one aspect, the disclosed method of treating a genetic disease or condition may further include administering one or more immunosuppressants. In one aspect, the immunosuppressant may be, but is not limited to, azathioprine, methotrexate, sirolimus, anti-thymocyte globulin (ATG), cyclosporine (CSP), mycophenolate mofetil (MMF), steroids, or combinations thereof. In one aspect, the disclosed treatment method may include administering one or more immunosuppressants more than once. In one aspect, the disclosed method may include repeatedly administering one or more immunosuppressants over time. In one aspect, the disclosed treatment method may include administering compounds that target or alter antigen presentation or humoral or cell-mediated or innate immune responses.

[0252] In one aspect, the disclosed method of treating a genetic disease or condition may further include administering compounds that exert a therapeutic effect against B cells and / or compounds that target or alter antigen presentation or humoral or cell-mediated immune responses.In one aspect, the disclosed compounds may be rituximab, methotrexate, intravenous gamma globulin, anti-CD4 antibody, anti-CD2, anti-FcRN antibody, BTK inhibitor, anti-IGFIR antibody, CD19 antibody (e.g., inebilizumab), anti-IL6 antibody (e.g., tocilizumab), antibody against CD40, IL2 mutant protein, or combinations thereof. Treg infusions (e.g., antigen-specific Treg cells against AAV) that can be administered as a means to aid immune tolerance are also disclosed herein.

[0253] In one aspect, the disclosed treatment method may further include the administration of lipid nanoparticles (LNPs). In one aspect, the LNPs may be organ-targeted. In one aspect, the LNPs may be liver-targeted or testis-targeted. For example, in one aspect, an LNP-encapsulated mRNA therapy for systemic delivery to a subject has the potential to restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes.

[0254] In one aspect, the disclosed method of treating a genetic disease or condition may further include treating a subject who has developed or may develop neutralizing antibodies (ABs) to the disclosed nucleic acid molecule, the disclosed vector, the disclosed AAV particles, the disclosed pharmaceutical formulation, or any combination thereof. In one aspect, treating a subject who has developed or may develop neutralizing antibodies may include plasmapheresis and immunosuppression. In one aspect, the disclosed method may include using immunosuppression to reduce T cell, B cell, and / or plasma cell populations in general, thereby reducing innate immune responses, inflammatory responses, and antibody levels. In one aspect, the disclosed method may include administering an IgG degrading agent that depletes pre-existing neutralizing antibodies. In one aspect, the disclosed method may include administering IdeS or IdeZ, rapamycin, and / or SVP-rapamycin to a subject. In one aspect, the disclosed treatment method may include administering tacrolimus. In one aspect, the disclosed IgG degrading agent is bacterial IdeS or IdeZ.

[0255] In one aspect, the disclosed treatment method may include repeated administration steps, such as repeated administration of a disclosed nucleic acid molecule, a disclosed carrier, a disclosed AAV particle, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, a disclosed immunomodulator, a disclosed proteasome inhibitor, a disclosed immunosuppressant, a disclosed compound that exerts a therapeutic effect against B cells, and / or a disclosed compound that targets or modifies antigen presentation or humoral or cell-mediated immune responses.

[0256] In one aspect, the disclosed treatment method may include modifications to one or more of the disclosed steps. For example, modifying one or more steps of the disclosed method may include modifying or altering one or more steps of the disclosed method.Multiple features or aspects. For example, in one aspect, the method can be changed by altering the amount of one or more disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed carriers, one or more disclosed pharmaceutical preparations, or any combination thereof administered to the subject, or by altering the frequency of administration of one or more disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed carriers, one or more disclosed pharmaceutical preparations, or any combination thereof to the subject, or by altering the duration of administration of one or more disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed carriers, one or more disclosed pharmaceutical preparations, or any combination thereof to the subject.

[0257] In one aspect, a disclosed treatment method may be modified by changing the amount administered to a subject of one or more disclosed therapeutic agents, disclosed immunomodulators, disclosed proteasome inhibitors, disclosed immunosuppressants, disclosed compounds that have a therapeutic effect on B cells and / or disclosed compounds that target or alter antigen presentation or humoral or cell-mediated immune responses, or by changing the frequency of administration of one or more of the disclosed therapeutic agents, disclosed immunomodulators, disclosed proteasome inhibitors, disclosed immunosuppressants, disclosed compounds that have a therapeutic effect on B cells and / or disclosed compounds that target or alter antigen presentation or humoral or cell-mediated immune responses.

[0258] In one aspect, the disclosed treatment methods may include the concurrent administration of one or more disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed vectors, one or more disclosed pharmaceutical preparations, one or more disclosed therapeutic agents, one or more disclosed immunomodulators, one or more disclosed proteasome inhibitors, one or more disclosed immunosuppressants, one or more disclosed compounds that exert therapeutic effects on B cells, one or more disclosed compounds that target or alter antigen presentation or humoral or cell-mediated immune responses, or any combination thereof. In one aspect, the disclosed immunomodulators may be administered before or after the administration of the disclosed therapeutic agents.

[0259] In one aspect, the disclosed method of treating a genetic disease or condition may further include generating the disclosed nucleic acid molecules, the disclosed vectors, the disclosed AAV particles, the disclosed pharmaceutical preparations, or any combination thereof.

[0260] In one aspect, the disclosed treatment methods may further include gene editing of one or more related genes (e.g., deleted, defective, and / or mutated proteins or enzymes), wherein the editing includes, but is not limited to, single gene knockout, single loss-of-function screening of multiple genes, gene knock-in, or combinations thereof.

[0261] In one aspect, the disclosed method of treating a genetic disease or condition may further include administering oligosaccharides to the subject.Nucleotide therapeutic agents. Disclosed oligonucleotide therapeutic agents may include single-stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA, non-coding RNA (ncRNA), antisense molecules, miRNA, morpholino, peptide-nucleic acid (PNA), or analogs or conjugates thereof. In one aspect, the disclosed oligonucleotide therapeutic agent may be ASO or RNAi. In one aspect, the disclosed oligonucleotide therapeutic agent may contain one or more modifications at any applicable position. In one aspect, the disclosed oligonucleotide therapeutic agent may include a CRISPR-based endonuclease. In one aspect, the disclosed endonuclease may be Cas9. In one aspect, the disclosed Cas9 may be derived from Staphylococcus aureus or Streptococcus pyogenes. In one aspect, the disclosed treatment method may include administering the disclosed RNA therapeutic agent to a subject.

[0262] In one aspect, the disclosed method of treating a genetic disease or condition may further include generating and / or validating one or more disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed vectors, one or more disclosed pharmaceutical preparations, or any combination thereof. In one aspect, the disclosed treatment methods can inhibit and / or minimize one or more aspects of disease progression in a subject (e.g., the genetic diseases or conditions described above).

[0263] In one aspect, the disclosed treatment methods can slow and / or reduce one or more aspects of disease progression in a subject (e.g., the genetic diseases or conditions described above).

[0264] In one aspect, the disclosed methods can be used specifically to treat subjects suffering from autosomal dominant genetic diseases or conditions (e.g., progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert's disease, hereditary hemorrhagic telangiectasia, hereditary elliptic polycythemia, hereditary spherocytosis, Huntington's disease, idiopathic hypoparathyroidism, intestinal polyposis, marble bone disease, Marfan syndrome, neurofibromatosis, polycystic kidney disease (adult), protein C deficiency, osteogenesis imperfecta, TracyCollins syndrome, tuberous sclerosis, Villebrand disease, etc.).

[0265] E. Method for Inhibiting and / or Minimizing Disease Progression 49 / 109 pages 53 CN 121195069 A This document discloses a method for inhibiting and / or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a disclosed nonviral vector or a disclosed viral vector or a pharmaceutical preparation thereof to a subject in need of the method, wherein the resulting chimeric RNA molecule can restore cellular homeostasis and / or one or more aspects of cellular functional and / or metabolic dysregulation.

[0266] This article discloses a method for inhibiting and / or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a publicly disclosed nonviral vector or a publicly disclosed viral vector or a pharmaceutical preparation thereof to a subject in need thereof, wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0267] This document discloses a method for inhibiting and / or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a nonviral or viral vector containing a nucleic acid molecule to a subject in need of the molecule, the nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA-targeting motifs; one or more RNA structures, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0268] This document discloses a method for inhibiting and / or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a nonviral or viral vector containing a nucleic acid molecule to a subject in need of the molecule; the nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; and one or more RNA structures comprising sequences of any one of SEQ ID NO: 01-SEQ ID NO: 09, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic dysregulation, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0269] This document discloses a method for inhibiting and / or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector containing a nucleic acid molecule to a subject in need of the molecule, said nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' hemiintron linked to the exogenous RNA to be trans-spliced; one or more RNA-targeting motifs; and one or more RNA structures, whereinNucleic acid molecules enable the 5' substitution of targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic dysregulation, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0270] This document discloses a method for inhibiting and / or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a nonviral or viral vector containing a nucleic acid molecule to a subject in need of the molecule, the nucleic acid molecule comprising an exogenous RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising sequences of any one of SEQ ID NO: 01-SEQ ID NO: 09, wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above). (See specification 50 / 109 pages 54 CN 121195069 A)

[0271] This document discloses a method for inhibiting and / or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector containing a nucleic acid molecule to a subject in need of the method, the nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' hemiintron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0272] This document discloses a method for inhibiting and / or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector containing a nucleic acid molecule to a subject in need of the method, the nucleic acid molecule comprising one or more RNA structures containing SEQ ID NO: 01-SEQ ID NO:The sequence of any one of 09; one or more RNA targeting motifs; a 3' half-intron linked to the exogenous RNA to be trans-spliced; and the exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0273] This document discloses a method for inhibiting and / or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector containing a nucleic acid molecule to a subject in need thereof, the nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0274] This document discloses a method for inhibiting and / or minimizing disease progression, the method comprising generating a chimeric RNA molecule in one or more cells by administering a therapeutically effective amount of a non-viral or viral vector containing a nucleic acid molecule to a subject in need of the molecule, the nucleic acid molecule comprising one or more RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to a targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA, wherein the resulting chimeric RNA molecule can restore one or more aspects of cellular homeostasis and / or cellular functionality and / or metabolic dysregulation, and / or wherein the resulting chimeric RNA molecule can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0275] This document discloses a method for inhibiting and / or minimizing disease progression, the method comprising administering a therapeutically effective amount of a nonviral vector, a viral vector, AAV particles, or a pharmaceutical preparation thereof to a subject in need, in one or moreChimeric RNA molecules are produced in cells, wherein the non-viral vector, viral vector, AAV particle, or pharmaceutical formulation thereof comprises (i) one or more 5' substitution constructs, (ii) one or more 3' substitution constructs, or (iii) one or more 5' substitution constructs and / or one or more 3' substitution constructs, wherein the resulting chimeric RNA molecules can restore one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic dysregulation, and / or wherein the resulting chimeric RNA molecules can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes (e.g., those encoded by one of the genes provided above).

[0276] In one aspect of the disclosed methods for inhibiting and / or minimizing disease progression, the disclosed 5' substitution construct to be used in combination with one or more other substitution constructs may comprise any of the disclosed 5' substitution constructs. In one aspect, the disclosed 5' substitution construct may comprise (i) a nucleic acid molecule containing exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures; (ii) a nucleic acid molecule containing exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising a sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; (iii) a nucleic acid molecule containing exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; a 5' half-intron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures, wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA; or (iv) a nucleic acid molecule containing exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA. The exogenous RNA; a 5' hemiintron linked to the exogenous RNA to be trans-spliced; one or more RNA targeting motifs; one or more RNA structures comprising sequences of any one of SEQ ID NO: 01-SEQ ID NO: 09, wherein the nucleic acid molecule enables 5' substitution of the targeted endogenous pre-mRNA, or (v) any combination thereof.

[0277] In one aspect of the disclosed method of inhibiting and / or minimizing disease progression, the disclosed 3' substitution construct to be used in combination with one or more other substitution constructs may comprise any disclosed 3' substitution construct. In one aspect, the disclosed 3' substitution construct may comprise (i) a nucleic acid molecule comprising one or more RNA structures; one or more(ii) A nucleic acid molecule comprising one or more RNA structures comprising any one of the sequences of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; (iii) A nucleic acid molecule comprising one or more RNA structures; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA; (iv) A nucleic acid molecule comprising one or more RNA structures comprising any one of SEQ ID NO: 01-SEQ ID NO: 09; one or more RNA targeting motifs; a 3' half-intron linked to a foreign RNA to be trans-spliced; and a foreign RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables 3' substitution of the targeted endogenous pre-mRNA; and (iv) A nucleic acid molecule comprising one or more RNA structures comprising any one of SEQ ID NO: 01-SEQ ID NO: 09; The sequence of any one of 09; one or more RNA targeting motifs; a 3' half-intron linked to the exogenous RNA to be trans-spliced; and the exogenous RNA to be trans-spliced ​​to the targeted endogenous pre-mRNA; wherein the nucleic acid molecule enables a 3' substitution of the targeted endogenous pre-mRNA, or (v) any combination thereof.

[0278] In one aspect of the disclosed methods for inhibiting and / or minimizing disease progression, the subject may suffer from a genetic disease or condition associated with and / or caused by one or more of the disclosed genes (including those disclosed in Section VII(B)(1). In one aspect of the disclosed methods for inhibiting and / or minimizing disease progression, the expression of the disclosed protein-coding gene may be restored and / or returned to wild-type, normal, or control expression levels. In one aspect, the disclosed methods for inhibiting and / or minimizing the progression of a genetic disease or condition can restore the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes. In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression may include one or more aspects of restoring cellular homeostasis and / or cellular function and / or metabolic disorders. In one aspect, restoring cellular homeostasis and / or cellular function and / or metabolic disorders includes restoring the functionality and / or structural integrity of missing, defective, and / or mutated proteins or enzymes. In one aspect, restoring cellular homeostasis and / or cellular function...One or more aspects may include one or more of the following: (i) correcting cellular starvation in one or more cell types; (ii) normalizing aspects of the autophagy pathway (e.g., correcting, preventing, reducing, and / or alleviating autophagy); (iii) improving, enhancing, restoring, and / or preserving mitochondrial functionality and / or structural integrity; (iv) improving, enhancing, restoring, and / or preserving organelle functionality and / or structural integrity; (v) correcting enzymatic dysregulation; (vi) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of a multisystemic manifestation of a genetic disease or condition; (vii) reversing, inhibiting, preventing, stabilizing, and / or slowing the rate of progression of a genetic disease or condition, or (viii) any combination thereof. In one aspect, restoring cellular homeostasis may include one or more aspects of improving, enhancing, restoring, and / or preserving cellular structural and / or functional integrity. In one aspect, the restoration of the activity and / or functionality of a missing, defective, and / or mutated protein or enzyme (e.g., encoded by a protein-coding gene as described above) may include a restoration of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount compared to a pre-existing level, such as a pre-treatment level. In one aspect, the amount of restoration may be 10–20%, 20–30%, 30–40%, 40–50%, 50–60%, 60–70%, 70–80%, 80–90%, or 90–100% higher than a pre-existing level, such as a pre-treatment level. In one aspect, restoration may be measured against a control or reference level (e.g., determined using one or more subjects without the missing, defective, and / or mutated protein or enzyme). In one aspect, restoration may be partial or incomplete. In one aspect, restoration may be complete or near-complete, such that the levels of expression, activity, and / or functionality are similar to wild-type or control levels.

[0280] In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression may further include monitoring metabolic and / or physiological improvements in the subject after one administration step and / or after multiple administration steps. In one aspect, clinicians may measure and / or determine the subject's metabolic and / or physiological status over time to identify one or more improvements and / or one or more attenuations. In one aspect of the disclosed methods, clinicians may use the subject's metabolic and / or physiological status and / or trends and / or tendencies in the subject's metabolic and / or physiological status to make treatment decisions and / or modify one aspect of the disclosed methods and / or continue treatment of the subject and / or continue administration of the disclosed AAV particles, the disclosed carrier, the disclosed nucleic acid molecule, the disclosed pharmaceutical formulation, the disclosed therapeutic agent, and / or the disclosed immunomodulator, or any combination thereof. In one aspect, metabolic and / or physiological data may be communicated to clinicians and to the treatment plan.

[0281] In one aspect of the disclosed methods for inhibiting and / or minimizing disease progression, techniques for monitoring, measuring, and / or assessing the restoration of one or more aspects of cellular homeostasis and / or cellular function may include qualitative (or subjective) means as well as quantitative (or objective) means. These means are known to those skilled in the art. For example, representative regulatory variables and sensors related to systemic homeostasis are provided as described above.

[0282] In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression may further include performing one or more invasive or non-invasive diagnostic assessments on the subject. Diagnostic assessments are known in the art. In one aspect, the disclosed non-invasive diagnostic assessments may include x-ray, computed tomography (CT) scan, magnetic resonance imaging (MRI) scan, ultrasound, positron emission tomography (PET) scan, or any combination thereof. In one aspect, the disclosed invasive diagnostic assessments may include tissue biopsy or exploratory surgery.

[0283] In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression may be used to repair diseased and / or dysfunctional cell types in one or more diseased and / or dysfunctional cells, tissues, and / or organs.

[0284] In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression, compared to pre-treatment levels, can be used to improve and / or enhance the quality of life of a subject. In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression, compared to the subject's pre-treatment quality of life, can improve the subject's quality of life by at least 50%. In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression, compared to pre-treatment levels, can be used to improve and / or enhance the quality of life of a subject. In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression, compared to the subject's pre-treatment quality of life, can improve the subject's quality of life by at least 50%.

[0285] In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression can be used to reduce and / or alleviate one or more symptoms related to and / or associated with a subject's genetic disease and / or genetic condition. In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression can be used to prevent undesirable physiological changes, diseases, pathological conditions, or symptoms in a subject. In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression can be used to inhibit physiological changes, diseases, pathological conditions, or symptoms in a subject, i.e., to halt their development. In another aspect, the disclosed methods for inhibiting and / or minimizing disease progression can be used to eliminate physiological changes, diseases, pathological conditions, or symptoms in a subject, i.e., to induce disease regression.

[0286] In one aspect of the disclosed methods for inhibiting and / or minimizing disease progression, administration to the subject may include...Contacting one or more cells with one or more disclosed nucleic acid molecules, disclosed carriers or disclosed AAV particles, disclosed pharmaceutical formulations or any combination thereof.

[0287] In one aspect of the disclosed methods for inhibiting and / or minimizing disease progression, administration of the disclosed nucleic acid molecules, disclosed carriers, disclosed AAV particles, disclosed pharmaceutical formulations or any combination thereof may include one or more routes. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intrauterine administration, intrahepatic administration, intravaginal administration, ocular administration, intraauricular administration, intracerebral administration, rectal administration, sublingual administration, buccal administration and parenteral administration, including injectable administration, such as intravenous administration, intra-CSF administration, intra-arterial administration, intramuscular administration and subcutaneous administration. Administration may also include intra-hepatic artery administration or administration via the portal vein (HPV). The administration of the disclosed nucleic acid molecule, the disclosed carrier, the disclosed AAV particles, the disclosed pharmaceutical formulation, or any combination thereof may include direct administration to the CNS (e.g., intraparenchymal, intraventricular, intrasheathic cisterns, intrasheath (lumbar), deep gray matter delivery, convection-enhanced delivery to deep gray matter) or PNS. Administration may be continuous or intermittent.

[0288] In one aspect, the disclosed methods for inhibiting and / or minimizing the progression of a genetic disease or condition may be administered to a subject via multiple routes of administration. In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression may employ a first route of administration, which may be the same as or different from a second and / or subsequent route of administration.

[0289] In one aspect of the disclosed methods for inhibiting and / or minimizing the progression of a genetic disease or condition, the therapeutically effective amount of the disclosed carrier or the disclosed pharmaceutical formulation may include a range from about 1 × 10¹⁰ vg / kg to about 2 × 10¹⁴ vg / kg. In one aspect, for example, the therapeutically effective amount of the disclosed carrier or the disclosed pharmaceutical preparation may include a dose of about 1 × 10¹¹ vg / kg to about 8 × 10¹³ vg / kg or about 1 × 10¹² vg / kg to about 8 × 10¹³ vg / kg. In one aspect, the therapeutically effective amount of the disclosed carrier or the disclosed pharmaceutical preparation may include a dose of about 1 × 10¹³ vg / kg to about 6 × 10¹³ vg / kg. In one aspect, the therapeutically effective amount of the disclosed carrier or the disclosed pharmaceutical preparation may include at least about 1 × 10¹⁰ vg / kg, at least about 5 × 10¹⁰ vg / kg, at least about 1 × 10¹¹ vg / kg, at least about 5 × 10¹¹ vg / kg, at least about 1 × 10¹² vg / kg, at least about 5 × 10¹² vg / kg, at least about 1 × 10¹³ vg / kg, at least about 5 × 10¹³ vg / kg, or at least about 1 × 10¹⁴ vg / kg.The therapeutically effective amount of the disclosed carrier or the disclosed pharmaceutical preparation may include a dose not exceeding about 1 × 10¹⁰ vg / kg, not exceeding about 5 × 10¹⁰ vg / kg, not exceeding about 1 × 10¹¹ vg / kg, not exceeding about 5 × 10¹¹ vg / kg, not exceeding about 1 × 10¹² vg / kg, not exceeding about 5 × 10¹² vg / kg, not exceeding about 1 × 10¹³ vg / kg, not exceeding about 5 × 10¹³ or not exceeding about 1 × 10¹⁴ vg / kg. In one aspect, the therapeutically effective amount of the disclosed carrier or the disclosed pharmaceutical preparation may include a dose of about 1 × 10¹² vg / kg or about 1 × 10¹¹ vg / kg. In one aspect, the therapeutically effective amount of the disclosed carrier or the disclosed pharmaceutical formulation may be administered in single or multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses) as required for the desired therapeutic outcome.

[0290] In one aspect, the therapeutically effective amount of the disclosed carrier or the disclosed pharmaceutical formulation may include a range from about 1×10¹² vg to about 1×10¹⁷ vg per subject in total. In one aspect, the therapeutically effective amount of the disclosed carrier or the disclosed pharmaceutical formulation may include a range of about 1×10¹², about 1×10¹³ vg, about 1×10¹⁴ vg, about 1×10¹⁵ vg, about 1×10¹⁶ vg, or about 1×10¹⁷ vg per subject in total.

[0291] In one aspect, the therapeutically effective amount of the disclosed carrier or the disclosed pharmaceutical preparation may include about 1 × 10⁶ DRP / mL to about 1 × 10¹⁴ DRP / mL. In one aspect, the therapeutically effective amount of the disclosed carrier or the disclosed pharmaceutical preparation may include about 1 × 10⁶ DRP / mL, 1 × 10⁷ DRP / mL, 1 × 10⁸ DRP / mL, 1 × 10⁹ DRP / mL, 1 × 10¹⁰ DRP / mL, 1 × 10¹¹ DRP / mL, 1 × 10¹² DRP / mL, 1 × 10¹³ DRP / mL, or 1 × 10¹⁴ DRP / mL. In one aspect, the therapeutically effective amount of the disclosed carrier or the disclosed pharmaceutical preparation may include a range determined by a person skilled in the art.

[0292] In one aspect, the disclosed method of inhibiting and / or minimizing the progression of a genetic disease or condition may further include administering a therapeutically effective amount of the therapeutic agent to a subject. The therapeutic agent may be any disclosed agent that achieves the desired clinical outcome.

[0293] In one aspect, methods for suppressing and / or minimizing the progression of genetic diseases or conditions may further includeThe method includes monitoring subjects for adverse reactions. In one aspect, in the absence of adverse reactions, the method may further include continuing treatment of the subject. In one aspect, in the presence of adverse reactions, the method may further include modifying the treatment steps. Methods for monitoring the health of subjects may include both subjective and objective criteria (and as described above). Such methods are known to those skilled in the art.

[0294] In one aspect, the disclosed method for inhibiting and / or minimizing disease progression may further include administering to the subject a therapeutically effective amount of an agent that can correct one or more aspects of a dysregulated metabolic or enzymatic pathway. In one aspect, such an agent may include an enzyme for enzyme replacement therapy. In one aspect, the disclosed enzyme may replace any enzyme in a dysregulated or dysfunctional metabolic or enzymatic pathway. In one aspect, the disclosed method for inhibiting and / or minimizing disease progression may include replacing one or more enzymes in a dysregulated or dysfunctional metabolic pathway.

[0295] In one aspect, the disclosed method for inhibiting and / or minimizing disease progression of a genetic disease or condition may further include administering one or more immunomodulators. In one aspect, the disclosed immunomodulator may be methotrexate, rituximab, intravenous gamma globulin, or bortezomib or a combination thereof. In one aspect, the disclosed immunomodulator may be bortezomib or SVP-rapamycin. In one aspect, the disclosed immunomodulator may be tacrolimus. In one aspect, the disclosed immunomodulator, such as methotrexate, may be administered in an instantaneous low to high dose. In one aspect, the disclosed immunomodulator may be administered at a dose of about 0.1 mg / kg body weight to about 0.6 mg / kg body weight. In one aspect, the disclosed immunomodulator may be administered at a dose of about 0.4 mg / kg body weight. In one aspect, the disclosed immunomodulator may be administered at a daily dose of about 0.4 mg / kg body weight for 3 to 5 or more cycles, each cycle lasting up to three days. In one aspect, the disclosed immunomodulator may be administered at a daily dose of about 0.4 mg / kg body weight for at least 3 cycles, each cycle lasting three days. In one aspect, those skilled in the art can determine the appropriate number of cycles. In one aspect, the disclosed immunomodulator may be administered multiple times as needed to achieve the desired clinical effect.

[0296] In one aspect, the disclosed immunomodulator may be administered orally about 1 hour before the disclosed therapeutic agent. In one aspect, the disclosed immunomodulatory agent may be administered subcutaneously approximately 15 minutes prior to the disclosed therapeutic agent. In another aspect, the disclosed immunomodulatory agent may be administered concurrently with the disclosed therapeutic agent. In another aspect, the disclosed immunomodulatory agent may be administered orally approximately one hour or several days prior to the disclosed nucleic acid molecule, disclosed carrier, disclosed pharmaceutical preparation, or a combination thereof. (See specification 55 / 109, page 59, CN 121195069 A)In one aspect, the disclosed immunomodulatory agent may be administered subcutaneously approximately 15 minutes or several days prior to the disclosed nucleic acid molecule, the disclosed carrier, the disclosed pharmaceutical preparation, or a combination thereof. In another aspect, the disclosed immunomodulatory agent may be administered in parallel with the disclosed nucleic acid molecule, the disclosed carrier, the disclosed pharmaceutical preparation, or a combination thereof.

[0297] In one aspect, the disclosed method of inhibiting and / or minimizing disease progression may further include administering one or more proteasome inhibitors (e.g., bortezomib, carfilzomib, marizomib, ixazomib, and opzomib). In one aspect, the proteasome inhibitor may be an agent that acts on plasma cells (e.g., daratumumab). In one respect, the reagents acting on plasma cells can be melphalan hydrochloride, melphalan, pamidronate disodium, carmustine, carfilzomib, cyclophosphamide, daratumumab, doxorubicin hydrochloride liposomes, doxorubicin hydrochloride liposomes, erlotuzumab, melphalan hydrochloride, pambistat, ixazomib citrate, carfilzomib, lenalidomide, melphalan hydrochloride, prasaf, ixazomib citrate, pamidronate disodium, pambistat, prasaf, pomalidomide, lenalidomide, celinido, thalidomide, thalidomide, bortezomib, celinido, zoledronic acid, or zoledronic acid.

[0298] In one aspect, the disclosed method for inhibiting and / or minimizing disease progression stability may further include administering one or more proteasome inhibitors or plasma cell-acting agents prior to the administration of the disclosed nucleic acid molecule, the disclosed carrier, the disclosed AAV particles, the disclosed pharmaceutical formulation, or any combination thereof. In one aspect, the disclosed method for inhibiting and / or minimizing disease progression may include administering one or more proteasome inhibitors or one or more plasma cell-acting agents in parallel with the administration of the disclosed nucleic acid molecule, the disclosed carrier, the disclosed AAV particles, the disclosed pharmaceutical formulation, or any combination thereof. In one aspect, the disclosed method for inhibiting and / or minimizing disease progression may include administering one or more proteasome inhibitors or one or more plasma cell-acting agents after the administration of the disclosed nucleic acid molecule, the disclosed carrier, the disclosed AAV particles, the disclosed pharmaceutical formulation, or any combination thereof. In one aspect, the disclosed method for inhibiting and / or minimizing disease progression may further include administering one or more proteasome inhibitors more than once. In one aspect, the disclosed method for inhibiting and / or minimizing disease progression may include repeatedly administering one or more proteasome inhibitors over time.

[0299] In one aspect, the disclosed methods for inhibiting and / or minimizing the progression of a genetic disease or condition may further include administering one or more immunosuppressants. In one aspect, the immunosuppressants may be, but are not limited to, azathioprine, methotrexate, sirolimus, anti-thymocyte globulin (ATG), cyclosporine (CSP), mycophenolate mofetil (MMF), or steroids.Or a combination thereof. In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression may include administering one or more immunosuppressants more than once. In one aspect, the disclosed methods may include repeatedly administering one or more immunosuppressants over time. In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression may include administering compounds that target or alter antigen presentation or humoral or cell-mediated or innate immune responses.

[0300] In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression of a genetic disease or condition may further include administering compounds that have a therapeutic effect against B cells and / or compounds that target or alter antigen presentation or humoral or cell-mediated immune responses. In one aspect, the disclosed compounds may be rituximab, methotrexate, intravenous gamma globulin, anti-CD4 antibody, anti-CD2, anti-FcRN antibody, BTK inhibitor, anti-IGF1R antibody, CD19 antibody (e.g., inelolizumab), anti-IL6 antibody (e.g., tocilizumab), antibody against CD40, IL2 mutant protein, or a combination thereof. This document also discloses Treg infusions (e.g., antigen-specific Treg cells for AAV) that can be administered as a means of aiding immune tolerance.

[0301] In one aspect, the disclosed methods of inhibiting and / or minimizing disease progression may further include the administration of lipid nanoparticles (LNPs). In one aspect, the LNPs may be organ-targeted. In one aspect, the LNPs may be liver-targeted or testis-targeted. For example, in one aspect, mRNA therapies with LNP encapsulation for systemic delivery to a subject have the potential to restore the functionality and / or structural integrity of a missing, defective, and / or mutated protein or enzyme.

[0302] In one aspect, the disclosed methods of inhibiting and / or minimizing disease progression of a genetic disease or condition may further include treating a subject who has developed or may develop neutralizing antibodies (ABs) to the disclosed nucleic acid molecule, the disclosed vector, the disclosed AAV particles, the disclosed pharmaceutical formulation, or any combination thereof. In one aspect, treating a subject who has developed or may develop neutralizing antibodies may include plasmapheresis and immunosuppression. In one aspect, the disclosed methods may include using immunosuppression to reduce overall T-cell, B-cell, and / or plasma cell populations, thereby decreasing innate immune responses, inflammatory responses, and antibody levels. In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression may include administering an IgG degrading agent that depletes pre-existing neutralizing antibodies. In one aspect, the disclosed methods may include administering IdeS or IdeZ, rapamycin, and / or SVP-rapamycin to a subject. In one aspect, the disclosed methods for inhibiting and / or minimizing disease progression...The method may include administering tacrolimus. In one aspect, the disclosed IgG degrading agent is bacterial IdeS or IdeZ.

[0303] In one aspect, the disclosed method of inhibiting and / or minimizing disease progression may include repeating the disclosed administration steps, for example, repeatedly administering the disclosed nucleic acid molecule, the disclosed carrier, the disclosed AAV particles, the disclosed pharmaceutical formulation, the disclosed therapeutic agent, the disclosed immunomodulator, the disclosed proteasome inhibitor, the disclosed immunosuppressant, the disclosed compound that exerts a therapeutic effect against B cells, and / or the disclosed compound that targets or modifies antigen presentation or humoral or cell-mediated immune responses.

[0304] In one aspect, the disclosed method of inhibiting and / or minimizing disease progression may include modifying one or more of the disclosed steps. For example, modifying one or more steps of the disclosed method may include modifying or altering one or more features or aspects of one or more steps of the disclosed method. For example, in one aspect, the method can be modified by changing the amount of one or more disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed carriers, one or more disclosed pharmaceutical preparations, or any combination thereof administered to the subject, or by changing the frequency of administration of one or more disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed carriers, one or more disclosed pharmaceutical preparations, or any combination thereof to the subject, or by changing the duration of administration of one or more disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed carriers, one or more disclosed pharmaceutical preparations, or any combination thereof to the subject.

[0305] In one aspect, the disclosed method of inhibiting and / or minimizing disease progression can be modified by changing the amount of one or more disclosed therapeutic agents, disclosed immunomodulators, disclosed proteasome inhibitors, disclosed immunosuppressants, disclosed compounds that act against B cells and / or disclosed compounds that target or modify antigen presentation or humoral or cell-mediated immune responses, or by changing the frequency of administration of one or more disclosed therapeutic agents, disclosed immunomodulators, disclosed proteasome inhibitors, disclosed immunosuppressants, disclosed compounds that act against B cells and / or disclosed compounds that target or modify antigen presentation or humoral or cell-mediated immune responses to the subject.

[0306] In one aspect, the disclosed method of inhibiting and / or minimizing disease progression may include the concurrent administration of one or more disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed carriers, one or more disclosed pharmaceutical formulations, one or more disclosed therapeutic agents, one or more disclosed immunomodulators, one or more disclosed proteasome inhibitors, one or more disclosed immunosuppressants, one or more disclosed compounds that act against B cells.The disclosed compounds, one or more disclosed compounds that target or modify antigen presentation or humoral or cell-mediated immune responses, or any combination thereof. In one aspect, the disclosed immunomodulators may be administered before or after administration of the disclosed therapeutic agent. In one aspect, the disclosed methods for inhibiting and / or minimizing the progression of a genetic disease or condition may further include generating the disclosed nucleic acid molecule, the disclosed vector, the disclosed AAV particles, the disclosed pharmaceutical formulation, or any combination thereof. Specification 57 / 109 pages 61 CN 121195069 A

[0307] In one aspect, the disclosed methods for inhibiting and / or minimizing the progression of a disease may further include gene editing of one or more related genes (e.g., those listed in Table 1 below).

[0308] Table 1 – Large Genes and Identification of Affected Chromosomes and Mutations # Specification 58 / 109 Page 62 CN 121195069 A Specification 59 / 109 Page 63 CN 121195069 A Specification 60 / 109 Page 64 CN 121195069 A Specification 61 / 109 Page 65 CN 121195069 A Specification 62 / 109 Page 66 CN 121195069 A Specification 63 / 109 Page 67 CN 121195069 A Specification 64 / 109 Page 68 CN 121195069 A

[0309] In one respect, the disclosed methods can be specifically used to treat subjects with autosomal dominant genetic diseases or conditions (e.g., progeria, achondroplasia, antithrombin III deficiency, Ehlers-Danlos syndrome, Gilbert's disease, hereditary hemorrhagic telangiectasia, hereditary elliptic polycythemia, hereditary spherocytosis, Huntington's disease, idiopathic hypoparathyroidism, intestinal polyposis, marble bone disease, Marfan syndrome, neurofibromatosis, polycystic kidney disease (adults), protein C deficiency, osteogenesis imperfecta, TracyCollins syndrome, tuberous sclerosis, Villebrand disease, etc.).

[0310] VIII. Examples Due to the presence of pathogenic mutations exceeding AAV packaging capacity in more than 500 genes, some efforts have been made to circumvent this barrier of large gene expression. While these techniques differ, general approaches remain broadly similar in strategy. In short, a dual AAV vector approach is employed; the DNA sequence encoding the target protein is split and packaged into separate vectors. After co-infecting target cells with the two vectors, the genomes of the two vectors recombine with each other via inverted repeat sequences or overlapping complementary sequences, forming a single genome carrying a reconstructed DNA sequence expressing the complete target protein. Although this strategy...While this is feasible, the efficiency of recombination between genomes limits its widespread adoption.

[0311] Additionally, other efforts to phenotypically correct genes unsuitable for classical AAV-mediated gene therapy have inspired a method involving the manipulation of endogenous messenger RNA (the conduit between DNA and protein). An RNA editing strategy called spliceosome-mediated RNA transsplicing (SMART) has been developed as a strategy to introduce large, precise modifications into the primary structure of RNA transcripts, independent of target transcript length. The aim of this method is to hijack the cellular RNA processing mechanism to incorporate the desired sequence into the endogenous transcript. In this strategy, a recombinant RNA molecule is introduced into the cell, comprising three essential components: an RNA-targeting motif, a hemiintron sequence, and the primary sequence of the desired RNA to be ligated into the endogenous RNA transcript. The RNA-targeting motif consists of an oligonucleotide segment antisense to the intron of the endogenous target pre-mRNA. After the RNA-targeting motif pairs with the Watson-Crick bases of the endogenous intron, the hemiintron is recognized by the spliceosome. Depending on the desired splicing response, a 5' hemiintron promotes the splicing of the trans-splicing molecule to the 3' exon immediately adjacent to the targeted intron, or a 3' hemiintron linked to the exogenous RNA to be trans-spliced ​​promotes the splicing of the trans-splicing molecule to the 5' exon immediately adjacent to the targeted intron. This produces a mature chimeric RNA transcript consisting of either a recombinant 5' initiator linked to an endogenous 3' sequence or an endogenous 5' initiator linked to a recombinant 3' sequence. The advantage of this strategy is that a single AAV vector only needs to package the genome capable of producing trans-spliced ​​RNA molecules containing a portion of the sequence for the gene, avoiding the need to deliver the full-length protein-coding sequence to the cell. Since the entire recombinant RNA region of the chimeric RNA product can be specified by the user, the trans-spliced ​​RNA can contain the wild-type sequence of the target RNA, an inactivating mutation in the target RNA, or a modified RNA sequence encoding a new protein. However, similar to the split AAV vector approach, the low specificity and efficiency of RNA targeting via antisense RNA sequences hinder the widespread use of this technology in research and clinical settings. Despite its promise, unrestricted expression of CRISPR proteins (e.g., long-term expression) as a therapy presents challenges due to immunogenicity and off-target effects. Furthermore, CRISPR-based methods typically require the delivery of two constructs (i.e., a CRISPR effector protein and an RNA trans-splicing construct), leading to high dose requirements and low correction efficiency. This paper discloses a system for transcriptome engineering that does not require a CRISPR system. Many endogenous RNA-binding ribonucleoproteins exist in human cells, and the system disclosed herein...The system utilizes several RNA structures that interact with known human ribonucleoproteins to enable trans-splicing. As detailed herein, by incorporating these RNA structures into trans-splicing RNA, efficient trans-splicing of many targeted endogenous pre-mRNAs is achieved. This allows for the rewriting of large segments of mRNA.

[0312] Example 1 Validation of Trans-Splicing Efficiency To determine trans-splicing efficiency, a green fluorescence-based screening system was designed and constructed. The first step was to construct a reporter in which the green fluorescent protein (EGFP) open reading frame was divided into two halves. Introns were inserted between the two halves. Thus, if the construct is cis-spliced, it will produce mature RNA encoding EGFP and will express a green fluorophore during translation.

[0313] Next, we eliminated fluorescence expression by introducing an early stop codon into the first “exon” of the EGFP open reading frame (Figure 1 (left)). If the construct is cis-spliced, there will be no green fluorescence expression because the premature stop codon in the first half of the EGFP open reading frame terminates the translation of the full-length protein.

[0314] Green fluorescence expression can be restored by delivering trans-splicing RNA encoding the correct open reading frame for the first half of EGFP, followed by a hemiintron and guide RNA sequence. Therefore, green fluorescence is used as an indirect reading of trans-splicing efficiency (Figure 1). This reporter subsystem is the mainstay of our downstream assays.

[0315] The first trans-splicing RNA delivered to these cells (hereinafter referred to as SMaRT or spliceosome-mediated RNA trans-splicing) contains only the first half of EGFP, a hemiintron, and a 30 bp antisense targeting domain. The sequence was then cloned into a trans-splicing RNA vector to determine if it could enhance trans-splicing efficiency. These new constructs were transfected into HEK293 cells with a splitting GFP reporter, and after 48 hours, the green fluorescence intensity (percentage GFP% and average fluorescence intensity) was measured by flow cytometry as a representative of trans-splicing efficiency (Fig. 2A and Fig. 2B). Through this process, three (3) RNA structures produced significantly enhanced trans-splicing efficiency (RNA structures 4, 5, and 7). In Fig. 2A–Fig. 2B, the RNA targeting base sequence for RYR2 is listed in SEQ ID NO: 23.

[0316] Next, it was examined whether any member of this set of RNA sequences enhanced the 3' trans-splicing method. For this purpose, the same splitting GFP reporter was used, but with a slight modification, i.e., the stop codon was moved from the first half of the open reading frame to the second half (Fig. 1 (right)). When delivered to HEK293 cells, the construct itself did not express green fluorescence because cis-splicing preserved the premature stop codon. To restore EGFP expression, the open reading frame of EGFP completed after proper splicing was used.Trans-splicing RNA. Similar to the 5' editing method, trans-splicing RNA containing a 30 bp antisense targeting motif, followed by a half-intron and the latter half of an EGFP open reading frame (this construct is SMaRT). The same set of RNA structures was then introduced into the RNA molecule to improve trans-splicing efficiency.

[0317] The new construct was co-transfected with a splitting GFP reporter into HEK 293 cells. Then, 48 hours later, the green fluorescence intensity (GFP% percentage and mean fluorescence intensity) (as a representative of trans-splicing efficiency) was measured by flow cytometry. (Fig. 3A-Fig. 3B). By this method, four RNA structures (RNA structures 1, 2, 10, and 11) that produced significantly enhanced trans-splicing efficiency were identified. This set was repeated on the second intron target intron (Fig. 3C-Fig. 3D), and a similar trend of fold changes in editing efficiency was observed. In Figures 3A-3B, the RNA targeting motif for RYR2 is in SEQ ID NO: 23, while in Figures 3C-3D, the RNA targeting motif for LMNA is in SEQ ID NO: 22.

[0318] Following these results, the trans-splicing assays for the highest hits (RNA structures 1 and 2) were repeated. A second control RNA structure (i.e., a direct repeat from Ruminococcus flavefaciens XPD3002 (RfxCas13d)) was also used. The basic principle of this control was to ensure that the enhancement of RNA editing was unique to these RNA structures, rather than an artificial phenomenon of including extra sequence length / complexity in the trans-splicing RNA. In fact, only RNA structures 1 and 2 showed enhanced editing efficiency, while the Cast13 direct repeat sequence had a slightly adverse effect on trans-splicing efficiency (Figures 4A-4B).

[0319] Finally, the new target intron was cloned into the split GFP reporter and the editing efficiency was determined using RNA structure 2 and several guide RNA candidates. Editing was observed at the new target in more than 50% of GFP-positive cells (Fig. 5A-Fig. 5B). In Fig. 5A-Fig. 5B, the RNA targeting motif for FXN is listed in SEQ ID NO: 24. In Table 2, structures 4 and 5 contain cloning sites in which guide RNA is inserted, which are bolded sequences.

[0320] Table 2 – List of RNA structures

[0321] Table 3 – List of plasmids used in the examples

[0322] Example 2 Trans-splicing of DMD (dystrophin) is known in the art (e.g., Gene ID 1756), and the nucleotide sequence may contain accession number NG.Nucleotides 5001-2225382 in 012232.1. DMD spans a genome greater than 2 Mb and is a large protein encoded by CN 121195069 A, containing an N-terminal actin-binding domain and multiple spectrin repeats. The encoded protein (SEQ ID NO: 17) forms a component of the dystrophin-glycoprotein complex (DGC), which bridges the internal cytoskeleton and extracellular matrix. Deletions, duplications, and point mutations at this gene locus can lead to Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), or cardiomyopathy. Approximately 1750 pathogenic mutations of DMD have been reported to date. Constructs addressing more than 1000 of these pathogenic mutations are described in detail below. Here, a 5' substitution construct for DMD is generated using a plasmid based on the schematic diagram presented in Figure 7A. The 5' substitution construct targets exons 1-23 of DMD (SEQ ID NO: 33). The 5' replacement construct corrected one or more of the 618 mutations identified in Table 4 below.

[0323] Table 4 - List of mutations in exons 1-23 of DMD (Page 68 / 109 of the specification) 72 CN 121195069 A (Page 69 / 109 of the specification) 73 CN 121195069 A (Page 70 / 109 of the specification) 74 CN 121195069 A (Page 71 / 109 of the specification) 75 CN 121195069 A (Page 72 / 109 of the specification) 76 CN 121195069 A (Page 73 / 109 of the specification) 77 CN 121195069 A (Page 74 / 109 of the specification) 78 CN 121195069 A (Page 75 / 109 of the specification) 79 CN 121195069 A (Page 76 / 109 of the specification) 80 CN 121195069 A (Page 77 / 109 of the specification) 81 CN 121195069 A Instruction Manual 78 / 109 pages 82 CN 121195069 A Instruction Manual 79 / 109 pages 83 CN 121195069 A Instruction Manual 80 / 109 pages 84 CN 121195069 A Instruction Manual 81 / 109 pages 85 CN 121195069 A Instruction Manual 82 / 109 pages 86 CN 121195069 A Instruction Manual 83 / 109 pages 87 CN 121195069 A Instruction Manual 84 / 109 pages 88 CN 121195069 A

[0324] The AAV vector based on the plasmid construct shown in Figure 7A was delivered to subjects with one or more 5' mutations in DMD (e.g., Table 4). Following administration of a therapeutically effective amount of the AAV vector (e.g., about 1 × 10¹⁰ vg / kg to about 2 × 10¹⁴ vg / kg), cells, tissues, and / or organs of one or more subjects produced chimeric RNA molecules encoding corrected and / or restored DMD. The production of chimeric RNA molecules was confirmed by obtaining samples from the subjects and verifying the expression levels of corrected and / or restored DMD (by comparing post-treatment levels of surgical / functional DMD with pre-treatment levels of surgical / functional DMD in the subjects). Subjects experienced suppression and / or minimization of DMD disease progression. The subjects' quality of life improved. Here, a 3' substitution construct for DMD was generated using a plasmid based on the schematic diagram presented in Figure 7B. The 3' substitution construct is targeting exons 53–79 of DMD (SEQ ID NO: 32). The 3' replacement construct corrected one or more of the 446 mutations identified in Table 5 below.

[0325] Table 5 - List of mutations in exons 53-79 of DMD (Manuscript 85 / 109 page 89 CN 121195069 A, Manuscript 86 / 109 page 90 CN 121195069 A, Manuscript 87 / 109 page 91 CN 121195069 A, Manuscript 88 / 109 page 92 CN 121195069 A, Manuscript 89 / 109 page 93 CN 121195069 A, Manuscript 90 / 109 page 94 CN 121195069 A, Manuscript 91 / 109 page 95 CN 121195069 A, Manuscript 92 / 109 page 96 CN 121195069 A, Manuscript 93 / 109 page 97 CN 121195069 A, Manuscript 94 / 109 page 98 CN) 121195069 A Instruction Manual 95 / 109 pages 99 CN 121195069 A Instruction Manual 96 / 109 pages 100 CN 121195069 A Instruction Manual 97 / 109 pages 101 CN 121195069 A Instruction Manual 98 / 109 pages 102 CN 121195069 A Instruction Manual 99 / 109 pages 103 CN 121195069 A Instruction Manual 100 / 109 pages 104 CN 121195069 A Instruction Manual 101 / 109 pages 105 CN 121195069 A Instruction Manual 102 / 109 pages 106CN 121195069 A Instruction Manual 103 / 109 Pages 107 CN 121195069 A Instruction Manual 104 / 109 Pages 108 CN 121195069 A Instruction Manual 105 / 109 Pages 109 CN 121195069 A Instruction Manual 106 / 109 Pages 110 CN 121195069 A Instruction Manual 107 / 109 Pages 111 CN 121195069 A

[0326] The AAV vector based on the plasmid construct shown in FIG7B was delivered to subjects with one or more 3' mutations (e.g., Table 5). After administration of a therapeutically effective amount of the AAV vector (e.g., about 1 × 10¹⁰ vg / kg to about 2 × 10¹⁴ vg / kg), cells, tissues and / or organs of one or more subjects produced chimeric RNA molecules encoding corrected and / or restored DMD. The generation of chimeric RNA molecules was confirmed by obtaining samples from subjects and verifying the expression levels of corrected and / or restored DMD (by comparing the post-treatment levels of surgical / functional DMD with the pre-treatment levels of the subjects' surgical / functional DMD). Subjects experienced suppression and / or minimization of DMD disease progression. Subjects experienced improved quality of life.

[0327] Summary of Examples The compositions and methods disclosed herein are superior to previously disclosed compositions and methods, such as SMART. Numerous advantages are imparted by the disclosed system. These surprising and unexpected advantages include: (i) the elimination of the need for additional effector proteins (e.g., CRISPR-based trans-splicing); (ii) the ability to modify effector RNA structures to bind to endogenous splicing mechanisms and enable 3' or 5' end substitution of pre-splicing mRNA; (iii) the ability to deliver constructs in a single AAV vector or as a trans-splicing RNA fragment using non-viral delivery systems (e.g., LNP); (iv) the ability to edit large segments of mRNA that constitute multiple exons; (v) the ability to correct mRNA in a single "knockdown and replacement" approach, particularly useful in autosomal dominant genetic disorders, using a single trans-splicing construct without having to provide two different constructs for knockdown and replacement; and (vi) the ability to prevent any gene overexpression, with correction based on the provided trans-splicing fragment, which depends on the level of the endogenous mutant transcript (therefore, the corrected mRNA will never be expressed at a level higher than that of the endogenous disease transcript). While comprehensive, the list of advantages in the specification (pages 108 / 109, CN 121195069 A) is not exhaustive.

[0328] In summary, this document describes a method that is independent of CRISPR-based targeting and outperforms simple antisense base pairing.An efficient RNA editing platform. This enables the efficient rewriting of large segments of RNA, with implications for human health and basic biology. As a therapeutic agent, this will allow for the rewriting of genes beyond AAV packaging capacity, correcting dominant-negative mutations while maintaining the expression of target transcripts at endogenous levels. Instruction Manual 109 / 109 Page 113 CN 121195069 A Figure 1 Instruction Manual Figure 1 / 20 Page 114 CN 121195069 A Figure 2A Instruction Manual Figure 2 / 20 Page 115 CN 121195069 A Figure 2B Instruction Manual Figure 3 / 20 Page 116 CN 121195069 A Figure 3A Instruction Manual Figure 4 / 20 Page 117 CN 121195069 A Figure 3B Instruction Manual Figure 5 / 20 Page 118 CN 121195069 A Figure 3C Instruction Manual Figure 6 / 20 Page 119 CN 121195069 A Fi...

Claims

1. Nucleic acid molecules, which include: One or more RNA structures comprising the sequence of any one of SEQ ID NO: 01-SEQ ID NO: 09; One or more RNA targeting motifs; 3' half intron; and The exogenous RNA is to be trans-spliced ​​to the targeted endogenous pre-mRNA.

2. Nucleic acid molecules, which include: The exogenous RNA is to be trans-spliced ​​to the targeted endogenous pre-mRNA; 5' hemi-intron; One or more RNA targeting motifs; One or more RNA structures comprising the sequence of any one of SEQ ID NO: 01-SEQ ID NO:

09.

3. The nucleic acid molecule of claim 1 or claim 2, wherein the targeted endogenous pre-mRNA comprises one or more mutations in one or more exons.

4. The nucleic acid molecule of claim 3, wherein one or more mutations are located in the 3' portion of the targeted endogenous pre-mRNA.

5. The nucleic acid molecule of claim 3, wherein one or more mutations are located in the 5' portion of the targeted endogenous pre-mRNA.

6. The nucleic acid molecule of claim 1, wherein the targeted endogenous pre-mRNA encodes a protein-encoding gene.

7. The nucleic acid molecule of claim 6, wherein the protein-coding gene comprises one or more coding regions of: ABCA1 , ABCA12 , ABCA13 , ABCA2 , ABCA3 , ABCA4 , ABCA5 , ABCC1 , ABCC2 , ABCC6 , ABCC8 , ABCC9 , ACAN , ADAMTS13 , ADCY10 , ADGRV1 , AGL , AGRN , AHDC1 , ALK , ALMS1 , ALPK3 , ALS2 , ANAPC1 , ANK1 , ANK2 , ANK3 , ANKRD11 , ANKRD26 , APC , APC2 , APOB , ARFGEF2 , ARHGAP31 , ARHGEF10 , ARHGEF18 , ARID1A , ARID1B , ARID2 , ASH1L , ASPM , ASXL1 , ASXL2 , ASXL3 , ATM , ATP7A , ATP7B , ATR , ATRX , BAZ1A , BAZ2B , BCOR , BCORL1 , BDP1 , BLM , BPTF , BRCA1 , BRCA2 , BRD4 , BRWD3 , C2CD3 , C3 , C5 , CACNA1A , CACNA1B , CACNA1C , CACNA1D , CACNA1E , CACNA1F 、 CACNA1G 、 CACNA1H 、 CACNA1S 、 CAD 、 CAMTA1 、 CARMIL2 、 CC2D2A 、 CCDC88A 、 CCDC88C 、 CCNB3 、 CDH23 、 CDK13 、 CDK5RAP2 、 CELSR1 、 CEMIP2 、 CENPE 、 CENPF 、 CENPJ 、 CEP152 、 CEP164 、 CEP250 、 CEP290 、 CFAP43 、 CFAP44 、 CFAP65 、 CFTR / ABCC7 、 CHD1 、 CHD2 、 CHD3 、 CHD4 、 CHD7 、 CHD8 、 CIC 、 CIT 、 CLIP1 、 CLTC 、 CNOT1 、 CNTNAP1 、 COL11A1 、 COL11A2 、 COL12A1 、 COL17A1 、 COL18A1 、 COL1A1 、 COL1A2 、 COL27A1 、 COL2A1 、 COL3A1 、 COL4A1 、 COL4A2 、 COL4A3 、 COL4A4 、 COL4A5 、 COL4A6 、 COL5A1 、 COL5A2 、 COL6A3 、 COL7A1 、 CPAMD8 、 CPLANE1 、 CPS1 、 CPSF1 、 CRB1 、 CREBBP 、 CUBN 、 CUL7 、 CUX1 、 DCC 、 DCHS1 、 DEPDC5 、 DICER1 、 DIP2B 、 DLC1 、 DMD 、 DMXL2 、 DNAH1 、 DNAH1 、 DNAH17 、 DNAH2 、 DNAH5 、 DNAH7 、 DNAH8 、 DNAH9 、 DNMBP 、 DNMT1 、 DOCK2 、 DOCK3 、 DOCK6 、 DOCK7 、 DOCK8 、 DSCAM 、 DSP 、 DST 、 DUOX2 、 DYNC1H1 、 DYNC2H1 、 DYSF 、 EIF2AK4 、 EP300 、 EPG5 、 ERCC6 、 ERCC6L2 、 EXPH5 、 EYS 、 F5 、 F8 、 FANCA 、 FANCD2 、 FANCM 、 FAT1 、 FAT4 、 FBN1 、 FBN2 、 FLG 、 FLG2 、 FLNA 、 FLNB 、 FLNC 、 FLT4 、 FMN2 、 FN1 、 FRAS1 、 FREM1 、 FREM2 、 FSIP2 、 FXN 、 FYCO1 、 GLI2 、 GLI3 、 GPR179 、 GREB1L 、 GRIN2A 、 GRIN2B 、 GRIN2D 、 HCFC1 、 HECW2 、 HERC1 、 HERC2 、 HFM1 、 HIVEP1 、 HIVEP2 、 HMCN1 、 HSPG2 、 HTT 、 HUWE1 、 HYDIN 、 IFT140 、 IFT172 、 IGF1R 、 IGF2R 、 IGSF1 、 INSR 、 INTS1 、 IQSEC2 、 ITGB4 、 ITPR1 、 ITPR2 、 JMJD1C 、 KALRN 、 KANK1 、 KAT6A 、 KAT6B 、 KDM3B 、 KDM5B 、 KDM5C 、 KDM6A 、 KDM6B 、 KDR 、 KIAA0586 、 KIAA1109 、 KIAA1549 、 KIDINS2 、 KIF1 、 KIF1A 、 KIF1B 、 KIF21A 、 KIF26B 、 KIF7 、 KMT2A 、 KMT2B 、 KMT2C 、 KMT2D 、 KMT2E 、 KNL1 、 LAMA1 、 LAMA2 、 LAMA3 、 LAMA4 、 LAMA5 、 LAMB1 、 LAMB2 、 LAMC3 、 LCT 、 LMNA 、 LOXHD1 、 LPA 、 LRBA 、 LRP1 、 LRP2 、 LRP4 、 LRP5 、 LRP6 、 LRPPRC 、 LRRK1 、 LRRK2 、 LTBP2 、 LTBP4 、 LYST 、 MACF1 、 MADD 、 MAGIC2 、 MAP1B 、 MAP3K1 、 MAPK8IP3 、 MAPKBP1 、 MAST1 、 MBD5 、 MCM3AP 、 MED12 、 MED12L 、 MED1 、 MED13L 、 MED23 、 MEGF8 、 ALSO 、 MLH3 、 MPDZ 、 MSH6 、 MTOR 、 MYH1 、 MYH1 、 MYH1 、 MYH2 、 MYH3 、 MYH6 、 MYH7 、 MYH7B 、 MYH8 、 MYH9 、 MYLK 、 MYO15A 、 MYO18B 、 MYO3A 、 MYO5A 、 MYO5B 、 MYO7A 、 MYO9A 、 NALCN 、 NBAS 、 NBEA 、 NBEAL2 、 NCAPD2 、 NCAPD3 、 NEB 、 NEXMIF 、 NEXMIF 、 NF1 、 NFASC 、 NHS 、 NIN 、 NIPBL 、 NLRP1 、 NOTCH1 、 NOTCH2 、 NOTCH3 、 NPHP4 、 NRXN1 、 NRXN3 、 NSD1 、 NSD2 、 NUP155 、 NUP188 、 NUP205 、 OBSCN 、 OBSL1 、 OTOF 、 OTOG 、 OTOGL 、 PARD3 、 PBRM1 、 PCDH15 、 PCLO 、 PCNT 、 PHIP 、 PI4KA 、 PIEZO1 、 PIEZO2 、 PIK3C2A 、 PIKFYVE 、 PKD1 、 PKD1L1 、 PKHD1 、 PLCE1 、 PLEC 、 PLEKHG2 、 PNPLA6 、 POGZ 、 POLA1 、 POLE 、 POLR1A 、 POLR2A 、 POLR3A 、 PRG4 、 PRKDC 、 PRPF8 、 PRR12 、 PRX 、 PTCH1 、 PTPN23 、 PTPRF 、 PTPRJ 、 PTPRQ 、 PXDN 、 QRICH2 、 RAB3GAP2 、 RAI1 、 RALGAPA1 、 RANBP2 、 RB1CC1 、 RELN 、 RERE 、 REV3L 、 RIC1 、 RIMS1 、 RIMS 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 、 ​ 8. The nucleic acid molecule of any one of claims 3-7, wherein one or more mutations in one or more exons contribute to the pathogenesis in one or more cells.

9. The nucleic acid molecule of claim 8, wherein one or more cells are in a subject.

10. The nucleic acid molecule of claim 1, wherein the 5' portion of the targeted endogenous pre-mRNA is trans-spliced ​​with the exogenous RNA.

11. The nucleic acid molecule of claim 2, wherein the 3' portion of the targeted endogenous pre-mRNA is trans-spliced ​​with the exogenous RNA.

12. The nucleic acid molecule of claim 1 or claim 2, wherein the RNA targeting motif binds to the targeted endogenous pre-mRNA.

13. The nucleic acid molecule of claim 12, wherein the RNA targeting motif comprises an antisense oligonucleotide.

14. The nucleic acid molecule of claim 13, wherein the antisense oligonucleotide comprises about 15 nucleotides to about 50 nucleotides.

15. The nucleic acid molecule of claim 1, wherein the RNA targeting motif binds to the 3' end of the targeted endogenous pre-mRNA.

16. The nucleic acid molecule of claim 2, wherein the RNA targeting motif binds to the 5' end of the targeted endogenous pre-mRNA.

17. The nucleic acid molecule of claim 3, wherein the RNA targeting motif is specific to endogenous pre-mRNA having one or more mutations.

18. The nucleic acid molecule of claim 1, wherein the RNA targeting motif is directed to an intron immediately adjacent to the 3' of an exon of the targeted endogenous pre-mRNA to be spliced.

19. The nucleic acid molecule of claim 2, wherein the RNA targeting motif is directed to an intron immediately adjacent to the 5' of the exon of the targeted endogenous pre-mRNA to be spliced.

20. The nucleic acid molecule of claim 1, wherein the 3' half-intron is linked to the exogenous RNA to be trans-spliced ​​with the endogenous mRNA.

21. The nucleic acid molecule of claim 1, wherein the 3' half-intron is recognized by nuclear splicing components in the host cell.

22. The nucleic acid molecule of claim 1, wherein the 3' half-intron comprises (i) a 3' splice region containing a branch point, (ii) a polypyrimidine string, and (iii) a 3' splice acceptor site.

23. The nucleic acid molecule of claim 2, wherein the 5' half-intron is linked to the exogenous RNA to be trans-spliced ​​with the endogenous mRNA.

24. The nucleic acid molecule of claim 2, wherein the 5' half-intron is recognized by the nuclear splicing component in the host cell.

25. The nucleic acid molecule of claim 2, wherein the 5' half-intron contains a 5' splicing site.

26. A nucleic acid molecule according to any one of the preceding claims, wherein one or more RNA structures are bound to one or more RNA-binding proteins.

27. A nucleic acid molecule according to any one of the preceding claims, wherein one or more RNA structures (i) improve and / or enhance trans-splicing efficiency, (ii) stabilize the resulting chimeric RNA transcript, (iii) localize the RNA to the cell nucleus, (iv) stabilize the interaction between the targeted endogenous pre-mRNA molecule and the exogenous RNA to be trans-spliced, or (v) any combination thereof.

28. A viral vector comprising: a nucleic acid molecule of any one of claims 1-27.

29. Pharmaceutical preparations comprising: The carrier of claim 28; and One or more pharmaceutically acceptable carriers.

30. A method for producing a chimeric RNA molecule, the method comprising: Contacting the targeted endogenous pre-mRNA in one or more cells with the nucleic acid molecule of any one of claims 1-27 or the vector of claim 28, wherein the resulting chimeric RNA molecule comprises a trans-spliced ​​nucleic acid sequence.

31. The method of claim 30, wherein one or more cells are in a subject.

32. The method of claim 31, wherein the subject has been diagnosed with or is suspected of having a genetic disease or genetic condition.

33. A method for treating a genetic disease or hereditary condition, the method comprising: By administering a therapeutically effective amount of the carrier of claim 28 or the pharmaceutical preparation of claim 29 to a subject in need, a chimeric RNA molecule is generated in one or more cells; The resulting chimeric RNA molecule contains a trans-spliced ​​nucleic acid sequence; and The resulting chimeric RNA molecules restore one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic disorders.

34. The method of claim 33, wherein restoring one or more aspects of cellular homeostasis and / or cellular functional and / or metabolic disorders includes restoring the functional and / or structural integrity of missing, defective and / or mutated proteins or enzymes.

35. The method of claim 33, wherein the therapeutically effective amount of the carrier comprises about 1 × 10⁻⁶. 10 vg to approximately 2×10 14 vg.

36. The method of claim 33, further comprising administering to the subject a therapeutically effective amount of one or more therapeutic agents.

37. The method of claim 33, further comprising administering to the subject a therapeutically effective amount of one or more immune modulators.

38. The method of any one of claims 33-37, further comprising the step of repeated application.

39. The method of any one of claims 33-38, further comprising monitoring subjects for adverse effects.

40. The method of any one of claims 33-39, wherein one or more aspects of restoring cellular homeostasis and / or cellular functional and / or metabolic dysfunction include (i) correcting cellular starvation in one or more cells; (ii) correcting, preventing, reducing and / or alleviating autophagy; (iii) improving, enhancing, restoring and / or preserving mitochondrial functional and / or structural integrity; (iv) improving, enhancing, restoring and / or preserving organelle functional and / or structural integrity; (v) correcting enzyme dysregulation; (vi) reversing, inhibiting, preventing, stabilizing and / or slowing the rate of progression of a multisystemic manifestation of a genetic disease or condition; (vii) reversing, inhibiting, preventing, stabilizing and / or slowing the rate of progression of a genetic disease or condition, or (viii) any combination thereof.

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