Engineered nucleic acids and uses thereof

Abstract

Provided herein are engineered nucleic acids. The engineered nucleic acids may comprise various structural elements or sequences. These structural elements or sequences may facilitate the engineered nucleic acids to have increased stability, potency, expression level, nuclear localization, or ability to be expressed from an engineered promoter to promote higher expression, expression on desired tissues only, and to limit toxicity. Further provided herein are compositions comprising the engineered nucleic acids for various applications. The methods for using the same are also provided.

Description

[0001] ENGINEERED NUCLEIC ACIDS AND USES THEREOF

[0002] SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on October 24, 2025, is named “51899-002W02_Sequence_Listing_10_24_25” and is 130,435 bytes in size.

[0004] FIELD OF THE INVENTION

[0005] The present disclosure relates to nucleic acid molecules and expression constructs. The nucleic acid molecules and expression constructs disclosed herein have improved stability, potency, and function to target mutated / non-functional target sequences present in disease.

[0006] BACKGROUND

[0007] Effective treatment of human genetic disease can comprise modulation of splicing of genes using engineered splicing regulators.

[0008] SUMMARY OF THE INVENTION

[0009] Provided herein are nucleic acid molecules. In an aspect, a nucleic acid molecule comprises: (a) an engineered promoter; and (b) a sequence encoding an engineered Sm-binding site. In some embodiments, the engineered promoter comprises a tissue-specific promoter. In some embodiments, the engineered Sm-binding site is configured to recruit a splicing regulator to a ribonucleic acid (RNA) molecule encoded by the nucleic acid molecule. In some embodiments, the splicing regulator comprises a Sm ring protein. In some embodiments, the engineered Sm-binding site comprises a U1 Sm-binding sequence, a U2 Sm-binding sequence, a U4 Sm-binding sequence, a U5 Sm-binding sequence, a sequence (e.g., an optimized sequence) derived from major spliceosomal U small nuclear ribonucleoprotein (SmOpt), or any combination thereof. In some embodiments, the engineered Sm-binding site comprises a Sm-binding site derived from a human, a murine, a plant, a virus, or any combination thereof. In some embodiments, the engineered Sm-binding site comprises any one of SEQ ID NOs: 61-67. In some embodiments, the nucleic acid molecule further comprises a sequence of an engineered terminator. In some embodiments, the engineered terminator is configured to facilitate: (1) transcriptional termination of the RNA molecule, (2) 3’end processing of the RNA molecule, or (3) stabilization of the RNA molecule. In some embodiments, the engineered terminator comprises at least a 3’ terminator sequence. In some embodiments, the 3’ terminator sequence comprises: (a) an engineered stabilizing sequence or a secondary structure sequence; (b) a nuclease cleavage site; (c) a transcription termination domain (TTD); (d) or any combinations thereof. In some embodiments, the engineered stabilizing sequence is configured to form at least one secondary structure. In some embodiments, the engineered stabilizing sequence comprises: (a) a stem-loop sequence; (b) a triple helix sequence; (c) a quadruplex sequence; (d) a tRNA-like sequence; (e) a pseudoknot sequence; or (f) any combinations thereof. In some embodiments, the engineered stabilizing sequence comprises: (a) a small nucleolar RNA snoRNA (H / ACA and C / D box) derived stem-loop sequence; (b) a viral snRNA derived stem-loop sequence; (c) a plant snRNA derived stem-loop sequence; (d) a non-coding or viral RNA derived triple helix sequence; (e) a clustered regularly interspaced short palindromic repeats (CRISPR) single-guide (sg) RNA derived stem-loop sequence; (f) a long non-coding RNA (LncRNA) derived stem-loop sequence; (g) a PlWI-interacting RNA (piRNA) derived stem-loop sequence; (h) a stem-bulge RNA (sbRNA) derived stem-loop sequence; (i) a transfer RNA (tRNA) derived stem-loop sequence; (j) a bacterial intergenic region derived stem-loop (IR); or (k) any combinations thereof. In some embodiments, the nuclease cleavage site comprises: (a) a RNAse P recognition sequence; (b) a RNAse Z recognition sequence; (c) a yeast U1 or U2 snRNA maturation sequence; (d) a CCA-adding enzyme recognition sequence; (e) a ribozyme sequence; (f) or any combinations thereof. In some embodiments, the ribozyme comprises: (a) a hammerhead ribozyme, (b) a twister ribozyme, (c) a hepatitis delta virus (HDV) ribozyme, (d) the mammalian CPEB3 ribozyme, (e) a pistol ribozyme, or (f) a combination thereof. In some embodiments, the TTD comprises: (a) snRNA promoter coupled 3’ box domain; (b) a transfer RNA (tRNA) derived stemloop sequence; (c) a mascRNA derived stem-loop sequence; or (d) any combinations thereof. In some embodiments, the engineered terminator is derived from: (a) a LncRNA; (b) an animal; (c) a plant; (d) a virus; (e) a CRISPR sequence; (f) a small nucleolar RNAs (snoRNA); (g) a histone messenger RNA (mRNA); or (h) any combinations thereof. In some embodiments, the engineered terminator comprises any one of SEQ ID NOs: 1-18. In some embodiments, the engineered stabilizing sequence or the secondary structure sequence comprises any one of SEQ ID NO: 19-60. In some embodiments, the nucleic acid molecule further encodes one or more antisense sequences complementary to one or more portions of one or more target RNA molecules. In some embodiments, the one or more antisense sequences are complementary to: (a) an intron of the one or more target RNA molecules; (b) an exon of the one or more target RNA molecules; (c) an intron-exon junction of the one or more target RNA molecules; (d) an untranslated region (UTR) of one or more the target RNA molecules; or (e) any combinations thereof. In some embodiments, the UTR is a 5’ UTR. In some embodiments, the UTR is a 3’ UTR. In some embodiments, the one or more antisense sequences comprise a steric-blocking antisense oligonucleotide, a small interfering RNA, a splice-switching oligonucleotide, or an RNA binding protein interacting decoy sequence. In some embodiments, the engineered Sm-binding site is located 3’ to the one or more antisense sequences. In some embodiments, the engineered Sm-binding site is located 5’ to the 3’ terminator sequence. In some embodiments, the engineered Sm-binding site is located 5’ to the nuclease site. In some embodiments, the nuclease site is located 3’ to the one or more antisense sequences. In some embodiments, the nuclease cleavage site is located 3’ to the engineered stabilizing sequence or the secondary structure sequence. In some embodiments, a 5’end of the nucleic acid molecule further comprises a second sequence configured to form at least one double-stranded region. In some embodiments, the nucleic acid molecule does not comprise a sequence that encodes a poly(A) tail. In some embodiments, the nucleic acid molecule does not comprise a sequence that encodes a native U7 stem-loop sequence. In some embodiments, the nucleic acid molecule does not comprise a sequence that encodes a native U7 Sm-binding sequence. In some embodiments, the nucleic acid molecule does not comprise a sequence or nucleotide that is configured to be inserted into the one or more target RNA molecules. In some embodiments, the nucleic acid molecule is configured not to delete a nucleotide or sequence of the one or more target RNA molecules.

[0010] Provided herein are nucleic acid molecules for targeting an engineered small nuclear ribonucleic acid (snRNA) to one or more target ribonucleic acid (RNA) molecules. In an aspect, a nucleic acid molecule for targeting an engineered small nuclear ribonucleic acid (snRNA) to one or more target ribonucleic acid (RNA) molecules comprises: (a) one or more antisense sequences complementary to one or more portion of the one or more target RNA molecules; (b) an engineered Sm-binding site; and (c) an engineered stabilizing sequence or a secondary structure sequence. In some embodiments, the nucleic acid molecule further comprises an engineered terminator.

[0011] Provided herein are nucleic acid molecules for targeting an engineered small nuclear ribonucleic acid (snRNA) to one or more target ribonucleic acid (RNA) molecules. In an aspect, a nucleic acid molecule for targeting an engineered small nuclear ribonucleic acid (snRNA) to one or more target ribonucleic acid (RNA) molecules comprises: (a) one or more antisense sequences complementary to one or more portion of the one or more target RNA molecule; (b) an engineered Sm-binding site; and (c) an engineered terminator. In some embodiments, the nucleic acid molecule further comprises an engineered stabilizing sequence or a secondary structure sequence.

[0012] In some embodiments, the engineered terminator is configured to facilitate: (1) transcriptional termination of the nucleic acid molecule, (2) 3’end processing of the nucleic acid molecule, or (3) stabilize the nucleic acid molecule. In some embodiments, the engineered terminator comprises at least a 3’ terminator sequence. In some embodiments, the 3’ terminator sequence comprises: (a) a stabilizing sequence that is configured to form at least a double-stranded region; (b) a nuclease cleavage site; (c) a transcription termination domain (TTD); or (d) any combinations thereof. In some embodiments, the stabilizing sequence forms the double-stranded region. In some embodiments, the stabilizing sequence comprises: (a) a stem-loop sequence; (b) a triple helix sequence; (c) a quadruplex sequence; (d) a tRNA-like sequence; (e) a pseudoknot sequence; or (f) any combinations thereof. In some embodiments, the stabilizing sequence comprises: (a) a small nucleolar RNA snoRNA (H / ACA and C / D box) derived stemloop sequence; (b) a viral snRNA derived stem-loop sequence; (c) a plant snRNA derived stem-loop sequence; (d) a non-coding or viral RNA derived triple helix sequence; (e) a clustered regularly interspaced short palindromic repeats (CRISPR) single-guide (sg) RNA derived stem-loop sequence; (f) a long non-coding RNA (LncRNA) derived stem-loop sequence; (g) a PlWI-interacting RNA (piRNA) derived stem-loop sequence; (h) a stem-bulge RNA (sbRNA) derived stem-loop sequence; (i) a transfer RNA (tRNA) derived stem-loop sequence; (j) a bacterial intergenic region derived stem-loop (IR); or (k) any combinations thereof. In some embodiments, the nucleic acid further comprises any one of: (a) a U1 promoter; (b) a U2 promoter; (c) a U4 promoter; (d) a U7 promoter; or (e) any derivatives thereof. In some embodiments, the nuclease cleavage site comprises: (a) a RNAse P recognition sequence; (b) a RNAse Z recognition sequence; (c) a yeast U1 or U2 snRNA maturation sequence; (d) a CCA-adding enzyme recognition sequence; (e) a ribozyme sequence; (f) or any combinations thereof. In some embodiments, the ribozyme comprises: (a) a hammerhead ribozyme; (b) a twister ribozyme; (c) a hepatitis delta virus (HDV) ribozyme; (d) a pistol ribozyme; (e) the mammalian CPEB3 ribozyme; or (f) any combinations thereof. In some embodiments, the TTD comprises: (a) snRNA promoter coupled 3’ box domain; (b) a transfer RNA (tRNA) derived stem-loop sequence; (c) a mascRNA derived stem-loop sequence; or (d) any combinations thereof. In some embodiments, the TTD is derived from: (a) a LncRNA; (b) an animal; (c) a plant; (d) a virus; (e) a CRISPR sequence; (f) a small nucleolar RNAs (snoRNA); (g) a histone messenger RNA (mRNA); or (h) any combinations thereof. In some embodiments, the TTD comprises any one of SEQ ID NOs: 1-18. In some embodiments, the engineered stabilizing sequence or the secondary structure sequence is derived from: (a) a LncRNA; (b) a plant; (c) a virus; (d) a CRISPR sequence; (e) a small nucleolar RNAs (snoRNA); (f) a histone messenger RNA (mRNA); or (g) any combinations thereof. In some embodiments, the engineered stabilizing sequence comprises any one of SEQ ID NOs: 19-60. In some embodiments, the engineered Sm-binding site is configured to recruit a splicing regulator to the nucleic acid molecule. In some embodiments, the engineered Sm-binding site is configured to recruit a splicing regulator to the one or more target RNA molecules. In some embodiments, the splicing regulator comprises the Sm ring protein. In some embodiments, the engineered Sm-binding site comprises a Sm-binding site derived from a human, murine, mammalian, plant, eucaryotic, or virus. In some embodiments, the engineered Sm-binding site comprises a U1 Sm-binding sequence, a U2 Sm-binding sequence, a U4 Sm-binding sequence, a U5 Sm-binding sequence, a sequence derived from major spliceosomal U small nuclear ribonucleoprotein (SmOpt), or any combination thereof. In some embodiments, the engineered Sm-binding site comprises the sequence derived from the SmOpt. In some embodiments, the engineered Sm-binding site comprises any one of SEQ ID NOs: 61-67. In some embodiments, the engineered Sm-binding site is located 3’ to the one or more antisense sequences. In some embodiments, the engineered Sm-binding site is located 5’ to the 3’ terminator sequence. In some embodiments, the engineered Sm-binding site is located 5’ to the nuclease cleavage site. In some embodiments, the nuclease cleavage site is located 3’ to the one or more antisense sequences. In some embodiments, the nuclease cleavage site is located 3’ to the engineered stabilizing sequence. In some embodiments, the nucleic acid molecule further comprises a 5’ methyl G cap. In some embodiments, the 5’ methyl G cap is configured to stabilize the nucleic acid molecule. In some embodiments, the 5’ methyl G cap comprises 2,2,7 trimethyl G (m3G) cap. In some embodiments, a 5’end of the nucleic acid molecule further comprises a second sequence configured to form at least one double-stranded region. In some embodiments, the one or more antisense sequences are complementary to: (a) an intron of the one or more target RNA molecules; (b) an exon of the one or more target RNA molecules; (c) an intron-exon junction of the one or more target RNA molecules; (d) an untranslated region (UTR) of the one or more target RNA molecules; or (e) any combinations thereof. In some embodiments, the UTR is a 5’ UTR. In some embodiments, the UTR is a 3’ UTR. In some embodiments, the nucleic acid is less than 500, less than 400, less than 300, less than 250, less than 200, less than 150 nucleotides, or less than 100 nucleotides in length. In some embodiments, the one or more antisense sequences comprise a steric-blocking antisense oligonucleotide, a small interfering RNA, a splice-switching oligonucleotide, or an RNA binding protein interacting decoy sequence. In some embodiments, the nucleic acid molecule does not comprise a poly(A) tail. In some embodiments, the nucleic acid molecule does not comprise a native U7 stem-loop sequence. In some embodiments, the nucleic acid molecule does not comprise a native U7 Sm-binding sequence. In some embodiments, the nucleic acid molecule does not comprise a sequence or nucleotide that is configured to be inserted into the one or more target RNA molecules. In some embodiments, the nucleic acid molecule is configured to not delete a nucleotide or sequence of the one or more target RNA molecules. In some embodiments, the nucleic acid molecule is RNA.

[0013] Provided herein are deoxyribonucleic acid (DNA) sequences. In an aspect, a DNA sequence encodes the nucleic acid molecule as described herein, wherein the DNA sequence comprises a promoter. In some embodiments, the promoter comprises an engineered promoter. In some embodiments, the promoter comprises a tissue-specific promoter. In some embodiments, the tissue- specific promoter comprises an eye-specific promoter, a brain-specific promoter, a muscle-specific promoter, or any combinations thereof. In some embodiments, the promoter comprises a constitutively active promoter. In some embodiments, the promoter comprises a CMV, RSV, SV40, CBA, CAG, truncated CAG, Cbh, EF-1a, EFS, PGK, UBC, GUSB, UCOE, hAAT, TBG, GRM6, 770En_454P, HSA, Desmin, SkCRM4 / Des, MCK, CK6, MHCK7, dMCK, tMCK, CK8, CK8e, C5-12, NSE, CMV-MyoD, SynM, Synapsin, aMHC, PDGF, MLC2v, cTnT, MecP2, CaMKII, mGluR2, NFL, NFH, np2, PPE, ENK, EAAT2, GFAP, Myo, AUSEx3, SPcA5-12, unc45b, SPc5-12, MBP, Cox-2, PCP2, CLDN5, NR2E1, PITX3, PDE6H, RHO, RHOK, CNGA1 , CNGB1 , PDE6B, GRK1 , SAG, ARR3, S-opsin, L / M-opsin, mopsin-500, CNGA3, CNGB3, RPGR, RPGRIP1, IRBP, NHPH5, PRPH2, CRX, STK38L, RPE65, BEST1, PR1.7, PRO.5, 3LCR-PRO.5, PR2.1 , CAR, IRBP, Pou4f3, Ocp1 , Mathl , Prestin, Myo7a, Opto-mGluR6, H1 , 7SK, U1 , U2, U4, U5, or U6 promoter. In some embodiments, the promoter comprises an engineered U7 promoter. In some embodiments, the promoter is a sequence isolated or derived from a promoter capable of driving expression of a transfer RNA (tRNA). In some embodiments, the nucleic acid molecule does not comprise an endogenous U7 snRNA promoter. In some embodiments, the promoter comprises an enhancer. In some embodiments, the enhancer is a constitutive or tissue-specific enhancer. In some embodiments, the enhancer is a SV40 enhancer or a CMV enhancer. In some embodiments the enhancer is a muscle specific enhancer (skCRM4) or an ocular specific enhancer (Rhodopsin or Rhodopsin kinase).

[0014] Provided herein are vectors. In an aspect, a vector comprises (1) the nucleic acid molecule as described herein or (2) the DNA sequence as described herein. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adenovirus-associated viral vector.

[0015] Provided herein are methods of targeting a splicing-regulatory element or a protein binding site of a target nucleic acid sequence. In an aspect, a method of targeting a splicing-regulatory element or a protein binding site of a target nucleic acid sequence comprises contacting the target nucleic acid sequence with (1) a nucleic acid molecule as described herein; (2) a nucleic acid molecule expressed from the DNA sequence as described herein; (3) a nucleic acid molecule expressed from the vector as described herein. In some embodiments, the one or more antisense sequences of the nucleic acid molecule bind to the corresponding sense sequence of the target nucleic acid sequence. In some embodiments, the target nucleic acid sequence is delivered to a subject with a disease. In some embodiments, the disease is Usher syndrome type II, Duchenne muscular dystrophy, Myotonic Dystrophy Type, or Dravet syndrome. In some embodiments, the target nucleic acid sequence comprises a sequence of Usherin (USH2a), DMPK, Sodium voltage-gated channel alpha subunit 9 (Navi .7), Peripherin-2 (Prph2), or a combination thereof.

[0016] Also provided herein are nucleic acid molecules comprising i) one or more antisense sequences, an engineered Sm-binding site, and a 3’ engineered element; ii) a 5’ engineered element, one or more antisense sequences, and an engineered Sm-binding site; or iii) a 5’ engineered element, one or more antisense sequences, an engineered Sm-binding site, and a 3’ engineered element.

[0017] In some embodiments, the engineered Sm-binding site is configured to recruit a splicing regulator to a ribonucleic acid (RNA) molecule encoded by the nucleic acid molecule. In some embodiments, the splicing regulator comprises a Sm ring protein. In some embodiments, the engineered Sm-binding site comprises a U1 Sm-binding sequence, a U2 Sm-binding sequence, a U4 Sm-binding sequence, a U5 Sm-binding sequence, a sequence from a major spliceosomal U small nuclear ribonucleoprotein (SmOpt), or any combination thereof. In some embodiments, the engineered Sm-binding site comprises a Sm-binding site from a human, a murine, a plant, a virus, or any combination thereof. In some embodiments, the engineered Sm-binding site comprises any one of SEQ ID NOs: 61-67 and 129.

[0018] In some embodiments, the 3’ engineered element comprises one or more of an engineered stabilizing sequence or a secondary structure sequence and a terminator. In some embodiments, the 3’ engineered element is configured to form at least one secondary structure. In some embodiments, the terminator is configured to facilitate: (1) 3’ end processing of the artificial nucleic acid molecule and / or (2) stabilization of the nucleic acid molecule.

[0019] In some embodiments, the 5’ engineered element comprises one or more of a methylation element, an engineered stabilizing sequence or a secondary structure, and a recruitment site. In some embodiments, the recruitment site comprises a binding site for RNA binding proteins (RBPs) or a protein recruitment domain. In some embodiments, the recruitment site comprises a binding site for hnRNPAI . In some embodiments, the methylation element comprises a 5’ methyl G cap. In some embodiments, the 5’ methyl G cap comprises 2,2,7 trimethyl G (m3G) cap.

[0020] In some embodiments, the engineered stabilizing sequence or the secondary structure sequence comprises a stem-loop sequence, a triple helix sequence, a quadruplex sequence, a tRNA-like sequence, a pseudoknot sequence, or any combinations thereof. In some embodiments, the stem-loop sequence comprises a small nucleolar RNA snoRNA (H / ACA and C / D box) derived stem-loop sequence, a viral snRNA derived stem-loop sequence, a plant snRNA derived stem-loop sequence, a non-coding or viral RNA derived triple helix sequence, a clustered regularly interspaced short palindromic repeats (CRISPR) single-guide (sg) RNA derived stem-loop sequence, an intergenic region forming a stem loop structure, a long non-coding RNA (LncRNA) derived stem-loop sequence, a PlWI-interacting RNA (piRNA) derived stem-loop sequence, a stem-bulge RNA (sbRNA) derived stem-loop sequence, a transfer RNA (tRNA) derived stem-loop sequence, or any combinations thereof. In some embodiments, the engineered stabilizing sequence or the secondary structure sequence comprises any one of SEQ ID NO: 19-60, 72-74, and 105-118.

[0021] In some embodiments, the one or more antisense sequences have a length of 15 to 50 nucleotides. In some embodiments, the one or more antisense sequences are complementary to a region of one or more target RNA molecules. In some embodiments, the one or more antisense sequences are complementary to an intron of the one or more target RNA molecules, an exon of the one or more target RNA molecules, an intron-exon junction of the one or more target RNA molecules, an untranslated region (UTR) of one or more the target RNA molecules, or any combinations thereof. In some embodiments, the UTR is a 5’ UTR and / or a 3’ UTR. In some embodiments, the one or more antisense sequences comprise a steric-blocking antisense oligonucleotide, a small interfering RNA, a splice-switching oligonucleotide, or an RNA binding protein interacting decoy sequence.

[0022] In some embodiments, the engineered Sm-binding site is located 3’ to the one or more antisense sequences. In some embodiments, the engineered Sm-binding site is located 5’ to the 3’ engineered element. In some embodiments, the engineered Sm-binding site is located 3’ to the 5’ engineered element.

[0023] In some embodiments, the nucleic acid is less than 500, less than 400, less than 300, less than 250, less than 200, less than 150 nucleotides, or less than 100 nucleotides in length. In some embodiments, the nucleic acid molecule does not comprise a native U7 stem-loop sequence. In some embodiments, the nucleic acid molecule does not comprise a native U7 Sm-binding sequence.

[0024] In some embodiments, the nucleic acid molecule is RNA.

[0025] In some embodiments, the nucleic acid RNA molecule comprises one or more synthetic or modified nucleobases or nucleotides. In some embodiments, the synthetic or modified nucleobase comprises hypoxanthine, xanthine, 7-methylguanine, 5, 6- dihydrouracil, 5-methylcytosine, or 5-hydroxymethoyl cytosine. In some embodiments, the synthetic or modified nucleotide comprises a 2’-0-methyl (2’-OMe) modification, a 2'-0-methoxyethyl (2'-O-MOE) modification, or a 2'-fluoro (2’-F) modification.

[0026] Also provided herein are expression cassettes encoding a nucleic acid molecule comprising: i) an engineered promoter, one or more antisense sequences, an engineered Sm-binding site, and a 3’ engineered terminator; or ii) an engineered promoter, a 5’ engineered element, one or more antisense sequences, an engineered Sm-binding site, and a 3’ engineered terminator.

[0027] In some embodiments, the 3’ engineered terminator comprises an engineered stabilizing sequence or a secondary structure sequence, an insulator, an engineered transcription terminator domain (TTD), an engineered cleavage / processing site, or any combinations thereof. In some embodiments, the 3’ engineered terminator is from a LncRNA, an animal, a plant, a virus, a CRISPR sequence, a small nucleolar RNAs (snoRNA), a histone messenger RNA (mRNA), or any combinations thereof. In some embodiments, the 3’ engineered terminator comprises any one SEQ ID NOs: 1-18, 119-124, and 130. In some embodiments, the engineered stabilizing sequence or the secondary structure sequence comprises a stem-loop sequence, a triple helix sequence, a quadruplex sequence, a tRNA-like sequence, a pseudoknot sequence, or any combinations thereof. In some embodiments, the stem-loop sequence comprises a small nucleolar RNA snoRNA (H / ACA and C / D box) derived stem-loop sequence, a viral snRNA derived stem-loop sequence, a plant snRNA derived stem-loop sequence, a non-coding or viral RNA derived triple helix sequence, a CRISPR sg RNA derived stem-loop sequence, an intergenic region forming a stem loop structure, a LncRNA derived stem-loop sequence, a piRNA derived stem-loop sequence, a sbRNA derived stem-loop sequence, a tRNA derived stem-loop sequence, or any combinations thereof. In some embodiments, the engineered stabilizing sequence or the secondary structure sequence comprises any one of SEQ ID NO: 19-60, 72-74, 105-118, and 125. In some embodiments, the engineered cleavage / processing site comprises a RNAse P recognition sequence, a RNAse Z recognition sequence, a yeast U1 or U2 snRNA maturation sequence, a CCA-adding enzyme recognition sequence, a ribozyme sequence, or any combinations thereof. In some embodiments, the ribozyme comprises: a hammerhead ribozyme, a twister ribozyme, a hepatitis delta virus (HDV) ribozyme, the mammalian CPEB3 ribozyme, a pistol ribozyme, or a combination thereof. In some embodiments, the ribozyme sequence comprises any one of SEQ ID NO: 68-71. In some embodiments, the TTD comprises one or more of distinct 3’ box domains, tRNA derived stem-loop sequence, MALAT 1 -associated small cytoplasmic RNAs (mascRNAs), or other transcription termination domains. In some embodiments, the TTD comprises any one SEQ ID NOs: 1-18, 119-124, and 130. In some embodiments, the 3’ engineered terminator is configured to facilitate: (1) transcriptional termination of the nucleic acid molecule, (2) 3’ end processing of the nucleic acid molecule, and / or (3) stabilization the nucleic acid molecule. In some embodiments, the engineered stabilizing sequence is configured to form at least one secondary structure. In some embodiments, the 5’ engineered element comprises one or more of a methylation element, an engineered stabilizing sequence or a secondary structure, and a recruitment site. In some embodiments, the recruitment site comprises a binding site for RNA binding proteins (RBPs) or protein recruitment domain. In some embodiments, the methylation element comprises a 5’ methyl G cap. In some embodiments, the 5’ methyl G cap comprises a m3G cap. In some embodiments, the engineered stabilizing sequence or the secondary structure sequence comprises a stem-loop sequence, a triple helix sequence, a quadruplex sequence, a tRNA-like sequence, a pseudoknot sequence, or any combinations thereof. In some embodiments, the stem-loop sequence comprises a small nucleolar RNA snoRNA (H / ACA and C / D box) derived stem-loop sequence, a viral snRNA derived stem-loop sequence, a plant snRNA derived stem-loop sequence, a non-coding or viral RNA derived triple helix sequence, a CRISPR sg RNA derived stem-loop sequence, an intergenic region forming a stem loop structure, a LncRNA derived stem-loop sequence, a piRNA derived stem-loop sequence, a sbRNA derived stem-loop sequence, a tRNA derived stem-loop sequence, or any combinations thereof. In some embodiments, the engineered stabilizing sequence or the secondary structure sequence comprises any one of SEQ ID NO: 19-60, 72-74, 105-118, and 125. In some embodiments, the engineered promoter comprises a U1 promoter, a U2 promoter, a U4 promoter, or a U7 promoter. In some embodiments, the engineered promoter comprises a tissue-specific promoter. In some embodiments, the tissue-specific promoter comprises an eye-specific promoter, a brain-specific promoter, a muscle-specific promoter, or any combinations thereof. In some embodiments, the promoter comprises a constitutively active promoter. In some embodiments, the promoter comprises a CMV, RSV, SV40, CBA, CAG, truncated CAG, Cbh, EFla, EFS, PGK, UBC, GUSB, UCOE, hAAT, TBG, GRM6, 770En_454P, HSA, Desmin, SkCRM4 / Des, MCK, CK6, MHCK7, dMCK, tMCK, CK8, CK8e, C5-12, NSE, CMV-MyoD, SynM, Synapsin, aMHC, PDGF, MLC2v, cTnT, MecP2, CaMKII, mGluR2, NFL, NFH, np2, PPE, ENK, EAAT2, GFAP, Myo, AUSEx3, SPcA5-12, unc45b, SPc5-12, MBP, Cox-2, PCP2, CLDN5, NR2E1, PITX3, PDE6H, RHO, RHOK, CNGA1, CNGB1, PDE6B, GRK1, SAG, ARR3, S-opsin, L / M-opsin, mopsin-500, CNGA3, CNGB3, RPGR, RPGRIP1 , IRBP, NHPH5, PRPH2, CRX, STK38L, RPE65, BEST1 , PR1.7, PR0.5, 3LCR-PRO.5, PR2.1, CAR, IRBP, Pou4f3, Ocp1, Mathl, Prestin, Myo7a, Opto-mGluR6, H1, 7SK, U1, U2, U4, U5, U6, or U7 promoter. In some embodiments, the promoter comprises any one of SEQ ID NOS: 77-92 and 132-139. In some embodiments, the engineered promoter further comprises regulatory elements. In some embodiments, the regulatory elements comprise enhancers or internal ribosomal entry sites (IRES). In some embodiments, the enhancer comprises any one of SEQ ID NOS: 93-104.

[0028] In some embodiments, the engineered Sm-binding site is configured to recruit a splicing regulator to an RNA molecule encoded by the nucleic acid molecule. In some embodiments, the splicing regulator comprises a Sm ring protein. In some embodiments, the engineered Sm-binding site comprises a U1 Sm-binding sequence, a U2 Sm-binding sequence, a U4 Sm-binding sequence, a U5 Sm-binding sequence, a sequence from SmOpt, or any combination thereof. In some embodiments, the engineered Sm-binding site comprises a Sm-binding site from a human, a murine, a plant, a virus, or any combination thereof. In some embodiments, the engineered Sm-binding site comprises any one of SEQ ID NOs: 61-67 and 129. In some embodiments, the one or more antisense sequences have a length of 15 to 50 nucleotides. In some embodiments, the one or more antisense sequences are complementary to a region of one or more target RNA molecules. In some embodiments, the one or more antisense sequences are complementary to an intron of the one or more target RNA molecules, an exon of the one or more target RNA molecules, an intron-exon junction of the one or more target RNA molecules, a UTR region of one or more the target RNA molecules, or any combinations thereof. In some embodiments, the UTR is a 5’ UTR and / or a 3’ UTR. In some embodiments, the one or more antisense sequences comprise a steric-blocking antisense oligonucleotide, a small interfering RNA, a splice-switching oligonucleotide, or an RNA binding protein interacting decoy sequence.

[0029] In some embodiments, the engineered Sm-binding site is located 3’ to the one or more antisense sequences. In some embodiments, the engineered Sm-binding site is located 5’ to the 3’ engineered element. In some embodiments, the engineered Sm-binding site is located 3’ to the 5’ engineered element. In some embodiments, the expression cassette is less than 5000, less than 4000, less than 3000, less than 2500, less than 2000, less than 1500 nucleotides, or less than 1000 nucleotides in length. In some embodiments, the nucleic acid is less than 500, less than 400, less than 300, less than 250, less than 200, less than 150 nucleotides, or less than 100 nucleotides in length. In some embodiments, the nucleic acid molecule does not comprise a native U7 stem-loop sequence. In some embodiments, the nucleic acid molecule does not comprise a native U7 Sm-binding sequence. In some embodiments, the encoded nucleic acid molecule is RNA.

[0030] Also provided herein are vectors comprising (1) the artificial nucleic acid as described herein or (2) the expression cassette as described herein. In some embodiments, the vector is a viral vector.

[0031] Also provided herein are methods of targeting a splicing-regulatory element or a protein binding site of a target nucleic acid sequence, the method comprising contacting the target nucleic acid sequence with (1) the nucleic acid molecule as described herein, (2) the expression cassette as described herein, or (3) the vector as described herein.

[0032] Also provided herein are methods of modulating expression of a protein, the method comprising contacting a target nucleic acid sequence encoding the protein with (1) the nucleic acid molecule as described herein, (2) or a nucleic acid molecule transcribed from the expression cassette as described herein, (3) or a nucleic acid molecule transcribed from the vector as described herein.

[0033] Also provided herein are methods of modulating levels of a protein, the method comprising contacting a target nucleic acid sequence encoding the protein with (1) the nucleic acid molecule as described herein, (2) or a nucleic acid molecule transcribed from the expression cassette as described herein, (3) or a nucleic acid molecule transcribed from the vector as described herein. In some embodiments, the target nucleic acid sequence is a mature mRNA.

[0034] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. INCORPORATION BY REFERENCE

[0035] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

[0036] BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (“FIG.” or “FIGs.” herein), of which:

[0038] FIGs. 1A-1F depict five non-limiting examples of engineered nucleic acids as described herein.

[0039] FIG. 1A shows the original unmodified nucleic acid molecule (snRNA), containing an antisense complementary sequence to the target, a SmOPT binding site, and a stem loop. FIGs. 1B and 1C depict two distinct non-limiting examples of nucleic acid engineered stem loops for increased efficiency and / or stability. FIGs. 1D and 1E depict two non-limiting examples of modifications of the nucleic acid molecule 5’ end, engineered to promote higher stability, blocking nuclease degradation, and / or recruiting additional splicing regulatory factors to further regulate different splicing modalities and promote increased efficiency, respectively. FIG. 1F depicts a non-limiting example of modifications of the Sm binding site to modulate recruitment of RNA binding proteins and promote higher stability and / or potency.

[0040] FIGs.2A-2J depict non-limiting examples of further engineering of the nucleic acid expression cassette, namely promoter and terminator sequences to improve and / or modulate expression levels, stability, and potency. FIG.2A shows the original, naturally occurring, transcriptional elements for the expression of a snRNA molecule, containing an endogenous U7 promoter, the snRNA molecule, and a U7 terminator. FIG.2B depicts the five modification domains of the engineered nucleic acid expression cassette: the promoter, the regulatory elements, the stabilizing domain, the cleavage / processing site and the transcription termination domain (TTD). FIG.2C depicts an engineered promoter and additional transcriptional regulatory elements to increase and / or modulate RNA expression. Those modifications can include, but are not limited to, the addition of more or distinct distal sequence elements sites (DSEs) and proximal sequence elements sites (PSEs) or addition of a TATA box or sequences to recruit distinct transcription factors. Those modifications can also include, but are not limited to, the use of distinct Pol II or Pol III promoter sequences, such as tissue specific promoters to limit RNA expression to specific tissues. Other modifications can include, but are not limited to, the use of other regulatory elements such as enhancers, introns, 5’ UTRs and internal ribosomal entry sites (IRESs). FIGs.2D-2F show examples of terminator domain engineering of the nucleic acid expression cassette. FIG.2D depicts an engineered stabilizing domain to increase and / or modulate RNA stability. Those modifications can include, but are not limited to, the use of a modified stem loop, a triple helix, a quadruplex sequence, a tRNA-like sequence, or a pseudoknot. FIG. 2E depicts an engineered cleavage / processing site to allow efficient and complete 3’ end processing of the engineered nucleic acid. Those modifications can include, but are not limited to, the use of distinct RNA cleavage sites, RNAse P cleavage sites, insulators, ribozyme structures, and other RNase cleavage sequences. FIG.2F depicts an engineered TTD to allow efficient and complete 3’ end processing of the engineered nucleic acid. Those modifications can include, but are not limited to, the use of distinct 3’ box domains, insulators, Poly-A signals, Poly-T signals, t-RNA or t-RNA-like structures, MALAT1 -associated small cytoplasmic RNAs (masc-RNAs), or other transcription termination domains.

[0041] FIGs.2G-2J depict examples of engineered nucleic acids expression cassettes. FIG.2G includes the use of a snoRNA stem loop stabilizing domain to confer stability and protection against 3’ exonucleases. FIG.

[0042] 2H includes the use of a strong CMV or any strong Pol II promoter to increase expression of the engineered nucleic acid, and a mascRNA cleavage and TTD to allow for proper 3’end processing that is uncoupled from the promoter choice. FIG.21 includes the use of a strong CMV or any strong Pol II promoter to increase expression of the engineered nucleic acid, and a ribozyme structure to allow for proper 3’end processing that is uncoupled from the promoter choice. FIG.2J includes the use of a strong CMV or any strong Pol II promoter to increase expression of the engineered nucleic acid, and a MALAT-1 terminator (triple helix stabilizer + mascRNA) structure to allow for proper 3’end processing that is uncoupled from the promoter choice, and increased stability and protection against 3’ exonucleases.

[0043] FIGs.3A-3D depict a schematic of an example RNA biogenesis, processing, or maturation pathway for the engineered nucleic acids as described herein. FIG.3A depicts the first steps of the engineered nucleic acid biogenesis that take place in the nucleus. The engineered stabilizing sequence / secondary structure sequence is highlighted by an oval. The asterisk depicts the endogenous U1 structure that may not be incorporated into the engineered nucleic acids, and that can instead be replaced by the antisense sequence as described herein. FIG.3B depicts example detailed components of a native DNA cassette as described herein. The DNA cassette may encode one or more components of an engineered RNA sequence. The engineered RNA sequence may be involved in endogenous snRNA biogenesis, processing, or maturation. FIG.3C depicts an example of maturation for an engineered nucleic acid as described herein, and the modification steps that take place in the cytoplasm. The engineered stabilizing sequence / secondary structure sequence is highlighted by oval. The asterisk shows the endogenous U1 structure that may not be incorporated into the engineered nucleic acids, and that can instead be replaced by the antisense sequence. FIG.3D depicts the engineered nucleic acid maturation or modification steps that take place in the nucleus once the engineered nucleic acids are imported back into the nucleus from the cytoplasm, subsequent to the maturation or modification steps described in FIG.

[0044] 3C.

[0045] FIGs.4A-4B depict schematics of the assay used to assess splicing modulation efficiency of modified snRNAs. As shown in FIG.4A, the human USH2A minigene contains exons 12 to 14, with 5’ and 3’ flanking intronic sequences of 190 bp. “F primer” and “R primer” indicate the positions of the RT-PCR primers used to amplify USH2A transcripts either including or excluding exon 13. As shown in FIG.4B, after transfection of modified snRNAs, splicing changes were measured using a semi-quantitative RT-PCR reaction (e.g., the smaller amplicon lacks exon 13).

[0046] FIG. 5 shows USH2A exon 13 skipping percentage after treatment with modified snRNAs. Tested modified snRNAs include engineered elements: mouse U7 snRNA stem-loop (WT), truncated human MALAT1 Triple Helix domain (hMTH), mouse MALAT1 Triple Helix domain (Mmth), comp 14 derivative of hMTH (MTH comp 14), comp 1 derivative of hMTH (MTH comp 1), human NEAT1 / MEN p Triple Helix domain (hMbTH), mouse NEAT1 / MEN p Triple Helix domain (mMbTH), KSHV PAN core Triple Helix (KTH), EHV2 triple helix (ETH), TYMV pseudoknot (TYMV), human MAT2A 3’UTR stem-loop structure Hairpin A (MAT2A HA), human MAT2A 3’UTR stem-loop structure Hairpin C (MAT2A HC), intergenic region sequence 1 truncated (IR1 ), intergenic region sequence 5 truncated (IR5), mouse U7 snRNA stemloop with modified loop (mut 1), mouse U7 snRNA stem-loop with modified loop (mut 2), 5’ end hnRNPAI binding site (hnRNPAI), Cas13d derived stem-loop (CasRX), or human U1 pseudogene sm binding site (Pseudo U1 sm). A non-targeting control (NT) was also included.

[0047] FIGs. 6A-6C the use of engineered nucleic acids with engineered 3’ terminator domains to modulate splicing. FIG. 6A shows the percentage of exon 13 skipping after mammalian cells (i.e., HEK-293T cells) were transfected with expression cassettes encoding modified snRNAs (i.e., pcDNA3.1 plasmid backbone) and a human USH2A minigene plasmid. Transcription was terminated with either a traditional 3’ Box domain, or a non-canonical pseudo-tRNA domain (e.g., FIG. 6B, mascRNA, FIG. 6C menRNA). Tested snRNAs include engineered elements: Wild type U7 terminator (WT), human mascRNA transcription termination (1), mouse mascRNA transcription termination (2), human menRNA transcription termination (3), mouse menRNA transcription termination (4), compl 4 derived mouse Malatl terminator (5), comp9 derived mouse Malatl terminator (6), compl 2 derived mouse Malatl terminator (7), compl 4 V2 derived mouse Malatl terminator (8), or mouse mutant U2.5 mascRNA transcription termination (9). A NT control was also included.

[0048] FIG. 7 demonstrates using engineered nucleic acids with engineered promoters and engineered 3’ terminator domains to modulate splicing. FIG. 7 shows the percentage of exon 13 skipping after mammalian cells (i.e., HEK-293T cells) were transfected with expression cassettes encoding modified snRNAs and a human USH2A minigene plasmid. The tested engineered elements include one or more of 5’ transcription start site stabilizer (1), SV40 promoter enhancer (2), human mascRNA sequence (3), CMV promoter enhancer (4), 2x proximal sequence element (5), 3x distal sequence element (6), CasRX stemloop with extra 5’ CA bases (7), extra CCU sequence 5’ of stem loop (8), insulator (9), compl 4 derived human MALAT1 triple helix (10), 3’ sl4 U1 stem-loop (11), 2x smOPT (12), and 2x truncated U7 terminator (13). A NT control and WT U7 control were also included.

[0049] FIGs. 8A-8D depict using AAV vectors containing expression cassettes encoding engineered nucleic acids to modulate splicing. Mammalian cells (i.e., HEK-293T cells) were transfected with AAV expression vectors containing expression cassettes encoding modified snRNAs and a human USH2A minigene plasmid. FIG. 8A shows a graphical representation of the percentage of exon 13 skipping and FIG. 8B shows the corresponding DNA electrophoresis gel of the RT-PCR products generated using primers that anneal to exons 12 and 14. FIG. 8C shows the modified snRNA (e.g., U7 Crank) elements including the engineered 3’ elements of a U7 stem-loop, an insulator, and the human mascRNA translation termination sequence. FIG. 8D shows the modified snRNA (e.g., U7 Crank) elements including an engineered promoter comprising a SV40 enhancer sequence upstream of the mouse U7 promoter. A NT control was also included. DETAILED DESCRIPTION

[0050] The splicing code is defined by cis splicing regulatory sequences and their trans binding partners. DNA / RNA are composed of exons that contain protein coding sequences and are interspersed with introns, the non-coding portion. Nascent (recently transcribed) ribonucleic acid (RNA) transcripts emerge from the RNA polymerase Pol II (Pol II) complex as pre-mRNA that contains both exons and introns. The process of pre-messenger ribonucleic acid (pre-mRNA) splicing removes introns, joining coding exons to produce mature mRNA. Further, the process of alternative splicing, which is regulated by cis sequences and separate RNA binding proteins, may remove or include one or more alternative exons within the mRNA molecule for added diversity. Pre-mRNA splicing takes place in the nucleus of eukaryotic cells and is mediated by the spliceosome. Examples of such splicing are provided at Wang & Burge, RNA. 2008 May;14(5):802-13, which is incorporated herein by reference in its entirety. A spliceosome is a ribonuclear protein complex (RNP) that is composed of five small nuclear (sn)RNAs (U1 , U2, U4, U5, and U6) and hundreds of associated proteins. Together the snRNAs and proteins form small nuclear ribonucleoproteins (snRNPs), and assemble on the pre-mRNA in a step-wise process by recognizing highly conserved sequences within exons and introns such as 5’ splice site (5’ ss) and 3’splice site (3’ ss), polypyrimidine tract (ppT) and the branchpoint adenosine (BpA) found upstream of the 3’ splice site. Additional RNA sequence elements located within exonic (exon splicing enhancer or ESE, exon splicing silencer or ESS) and intronic (intronic splicing enhancer or ISE, intronic splicing silencer or ISS) regions may be used for binding of RNA binding proteins (RBP) to further regulate spliceosome recruitment and splicing events. Following spliceosomal assembly, a series of ATP-dependent steps, can lead to RNA structure rearrangements within the spliceosome to catalyze two trans-esterification reactions resulting in the removal of an intron in the form of a lariat and the ligation of the two exons. Although a plethora of the aforementioned splicing regulatory sequences may allow for the opportunity of pathogenic mutations, these sequences also allow for the modulation of splicing for therapeutic purposes in a programmable manner.

[0051] Many genetic diseases can be addressed by modulation of the splicing code at the pre-mRNA level. However, existing splicing modulators when used as therapeutics have several limitations. One example of existing splicing regulators being used as therapeutics include Antisense oligonucleotides (ASOs), which have been approved by the United States Food and Drug Administration (FDA) for modulation of exon inclusion or exon skipping by blocking splicing regulatory elements (SREs) for the treatments for spinal muscular atrophy (SMA), Duchenne muscular dystrophy (DMD), and other diseases. ASOs are short oligonucleotides that bind to RNA in a target-specific manner to regulate pre-mRNA splicing (by targeting SREs) or mRNA levels (via splice modulation or RNase H recruitment). Despite the therapeutic potential and compelling efficacy in animals, ASOs are a poor therapeutic option for humans due to their poor biodistribution in relevant tissues, poor translation of efficacy, need for repeat or recurrent administration, and dose limiting tolerability that limits combination treatments (more than one ASO per dose). Examples of such methods are provided at Lauffer et al., 2024, which is herein incorporated by reference in its entirety. In particular, ASOs for the treatment of Duchenne Muscular Dystrophy (DMD), show very low levels of exon skipping in patients at the translated doses. FDA approved ASOs for mutations amenable to skipping of dystrophin exons 45 (casimersen or Amondys 45), 51 (eteplirsen or Exondys 51) and 53 (golodirsen or Vyondys 53) require chronic weekly intravenous dosing and exhibit low levels of exon skipping and dystrophin protein restoration (< 1% for exon 51). Nextgeneration ASOs employ conjugation with receptor-targeted antibodies and peptides for better tissue penetration but clinical efficacy and safety with chronic dosing remains to be demonstrated. Current clinical trials with 2ndgeneration ASOs show <5% exon skipping for different DMD exons.

[0052] Recognized herein is an industry-wide problem where existing splicing modulators lack efficacy and require recurrent delivery; suffer from limited tissue bioavailability in target tissues; and / or target only one splicing regulatory element with poor efficacy. The existing splicing code modulators are ineffective and inefficient and lack applicability for various diseases and / or subject populations. Accordingly, recognized herein is a need for development of splicing code modulators that can overcome such issues.

[0053] Engineered U7 small nuclear RNA (snRNA) have recently been tested as therapeutics. U7 snRNAs have an antisense sequence that may be programmed and used for targeting, e.g., SREs in or near the exons to be skipped. U7 snRNAs have been effectively used in both pre-clinical and clinical studies.

[0054] U7 snRNAs are a family of small non-coding Pol II transcripts that play an important role in the processing of pre-mRNAs. The major components of the spliceosome are small nuclear ribonucleoprotein particles (snRNPs) U1 , U2, U5, U4 / U6, U11 , U12, and U4atac / U6atac, each of which comprises one U snRNA molecule, a common core of seven Sm proteins (B / B’, D1 , D2, D3, E, F and G), and several snRNP-specific proteins. The biogenesis of snRNPs occurs in the cytoplasm shortly after the nuclear export of nascent snRNAs. A properly assembled Sm core, cap hypermethylation, and 3’-end processing are required for the translocation of the mature snRNPs into the nucleus, where they function in splicing.

[0055] Most U-rich snRNAs are part of snRNP complexes that mediate endogenous RNA splicing. U7 snRNA is an exception. It is involved in the 3’ end processing of replication-dependent histone mRNAs, a process that also takes place in the nucleus. Both spliceosomal and U7 snRNAs share a similar biogenesis pathway with few distinct differences. U7 snRNA contains a unique mixed Sm / LSm core comprising Sm proteins B, D3, E, F, G, and two LSm proteins instead of the D1 and D2 of spliceosomal snRNPs. Additionally, U7 snRNA 5’ antisense region contains a conserved sequence used for basepairing with histone pre-mRNA substrates.

[0056] U7 snRNAs can be engineered by programming antisense sequence complementary to the target sequence and replacing the U7 Sm-binding sequence with an SmOPT sequence that recruits the heptameric core of spliceosomal Sm proteins (Gorman et al., PNAS. 1998 Apr 28; 95(9): 4929-4934.; which is herein incorporated by reference in its entirety). This arrangement allows for spliceosomal localization of engineered or modified U7 snRNAs (MsnRNAs; important for splicing regulation) and greater stability compared to endogenous U7 snRNAs. Efficient and complete processing of engineered or modified U7 snRNA precursors may require a 13-16 nucleotide cis-acting element named the 3’box, located 9-19 nucleotides downstream of the 3’ end of the RNA-encoding region (Hernandez, EMBO J. 1985 Jul ;4(7) :1827-37. ; which is herein incorporated by reference in its entirety).

[0057] However, currently available U7 snRNA-based splicing modulators may suffer from sufficient expression level or stability for sufficient efficacy to generate a treatment effect. Multiple MsnRNAs can be delivered in a single AAV vector for targeting multiple splice regulatory sites to provide effective splicing modulation treatments for genetic disorders. However, packaging of multiple small RNA expression cassettes into AAV genomes can result in genome recombination and truncations causing product heterogeneity and low productivity precluding rapid development of modified U7 snRNAs based clinical products. Additionally, existing modified U7 snRNAs do not allow for tissue-specific expression since the used endogenous promoter is expressed constitutively in all tissues after intravenous administration.

[0058] A compatible pol II snRNA promoter can also be required for proper 3’ end formation of pre-snRNAs, highlighting the tight link between transcription and the function of the 3’ box. A 3’ poly(A) tail can provide stability of RNA pol II transcripts. However, a significant fraction (>25%) of long Pol II transcripts present in cells, such as non-coding RNAs or snRNAs, may lack a canonical poly(A) tail. Further, the substrates of snRNAs such as histone mRNAs also lack a poly(A) tail. These RNAs that lack a canonical poly(A) tail can often have conserved stem-loop structures in their 3’ untranslated regions (UTRs) or ensuring transcription termination and enhancing RNA stability & translational efficiency. Additional Pol II transcripts may be subjected to other noncanonical 3’ end processing mechanisms. In particular, enzymes with well-known roles in RNA processing events, such as pre-mRNA splicing and tRNA biogenesis, have been shown to cleave certain nascent transcripts to generate mature 5’ and 3’ ends. For example, RNase P can endonucleolytically cleave tRNA precursors to produce the mature 5’ termini of functional tRNAs. RNase P can also generate the mature 3’ end of the long non-coding RNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1). Cleavage by RNase P can simultaneously generate the mature 3’ end of the 6.7-kb MALAT 1 non-coding RNA and the 5’ end of a small tRNA-like transcript. tRNA biogenesis enzymes including RNase Z and the CCA-adding enzyme, further process the small RNA to generate the mature 61 -nucleotide transcript known as mascRNA (MALAT1 -associated small cytoplasmic RNA). Despite lacking a canonical poly(A) tail, MALAT1 is one of the most abundant long non-coding RNAs in mouse and human cells. Although the 3’ end of MALAT1 is generated via a mechanism distinct from canonical cleavage / polyadenylation, the mature MALAT1 transcript contains an A-rich region, U-rich motifs and a highly conserved stem-loop region that can enhance MALAT1 stability.

[0059] Accordingly, recognized herein is a long-felt need for engineered nucleic acid splicing regulators that have sufficient expression level or stability (or both); tissue-specific expression; and / or flexibility in target designs. The present disclosure provides engineered nucleic acids to address these long-felt needs. In some embodiments, the engineered nucleic acid provided herein can comprise modified U7 snRNAs that have various sequence or structural elements not found in the existing technologies, rendering these modified U7 snRNAs to have beneficial expression level or stability and tissue-specificity over the existing technology. The present disclosure provides compositions or pharmaceutical compositions (such as for gene therapy / therapeutic purposes) comprising engineered RNA or snRNA (or the DNA encoding the same) comprising Sm-binding sequences; one or more engineered stem-loops; engineered promoters and / or engineered 3’ ends for proper expression, termination, processing, localization, and increased stability. Such modified nucleic acids are beneficial in the modulation of target RNA and their respective protein levels for treatment of rare and common diseases that may benefit from exon-skipping or inclusion, modulation of intronic mis-spliced regions, sterically blocking protein-RNA interaction or modulation of protein levels.

[0060] Moreover, the use of different snRNA promoters and terminators to reduce homology and structural elements in the AAV genome can address modified U7 snRNAs packaging challenges leading to high titer full-length AAV genomes. These well-packaged self-complementary (sc) AAV-MsnRNAs can show dose-dependent snRNA expression and exon skipping activity with low levels of off-targets (see, for example, PCT Publication No. W02024 / 086650 and PCT Publication No. WO2024 / 119102, each of which is incorporated by reference in its entirety). Thus, the present disclosure provides nucleic acid molecules comprising sufficient efficacy and applicability for treating various diseases or conditions. Also provided herein are various packing modalities of the nucleic acid molecules. Further provided here are methods of using the same.

[0061] Nucleic Acid Molecules

[0062] Provided herein are nucleic acid molecules modified for improved stability, potency, and function to target mutated / non-functional target sequences present in disease. In some embodiments, the nucleic acid molecules comprise the engineered nucleic acids. In some embodiments, the nucleic acid molecules encode the engineered nucleic acids. In some embodiments, the engineered nucleic acids comprise the engineered elements. In some embodiments, the engineered nucleic acids encode the engineered elements. In some embodiments, the nucleic acid molecules can comprise the engineered nucleic acids. “Engineered” or “engineering” as used herein, when referring to a nucleic acid or a component thereof, refers to a non-naturally occurring nucleic acid or a component thereof. When referring to a component of a nucleic acid, the component is either present with another component in a combination that is not naturally occurring, or present without another component that is naturally occurring, or both. In some embodiments, provided herein are nucleic acid molecules for targeting a modified snRNA to a target nucleic acid. In some embodiments, the nucleic acid molecules comprise the modified snRNA. In some embodiments, the nucleic acid molecules encode the modified snRNA. In some embodiments, an expression cassette comprises the nucleic acid molecules encoding the modified snRNA. In some embodiments, the nucleic acid molecules comprise the modified snRNA.

[0063] The present disclosure provides the considerations for inclusion and / or exclusion of a particular structural element or sequence or nucleotide within the engineered nucleic acid, based at least in part of whether such a particular structural element or sequence or nucleotide would facilitate the engineered nucleic acid to process certain property(ies), as described herein. The properties can comprise the stabilization of the engineered nucleic acids, the localization of the engineered nucleic acids, the transcriptional regulation (via the engineered promoters or transcription termination domain (TTD) as described herein), as exemplified in FIGs. 1 A-2J, the biogenesis, processing, or maturation of the engineered nucleic acids, or the splicing regulation functions and potency as described herein, or any combinations thereof.

[0064] In some embodiments, the types of elements that an engineered nucleic acid molecule comprises depends on the biogenesis, maturation, or modification stage of the engineered nucleic acid molecule. In some embodiments, the types of elements that an engineered nucleic acid molecule comprises are engineered post-transcriptional elements. In some embodiments, the types of elements that an engineered nucleic acid molecule comprises are engineered pre-transcriptional elements. In some embodiments, an expression cassette encoding an engineered nucleic acid molecule comprises engineered pre-transcriptional elements.

[0065] The engineered nucleic acids described herein can make use of the endogenous transcriptional and co- and / or post-transcriptional pathway of the snRNA for its biogenesis, processing, or maturation. As an example, FIG. 3A depicts the first four sub steps of snRNA biogenesis that take place in the nucleus for the biogenesis, processing, or maturation of the engineered nucleic acid. In sub-step 1 of FIG.3A, RNA polymerase II (Pol II) can find the promoter and assembles various processing factors. In sub-step 2 of FIG.3A, RNA pol II can initiate the transcription of the pre-transcription engineered nucleic acid elements. In sub-step 3 of FIG.3A, the RNA pol II can transcribe the 3’ box and recruit the integrator (see FIG.3B, which shows the integrator and also the other biogenesis, maturation, or processing factors). In sub-step 4 of FIG. 3A, an integrator, with the help of other factors can cleave the engineered nucleic acid at the region upstream of the 3’ box, terminate the transcription, and create the 3’ end of the engineered nucleic acid. In some embodiments, an engineered nucleic acid with a 7-methylguanosine (m7G) cap can thus be generated. In FIG. 3A, the engineered nucleic acid is shown with an additional secondary structure (denoted as an asterisk in FIGs.3A and 3C). This structure may not be present in the engineered nucleic acid but present in endogenous snRNAs. In the engineered nucleic acid described herein, the structure denoted by an asterisk can also be replaced by other structural and / or sequence elements described herein, such as the antisense sequence. As an example, FIG.3C depicts how, following the sub-steps described herein FIG.3A, the engineered nucleic acid with the m7G cap can then be exported into the cytoplasm. In this particular example, the step of being exported to the cytoplasm is denoted as “1” in FIG.3C for further processing or maturation steps. In the cytoplasm, the engineered nucleic acid can be assembled with various Sm proteins (snRNP complex) that can facilitate the hypermethylation and trafficking to the splicing factories (pathway denoted as “2” in FIG.3C).

[0066] Subsequently, the m7G cap can undergo hypermethylation to form the m3G (pathway denoted as “3” in FIG.3C) that is recognizable by nuclear pores for importing the engineered nucleic acid back into the nucleus (pathway denoted as “4” in FIG.3C). In FIG.3C, the engineered nucleic acid is shown with an additional secondary structure (denoted as an asterisk in FIGs.3A and 3C) that may not be present in the engineered nucleic acid but present in endogenous snRNAs.

[0067] Subsequent to the steps described in FIG.3C, the engineered nucleic acid can undergo further processing or maturation steps, as depicted in FIG. 3D. For endogenous snRNAs, additional modifications and loading of the snRNPs can occur (denoted as “5” in FIG.3D). The engineered nucleic acid described herein may not undergo these modifications nor recruit these snRNPs. Subsequently, the snRNP complex can be incorporated into spliceosome (denoted as “7” in FIG.3D). The engineered nucleic acid described herein may not comprise or encode all the protein components described in step 7 of FIG.3D. For example, ** denotes a U1 structure (bound with the U1 A protein) that may not be present in the engineered nucleic acid as described herein. Such structure may be replaced with the antisense sequence as described herein.

[0068] The engineered nucleic acids may comprise or encode one or more of an engineered Sm-binding site; one or more stabilizing sequence or secondary nucleic acid structure; TTD; one or more antisense sequences; a 5’ methyl G cap; an engineered promoter; a linker, an engineered regulatory sequence, any functional derivatives thereof; or any combinations thereof. The engineered nucleic acids may comprise or encode at least 2, 3, 4, 5 or 6 of: an engineered Sm-binding site; one or more stabilizing sequence or secondary nucleic acid structure; a TTD; one or more antisense sequences; a linker; an engineered regulatory sequence; a sequence for recruitment of splicing regulatory factors; or a 5’ methyl G cap. Any combinations of the elements described herein are configured to allow the engineered nucleic acids to process sufficient expression level, stability, intracellular localization, ability to regulate the co- or post-transcriptional modification of a target nucleic acid molecule, or tissue-specificity of the engineered nucleic acids for various application purposes, such as the therapeutic purposes as described herein.

[0069] In some embodiments, a nucleic acid may comprise a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In some embodiments, a nucleic acid may be a species or type of nucleic acid. In some embodiments, a nucleic acid may comprise a polymeric form of nucleotides. In some embodiments, a nucleic acid may comprise a polynucleotide. In some embodiments, the nucleic acid may comprise a sequence of nucleotides (i.e., a nucleic acid sequence). In some embodiments, a nucleic acid may comprise a modified polynucleotide. In some embodiments, a nucleic acid may comprise a canonical or non-canonical nucleotide. A canonical nucleotide may comprise adenosine with base types (A), cytosine (C), guanine (G), thymine (T), uracil (U), or variants thereof. When referring to a base type of a nucleotide or a polynucleotide, T and U may be interchangeable. When referring to a sequence of a nucleic acid, the sequence may comprise the complementary form of the sequence. The complementary of the nucleic acid sequence may be based on canonical base-pairing of the nucleotides or nucleic acids. A nucleic acid may comprise one or more modified nucleotides or nucleotide analogs. When referring to a sequence of a nucleic acid, the sequence may comprise the DNA or RNA form of the nucleic acid.

[0070] In some embodiments, a nucleic acid may be linear. In some embodiments, a nucleic acid may be closed linear double-stranded. In some embodiments, a nucleic acid may be circular. In some embodiments, a nucleic acid may be branched. In some embodiments, a nucleic acid may be singlestranded, double-stranded, triple stranded, or a combination thereof. In some embodiments, an engineered nucleic acid may be single-stranded. In some embodiments, an engineered nucleic acid may be double-stranded. In some embodiments, an engineered nucleic acid may comprise single-stranded and double-stranded regions or portions.

[0071] In some embodiments, the engineered nucleic acid may comprise or encode a structural element or sequence (or complementary thereof) of a messenger ribonucleic acid (mRNA), a micro ribonucleic acid (miRNA), a transfer ribonucleic acid (tRNA), a long non-coding RNA (IncRNA), a ribosomal ribonucleic acid (rRNA), snRNA, a piwi-interacting RNA (piRNA), a small nucleolar RNA (snoRNA), a small Cajal body-specific RNA (scaRNA), a silencing ribonucleic acid (siRNA), a short hairpin RNA (shRNA), a heterogeneous nuclear RNA (HnRNA), an endless / circular RNA (eRNA), a cis-natural antisense transcript (cis-NAT), a circularized RNA (ciRNA or circRNA), or a combination thereof. In some embodiments, the engineered nucleic acid may comprise or encode a structural element or sequence (or complementary thereof) of a shRNA. In some embodiments, the engineered nucleic acid may comprise or encode a structural element or sequence (or complementary thereof) of a ciRNA. In some embodiments, the engineered nucleic acid may comprise or encode a structural element or sequence (or complementary thereof) of an mRNA. In some embodiments, the engineered nucleic acid may comprise or encode a structural element or sequence (or complementary thereof) of a miRNA. In some embodiments, the engineered nucleic acid may comprise or encode a structural element or sequence (or complementary thereof) of a tRNA. In some embodiments, the engineered nucleic acid may comprise or encode a structural element or sequence (or complementary thereof) of a rRNA. In some embodiments, the engineered nucleic acid may comprise or encode a structural element or sequence (or complementary thereof) of a piRNA. In some embodiments, the engineered nucleic acid may comprise or encode a structural element or sequence (or complementary thereof) of a snoRNA. In some embodiments, the engineered nucleic acid may comprise or encode a structural element or sequence (or complementary thereof) of a snRNA. In some embodiments, the engineered nucleic acid may comprise or encode a structural element or sequence (or complementary thereof) of a scaRNA. In some embodiments, the engineered nucleic acid may comprise or encode a structural element or sequence (or complementary thereof) of a IncRNA. In some embodiments, the engineered nucleic acid may comprise or encode a structural element or sequence (or complementary thereof) of a HnRNA. In some embodiments, the engineered nucleic acid may comprise or encode a structural element or sequence (or complementary thereof) of a ci-NAT.

[0072] In some embodiments, the engineered nucleic acid (or a transcript expressed therefrom or a transcript expressed from an expression cassette encoding the engineered nucleic acid) may increase the exon-skipping, exon-inclusion, intron-skipping, or intron-inclusion of a target nucleic acid molecule, relative to a control. In some embodiments, the engineered nucleic acid (or a transcript expressed therefrom) may decrease the exon-skipping, exon-inclusion, intron-skipping, or intron-inclusion of a target nucleic acid molecule, relative to a control. The control may comprise the level of exon-skipping, exoninclusion, intron-skipping, or intron-inclusion of a target nucleic acid molecule when the engineered nucleic acid is not present or when the target nucleic acid molecule is contacted with control nucleic acid in which a particular structural element or sequence is not present. In some embodiments, the target nucleic acid molecule is a pre-mRNA of a gene of interest. In some embodiments, the target nucleic acid molecule is a mature mRNA of a gene of interest.

[0073] The exon-skipping, exon-inclusion, intron-skipping, or intron-inclusion may be referred to the region of the target nucleic acid molecule that is complementary to the antisense sequence. The exonskipping, exon-inclusion, intron-skipping, or intron-inclusion may be referred to the region of the target nucleic acid molecule that is within about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 250, 500 or 1000 nucleotides from the region that is complementary to the antisense sequence. The level of exon-skipping, exon-inclusion, intron-skipping, or intron-inclusion of a target nucleic acid molecule can be assayed by measuring the expression level of the target nucleic acid molecule populations comprising or not comprising a particular intron or exon.

[0074] In some embodiments, the engineered nucleic acid (or a transcript expressed therefrom) may increase the exon-skipping level of a target nucleic acid molecule that is at least: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of control. In some embodiments, the engineered nucleic acid (or a transcript expressed therefrom) may increase the exon-skipping level of a target nucleic acid molecule that is at most: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of control.

[0075] In some embodiments, the engineered nucleic acid (or a transcript expressed therefrom) may increase the exon-inclusion level of a target nucleic acid molecule that is at least: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of control. In some embodiments, the engineered nucleic acid (or a transcript expressed therefrom) may increase the exon-inclusion level of a target nucleic acid molecule that is at most: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of control.

[0076] In some embodiments, the engineered nucleic acid (or a transcript expressed therefrom) may increase the intron-skipping level of a target nucleic acid molecule that is at least: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of control. In some embodiments, the engineered nucleic acid (or a transcript expressed therefrom) may increase the intron-skipping level of a target nucleic acid molecule that is at most: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of control.

[0077] In some embodiments, the engineered nucleic acid (or a transcript expressed therefrom) may increase the intron-inclusion level of a target nucleic acid molecule that is at least: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of control. In some embodiments, the engineered nucleic acid (or a transcript expressed therefrom) may increase the intron-inclusion level of a target nucleic acid molecule that is at most: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of control.

[0078] Engineered Elements

[0079] Provided herein are nucleic acid molecules comprising engineered elements for improved stability and potency of engineered U7 snRNA-based splicing modulators. Also, provided herein are nucleic acid molecules comprising engineered elements for improved function to target mutated / non-functional target sequences present in disease. In some embodiments, the nucleic acid molecules comprise one or more antisense sequences, an engineered Sm-binding site, and a 3’ engineered element. In some embodiments, the nucleic acid molecules comprise a 5’ engineered element, one or more antisense sequences, and an engineered Sm-binding site. In some embodiments, the nucleic acid molecules comprise a 5’ engineered element, one or more antisense sequences, an engineered Sm-binding site, and a 3’ engineered element.

[0080] Also provided herein are expression cassettes encoding nucleic acid molecules comprising engineered elements for improved function to target mutated / non-functional target sequences present in disease. In some embodiments, the expression cassettes encoding the nucleic acid molecules comprise an engineered promoter, one or more antisense sequences, an engineered Sm-binding site, and a 3’ engineered terminator. In some embodiments, the nucleic acid molecules comprise an engineered promoter, a 5’ engineered element, one or more antisense sequences, an engineered Sm-binding site, and a 3’ engineered terminator. In some embodiments, the nucleic acid molecules comprise an engineered promoter and enhancer, a 5’ engineered element, one or more antisense sequences, an engineered Sm-binding site, and a 3’ engineered terminator.

[0081] The types of elements that an engineered nucleic acid molecule comprises depends on the biogenesis, maturation, or modification stage of the engineered nucleic acid molecule. Examples of this can be found in FIGs. 3A-3D. In some embodiments, the types of elements that an engineered nucleic acid molecule comprises are engineered post-transcriptional elements. In some embodiments, the engineered nucleic acid molecule or the post-transcribed form therefrom comprises an antisense sequence as described herein, an engineered element 5’ to the one or more antisense sequence as described herein, and one or more engineered Sm-binding sequences as described herein. In some embodiments, the engineered nucleic acid molecule or the post-transcribed form therefrom comprises an antisense sequence as described herein, an engineered element 3’ to the one or more antisense sequence as described herein, and engineered Sm-binding sequences as described herein. In some embodiments, the engineered nucleic acid molecule or the post-transcribed form therefrom comprises an antisense sequence as described herein, an engineered element 5’ to the one or more antisense sequence as described herein, an engineered element 3’ to the one or more antisense sequence as described herein, and engineered Sm-binding sequences as described herein. In some embodiments, the engineered nucleic acid molecule or the post-transcribed form therefrom comprises an antisense sequence as described herein, an engineered element 5’ to the one or more antisense sequence as described herein, an engineered element 3’ to the one or more antisense sequence as described herein, an engineered Sm-binding sequences, and a stabilizing sequence 3’ to the Sm-binding sequences as described herein.

[0082] In some embodiments, the types of elements that an engineered nucleic acid molecule comprises are engineered pre-transcriptional sequence elements. In some embodiments, the pre-transcribed form of the engineered nucleic acid molecule comprises an engineer promoter as described herein, an antisense sequence as described herein, an engineered element 5’ to the one or more antisense sequence as described herein, engineered Sm-binding sequences as described herein, and a 3’ engineered terminator. In some embodiments, the pre-transcribed form of the engineered nucleic acid molecule comprises an engineer promoter as described herein, an antisense sequence as described herein, an engineered element 5’ to the one or more antisense sequence as described herein, engineered Sm-binding sequences as described herein, and a 3’ engineered terminator. In some embodiments, a 3’ engineered element comprises a 3’ engineered terminator. In some embodiments, a 3’ engineered element is a 3’ engineered terminator. In some embodiments, the 3’ engineered terminator comprises an engineered stabilizing sequence or a secondary structure sequence, an insulator, an engineered transcription terminator domain (TTD), an engineered cleavage / processing site, or any combinations thereof. In some embodiments, the engineered stabilizing sequence or the secondary structure sequence is 3’ to the Sm-binding sequences as described herein.

[0083] In some embodiments, the types of 5’ engineered elements that an engineered nucleic acid molecule comprises are pre-transcriptional sequence elements. In some embodiments, the 5’ engineered element comprises one or more of a methylation element, an engineered stabilizing sequence or a secondary structure, and a recruitment site. In some embodiments, the recruitment site comprises a binding site for RNA-binding proteins (RBPs) or protein recruitment domain. In some embodiments, the methylation element comprises a sequence encoding a 5’ methyl G cap. In some embodiments, sequence encoding the 5’ methyl G cap comprises a 2,2,7 trimethyl G (m3G) cap. In some embodiments, the engineered stabilizing sequence or the secondary structure sequence comprises a stem-loop sequence, a triple helix sequence, a quadruplex sequence, a tRNA-like sequence, a pseudoknot sequence, or any combinations thereof.

[0084] In some embodiments, the types of 3’ engineered elements that an engineered nucleic acid molecule comprises are pre-transcriptional sequence elements. In some embodiments, the 3’ engineered terminator comprises an engineered stabilizing sequence or a secondary structure sequence as described herein, an insulator as described herein, an engineered TTD as described herein, an engineered cleavage / processing site as described herein, or any combinations thereof. In some embodiments, the 3’ engineered terminator sequence is from a LncRNA, an animal, a plant, a virus, a CRISPR sequence, a snoRNA, a histone mRNA, or any combinations thereof.

[0085] In some embodiments, the types of elements that an engineered nucleic acid molecule comprises are engineered post-transcriptional elements. In some embodiments, the engineered nucleic acid molecule or the post-transcribed form therefrom does not comprise the engineered promoters as described herein, the engineered regulatory elements as described herein, the engineered TTDs as described herein, or any combinations thereof.

[0086] In some embodiments, the engineered post-transcriptional nucleic acid molecule comprises a 3’ engineered element comprising one or more of an engineered stabilizing sequence or a secondary structure sequence and a terminator. In some embodiments, the 3’ engineered element is configured to form at least one secondary structure. In some embodiments, the terminator is configured to facilitate: (1) 3’ end processing of the nucleic acid molecule or (2) stabilization of the nucleic acid molecule. In some embodiments, the types of 5’ engineered elements that an engineered nucleic acid molecule comprises are post-transcriptional elements. In some embodiments, the 5’ engineered element comprises one or more of a methylation element, an engineered stabilizing sequence or a secondary structure, and a recruitment site. In some embodiments, the recruitment site comprises a binding site for one or more RBPs and / or a protein recruitment domain. In some embodiments, the methylation element comprises a 5’ methyl G cap. In some embodiments, the 5’ m3G cap. In some embodiments, the engineered stabilizing sequence or the secondary structure sequence comprises a stem-loop sequence, a triple helix sequence, a quadruplex sequence, a tRNA-like sequence, a pseudoknot sequence, or any combinations thereof.

[0087] Engineered Promoters

[0088] In some embodiments, the types of elements that a nucleic acid molecule comprises are engineered pre-transcriptional elements. Engineered nucleic acids may comprise an engineered promoter. In some embodiments, the pre-transcribed form of the engineered nucleic acid molecule comprises an engineer promoter as described herein. In some aspects, an expression cassette encoding a nucleic acid molecule comprises an engineered promoter. For instance, a DNA cassette as described herein may comprise an engineered promoter. A DNA cassette encoding a stabilizing sequence as described herein may further comprise an engineered promoter. In some embodiments, the engineered promoter may comprise any of those described herein, such as those of promoter coupled termination sequence. In some embodiments, the engineered promoter may comprise a tissue-specific promoter, a constitutively active promoter, a snRNA promoter, any functional derivatives thereof; or any combinations thereof. In some embodiments, the engineered promoter may comprise a tissue-specific promoter. In some embodiments, the engineered promoter may comprise a constitutively active promoter. In some embodiments, the engineered promoter may comprise a snRNA promoter. The snRNA promoter may comprise a U1 promoter; a U2 promoter; a U4 promoter; a U7 promoter; any functional derivatives thereof; or any combinations thereof. The snRNA promoter may comprise a U1 promoter. The snRNA promoter may comprise a U2 promoter. The snRNA promoter may comprise a U4 promoter. The snRNA promoter may comprise a U7 promoter. In some embodiments, the engineered promoter may comprise a Pol II promoter. In some embodiments, the engineered promoter may comprise a Pol I promoter. In some embodiments, the engineered promoter may comprise a Pol III promoter.

[0089] In some embodiments, the tissue-specific promoter may be any one of a(an): adrenal glands-specific promoter, anus-specific promoter, appendix-specific promoter, artery-specific promoter, bladderspecific promoter, blood cell-specific promoter, bone marrow-specific promoter, bone-specific promoter, brain-specific promoter, bronchi-specific promoter, bulbourethral gland-specific promoter, capillary-specific promoter, cerebellum-specific promoter, diaphragm-specific promoter, ear-specific promoter, esophagusspecific promoter, eye-specific promoter, fallopian tube-specific promoter, gallbladder-specific promoter, genital-specific promoter, hair-specific promoter, heart-specific promoter, human skeleton-specific promoter, hypothalamus-specific promoter, joint-specific promoter, kidney-specific promoter, large intestine-specific promoter, larynx-specific promoter, ligaments-specific promoter, liver-specific promoter, lung-specific promoter, lymph node-specific promoter, lymphatic vessel-specific promoter, mammary gland-specific promoter, mesentery-specific promoter, mouth-specific promoter, nail-specific promoter, nasal cavity-specific promoter, nerve-specific promoter, nose-specific promoter, olfactory epitheliumspecific promoter, ovary-specific promoter, pancreas-specific promoter, parathyroid gland-specific promoter, parathyroid gland-specific promoter, penis-specific promoter, pharynx-specific promoter, pineal gland-specific promoter, pituitary gland-specific promoter, placenta-specific promoter, prostate-specific promoter, rectum-specific promoter, salivary gland-specific promoter, scrotum-specific promoter, seminal vesicle-specific promoter, skeletal muscle-specific promoter, skin-specific promoter, small intestinespecific promoter, spinal cord-specific promoter, spleen-specific promoter, stomach-specific promoter, subcutaneous tissue-specific promoter, teeth-specific promoter, tendons-specific promoter, testis-specific promoter, the vestibular system of the ear-specific promoter, thoracic duct-specific promoter, thymus gland-specific promoter, thyroid-specific promoter, tongue-specific promoter, tonsils-specific promoter, trachea-specific promoter, ureter-specific promoter, urethra-specific promoter, uterus-specific promoter, vagina-specific promoter, vas deferens-specific promoter, vein-specific promoter, or any combinations thereof. In some embodiments, the tissue-specific promoter may comprise an adrenal gland-specific promoter. In some embodiments, the tissue-specific promoter may comprise an anus-specific promoter. In some embodiments, the tissue-specific promoter may comprise an appendix-specific promoter. In some embodiments, the tissue-specific promoter may comprise an artery-specific promoter. In some embodiments, the tissue-specific promoter may comprise a bladder-specific promoter. In some embodiments, the tissue-specific promoter may comprise a blood cell-specific promoter. In some embodiments, the tissue-specific promoter may comprise a bone marrow-specific promoter. In some embodiments, the tissue-specific promoter may comprise a bone-specific promoter. In some embodiments, the tissue-specific promoter may comprise a brain-specific promoter. In some embodiments, the tissue-specific promoter may comprise a bronchi-specific promoter. In some embodiments, the tissue-specific promoter may comprise a bulbourethral gland-specific promoter. In some embodiments, the tissue-specific promoter may comprise a capillary-specific promoter. In some embodiments, the tissue-specific promoter may comprise a cerebellum-specific promoter. In some embodiments, the tissue-specific promoter may comprise a diaphragm-specific promoter. In some embodiments, the tissue-specific promoter may comprise an ear-specific promoter. In some embodiments, the tissue-specific promoter may comprise an esophagus-specific promoter. In some embodiments, the tissue-specific promoter may comprise an eye-specific promoter. In some embodiments, the tissue-specific promoter may comprise a fallopian tube-specific promoter. In some embodiments, the tissue-specific promoter may comprise a gallbladder-specific promoter. In some embodiments, the tissue-specific promoter may comprise a genital-specific promoter. In some embodiments, the tissue-specific promoter may comprise a hair-specific promoter. In some embodiments, the tissue-specific promoter may comprise a heart-specific promoter. In some embodiments, the tissuespecific promoter may comprise a human skeleton-specific promoter. In some embodiments, the tissuespecific promoter may comprise a hypothalamus-specific promoter. In some embodiments, the tissuespecific promoter may comprise a joint-specific promoter. In some embodiments, the tissue-specific promoter may comprise a kidney-specific promoter. In some embodiments, the tissue-specific promoter may comprise a large intestine-specific promoter. In some embodiments, the tissue-specific promoter may comprise a larynx-specific promoter. In some embodiments, the tissue-specific promoter may comprise a ligaments-specific promoter. In some embodiments, the tissue-specific promoter may comprise a liverspecific promoter. In some embodiments, the tissue-specific promoter may comprise a lung-specific promoter. In some embodiments, the tissue-specific promoter may comprise a lymph node-specific promoter. In some embodiments, the tissue-specific promoter may comprise a lymphatic vessel-specific promoter. In some embodiments, the tissue-specific promoter may comprise a mammary gland-specific promoter. In some embodiments, the tissue-specific promoter may comprise a mesentery-specific promoter. In some embodiments, the tissue-specific promoter may comprise a mouth-specific promoter. In some embodiments, the tissue-specific promoter may comprise a nail-specific promoter. In some embodiments, the tissue-specific promoter may comprise a nasal cavity-specific promoter. In some embodiments, the tissue-specific promoter may comprise a nerve-specific promoter. In some embodiments, the tissue-specific promoter may comprise a nose-specific promoter. In some embodiments, the tissue-specific promoter may comprise an olfactory epithelium-specific promoter. In some embodiments, the tissue-specific promoter may comprise an ovary-specific promoter. In some embodiments, the tissue-specific promoter may comprise a pancreas-specific promoter. In some embodiments, the tissue-specific promoter may comprise a parathyroid gland-specific promoter. In some embodiments, the tissue-specific promoter may comprise a parathyroid gland-specific promoter. In some embodiments, the tissue-specific promoter may comprise a penis-specific promoter. In some embodiments, the tissue-specific promoter may comprise a pharynx-specific promoter. In some embodiments, the tissue-specific promoter may comprise a pineal gland-specific promoter. In some embodiments, the tissue-specific promoter may comprise a pituitary gland-specific promoter. In some embodiments, the tissue-specific promoter may comprise a placenta-specific promoter. In some embodiments, the tissue-specific promoter may comprise a prostate-specific promoter. In some embodiments, the tissue-specific promoter may comprise a rectum-specific promoter. In some embodiments, the tissue-specific promoter may comprise a salivary gland-specific promoter. In some embodiments, the tissue-specific promoter may comprise a scrotum-specific promoter. In some embodiments, the tissue-specific promoter may comprise a seminal vesicle-specific promoter. In some embodiments, the tissue-specific promoter may comprise a skeletal muscle-specific promoter. In some embodiments, the tissue-specific promoter may comprise a skin-specific promoter. In some embodiments, the tissue-specific promoter may comprise a small intestine-specific promoter. In some embodiments, the tissue-specific promoter may comprise a spinal cord-specific promoter. In some embodiments, the tissuespecific promoter may comprise a spleen-specific promoter. In some embodiments, the tissue-specific promoter may comprise a stomach-specific promoter. In some embodiments, the tissue-specific promoter may comprise a subcutaneous tissue-specific promoter. In some embodiments, the tissue-specific promoter may comprise a teeth-specific promoter. In some embodiments, the tissue-specific promoter may comprise a tendons-specific promoter. In some embodiments, the tissue-specific promoter may comprise a testis-specific promoter. In some embodiments, the tissue-specific promoter may comprise a vestibular system of the ear-specific promoter. In some embodiments, the tissue-specific promoter may comprise a thoracic duct-specific promoter. In some embodiments, the tissue-specific promoter may comprise a thymus gland-specific promoter. In some embodiments, the tissue-specific promoter may comprise a thyroid-specific promoter. In some embodiments, the tissue-specific promoter may comprise a tongue-specific promoter. In some embodiments, the tissue-specific promoter may comprise a tonsils-specific promoter. In some embodiments, the tissue-specific promoter may comprise a trachea-specific promoter. In some embodiments, the tissue-specific promoter may comprise a ureter-specific promoter. In some embodiments, the tissue-specific promoter may comprise a urethra-specific promoter. In some embodiments, the tissue-specific promoter may comprise a uterus-specific promoter. In some embodiments, the tissue-specific promoter may comprise a vagina-specific promoter. In some embodiments, the tissue-specific promoter may comprise a vas deferens-specific promoter.

[0090] In some embodiments, the engineered promoter may comprise a CMV, RSV, SV40, CBA, CAG, truncated CAG, Cbh, EF-1a, EFS, PGK, UBC, GUSB, UCOE, hAAT, TBG, GRM6, 770En_454P, HSA, Desmin, SkCRM4 / Des, MCK, CK6, MHCK7, dMCK, tMCK, CK8, CK8e, C5-12, NSE, CMV-MyoD, SynM, Synapsin, aMHC, PDGF, MLC2v, cTnT, MecP2, CaMKII, mGluR2, NFL, NFH, np2, PPE, ENK, EAAT2, GFAP, Myo, AUSEx3, SPcA5-12, unc45b, SPc5-12, myelin basic protein (MBP), Cox-2, PCP2, CLDN5, NR2E1, PITX3, PDE6H, RHO, RHOK, CNGA1, CNGB1, PDE6B, GRK1, SAG, ARR3, S-opsin, L / M-opsin, mopsin-500, CNGA3, CNGB3, RPGR, RPGRIP1, NHPH5, PRPH2, CRX, STK38L, RPE65, BEST1, PR1.7, PRO.5, 3LCR-PRO.5, PR2.1 , CAR, IRBP, Pou4f3, Ocp1 , Mathl , Prestin, Myo7a, Opto-mGluR6, H1 , 7SK, U1 , U2, U4, U5, or U6 promoter. In some embodiments, the promoter is a sequence isolated or derived from a promoter capable of driving expression of a transfer RNA (tRNA). In some embodiments, the engineered promoter may comprise an H1 promoter. In some embodiments, the engineered promoter may comprise a MBP promoter. In some embodiments, the engineered promoter may comprise a PR2.1 promoter. In some embodiments, the engineered promoter may comprise a 3LCR-PRO.5 promoter. In some embodiments, the engineered promoter may comprise a 770En_454P promoter. In some embodiments, the engineered promoter may comprise a 7SK promoter. In some embodiments, the engineered promoter may comprise an ARR3 promoter. In some embodiments, the engineered promoter may comprise a BEST1 promoter. In some embodiments, the engineered promoter may comprise a C5- 12 promoter. In some embodiments, the engineered promoter may comprise a CAG promoter. In some embodiments, the engineered promoter may comprise a CaMKII promoter. In some embodiments, the engineered promoter may comprise a CAR promoter. In some embodiments, the engineered promoter may comprise a CBA promoter. In some embodiments, the engineered promoter may comprise a Cbh promoter. In some embodiments, the engineered promoter may comprise a CK6 promoter. In some embodiments, the engineered promoter may comprise a CK8 promoter. In some embodiments, the engineered promoter may comprise a CK8e promoter. In some embodiments, the engineered promoter may comprise a CLDN5 promoter. In some embodiments, the engineered promoter may comprise a CMV promoter. In some embodiments, the engineered promoter may comprise a CMV-MyoD promoter. In some embodiments, the engineered promoter may comprise a CNGA1 promoter. In some embodiments, the engineered promoter may comprise a CNGA3 promoter. In some embodiments, the engineered promoter may comprise a CNGB1 promoter. In some embodiments, the engineered promoter may comprise a CNGB3 promoter. In some embodiments, the engineered promoter may comprise a Cox-2 promoter. In some embodiments, the engineered promoter may comprise a CRX promoter. In some embodiments, the engineered promoter may comprise a cTnT promoter. In some embodiments, the engineered promoter may comprise a Desmin promoter. In some embodiments, the engineered promoter may comprise a dMCK promoter. In some embodiments, the engineered promoter may comprise an EAAT2 promoter. In some embodiments, the engineered promoter may comprise an EF-1a promoter. In some embodiments, the engineered promoter may comprise an EFS promoter. In some embodiments, the engineered promoter may comprise an ENK promoter. In some embodiments, the engineered promoter may comprise a GFAP promoter. In some embodiments, the engineered promoter may comprise a GRK1 promoter. In some embodiments, the engineered promoter may comprise a GRM6 promoter. In some embodiments, the engineered promoter may comprise a GUSB promoter. In some embodiments, the engineered promoter may comprise a hAAT promoter. In some embodiments, the engineered promoter may comprise an HSA promoter. In some embodiments, the engineered promoter may comprise an IRBP promoter. In some embodiments, the engineered promoter may comprise an L / M-opsin promoter. In some embodiments, the engineered promoter may comprise a Mathl promoter. In some embodiments, the engineered promoter may comprise a MCK promoter. In some embodiments, the engineered promoter may comprise a MecP2 promoter. In some embodiments, the engineered promoter may comprise an mGluR2 promoter. In some embodiments, the engineered promoter may comprise an MHCK7 promoter. In some embodiments, the engineered promoter may comprise an MLC2v promoter. In some embodiments, the engineered promoter may comprise a mopsin-500 promoter. In some embodiments, the engineered promoter may comprise a Myo promoter. In some embodiments, the engineered promoter may comprise a Myo7a promoter. In some embodiments, the engineered promoter may comprise an NFH promoter. In some embodiments, the engineered promoter may comprise an NFL promoter. In some embodiments, the engineered promoter may comprise an NHPH5 promoter. In some embodiments, the engineered promoter may comprise an NR2E1 promoter. In some embodiments, the engineered promoter may comprise an NSE promoter. In some embodiments, the engineered promoter may comprise an np2 promoter. In some embodiments, the engineered promoter may comprise an Ocp1 promoter. In some embodiments, the engineered promoter may comprise an Opto-mGluR6 promoter. In some embodiments, the engineered promoter may comprise a PCP2 promoter. In some embodiments, the engineered promoter may comprise a PDE6B promoter. In some embodiments, the engineered promoter may comprise a PDE6H promoter. In some embodiments, the engineered promoter may comprise a PDGF promoter. In some embodiments, the engineered promoter may comprise a PGK promoter. In some embodiments, the engineered promoter may comprise a PITX3 promoter. In some embodiments, the engineered promoter may comprise a Pou4f3 promoter. In some embodiments, the engineered promoter may comprise a PPE promoter. In some embodiments, the engineered promoter may comprise a PRO.5 promoter. In some embodiments, the engineered promoter may comprise a PR1.7 promoter. In some embodiments, the engineered promoter may comprise a Prestin promoter. In some embodiments, the engineered promoter may comprise a PRPH2 promoter. In some embodiments, the engineered promoter may comprise an RHO promoter. In some embodiments, the engineered promoter may comprise an RHOK promoter. In some embodiments, the engineered promoter may comprise an RPE65 promoter. In some embodiments, the engineered promoter may comprise an RPGR promoter. In some embodiments, the engineered promoter may comprise an RPGRIP1 promoter. In some embodiments, the engineered promoter may comprise an RSV promoter. In some embodiments, the engineered promoter may comprise a SAG promoter. In some embodiments, the engineered promoter may comprise a SkCRM4 / Des promoter. In some embodiments, the engineered promoter may comprise a S-opsin promoter. In some embodiments, the engineered promoter may comprise a SPc5-12 promoter. In some embodiments, the engineered promoter may comprise a SPcA5-12 promoter. In some embodiments, the engineered promoter may comprise a STK38L promoter. In some embodiments, the engineered promoter may comprise an SV40 promoter. In some embodiments, the engineered promoter may comprise a Synapsin promoter. In some embodiments, the engineered promoter may comprise a SynM promoter. In some embodiments, the engineered promoter may comprise a TBG promoter. In some embodiments, the engineered promoter may comprise a tMCK promoter. In some embodiments, the engineered promoter may comprise a truncated CAG promoter. In some embodiments, the engineered promoter may comprise a U1 promoter. In some embodiments, the engineered promoter may comprise a U2 promoter. In some embodiments, the engineered promoter may comprise a U4 promoter. In some embodiments, the engineered promoter may comprise a U5 promoter. In some embodiments, the engineered promoter may comprise a U6 promoter. In some embodiments, the engineered promoter may comprise a UBC promoter. In some embodiments, the engineered promoter may comprise a UCOE promoter. In some embodiments, the engineered promoter may comprise an unc45b promoter. In some embodiments, the engineered promoter may comprise an aMHC promoter. In some embodiments, the engineered promoter may comprise a AUSEx3 promoter.

[0091] In some embodiments, a promoter described herein may have at least: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, 250, 251 , 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 , 262, 263, 264, 265, 266, 267, 268, 269, 270, 271 , 272, 273, 274, 275, 276, 277, 278, 279, 280, 281 , 282, 283, 284, 285, 286, 287, 288, 289, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 , 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321 , 322, 323, 324, 325, 326, 327, 328, 329, 330, 331 , 332, 333, 334, 335, 336, 337, 338, 339, 340, 341 , 342, 343, 344, 345, 346, 347, 348, 349, 350, 351 , 352, 353, 354, 355, 356, 357, 358, 359, 360, 361 , 362, 363, 364, 365, 366, 367, 368, 369, 370, 371 , 372, 373, 374, 375, 376, 377, 378, 379, 380, 381 , 382, 383, 384, 385, 386, 387, 388, 389, 390, 391 , 392, 393, 394, 395, 396, 397, 398, 399, 400, 401 , 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441 , 442, 443, 444, 445, 446, 447, 448, 449, 450, 451 , 452, 453, 454, 455, 456, 457, 458, 459, 460, 461 , 462, 463, 464, 465, 466, 467, 468, 469, 470, 471 , 472, 473, 474, 475, 476, 477, 478, 479, 480, 481 , 482, 483, 484, 485, 486, 487, 488, 489, 490, 491 , 492, 493, 494, 495, 496, 497, 498, 499, 500, 1000, 2000, 5000 or more nucleotides (nt; or base pair / bp). In some embodiments, a promoter described herein may have at most: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 , 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, 250, 251 , 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 , 262, 263, 264, 265, 266, 267, 268, 269, 270, 271 , 272, 273, 274, 275, 276, 277, 278, 279, 280, 281 , 282, 283, 284, 285, 286, 287, 288, 289, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 , 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331 , 332, 333, 334, 335, 336, 337, 338, 339, 340, 341 , 342, 343, 344, 345, 346, 347, 348, 349, 350, 351 , 352, 353, 354, 355, 356, 357, 358, 359, 360, 361 , 362, 363, 364, 365, 366, 367, 368, 369, 370, 371 , 372, 373, 374, 375, 376, 377, 378, 379, 380, 381 , 382, 383, 384, 385, 386, 387, 388, 389, 390, 391 , 392, 393, 394, 395, 396, 397, 398, 399, 400, 401 , 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441 , 442, 443, 444, 445, 446, 447, 448, 449, 450, 451 , 452, 453, 454, 455, 456, 457, 458, 459, 460, 461 , 462, 463, 464, 465, 466, 467, 468, 469, 470, 471 , 472, 473, 474, 475, 476, 477, 478, 479, 480, 481 , 482, 483, 484, 485, 486, 487, 488, 489, 490, 491 , 492, 493, 494, 495, 496, 497, 498, 499, 500, 1000, 2000, or 5000 nucleotides (nt; or base pair / bp).

[0092] In some embodiments, a promoter described herein may comprise at least 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, 250, 251 , 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 , 262, 263, 264, 265, 266, 267, 268, 269, 270, 271 , 272, 273, 274, 275, 276, 277, 278, 279, 280, 281 , 282, 283, 284, 285, 286, 287, 288, 289, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 , 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341 , 342, 343, 344, 345, 346, 347, 348, 349, 350, 351 , 352, 353, 354, 355, 356, 357, 358, 359, 360, 361 , 362, 363, 364, 365, 366, 367, 368, 369, 370, 371 , 372, 373, 374, 375, 376, 377, 378, 379, 380, 381 , 382, 383, 384, 385, 386, 387, 388, 389, 390, 391 , 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421 , 422, 423, 424, 425, 426, 427, 428, 429, 430, 431 , 432, 433, 434, 435, 436, 437, 438, 439, 440, 441 , 442, 443, 444, 445, 446, 447, 448, 449, 450, 451 , 452, 453, 454, 455, 456, 457, 458, 459, 460, 461 , 462, 463, 464, 465, 466, 467, 468, 469, 470, 471 , 472, 473, 474, 475, 476, 477, 478, 479, 480, 481 , 482, 483, 484, 485, 486, 487, 488, 489, 490, 491 , 492, 493, 494, 495, 496, 497, 498, 499, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 nucleotides (nt; or base pair / bp).

[0093] In some embodiments, the engineered promoter may comprise or encode a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or more sequence identity to any sequence disclosed in Table 1. In some embodiments, the engineered nucleic acid may comprise or encode a sequence having at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, at most 99%, at least 99.5% sequence identity to any sequence disclosed in Table 1. In some embodiments, the engineered nucleic acid may comprise or encode a sequence having 100% sequence identity to any sequence disclosed in Table 1.

[0094] In some embodiments, the engineered promoter may comprise or encode a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or more sequence identity to any sequence disclosed in SEQ ID NOS: 77-92 and 132-139. In some embodiments, the engineered nucleic acid may comprise or encode a sequence having at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, at most 99%, at least 99.5% sequence identity to any sequence disclosed in SEQ ID NOS: 77-92 and 132-139. In some embodiments, the engineered nucleic acid may comprise or encode a sequence having 100% sequence identity to any sequence disclosed in SEQ ID NOS: 77-92 and 132-139. Table 1 : Exemplar Engineered Promoter Sequences

[0095] Name of SEQUENCE SEQ ID

[0096] Notes sequence NO TAACAACATAGGAGCTGTGATTGGCTGTTTTCAGCCAATCAGCACT GAC T C AT T T GC AT AGC C T T T AC AAGC GGT C AC AAAC T C AAGAAAC G

[0097] U7 mouse AGCGGTTTTAATAGTCTTTTAGAATATTGTTTATCGAACCGAATAA GGAACTGTGCTTTGTGATTCACATATCAGTGGAGGGGTGTGGAAAT 77 Pol II promoter

[0098] GGCACCTTGATCTCACCCTCATCGAAAGTGGAGTTGATGTCCTTCC C T GGC T C GC T AC AGAC GC AC T T C C GC AA CATGATGAACGTTACGGTGGAACAGGATAAACAGTACGTCGCACCG AATGTCTAAATACACGTTAAAACACCGGTTTACTGTACATACTGTA ACTTGTCACGATCCGAAAATTAATCTTTATCAATCAACAACAATAC TGCGTCGCAATAGAACACCAATTGCAAGTTTTTCAAGCTTGATAAA CACAATTAGACTGTTTACAACAGCAAAATTAATCTTTATGTCTGCT

[0099] U1 Zebrafish T AGT C AAT GAT T T GAGT AGT AAAAT C AAC AGAT GT GGAC T C AC C T T 78 Pol II promoter CAAAGCAACGCCGTTGTATGCTGGTTGTAGGTTATTTAAGCTGAAG AAATCCAGTGTTTATGCATTTACATTTGTTTAAAACAGAAAAAGCA AC AAAC T AAAGT T T GAAT CCTTTGATTGCGGC GAGAAGAGC GAAAG GAT GAC GCCCCCTTGC AGC GATT GGAC GGCTGGATTCACAATCCGC TGT T C AAGAAT T T C AAC C T GGAAT AAAT AT AT AAAAAC TTAATAGCATA TTTTGCAAATGTCAATTAACAGTATGAATAATTGGTAATAAGTGAC

[0100] U7 Zebrafish ATCATTGAATGAATGTTGGAATTTGCATAATGTACAATGCATTTCA 79 Pol II promoter CACTTTCAGCATTCATTAGATTTAGGCACCATCAGTGAGATACTCT

[0101] T T C AAAGT GAT GGGGGAGAAT T C C T GAAAAC AC AT T ATTTTCGTGGCTTGTAAAACTATTGTGGGCCTCTCAGATTGTTTAG TTTTAATTAAAATTATTCTTATCATTATTCGCTCGTTAATATAATC

[0102] U1 drosophila ATTTAAATGAAACAGTAAAATCCAAAGGTTACATATAATATGTATA

[0103] melanogaster AGGGT GGAAGC AGAT AAGAAC C AC AAT AT C AAC GC AC AGT T T AC AA 80 Pol II promoter AAATCAACCCAACTGCAGATGTAACATTTTGCTAACATACTTTCGG

[0104] GGGT AAGAC T T T C AC GC AC T T T C AC T GC AAT AAT T C C C AAC T GC T T CTGGCCATCAGCTCAT GGAAAC CCATTCCCT GAGC T GAGGAAAGC TTGGAAATGATTGCCCCTATAATTTATATGGCAAAATCCATTTTGG AGTTCTTTGGGATTCCATTAAAATAAAGTCTTCATGTGAACAATTA TCTTATCTTTTTGTTATTATTCAATAGGAACACTTTTCTTTTTATT TTTTAAAGCATTCCGGAGTTGCAAGTAGATTTTATTTGAATTGGTT

[0105] U1 drosophila GAT C T AAAAAGC AT AC TTTATTCATTTACGATCC T AAAT AT T T AT T

[0106] melanogaster ACTACATATATACATACATATCTGGCCAGAACATTTGTTTACCTCC 81 Pol II promoter TATCGACTCAGCTGGGTCTGAAATGCGAACGTTTAGTGCGCATGCT AGTGAATTTTGTGTGGCATACTTATAGGGGTGCTTTATTTCGCCAC GCGTTCGTTGCAATTCCCAACTGGTTTTAGCTGCTCAGCCATGGAA ACCCTGCTGCCGAGCATCGAAAAGC ATTTGAAGAAGGATGTCCAGGCTTCATTGGGCCGCCGAAAAGTTGT T AC AC AAC AC AGGT T AAGC AC CAGCTGCTTT GGGAGAGAAC AGAT G C GGGGGC GGAGGAAAAAAGGGAGAGGC AAAC GT C AC TTCCCCTTGT

[0107] U1 GGGC T C C T GC AGC GGGT T GGT C C GC T GAGT GGAAGAAAGGCAGAAG

[0108] Macacajasci GGGAC T GGGAAAGGC AC T GT C GGT GAC AT C AC GGAGAGGGC GAC T T

[0109] cularis_6.0:C CTATGTAGATGAGGCAGCGCAGGGGCTGCTGCTTCGACATCTGCTG 82 Pol II M021939.1 CTTCGCCACGAAGGAGTTCCCCTGCCGTGGGAGCGGGTTCAGGACC

[0110] promoter GCTAGTCGGACCTGAGAGTCCCAGCTGCGTGTCAGGGCTAGGAGGG CTCGAGAGTGCGCGGGGCAAGTGACCGTACGTGTAAAGGGTGAGAC GTATGAGGCTGTGGCAGGGCGGAGGTGCTGGAGCTC GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACA AGGC T GT T AGAGAGAT AAT T GGAAT T AAT T T GAC T GT AAAC AC AAA GATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGG

[0111] U6 promoter TAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGC 83 Pol III

[0112] TTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTT

[0113]

[0114] GT GGAAAGGAC GAAAC AC C Name of SEQUENCE SEQ ID

[0115] Notes sequence NO GAACGCTGACGTCATCAACCCGCTCCAAGGAATCGCGGGCCCAGTG T C AC T AGGC GGGAAC AC C C AGC GC GC GT GC GC C C T GGC AGGAAGAT

[0116] H1 promoter GGCTGTGAGGGACAGGGGAGTGGCGCCCTGCAATATTTGCATGTCG 84 Pol III CTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATT TGGGAATCTTATAAGTTCTGTATGAGACCAC CTGAGGCGGAAAGAACCAGCTGTGGAATGTGTGTCAGTTAGGGTGT GGAAAGT C C C C AGGC T C C C C AGC AGGC AGAAGT AT GC AAAGC AT GC ATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCC SV40 AGC AGGC AGAAGT AT GC AAAGC AT GC AT C T CAAT TAGT C AGC AAC C

[0117] (enhancer and ATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCA 85 Pol II promoter) GTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTA

[0118] T GC AGAGGC C GAGGC CGCCTCGGCCTCT GAGC T AT T C C AGAAGT AG T GAGGAGGC T T T T T T GGAGGC CTAGGCTTTT GC AAA GC AGGC AGAAGT AT GC AAAGC AT GC AT C T CAAT TAGT C AGC AAC C A SV40 (early TAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAG

[0119] promoter TTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTAT 86 Pol II short) GCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGT

[0120] GAGGAGGC T T T T T T GGAGGC C T AGGC T TCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCC CTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGT

[0121] Chicken betaGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGGGGCGAGGGGCGGG GCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGC 87 Pol II actin promoter

[0122] GCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCC TATAAAAAGCGAAGCGCGCGGCGGGCG GTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTG ACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAG CMV promoter TTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAAC 88 Pol II AACTGCGATCGCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGT ACGGTGGGAGGTCTATATAAGCAGAGCT CCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCG

[0123] Truncated TTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAA 89 Pol II CMV promoter GCAGAGCT

[0124] Minimal CMV GACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGC

[0125] prom t AGAGCT 90 Pol II o er

[0126] GGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGG

[0127] Core promoter

[0128] GGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAAC

[0129] for human TGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTG 91 Pol II elongation GGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTT

[0130] factor EF-1a

[0131]

[0132] T C GC AAC GGGT T T GC C GC C AGAAC AC AG Name of SEQUENCE SEQ ID

[0133] Notes sequence NO GGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCC CCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAG AAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTC CGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAG TCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACA GGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGT TATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTCCAGTACG TGATTCTTGATCCCGAGCTGGAGCCAGGGGCGGGCCTTGCGCTTTA GGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCT GGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGC TGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCT GCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAGG EF-1a ATCTGCACACTGGTATTTCGGTTTTTGGGCCCGCGGCCGGCGACGG GGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGA 92 Pol II promoter

[0134] GCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGC CTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTG GGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGA TGGCCGCTTCCCGGCCCTGCTCCAGGGGGCT C AAAAT GGAGGAC GC GGC GC T C GGGAGAGC GGGC GGGT GAGT C AC C C AC AC AAAGGAAAAG GGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTAC CGGGCGCCGTCCAGGCACCTCGATTAGTTCTGGAGCTTTTGGAGTA CGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCC CCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTG ATGTAATTCTCCTTGGAATTTGGCCTTTTTGAGTTTGGATCTTGGT TCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATT TCAGGTGTCGTGA TTGCGCCTTTTCCAAGGCAGCCCTGGGTTTGCGCAGGGACGCGGCT GCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTC TCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCC GCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTGCTCCGCCCCTAA GTCGGGAAGGTTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAAC

[0135] hPGK GGAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAG 132

[0136] GGAGCAATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAATAGCG GCTGCTCAGCAGGGCGCGCCGAGAGCAGCGGCCGGGAAGGGGCGGT GCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCCTGTTCCTGCCC GCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAG TCGGCTCCCTCGTTGACCGAATCACCGACCTCTCTCCCCAG AGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCC T AC C T GAC GAC C GAC C C C GAC C C AC T GGAC AAGC AC C C AAC C C C C A T T C C C C AAAT TGCGCATCCCCTAT C AGAGAGGGGGAGGGGAAAC AG GATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACA GTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCA

[0137] hsynapsin CTGAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCT 133 Pol II TCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGA CCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATC TGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGG CAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAG GGGCCCCAGAAGCCTGGTGGTTGTTTGTCCTTCTCAGGGGAAAAGT GAGGC GGCCCCTT GGAGGAAGGGGC C GGGC AGAAT GATCTAATCGG ATTCCAAGCAGCTCAGGGGATTGTCTTTTTCTAGCACCTTCTTGCC

[0138] hRhodopsin ACTCCTAAGCGTCCTCCGTGACCCCGGCTGGGATTTAGCCTGGTGC 134 Pol II Kinase TGTGTCAGCCCCGGTCTCCCAGGGGCTTCCCAGTGGTCCCCAGGAA CCCTCGACAGGGCCCGGTCTCTCTCGTCCAGCAAGGGCAGGGACGG GCCACAGGCCAAGGGCGGTAC CCTCTCCTCCCTGACCTCAGGCTTCCTCCTAGTGTCACCTTGGCCC C T C T T AGAAGC C AAT T AGGC C C T C AGT T T C T GC AGC GGGGAT T AAT

[0139] hRhodpsin AT GAT TAT GAAC AC CCCCAATCTCC C AGAT GC T GAT T C AGC C AGGA 135 Pol II (short) GC T T AGGAGGGGGAGGT C AC T T T AT AAGGGT C T GGGGGGGT C AGAA

[0140]

[0141] CCCAGAGTCATC Name of SEQUENCE SEQ ID

[0142] Notes sequence NO TATGTGTCTGGCACCAGAAACGGAAGCTGCAGGTTGCAGCCCCTGC C C T C AT GGAGC T C C T C C T GT C AGAGGAGT GT GGGGAC T GGAT GAC T CCAGAGGTAACTTGTGGGGGAACGAACAGGTAAGGGGCTGTGTGAC GAGAT GAGAGAC T GGGAGAAT AAAC C AGAAAGT C TCTAGCTGTCCA

[0143] hRhodpsin GAGGAC AT AGC AC AGAGGC C C AT GGT C C C T AT T T C AAAC C C AGGC C

[0144] (500bp AC C AGAC T GAGC T GGGAC C T T GGGAC AGAC AAGT C AT GC AGAAGT T 136 Pol II upstream) AGGGGACCTTCTCCTCCCTTTTCCTGGATCCTGAGTACCTCTCCTC CCTGACCTCAGGCTTCCTCCTAGTGTCACCTTGGCCCCTCTTAGAA GCCAATTAGGCCCTCAGTTTCTGCAGCGGGGATTAATATGATTATG AAC AC CCCCAATCTCC C AGAT GCTGATTCAGC C AGGAGC T TAGGAG GGGGAGGTCACTTTATAAGGGTCTGGGGGGGTCAGAACCC TATGTGTCTGGCACCAGAAACGGAAGCTGCAGGTTGCAGCCCCTGC C C T C AT GGAGC T C C T C C T GT C AGAGGAGT GT GGGGAC T GGAT GAC T CCAGAGGTAACTTGTGGGGGAACGAACAGGTAAGGGGCTGTGTGAC GAGAT GAGAGAC T GGGAGAAT AAAC C AGAAAGT C TCTAGCTGTCCA GAGGAC AT AGC AC AGAGGC C CAT GGT C C C TAT T T C AAAC C C AGGC C

[0145] hRhodpsin AC C AGAC T GAGC T GGGAC C T T GGGAC AGAC AAGT CAT GC AGAAGT T

[0146] (500bp AGGGGACCTTCTCCTCCCTTTTCCTGGATCCTGAGTACCTCTCCTC 137 Pol II upstream+UT CCTGACCTCAGGCTTCCTCCTAGTGTCACCTTGGCCCCTCTTAGAA

[0147] R) GCCAATTAGGCCCTCAGTTTCTGCAGCGGGGATTAATATGATTATG

[0148] AAC AC CCCCAATCTCC C AGAT GCTGATTCAGC C AGGAGC T TAGGAG GGGGAGGTCACTTTATAAGGGTCTGGGGGGGTCAGAACCCAGAGTC ATCCAGCTGGAGCCCTGAGTGGCTGAGCTCAGGCCTTCGCAGCATT CTTGGGTGGGAGCAGCCACGGGTCAGCCACAAGGGCCACAGCC AGAC T GAGC T GGGAC C T T GGGAC AGAC AAGT CAT GC AGAAGT TAGG GGACCTTCTCCTCCCTTTTCCTGGATCCTGAGTACCTCTCCTCCCT

[0149] hRho GACCTCAGGCTTCCTCCTAGTGTCACCTTGGCCCCTCTTAGAAGCC

[0150] (proximal AAT T AGGC C C T C AGT T T C T GC AGC GGGGAT T AAT AT GAT T AT GAAC 138 Pol II promoter) AC CCCCAATCTCC C AGAT GCTGATTCAGC C AGGAGC T T AGGAGGGG GAGGTCACTTTATAAGGGTCTGGGGGGGTCAGAACCC GGGCCCCAGAAGCCTGGTGGTTGTTTGTCCTTCTCAGGGGAAAAGT GAGGC GGCCCCTT GGAGGAAGGGGC C GGGC AGAAT GATCTAATCGG ATTCCAAGCAGCTCAGGGGATTGTCTTTTTCTAGCACCTTCTTGCC

[0151] hRhodopsin ACTCCTAAGCGTCCTCCGTGACCCCGGCTGGGATTTAGCCTGGTGC

[0152] Kinase_full TGTGTCAGCCCCGGTCTCCCAGGGGCTTCCCAGTGGTCCCCAGGAA 139

[0153] 5'UTR CCCTCGACAGGGCCCGGTCTCTCTCGTCCAGCAAGGGCAGGGACGG GCCACAGGCAAGGGCAGCAGTCAGGCCTGCTCTGTCTGTGAACGCT CCCGGCTTGGCCTCGGCTGATGGGCCCTCACGCCTGAAGCGGGCAG

[0154]

[0155] GAAGCTCCGGG

[0156] In some embodiments, the engineered promoter may also comprise (1) a basic promoter or (2) an otherwise-constitutively active promoter, that is coupled to a regulatory (such as an enhancer or a repressor) that facilitate the expression from the promoter to be specific to a tissue, similarly to the tissue-specific promoter as described herein. In some embodiments, the enhancer is a short (e.g., 50-1500 bp) region of DNA that can be bound by proteins (activators) to increase the likelihood that transcription of a particular gene will occur. In some embodiments, the enhancer comprises a cytomegalovirus (CMV) immediate / early enhancer. In some embodiments, the enhancer comprises a Simian Virus 40 (SV40) enhancer.

[0157] In some embodiments, an enhancer or a repressor described herein may have at least: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, 250, 251 , 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 , 262, 263, 264, 265, 266, 267, 268, 269, 270, 271 , 272, 273, 274, 275, 276, 277, 278, 279, 280, 281 , 282, 283, 284, 285, 286, 287, 288, 289, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 , 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331 , 332, 333, 334, 335, 336, 337, 338, 339, 340, 341 , 342, 343, 344, 345, 346, 347, 348, 349, 350, 351 , 352, 353, 354, 355, 356, 357, 358, 359, 360, 361 , 362, 363, 364, 365, 366, 367, 368, 369, 370, 371 , 372, 373, 374, 375, 376, 377, 378, 379, 380, 381 , 382, 383, 384, 385, 386, 387, 388, 389, 390, 391 , 392, 393, 394, 395, 396, 397, 398, 399, 400, 401 , 402, 403, 404, 405, 406, 407, 408, 409, 410, 411 , 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431 , 432, 433, 434, 435, 436, 437, 438, 439, 440, 441 , 442, 443, 444, 445, 446, 447, 448, 449, 450, 451 , 452, 453, 454, 455, 456, 457, 458, 459, 460, 461 , 462, 463, 464, 465, 466, 467, 468, 469, 470, 471 , 472, 473, 474, 475, 476, 477, 478, 479, 480, 481 , 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 1000, 2000, 5000 or more nucleotides (nt; or base pair / bp) . In some embodiments, an enhancer or a repressor described herein may have at most: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, 250, 251 , 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 , 262, 263, 264, 265, 266, 267, 268, 269, 270, 271 , 272, 273, 274, 275, 276, 277, 278, 279, 280, 281 , 282, 283, 284, 285, 286, 287, 288, 289, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 , 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321 , 322, 323, 324, 325, 326, 327, 328, 329, 330, 331 , 332, 333, 334, 335, 336, 337, 338, 339, 340, 341 , 342, 343, 344, 345, 346, 347, 348, 349, 350, 351 , 352, 353, 354, 355, 356, 357, 358, 359, 360, 361 , 362, 363, 364, 365, 366, 367, 368, 369, 370, 371 , 372, 373, 374, 375, 376, 377, 378, 379, 380, 381 , 382, 383, 384, 385, 386, 387, 388, 389, 390, 391 , 392, 393, 394, 395, 396, 397, 398, 399, 400, 401 , 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431 , 432, 433, 434, 435, 436, 437, 438, 439, 440, 441 , 442, 443, 444, 445, 446, 447, 448, 449, 450, 451 , 452, 453, 454, 455, 456, 457, 458, 459, 460, 461 , 462, 463, 464, 465, 466, 467, 468, 469, 470, 471 , 472, 473, 474, 475, 476, 477, 478, 479, 480, 481 , 482, 483, 484, 485, 486, 487, 488, 489, 490, 491 , 492, 493, 494, 495, 496, 497, 498, 499, 500, 1000, 2000, or 5000 nucleotides (nt; or base pair / bp).

[0158] In some embodiments, an enhancer or a repressor described herein comprises at least 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, 250, 251 , 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 , 262, 263, 264, 265, 266, 267, 268, 269, 270, 271 , 272, 273, 274, 275, 276, 277, 278, 279, 280, 281 , 282, 283, 284, 285, 286, 287, 288, 289, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 , 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341 , 342, 343, 344, 345, 346, 347, 348, 349, 350, 351 , 352, 353, 354, 355, 356, 357, 358, 359, 360, 361 , 362, 363, 364, 365, 366, 367, 368, 369, 370, 371 , 372, 373, 374, 375, 376, 377, 378, 379, 380, 381 , 382, 383, 384, 385, 386, 387, 388, 389, 390, 391 , 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421 , 422, 423, 424, 425, 426, 427, 428, 429, 430, 431 , 432, 433, 434, 435, 436, 437, 438, 439, 440, 441 , 442, 443, 444, 445, 446, 447, 448, 449, 450, 451 , 452, 453, 454, 455, 456, 457, 458, 459, 460, 461 , 462, 463, 464, 465, 466, 467, 468, 469, 470, 471 , 472, 473, 474, 475, 476, 477, 478, 479, 480, 481 , 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 1000, 1500 nucleotides (nt; or base pair / bp). In some embodiments, the engineered promoter may comprise or encode an enhancer sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or more sequence identity to any sequence disclosed in Table 2. In some embodiments, the engineered nucleic acid may comprise or encode a sequence having at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, at most 99%, at least 99.5% sequence identity to any sequence disclosed in Table 2. In some embodiments, the engineered nucleic acid may comprise or encode a sequence having 100% sequence identity to any sequence disclosed in Table 2.

[0159] In some embodiments, the engineered promoter may comprise or encode an enhancer sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or more sequence identity to any sequence disclosed in SEQ ID NOS: 93-104. In some embodiments, the engineered nucleic acid may comprise or encode a sequence having at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, at most 99%, at least 99.5% sequence identity to any sequence disclosed in SEQ ID NOS: 93-104. In some embodiments, the engineered nucleic acid may comprise or encode a sequence having 100% sequence identity to any sequence disclosed in SEQ ID NOS: 93-104. Table 2: Exemplar Engineered Enhancer Sequences

[0160] Name of SEQUENCE SEQ ID

[0161] Notes sequence NO

[0162] GC GC AGC AC CAT GGC C T G AAAT AAC C T C T GAAAG AGGAAC T T GGT T AGGTACCTTCTGAGGCGGAAAGAACCAGCTGTGGAATGTGTGTCAG SV40 I T AGGGTGT G GAAAG T CCCC AGGC TCCCC AGC AG GC AGAAG T AT GC AAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCC 93 enhancer

[0163] AGGC T C C C C AGC AGGC AGAAGT AT GC AAAGC AT GC AT C T C AAT TAG T C AGC AAC CAT AGT C C C GC C C C T AAC T C C GC C C GAC AT TGAT T AT TGAC TAGT T AT T AAT AGT AATCAAT T AC GGGGTC ATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACG GT AAAT GGC C C GC C T GGC T GAC C GC C C AAC GAC C C C C GC C C AT T GA CGTCAATAAT GAC GTATGTTCC CAT AGT AAC GC C AAT AGGGAC T T T CMV C C AT T G AC GT C AAT GGGT GGAG T AT T T AC G GT AAAC T G C C C AC T T G 94

[0164] enhancer

[0165] GCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACG T C AAT GAC GG T AAAT GGC C C GC C T GGC AT TAT GC C C AGT AC AT GAC CTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATC GCTATTACCATG C GT T AC AT AAC T T AC GGT AAAT GGC C C GC C T GGC T GAC C GC C C AAC GACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAA CGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACG CMV GTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGT 95

[0166] enhancer 2 ACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATT ATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACAT CTACGTATTAGTCATCGCTATTACCATG C T C AC T C C C C GC C C AGGC AGC AAGGAGC C C AC AC C C T C AT GC C C C TCAGCTTCAGCCCCCACCTCCAGGAGGCCCT ACCCACGCTCA TGACCTTGCTATTCTGGGCCTTGTGTCCTGTAGGGAGATGGACAGG AGACAGCTGGGCTTCCAGGCCACCCAGGCGGGGGGCTAGCCGAGGG AAGCCTGCTGGCTCTCCTGCTTGCTCTAATTTCTGGGGCTCCCCAA CRM1 ACCTTGGCCT C AGGAGAC T GGGGAT AGGAC C GGC C T T GAAAGT GGG 2,538 bp to 96

[0167] (muscle) GGAAGCTTTGGAGAGCCGGGTGCTGGGTTCTTAGTGAGATGGCCAG ATP2A1TSS TGAAGGCTGTGGTGCCCCGAGGTAAGCAGGGCCTGATCCCCTCCTA ATCTTCCAGCAGCAACTGGTGCTCTGAGGCTCCCCCTCCCCCAGCC C T GC C AGC C T T C AGGGAC C T GC C T T C CAAAGAT GGGCAGGGGAGGG GGAC GAGGAC AC CCACCCACTCCT C AGAC CAGCATGTCTT CCCTCCAGATGGGTTTCCTGGAATCTAGATTTCCCAGGTTCCAAAG GAC AC C C GAGT C TCATGCCT GGAAC T C AGT GAGAC TAATTCACCTC TCCTCTGCCCTAATCTTCATCTCCAGCCAGAAGCCAACAGATCCCA CRM2 G GGGACTGGAGCCACAGGGGCTGCACCTGTTTACCGGGTATTTTT 701 bp to AGGAT GGT T GAT GAAC AC AT AAT AC C C AC C C T AT AGT C AGAGAAAG 97 (muscle) TNNI1 TSS ACAATGCCTGCTATGTTAATCCTGTGGCTATTATAGTCTGTCATCT CATGGGTT GGGGC AGGAC AC T GAC C C T C T C AGAGGC C AGAGAGAGG CCTCGCAAGCAGGAGGTTAGGGA AT GGAGAC AAT C C AT GAAT T C C T GAGAT GCTTGGCTGGTAT T AGAT T T T AT GGGC AGC T GC T T AT T C T T AGGGC T C T GC T T C T C C AAAGAC A C T GAGGAAGT C C AAAGGAAC AC C AGC T GGC GAAGAGC C AC C T C C AG GCCCATCTGTCCATCATCAGCCTCCAGGAATGCCAGTGTCCAGAGG CRM3 GCACCAGGTCTGCGTCTGTCTCCCTGGGATGTGCCTTGTCCTTGGT 1 ,438 bp to GGGCATTTGGCAGTGATCATGCCTCCCTGTCTCCCTCAGAGATCCA 98 (muscle) TNNI1 TSS ACTGTCCCCATTGTGGGGCCCTACCTTCCAAGGCCGGTTTACACCT CCTGCCAAGCTCCGGGGCCTGCCCCCAGCCTGCCTCACTGACAAAT GCCAGACCAAGGGGTCCCACGTCAGGCAAGAGGCCTCAGCCTGTGC

[0168]

[0169] TCTGACACCCCTCAG Name of SEQUENCE SEQ ID

[0170] Notes sequence NO TTCTGAGTCCTCTAAGGTCCCTCACTCCCAACTCAGCCCCATGTCC TGTCAATTCCCACTCAGTGTCTGATCTCCTTCTCCTCACCTTTCCC ATCTCCCGTTTGACCCAGCTTCCTGAGCTCTCCTCCCATTCCCCTT TTTGGAGTCCTCCTCCTCTCCCAGAACCCAGTAATAAGTGGGCTCC CRM4 TCCCTGGCCTGGACCCCCGTGGTAACCCTATAAGGCGAGGCAGCTG 2,368 bp to CTGTCTGAGGCAGGGAGGGGCTGGTGTGGGAGGCTAAGGGCAGCTG 99 (muscle) MYLPF TSS CTAAGTTTAGGGTGGCTCCTTCTCTCTTCTTAGAGACAACAGGTGG CTGGGGCCTCAGTGCCCAGAAAAGAAAATGTCTTAGAGGTATCGGC ATGGGCCTGGAGGAGGGGGGACAGGGCAGGGGGAGGCATCTTCCTC AGGAC AT C GGGT C C T AGAGG GACTAGGAATAAATCACATATCCTCAATCCCTGGACAACTTGTTTA CTTCTAGTGTTAGTTTTTTCTTAAAAAAAAAATTGAAATCATTCTG AGGCTGGAATACTTTGGACATGCCCAGCAGTTCCTGGCAGTTCCCA CAGAAGCATTACCTCATGACTGGAGTGGGTAAAGCATACTGTGGGC TAT GGAT AAGAC T GAC AT T AAC C AC AAGC AT GT T T GGC AGC AGAC T

[0171] 4,772 bp to CRM5 GGTGCTTTACAAGCTCCATGTTCAGCAGGAGCTGCAAAGTGTTCCT 100

[0172] MYH1 TSS (muscle) CCAAACCAATATTTGTCATTCTTGGATTCTATTTAGGAGGTCCTGT TACTCACATGTTTCAATATCAGCAGAAGCCAGTTTCCCTGTGGTAC C GAAGT GGAT C C T GAT GAAT T T AC C C T T GT AAGT AAAAAAAAT GAT GTTATACCCAAAGCTTGAAGTACGTAGTGGGGATGCCACTGAAATA ATT C AGAC AT GC T T GTGCTCATAGCTCCACCTTTTGTTCCTAATATGGTCTTTCCAGCTC CCTCCACCCCATCATTGTTCTCCTGGGGGAACACAGGGTGAGACGC T T T GAT GAAC T GAC AT C AC C AGC AAAAAAAAT AT C T AGC AAC AGC T GAGGC T GAT T T T AGAC AAT GGAAAGT GGGGGAGGGAAGAGGT T C T C CCTGACCCTGAAACTTTCCACTCATTCTGGGCAGCTCTATGGATGT T T T AAAAGAAGAGGAAGAGGGGAGGGAAGAAC AT T GAAAT AGAGAA CRM6 517 bp to 101 GTGTACTTTGGCAATTCTAGGTTGGCAGTTTGCATCCAGGGGGTCC (muscle) TPM3 TSS TGGTTGCCTTTCAGCTTCCCGTTTCACTCTCCCCCAGACTGTGTTG AATGCTGGTCAAACTCCGTTAGTTGAGTTTTAGCTTTTGATTCCTG GTATTCAAGGAGCTTGGGCACAGGGAAGAGGGGAGGTCACTCATGA TCCTTAACAATTCTCCCAGATCCCCAGATCAAATTGCTGTGCTATT CTGGGAGTCTCCG ATCGTGTGTCAGAGGTTTGTGTCAGCTTCCCAGCAAGGGAACCAGA AAGGAAAAGGAACCGGTTCCTCATGCTTCCTAGGGGAATGCATGCA T AT C T GAAGAGAAGGGAAT C T T AT AT AAGGC T GT T T AGC T AAGGGC AGCCACCAGCCAGGTGAGCCTTACAGAAGCACAGGGCTGGGTGTCT CRM7 507 bp to GCAGTTCCCTAGCAGATTAACCTGGGTCACAGTGACTCAGAGCTCC 102

[0173] (muscle) ANKRD2TSS AGCATGCGAGTTCCAGGTGTGGAACTGAGCAAGTACAGATCTGCTT TTGCTCCACTTGGGAGTATTTTTCCTTCTTAGTGAGCATGGGCAGC C T C C T GGC C AGGGAAGT C T GGC AC T GT C T GGGC C T GAC AGGGAAAC CCTG GC AGGGAAGGGGGC AC T C T T C T GAGC AGAC AGAT C T GGGAAT CCTG GGT GGGAAGAGAGAC AGT GAGAGAGAGAT T AAGGGAT AT T T C C C AG GCATCAGGGCTTTGCACTCTCAGGGGTCCTTCCGCCTGGATGTCCTho enhancer

[0174] Rho enhancer 103 TCCCCTGAAGCTTCCTCCTGTTGTTCCGTTCTCAGCTCAAGCTCCA region

[0175] GCTTCTCAGAGAAGCCTCCTGTGTTGGGAGTGGCTGCGACTGAACT GTCCCTACTGTTATTCGC C T AC C C C AC AC T GAGGC TTCCTCGTCT GAGC AAAC T GAGGC C C AGA GAGGGGAAGGAAGC AGGAC T AC CAT GGT GAC T C AAAGAC C AGC TAG AATCCAGCCTCCTCTCCTCGAGGCTTCCACTGCCCCACGCCAGGCC TGTGTGACTCAGTCTAGGGCCTTTCCATTACCCCAGCTAAACCTTT Cone-Rod CTTTAGTCATTTATACCATGGTGTGAATGGCTGGCTGGTCTTTCCT Rho CBR 104 Homeobox bo GAGAGC TATCTTTGAT GAGGGGAGGGAGGC AT AGC CAGGTTTGGGA und region 1 AGCTGATACCCCAGGAAGCCCAGTTGACTGTGTGGGTTATAGCCCA GGCTGTC ACT GATT T GT AAC GGGAC C T GAGC AAC T C T GC AGAGC TA GGCCTCAGTCTTTTCATCTGCAAAAT

[0176]

[0177] Engineered Regulatory Elements

[0178] Engineered nucleic acids as described herein may comprise at least one engineered regulatory sequence. In some cases, the engineered regulatory sequence may be configured to facilitate the generation of the engineered nucleic acid. For example, the engineered regulatory sequence may be configured to facilitate the transcription of the engineered nucleic acid. In some embodiments, the engineered regulatory sequence may comprise a non-coding sequence. In some embodiments, the noncoding sequence may comprise an intron, a UTR, a ribosomal entry site, or a combination thereof. In some embodiments, the UTR may comprise a 5’UTR, a 3’UTR, or a combination thereof. In some embodiments, the non-coding sequence may comprise an intron. In some embodiments, the non-coding sequence may comprise a UTR. In some embodiments, the non-coding sequence may comprise a ribosomal entry site.

[0179] The engineered nucleic acid may comprise at least: 1 , 2, 3, 4, 5 or more engineered regulatory sequence(s). The engineered nucleic acid may comprise at most: 1 , 2, 3, 4, or 5 engineered regulatory sequence(s).

[0180] In some embodiments, the engineered regulatory sequence may have at least: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, 250, 251 , 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 , 262, 263, 264, 265, 266, 267, 268, 269, 270, 271 , 272, 273, 274, 275, 276, 277, 278, 279, 280, 281 , 282, 283, 284, 285, 286, 287, 288, 289, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 , 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331 , 332, 333, 334, 335, 336, 337, 338, 339, 340, 341 , 342, 343, 344, 345, 346, 347, 348, 349, 350, 351 , 352, 353, 354, 355, 356, 357, 358, 359, 360, 361 , 362, 363, 364, 365, 366, 367, 368, 369, 370, 371 , 372, 373, 374, 375, 376, 377, 378, 379, 380, 381 , 382, 383, 384, 385, 386, 387, 388, 389, 390, 391 , 392, 393, 394, 395, 396, 397, 398, 399, 400, 401 , 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431 , 432, 433, 434, 435, 436, 437, 438, 439, 440, 441 , 442, 443, 444, 445, 446, 447, 448, 449, 450, 451 , 452, 453, 454, 455, 456, 457, 458, 459, 460, 461 , 462, 463, 464, 465, 466, 467, 468, 469, 470, 471 , 472, 473, 474, 475, 476, 477, 478, 479, 480, 481 , 482, 483, 484, 485, 486, 487, 488, 489, 490, 491 , 492, 493, 494, 495, 496, 497, 498, 499, 500, 1000, 2000, 5000 or more nucleotides (nt; or base pair / bp). In some embodiments, the engineered regulatory sequence may have at most: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, 250, 251 , 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 , 262, 263, 264, 265, 266, 267, 268, 269, 270, 271 , 272, 273, 274, 275, 276, 277, 278, 279, 280, 281 , 282, 283, 284, 285, 286, 287, 288, 289, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 , 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331 , 332, 333, 334, 335, 336, 337, 338, 339, 340, 341 , 342, 343, 344, 345, 346, 347, 348, 349, 350, 351 , 352, 353, 354, 355, 356, 357, 358, 359, 360, 361 , 362, 363, 364, 365, 366, 367, 368, 369, 370, 371 , 372, 373, 374, 375, 376, 377, 378, 379, 380, 381 , 382, 383, 384, 385, 386, 387, 388, 389, 390, 391 , 392, 393, 394, 395, 396, 397, 398, 399, 400, 401 , 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441 , 442, 443, 444, 445, 446, 447, 448, 449, 450, 451 , 452, 453, 454, 455, 456, 457, 458, 459, 460, 461 , 462, 463, 464, 465, 466, 467, 468, 469, 470, 471 , 472, 473, 474, 475, 476, 477, 478, 479, 480, 481 , 482, 483, 484, 485, 486, 487, 488, 489, 490, 491 , 492, 493, 494, 495, 496, 497, 498, 499, 500, 1000, 2000, or 5000 nt / bp.

[0181] In some embodiments, the engineered nucleic acid comprising at least one engineered regulatory sequence may be expressed at a level that is at least: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart. In some embodiments, the engineered nucleic acid comprising at least one engineered regulatory sequence may be expressed at a level that is at most: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart.

[0182] RNA Capping

[0183] Engineered nucleic acids as described herein may comprise a 5’ methyl G cap also known as a 7-methylguanosine (m7G) cap. In some embodiments, a DNA cassette may be transcribed into an engineered RNA molecule comprising a 5’ methyl G cap. In some embodiments, an expression cassette comprising the engineered nucleic acid molecule that is transcribed comprises a 5’ methyl G cap. The 5’ methyl G cap may comprise a 2,2,7 trimethyl G (m3G) cap also known as a 5'-trimethylguanosine (TMG) cap. In some embodiments, various snRNAs and certain snoRNAs as described herein can comprise an m7G cap when newly synthesized. The m7G cap structures of these snRNAs or snoRNAs (but not mRNAs) can be converted post-transcriptional ly to 2,2,7-trimethylguanosine (m3G) cap structures via the addition of two extra methyl groups to the guanine base. In some embodiments, the hypermethylation of the 5’ methyl G cap converts it to a m3G-cap. In some embodiments, the RNA cap (e.g., 5’ methyl G cap or m3G) of an engineered nucleic acid molecule is a post-transcriptional element. In some embodiments, the 5’ methyl G cap or 2,2,7 trimethyl G (m3G) cap is configured to stabilize the engineered nucleic acid. For example, the stabilization may allow to have an increased expression level or half-life, relative to a control counterpart. In some embodiments, the 5’ methyl G cap or 2,2,7 trimethyl G (m3G) cap increases nuclear localization of the engineered nucleic acid molecule.

[0184] In some embodiments, a 5’ methyl G cap or 2,2,7 trimethyl G (m3G) cap may prevent or protect an engineered nucleic acid to be degraded by a nucleic acid degradation enzyme such as a 5’ exonuclease. In some embodiments, the 5’ methyl G cap or 2,2,7 trimethyl G (m3G) cap may block the nucleic acid degradation enzyme from accessing the engineered nucleic acid.

[0185] In some embodiments, the engineered nucleic acid comprising a 5’ methyl G cap or 2,2,7 trimethyl G (m3G) cap may be expressed at a level that is at least: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart. In some embodiments, the engineered nucleic acid comprising a 5’ methyl G cap or 2,2,7 trimethyl G (m3G) cap may be expressed at a level that is at most: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart. In some embodiments, the engineered nucleic acid comprising a 5’ methyl G cap or 2,2,7 trimethyl G (m3G) cap may have a half-life that is at least: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart. In some embodiments, the engineered nucleic acid comprising a 5’ methyl G cap or 2,2,7 trimethyl G (m3G) cap may have a half-life that is at most: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart.

[0186] Recruitment Sites

[0187] Engineered nucleic acid molecules as described herein may comprise or encode protein recruitment sites. In some embodiments, the 5’ engineered element comprises a recruitment site. In some embodiments, the recruitment site comprises a binding site for one or more RBPs or one or more protein recruitment domain. In some embodiments, the engineered nucleic acid molecule comprises a recruitment site 5’ to the one or more antisense sequences. In some embodiments, the engineered nucleic acid molecule comprises a recruitment site 3’ to the m7G cap or m3G cap. In some embodiments, engineered nucleic acid molecules comprising recruitment sites recruit splicing regulatory factors. In some embodiments, recruiting splicing regulatory factors further regulates splicing.

[0188] In some embodiments, the recruitment site comprises a binding site for an RBP. In some embodiments, the recruitment site comprises a binding site for one or more RBPs. In some embodiments, the recruitment site comprises more than one binding site for one or more RBP. In some embodiments, the one or more RBP is selected from: PCBP4, PPARGC1 A, LSM7, RBM6, PABPC4, CPSF6, LSM4, TNRC6A, KRR1 , ZC3H4, YBX2, RBM27, SFRS9, EIF3G, CSDE1 , HNRNPC, SFRS3, DKC1 , PTBP1 , DAZL, RBM24, PPIL4, ZC3H3, HNRNPH3, KHDRBS2, RBM39, MATR3, SETD1A, QKI, ANKHD1, SAMD4A, CIRBP, CPEB4, KHDRBS3, YTHDC2, HNRNPM, FXR1 , RBM8A, CUGBP2, TRMT2A, NCL, LIN28, PUM2, SF3A1, GRSF1, RC3H2, SNRPD3, HDLBP, ANKRD17, ZC3H11A, P0LDIP3, SFRS7, UNK, PARP12, ZMAT5, PN01 , NIP7, CSDA, RBM9, TIA1 , T0E1 , SFRS8, ZC3H7B, SFRS4, RBM38, EIF4B, RBM23, SFPQ, SRRM1, U2AF2, SFRS5, SFRS11, MKRN1 , SFRS14, ZC3H14, PRPF3, EIF2S1 , SPEN, ACIN1, PTBP2, THUMPD3, ZC3H15, PABPN1, R0D1, RBMX2, YBX1, PABPC1L, RBM18, PUM1, ELAVL1, BRUN0L4, C14orf156, MSI1 , THUMPD1 , CSTF2, RBM25, SYNCRIP, DHX8, ZC3H12B, EN0X1 , ZC3H10, SRBD1 , FMR1 , TARDBP, HNRNPA1 , PABPC1 , HTATSF1 , AKAP1 , RC3H1 , DAZAP1 , RBM3, PSPC1 , IGF2BP3, IGF2BP2, ESRP2, ZCCHC17, NUPL2, ZNF638, MTHFSD, KHDRBS1 , SFRS1, SART3, DNAJC17, ZC3H7A, TRA2B, MKRN2, MYEF2, HNRNPA2B1 , CPEB2, RBM7, ESRP1, BICC1 , ALKBH8, RBMS2, HNRNPL, RBM19, SLTM, MBNL3, SNRNP70, ZC3H13, PNPT1 , SNRPA, SFRS16, SFRS6, THUMPD2, SYNJ2, TRMT1 , RNF113A, ASCC1 , A2BP1 , N0VA2, SNRPD2, SSB, SF4, SNRPB, HNRNPD, DDX43, ZC3HAV1 , SNRPB2, LARP1 B, KIAA0020, EIF3B, HNRNPR, G3BP2, CN0T4, RBM28, RALY, ZCRB1 , YTHDC1 , LSM5, RBM42, SNRPF, PPIE, EIF4H, HNRNPH2, CHERP, ELAVL2, ZFP36, SETD1B, RBM22, FUBP3, NAA38, RBM26, KHSRP, CPEB3, SNRPN, MBNL2, FUS, LARP4B, FXR2, RNF113B, RBM41 , SUPT6H, SAFB2, and N0VA1. In some embodiments, the recruitment site comprises a binding site for HNRNPA1. In some embodiments, the binding site for HNRNPA1 comprises SEQ ID NO: 131 (TATGATAGGGACTTAGGGTG).

[0189] Engineered Stabilizing Sequences / Secondary Structure Sequences

[0190] Engineered nucleic acids as described herein may comprise or encode an engineered stabilizing sequence or a secondary structure sequence. In some embodiments, the engineered stabilizing sequence is configured to stabilize the engineered nucleic acid. For example, the stabilization may allow to have an increased expression level or half-life, relative to a control counterpart. When comparing an engineered nucleic acid comprising a particular sequence or element, a control counterpart of the engineered nucleic acid may have the same nucleic acid sequence as the engineered nucleic acid except for the particular sequence or element.

[0191] In some embodiments, an engineered stabilizing sequence or secondary structure sequence may prevent or protect an engineered nucleic acid to be degraded by a nucleic acid degradation enzyme. The nucleic acid degradation enzyme may comprise a nuclease. The nuclease may comprise an endonuclease or an exonuclease. The exonuclease may comprise a 5’ exonuclease or 3’ exonuclease. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise a sequence configured to form the at least one double-stranded region. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may block the nucleic acid degradation enzyme from accessing the engineered nucleic acid. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may block the access of the nucleic acid degradation enzyme from accessing the engineered nucleic acid via recruitment of a nucleic acid-binding protein. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may form a secondary structure that blocks the nucleic acid degradation enzyme from accessing the engineered nucleic acid. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise a sequence that is configured to form at least a double-stranded region or at least two nucleotides that base-paired with each other.

[0192] In some cases, the engineered stabilizing sequence or secondary structure sequence may be configured to facilitate the nuclear localization of the engineered nucleic acid. In some embodiments, the secondary structure sequence may be configured to form at least one secondary structure, as described herein. In some embodiments, the engineered stabilizing sequence and the secondary structure sequence may be the same. In some embodiments, the engineered stabilizing sequence and the secondary structure sequence may be different.

[0193] The engineered nucleic acid may comprise or encode at least: 1 , 2, 3, 4, 5 or more engineered stabilizing sequence(s) or secondary structure sequence(s). The engineered nucleic acid may comprise or encode at most: 1 , 2, 3, 4, or 5 engineered stabilizing sequence(s) or secondary structure sequence(s). The engineered nucleic acid may comprise or encode 1 engineered stabilizing sequence or secondary structure sequence. The engineered nucleic acid may comprise or encode 2 engineered stabilizing sequences or secondary structure sequences. The engineered nucleic acid may comprise or encode 3 engineered stabilizing sequences or secondary structure sequences. The engineered nucleic acid may comprise or encode 4 engineered stabilizing sequences or secondary structure sequences. The engineered nucleic acid may comprise or encode 5 engineered stabilizing sequences or secondary structure sequences.

[0194] In some embodiments, an engineered nucleic acid molecule comprises a 5’ engineered element. In some embodiments, the 5’ engineered element comprises one or more of an engineered stabilizing sequence or a secondary structure sequence. In some embodiments, an engineered nucleic acid molecule comprises a 3’ engineered element. In some embodiments, the 3’ engineered element comprises one or more of an engineered stabilizing sequence or a secondary structure sequence. In some embodiments, an engineered nucleic acid molecule comprises a 5’ engineered element and a 3’ engineered element. In some embodiments, the 5’ engineered element comprises one or more of an engineered stabilizing sequence or a secondary structure sequence and the 3’ engineered element comprises one or more of an engineered stabilizing sequence or a secondary structure sequence.

[0195] In some embodiments, an expression cassette encodes an engineered nucleic acid molecule. In some embodiments, the engineered nucleic acid molecule comprises a 5’ engineered element. In some embodiments, the 5’ engineered element comprises one or more of an engineered stabilizing sequence or a secondary structure sequence. In some embodiments, the engineered nucleic acid molecule comprises a 3’ engineered terminator. In some embodiments, the 3’ engineered terminator comprises one or more of an engineered stabilizing sequence or a secondary structure sequence. In some embodiments, an engineered nucleic acid molecule comprises a 5’ engineered element and a 3’ engineered terminator. In some embodiments, the 5’ engineered element comprises one or more of an engineered stabilizing sequence or a secondary structure sequence and the 3’ engineered terminator comprises one or more of an engineered stabilizing sequence or a secondary structure sequence.

[0196] In some embodiments, the engineered stabilizing sequence or secondary structure sequence may have at least: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, 250 or more nucleotides (nt; or base pair / bp) . In some embodiments, the engineered stabilizing sequence or secondary structure sequence may have at most: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, or 250 nt / bp. In some embodiments, at least: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, 250 or more nucleotides of the engineered stabilizing sequence or secondary structure sequence may be singlestranded. In some embodiments, at most: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, or 250 nucleotides of the engineered stabilizing sequence or secondary structure sequence may be single-stranded.

[0197] In some embodiments, at least: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 156, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 , 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, 250 or more nucleotides of the engineered stabilizing sequence or secondary structure sequence may be base-paired with another nucleotide(s) of the engineered stabilizing sequence or secondary structure sequence. In some embodiments, at most: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 156, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 , 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, or 250 nucleotides of the engineered stabilizing sequence or secondary structure sequence may be basepaired with another nucleotide(s) of the engineered stabilizing sequence or secondary structure sequence.

[0198] In some embodiments, the engineered nucleic acid comprising at least one engineered stabilizing sequence or secondary structure sequence (or a transcript encoded therefrom) may be expressed at a level that is at least: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart. In some embodiments, the engineered nucleic acid comprising at least one engineered stabilizing sequence or secondary structure sequence (or a transcript encoded therefrom) may be expressed at a level that is at most: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart. In some embodiments, the engineered nucleic acid comprising at least one engineered stabilizing sequence or secondary structure sequence (or a transcript encoded therefrom) may have a half-life that is at least: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart. In some embodiments, the engineered nucleic acid comprising at least one engineered stabilizing sequence or secondary structure sequence (or a transcript encoded therefrom) may have a half-life that is at most: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart. In some embodiments, the amount of the engineered nucleic acid comprising at least one engineered stabilizing sequence or secondary structure sequence (or a transcript encoded therefrom) that is localized to the nucleus may be at least: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart. In some embodiments, the amount of the engineered nucleic acid comprising at least one engineered stabilizing sequence or secondary structure sequence (or a transcript encoded therefrom) that is localized to the nucleus may be at most: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart.

[0199] In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise a stem-loop sequence; a triple helix sequence; a quadruplex sequence; a tRNA-like sequence; a pseudoknot sequence; a loop-loop kiss sequence; any functional derivatives thereof; or any combinations thereof. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise a stem-loop sequence. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise a triple helix sequence. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise a quadruplex sequence. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise a tRNA-like sequence. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise a pseudoknot sequence. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise a loop-loop kiss sequence. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may also comprise a stem, hairpin loop, pseudoknot, loop-loop kiss, bulge(s), internal loop, multiloop, single-stranded region, double-stranded region, or any combinations thereof. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise a poly-U region. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise a poly-A region. In some embodiments, a triple helix sequence may comprise one or more poly-U region and one or more poly-A region. For example, in one embodiment, the triple helix sequence can comprise two poly-U regions and one poly-A region.

[0200] In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise a small nucleolar RNA snoRNA (H / ACA and C / D box) derived stem-loop sequence; a modified snRNA stem loop from other species, a viral snRNA derived stem-loop sequence; a plant snRNA derived stem-loop sequence; a non-coding or viral RNA derived triple helix sequence; a direct repeat (DR) sequence from a clustered regularly interspaced short palindromic repeats (CRISPR) single-guide (sg) RNA derived stem-loop sequence; an intergenic region forming a stem loop, a long non-coding RNA (LncRNA) derived stem-loop sequence; a PlWI-interacting RNA (piRNA) derived stem-loop sequence; a stem-bulge RNA (sbRNA) derived stem-loop sequence; a transfer RNA (tRNA) derived stem-loop sequence; any functional derivatives thereof; or any combinations thereof. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise a small nucleolar RNA snoRNA (H / ACA and C / D box) derived stem-loop sequence. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise a small nucleolar RNA snoRNA (H / ACA and C / D box) derived stem-loop sequence. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise a viral snRNA derived stem-loop sequence. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise a plant snRNA derived stem-loop sequence. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise a non-coding or viral RNA derived triple helix sequence. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise a clustered regularly interspaced short palindromic repeats (CRISPR) single-guide (sg) RNA derived stem-loop sequence. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise an intergenic region forming a stem loop. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise a long non-coding RNA (LncRNA) derived stem-loop sequence. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise a PlWI-interacting RNA (piRNA) derived stem-loop sequence. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise a stem-bulge RNA (sbRNA) derived stem-loop sequence. In some embodiments, the viral snRNA derived stem-loop sequence may comprise a WPRE (viral stabilizing sequence).

[0201] In some embodiments, the engineered stabilizing sequence or secondary structure sequence may be derived from a LncRNA; a plant; a virus; a DR from a CRISPR single guide RNA sequence; a small nucleolar RNAs (snoRNA); a histone messenger RNA (mRNA); any functional derivatives thereof; or any combinations thereof. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may be derived from a LncRNA. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may be derived from a plant. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may be derived from a virus. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may be derived from a direct repeat (DR) CRISPR guide RNA sequence. In some embodiments, the engineered stabilizing sequence or secondary structure sequence comprising the CRISPR sequence comprises one or more stem loops or optimized secondary structures. In some embodiments, the DR has a minimum length of 16 nts and a single stem loop. In some embodiments, the DR has a length longer than 16 nts, and has more than one stem loops or optimized secondary structures. In some embodiments, the stem loop sequence from the DR CRISPR sequence comprises a sequence set forth in Table 3.

[0202] In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise or encode a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or more sequence identity to any sequence disclosed in Table 3. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise or encode a sequence having at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, at most 99%, at least 99.5% sequence identity to any sequence disclosed in Table 3. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise or encode a sequence having 100% sequence identity to any sequence disclosed in Table 3.

[0203] In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise or encode a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or more sequence identity to any sequence disclosed in SEQ ID NOS: 72-74 and 105-118. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise or encode a sequence having at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, at most 99%, at least 99.5% sequence identity to any sequence disclosed in SEQ ID NOS: 72-74 and 105-118. In some embodiments, the engineered stabilizing sequence or secondary structure sequence may comprise or encode a sequence having 100% sequence identity to any sequence disclosed in SEQ ID NOS: 72-74 and 105-118.

[0204] Table 3: Exemplar Stem-loop Structure Sequences

[0205] Name of SEQ ID

[0206] Sequence Notes sequence NO

[0207] L GATT T AGAC T AC C C C AAAAAC GAAGGGGAC T AAAAC Leptotrichia wCas13a 72

[0208] wadei PspCas13b GTTGTGGAAGGTCCAGTTTTGAGGGGCTATTACAAC 73 Prevotella sp.

[0209] Ruminococcus RxCas13d CCCCTACCAACTGGTCGGGGTTT 74 flavefaciens XPD3002 CASRX AACCCCTACCAACTGGTCGGGGTTTGAAAC 105

[0210] CASRX long AACCCCTACCAACTGGTCGGGGTTTGAAACCA 106

[0211] U7 Stem loop GGTTTTCTGACTGTGGTCGGAAAACC 107

[0212] mut1

[0213] U7 Stem loop GGTTTTCTGACTGTGTGGTCGGAAAACC 108

[0214] comp mut 2

[0215] CAS13D

[0216] uncultured CACUAGUGCGAAUUUGCACUAGUCUAAAAC 109

[0217] Ruminococcu

[0218] s sp

[0219] CAST l-F GUGAAC UGC C GAGU AGGU AGC UGAUAAC 110

[0220] Caslll-B GUUCACUGCCGCACAGGCAGCUUAGAAA 111

[0221] CASI-E GUGUUCCCUGUAAUCACGGGGAUAAACC 112 GTTTTAGTCCCTGAAGGGACTAAAATAAAGAGTTTGCGGGA

[0222] Cje Cas9 CTCTGCGGGGTTACAATCCCCTAAAACCGCTTTTTT 113

[0223] GT T T AAGAGC T AT GC T GGAAAC AGC AT AGC AAGT T T AAAT A

[0224] spCas9 AGGC T AGT C C GT T AT C AAC T T GAAAAAGT GGC AC C GAGT C G 114

[0225]

[0226] GTGCTTTTTTT GTTTTAGTACTCT GGAAAC AGAAT C T AC T AAAAC AAGGC AA

[0227] SaCas9 AATGCCGTGTTTATCTCGTCAACTTGTTGGCGAGATTTTT 115

[0228] TTATTGAATTTAAATTTTCCATACTTTATGGCATCAAGCCA IR1 Truncated T AAAGT AT GGAGT AAAAAAAC AT AT GAGC AGAT T T T AC T C T 116

[0229] GCCATAA T T T T GAAGT T T T AT T T GGAAC AAT AGGAAT GC AAC T AGT GC IR5 truncated ATTCCTATTGTTCCTATCCTATTAGTTTCAGATTTATGATT 117

[0230] TGAACG

[0231] Truncated CTCTTCAGTAGGGTCATGAAGGTTTTTCTTTTCCTGAGAAA

[0232] Human ACAACACGTATTGTTTTCTCAGGTTTTGCTTTTTGGCCTTT 118

[0233] MALAT1 triple T T C T AGC T T AAAAAAAAAAAAAGC AAAA

[0234]

[0235] helix

[0236] In some embodiments, the engineered stabilizing sequence or secondary structure sequence may be derived from a small nucleolar RNAs (snoRNA). In some embodiments, the engineered stabilizing sequence or secondary structure sequence may be derived from a histone messenger RNA (mRNA).

[0237] Engineered 3’ Terminator Domain

[0238] Engineered nucleic acids as described herein may comprise or encode an engineered terminator. “Engineered terminator” or “terminator” or “engineered terminator domain” as used herein refers to a non-naturally occurring sequence or structural element of the engineered nucleic acid that is configured to (1) provide increased stability of the engineered nucleic acid relative to a control counterpart; (2) facilitate terminal end (e.g., 3’ end) processing (or maturation during or subsequent to the transcription) of the engineered nucleic acid; (3) facilitate the transcriptional termination of the engineered nucleic acid; (4) facilitate the transcription of the engineered nucleic acid; or any combination thereof.

[0239] In some aspects, an expression cassette encoding a nucleic acid molecule comprises an engineered terminator. The engineered terminator is 3’ to the one or more antisense sequences, and 3’ to an engineered Sm-binding site. In some embodiments, a 3’ engineered terminator comprises an engineered stabilizing sequence or a secondary structure sequence, an insulator, an engineered TTD, an engineered cleavage / processing site, or any combinations thereof.

[0240] In some embodiments, the engineered terminator may have at least: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, 250 or more nucleotides (nt; or base pair / bp). In some embodiments, the engineered terminator may have at most: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, or 250 nt / bp.

[0241] In some embodiments, the engineered terminator is configured to stabilize the engineered nucleic acid (or a transcript encoded therefrom). For example, the stabilization may allow the engineered nucleic acid (or a transcript encoded therefrom) to have an increased expression level or half-life, relative to a control counterpart. In some embodiments, an engineered terminator (or a portion, a processed derivative, or a fragment of the engineered terminator) may prevent or protect an engineered nucleic acid to be degraded by a nucleic acid degradation enzyme as disclosed herein. The nucleic acid degradation enzyme may comprise a nuclease. The nuclease may comprise an endonuclease or an exonuclease. In some embodiments, the engineered terminator (or a portion, a processed derivative, or a fragment of the engineered terminator) may block the nucleic acid degradation enzyme from accessing the engineered nucleic acid. In some embodiments, the engineered terminator (or a portion, a processed derivative, or a fragment of the engineered terminator) may block the access of the nucleic acid degradation enzyme from accessing the engineered nucleic acid via recruitment of a nucleic acid-binding protein. In some embodiments, the engineered terminator (or a portion, a processed derivative, or a fragment of the engineered terminator) may form a secondary structure that blocks the nucleic acid degradation enzyme from accessing the engineered nucleic acid. In some embodiments, the engineered terminator (a portion, a processed derivative, or a fragment of the engineered terminator) may comprise any of the engineered stabilizing sequence or secondary structure sequence as described herein.

[0242] In some embodiments, the engineered nucleic acid comprising at least one engineered terminator (or a transcript encoded therefrom) may be expressed at a level that is at least: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart. In some embodiments, the engineered nucleic acid comprising at least one engineered terminator (or a transcript encoded therefrom) may be expressed at a level that is at most: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart.

[0243] In some embodiments, the engineered terminator is configured to facilitate the terminal processing of the engineered nucleic acid. The terminal processing may comprise 3’ end processing during or subsequent to the transcription of the engineered nucleic acid. In some embodiments, the engineered terminator may be processed into a fragment during the terminal end processing. Thus, in some embodiments, the engineered nucleic acid comprising the engineered terminator may comprise at least a portion, fragment, or processed derivate of the engineered terminator during or subsequent to the transcription or during or subsequent to the terminal end processing. Accordingly, the engineered terminator can comprise (1) a full-length (or all elements) of the engineered terminator or (2) a portion, a processed derivative, or a fragment of the full-length of the engineered terminator. In some embodiments, the engineered terminator (a portion, a processed derivative, or a fragment of the engineered terminator) may comprise any of the engineered stabilizing sequence or secondary structure sequence as described herein. In some embodiments, the engineered terminator is configured to facilitate the terminal processing of the engineered nucleic acid. In some embodiments, the terminal end processing of the engineered nucleic acid may be the same as the terminal end processing of the engineered terminator. In some embodiments, the terminal end processing of the engineered nucleic acid may be different from the terminal end processing of the engineered terminator.

[0244] In some embodiments, the 3’ terminator may comprise at least a TTD sequence. In some embodiments, the 3’ terminator sequence may comprise a promoter coupled termination sequence; a nuclease cleavage site; a ribozyme; a tRNA processing nuclease site, any functional derivatives thereof; or any combinations thereof. In some embodiments, the 3’ terminator comprises an engineered stabilizing sequence or a secondary structure, a cleavage site, and a TTD. In some embodiments, the 3’ terminator sequence may comprise an insulator. In some embodiments, the insulator is positioned between the engineered stabilizing sequence or the secondary structure (e.g., stem loop) and the cleavage site. In some embodiments, the insulator is positioned upstream of the engineered stabilizing sequence or the secondary structure (e.g., stem loop).

[0245] In some embodiments, the 3’ terminator sequence may comprise a promoter coupled termination sequence. In some embodiments, the promoter coupled termination sequence may comprise a U1 promoter; a U2 promoter; a U4 promoter; a U7 promoter; any functional derivatives thereof; or any combinations thereof. In some embodiments, the promoter coupled termination sequence may comprise a U1 promoter. In some embodiments, the promoter coupled termination sequence may comprise a U2 promoter. In some embodiments, the promoter coupled termination sequence may comprise a U4 promoter. In some embodiments, the promoter coupled termination sequence may comprise a U7 promoter.

[0246] In some embodiments, the TTD may facilitate the transcription of the engineered nucleic acid (or a transcript encoded therefrom) from a non-naturally occurring promoter. In some embodiments, the TTD may facilitate the transcription of the engineered nucleic acid (or a transcript encoded therefrom) from a Pol II promoter. In some cases, the transcription of the engineered nucleic acid (or a transcript encoded therefrom) from the Pol II promoter is facilitated by the coupling of the transcription and 3’ end processing of the transcripts. The non-naturally occurring promoter may comprise the engineered promoter as described herein.

[0247] In some embodiments, the 3’ terminator sequence comprises an insulator. An insulator may be used to protect genes from inappropriate signals emanating from their surrounding environment. In some embodiments, the insulator protects the stabilizing sequence or secondary structure sequence from off-target cleavage stemming from processing of the nuclease cleavage site.

[0248] In some embodiments, the engineered insulator comprises at least: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, 250 or more nucleotides (nt; or base pair / bp) .

[0249] In some embodiments, the engineered insulator comprises at least: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides (nt; or base pair / bp). In some embodiments, the engineered insulator comprises a sequence set forth in SEQ ID NO: 119 (OAAAO AAAOAAA) .

[0250] In some embodiments, the engineered insulator may comprise or encode a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or more sequence identity to any sequence disclosed in Table 4. In some embodiments, the engineered insulator may comprise or encode a sequence having at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, at most 99%, at least 99.5% sequence identity to any sequence disclosed in Table 4. In some embodiments, the engineered insulator may comprise or encode a sequence having 100% sequence identity to any sequence disclosed in Table 4.

[0251] In some embodiments, the engineered insulator may comprise or encode a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or more sequence identity to any sequence disclosed in SEQ ID NOS: 119-122. In some embodiments, the engineered insulator may comprise or encode a sequence having at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, at most 99%, at least 99.5% sequence identity to any sequence disclosed in SEQ ID NOS: 119-122. In some embodiments, the engineered insulator may comprise or encode a sequence having 100% sequence identity to any sequence disclosed in SEQ ID NOS: 119-122.

[0252] Table 4: Exemplar Engineered Insulator Sequences

[0253] Name of SEQUENCE SEQ ID

[0254] Notes sequence NO

[0255] Insulator 1 CAAACAAACAAA 119

[0256] Insulator 2 GAAAGAAAGAAA 120

[0257] Insulator 3 CTTTCTTTCTTT 121

[0258] Insulator 4 GTTTGTTTGTTT

[0259]

[0260] 122 In some embodiments, the 3’ terminator sequence may comprise a processing nuclease cleavage site. In some embodiments, the nuclease cleavage site may comprise a RNAse P recognition sequence; a RNAse Z recognition sequence; a yeast U1 or U2 snRNA maturation sequence; a CCA-adding enzyme recognition sequence; any functional derivatives thereof; or any combinations thereof. In some embodiments, the nuclease cleave site may comprise a RNAse P recognition sequence. In some embodiments, the nuclease cleave site may comprise a RNAse Z recognition sequence. In some embodiments, the nuclease cleave site may comprise a yeast U1 maturation snRNA sequence recognition sequence. In some embodiments, the nuclease cleave site may comprise a yeast U2 maturation snRNA sequence recognition sequence, a CCA-adding enzyme recognition sequence a CCA-adding enzyme recognition sequence. Any enzyme recognition sequence may be configured to be recognized by an enzyme such that an enzyme can enzymatically process the enzyme recognition sequence and / or the nucleic acid comprising thereof.

[0261] In some embodiments, the 3’ terminator sequence may comprise a 3’ sequence of human Malatl Triple helix with RNase P cleavage site and t-RNA like structure, mouse Malat1_3’ WT+mascRNA, truncated version of mouse MALAT1 triple helix comp14 +mascRNA, truncated version of mouse MALAT1 triple helix comp.1 +mascRNA, truncated version of mouse MALAT1 triple helix comp.6 +mascRNA, truncated version of mouse MALAT1 triple helix comp.7 +mascRNA, truncated version of mouse MALAT1 triple helix comp.9 +mascRNA, truncated version of mouse MALAT1 triple helix comp.10 +mascRNA, truncated version of mouse MALAT1 triple helix comp.11 +mascRNA, truncated version of mouse MALAT1 triple helix comp.12 +mascRNA, truncated version of mouse MALAT1 triple helix comp.14 +mascRNA, Mut U2.5 +mascRNA, Human NEAT1 / MEN p triple helix + mascRNA, Mouse MEN p triple helix + mascRNA, Human MALAT1 mascRNA, Mouse MALAT1 mascRNA, Human MEN beta mascRNA, Mouse MEN beta mascRNA, or a combination thereof.

[0262] In some embodiments, the 3’ terminator sequence comprises a ribozyme. In some embodiments, the ribozyme comprises a hammerhead ribozyme; a twister ribozyme; a hepatitis delta virus (HDV) ribozyme; a hairpin ribozyme, a pistol ribozyme, a CPEB3 mammalian ribozyme; any functional derivatives thereof; or any combinations thereof. In some embodiments, the ribozyme comprises a hammerhead ribozyme. In some embodiments, the ribozyme comprises a twister ribozyme. In some embodiments, the ribozyme comprises a HDV ribozyme. In some embodiments, the ribozyme comprises a hairpin ribozyme. In some embodiments, the ribozyme comprises a pistol ribozyme. In some embodiments, the ribozyme comprises a CPEB3 mammalian ribozyme.

[0263] In some embodiments, the engineered TTD may comprise a snRNA promoter coupled 3’ box domain; a transfer RNA (tRNA) derived stem-loop sequence; a mascRNA derived stem-loop sequence; or any combinations thereof. In some embodiments, the engineered TTD may comprise a snRNA promoter coupled 3’ box domain. In some embodiments, the engineered TTD may comprise a tRNA derived stemloop sequence. In some embodiments, the engineered TTD may comprise a mascRNA derived stem-loop sequence.

[0264] In some embodiments, the engineered terminator may be derived from a LncRNA; an animal; a plant; a virus; a CRISPR sequence; a small nucleolar RNAs (snoRNA); a histone messenger RNA (mRNA); any functional derivatives thereof; or any combinations thereof. In some embodiments, the engineered terminator may be derived from a LncRNA. In some embodiments, the engineered terminator may be derived from a plant. In some embodiments, the engineered terminator may be derived from a virus. In some embodiments, the engineered terminator may be derived from a CRISPR sequence. In some embodiments, the engineered terminator may be derived from a snoRNA. In some embodiments, the engineered terminator may be derived from a histone mRNA. In some embodiments, the engineered terminator may be derived from an animal.

[0265] In some embodiments, the engineered 3’ terminator may comprise or encode a pseudo tRNA structure sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or more sequence identity to any sequence disclosed in Table 5. In some embodiments, the engineered 3’ terminator may comprise or encode a sequence having at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, at most 99%, at least 99.5% sequence identity to any sequence disclosed in Table 5. In some embodiments, the engineered 3’ terminator may comprise or encode a sequence having 100% sequence identity to any sequence disclosed in Table 5.

[0266] In some embodiments, the engineered 3’ terminator may comprise or encode a pseudo tRNA structure sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or more sequence identity to any sequence disclosed in SEQ ID NOS: 123-124. In some embodiments, the engineered 3’ terminator may comprise or encode a sequence having at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, at most 99%, at least 99.5% sequence identity to any sequence disclosed in SEQ ID NOS: 123-124. In some embodiments, the engineered 3’ terminator may comprise or encode a sequence having 100% sequence identity to any sequence disclosed in SEQ ID NOS: 123-124.

[0267] Table 5: Exemplar Engineered t-RNA Like Sequences

[0268] Name of SEQUENCE SEQ ID

[0269] Notes sequence NO

[0270] tRNA-Phe- GC C AAAAT AGC T C AGC T GGGAGAGC AT T AGAC T GAAGAT C T AAAGG

[0271] GAA-8-1 TCTCTGGTTTGATCCTGGGTTTCAGAA 123

[0272] tRNA-iMet- AGCAGAGTGGTGCAGTGGAAGCATACCTATGGGCCCATAACCCAGA

[0273] CAT- GGTTGATGGATGGAAACCATCCTCTGCTA 124

[0274]

[0275] 3-1

[0276] Antisense Sequences

[0277] Engineered nucleic acids as described herein may comprise or encode one or more antisense sequences. The antisense sequence may be engineered. In some embodiments, the antisense sequence may be complementary to at least a portion of a target nucleic acid molecule. In some cases, the antisense sequence may be complementary to at least a portion of a target RNA molecule.

[0278] In some embodiments, the antisense sequence may facilitate the binding of the engineered nucleic acid (or a transcript encoded therefrom) to a target nucleic acid. In some embodiments, the target nucleic acid is an RNA molecule. In some embodiments, the antisense sequence may facilitate the hybridization of the engineered nucleic acid (or a transcript encoded therefrom) to a target nucleic acid. In some embodiments, the antisense sequence may facilitate the localization of the engineered nucleic acid (or a transcript encoded therefrom) to a target nucleic acid. In some embodiments, the antisense sequence may facilitate the regulation of the splicing of a target nucleic acid.

[0279] In some embodiments, the antisense sequence may comprise a steric-blocking antisense oligonucleotide, a small interfering RNA, a splice-switching oligonucleotide, an RNA binding protein interacting decoy sequence, any functional derivatives thereof; or any combinations thereof. In some embodiments, the antisense sequence may comprise a steric-blocking antisense oligonucleotide. In some embodiments, the antisense sequence may comprise a small interfering RNA. In some embodiments, the antisense sequence may comprise a splice-switching oligonucleotide. In some embodiments, the antisense sequence may comprise an RNA binding protein interacting decoy sequence.

[0280] In some embodiments, the antisense sequence may comprise a sequence complementary to at least a portion of: an intron of the target RNA molecule; an exon of the target RNA molecule; an intronexon junction of the target RNA molecule; an untranslated region (UTR) of the target RNA molecule; any functional derivatives thereof; or any combinations thereof. In some embodiments, the antisense sequence may comprise a sequence complementary to at least a portion of an intron of the target RNA molecule. In some embodiments, the antisense sequence may comprise a sequence complementary to at least a portion of an exon of the target RNA molecule. In some embodiments, the antisense sequence may comprise a sequence complementary to at least a portion of intron-exon junction of the target RNA molecule. In some embodiments, the antisense sequence may comprise a sequence complementary to at least a portion of an UTR of the target RNA molecule. In some embodiments, the antisense sequence may comprise a sequence complementary to at least a portion of a 5’ UTR of the target RNA molecule. In some embodiments, the antisense sequence may comprise a sequence complementary to at least a portion of a 3’ UTR region of the target RNA molecule.

[0281] The engineered nucleic acid may comprise at least: 1 , 2, 3, 4, 5 or more antisense sequence(s). The engineered nucleic acid may comprise at most: 1 , 2, 3, 4, or 5 antisense sequence(s). The engineered nucleic acid may comprise 1 antisense sequence. The engineered nucleic acid may comprise 2 antisense sequences. The engineered nucleic acid may comprise 3 antisense sequences. The engineered nucleic acid may comprise 4 antisense sequences. The engineered nucleic acid may comprise 5 antisense sequences. In some cases, the more than one antisense sequences may be different sequences. In some cases, the more than one antisense sequences may be the same sequence. In some cases, the more than one antisense sequences may be complementary to different portions of the same target RNA molecule. The different portions of the target RNA molecule may comprise UTRs, exons, introns, or exon-intron junctions, or any combinations thereof. In some cases, the more than one antisense sequences may be complementary to different target RNA molecules.

[0282] In some embodiments, the one or more antisense sequences have a length of 15 to 50 nucleotides. In some embodiments, the antisense sequence may be at least: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 80 or more nucleotides (nt) or basepairs (bp) long. In some embodiments, the antisense sequence may be at most: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50 , 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70 or 80 nt / bp. In some embodiments, the antisense sequence may comprise a sequence that has at least: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity / complementarity to a target nucleic acid molecule. In some embodiments, the antisense sequence may comprise a sequence that has at most: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity / complementarity to a target nucleic acid molecule.

[0283] In some embodiments, the engineered nucleic acid comprising at least one antisense sequence may increase the exon-skipping, exon-inclusion, intron-skipping, or intron-inclusion of a target nucleic acid molecule, relative to that of a control counterpart. In some embodiments, the engineered nucleic acid comprising at least one antisense sequence may decrease the exon-skipping, exon-inclusion, intronskipping, or intron-inclusion of a target nucleic acid molecule, relative to that of a control counterpart.

[0284] In some embodiments, the engineered nucleic acid comprising at least one antisense sequence (or a transcript encoded therefrom) may increase the exon-skipping level of a target nucleic acid molecule that is at least: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart. In some embodiments, the engineered nucleic acid comprising at least one antisense sequence (or a transcript encoded therefrom) may increase the exonskipping level of a target nucleic acid molecule that is at most: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart.

[0285] In some embodiments, the engineered nucleic acid comprising at least one antisense sequence (or a transcript encoded therefrom) may increase the exon-inclusion level of a target nucleic acid molecule that is at least: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart. In some embodiments, the engineered nucleic acid comprising at least one antisense sequence (or a transcript encoded therefrom) may increase the exoninclusion level of a target nucleic acid molecule that is at most: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart.

[0286] In some embodiments, the engineered nucleic acid comprising at least one antisense sequence (or a transcript encoded therefrom) may increase the intron-skipping level of a target nucleic acid molecule that is at least: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart. In some embodiments, the engineered nucleic acid comprising at least one antisense sequence (or a transcript encoded therefrom) may increase the intron-skipping level of a target nucleic acid molecule that is at most: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart.

[0287] In some embodiments, the engineered nucleic acid comprising at least one antisense sequence (or a transcript encoded therefrom) may increase the intron-inclusion level of a target nucleic acid molecule that is at least: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart. In some embodiments, the engineered nucleic acid comprising at least one antisense sequence (or a transcript encoded therefrom) may increase the intron-inclusion level of a target nucleic acid molecule that is at most: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart.

[0288] In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of Usherin (USH2a), USH1C, Sodium voltagegated channel alpha subunit 9 (Nav1.7), Peripherin-2 (Prph2), Dystrophin (DMD), Myotonic Dystrophy Protein Kinase (DMPK), ZNF9, SCN1A, SCN2A, SCN8A, SNRPB, ARMS2, CFH, PRMT6, LSS, C2, C3, CFI, ApoE, MAPT, LRRK2, a-SNCA, HTRA1 , SMN2, CEP290, COL7A1 , NF1 , TTN, HTT, PCSK9, TTR, STMN2, SOD1, APP, SERPINA1 , LPA, HP, HSD17B13, LDLR, RHO, or any combinations thereof. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of USH2a. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of USH1 C. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of Navi .7. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of Prph2. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of DMD. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of DMPK. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of ZNF9. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of SCN1 A. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of SCN2A. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of SCN8A. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of SNRPB. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of ARMS2. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of CFH. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of PRMT6. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of LSS. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of C2. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of C3. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of CFI. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of APOE. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of MAPT. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of HTRA1. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of LRRK2. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of a-SNCA. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of SMN2. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of CEP290. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of COL7A1. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of NF1. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of TTN. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of HTT. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of PCSK9. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of TTR. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of STMN2. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of SOD1. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of APP. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of SERPINA1 . In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of LPA. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of HP. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of HSD17B13. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of LDLR. In some embodiments, the antisense sequence may comprise a sequence of or a sequence complementary of the transcribed nucleotide sequence of RHO.

[0289] Engineered Sm-binding Sequences

[0290] Engineered nucleic acids as described herein may comprise or encode an engineered Sm-binding sequence. In some embodiments, the engineered stabilizing sequence is configured to recruit a spliceosomal small nuclear ribonucleoprotein (snRNP) to the engineered nucleic acid. In some embodiments, engineered Sm-binding sequence may stabilize the engineered nucleic acid. Endogenous U7 snRNA is expressed at a low level, approximately 2-15x10A3 molecules per cell. However, the expression level and the nuclear concentration of U7 snRNA can be increased significantly by converting the wild-type U7 Sm-binding site (AAUUUGUCUAG; SEQ ID NO: 75) to an engineered Sm-binding sequence derived from the major spliceosomal snRNPs, for example, but not limited to SmOPT, AAUUUUUGGAG (SEQ ID NO: 76). See, for example, Forman et al., Proc Natl Acad Sci. 1998 Apr 28;95(9):4929-34., which is herein incorporated by reference in its entirety. Moreover, the engineered Sm-binding sequence can render the particle functionally inactive in histone pre-mRNA processing. This arrangement can have at least two beneficial effects: (i) the target nucleic acid molecule, such as betaglobin pre-mRNA, will not be cleaved by the histone 3’ end processing machinery, and (ii) because of the inability of engineered Sm-binding sequence to bind one or more U7-specific proteins, the engineered nucleic acid will not compete with endogenous U7 snRNP for potentially limiting U7-specific proteins. Additionally, whereas the wild-type U7 snRNPs are sequestered in coiled bodies, those with the engineered Sm-binding sequence are not and therefore may be redirected to the sites of pre-mRNA splicing. Thus, an engineered nucleic acid comprising the engineered Sm-binding sequence can have increased stability or nuclear localization (and thus have increased therapeutic effects), relative to the control counterpart.

[0291] In some cases, the engineered Sm-binding sequence may be configured to facilitate the nuclear localization of the engineered nucleic acid.

[0292] The engineered nucleic acid may comprise or encode at least: 1 , 2, 3, 4, 5 or more engineered Sm-binding sequence(s). The engineered nucleic acid may comprise or encode at most: 1 , 2, 3, 4, or 5 engineered Sm-binding sequence(s). The engineered nucleic acid may comprise or encode 1 Sm-binding sequence. The engineered nucleic acid may comprise or encode 2 Sm-binding sequences. The engineered nucleic acid may comprise or encode 3 engineered Sm-binding sequences. The engineered nucleic acid may comprise or encode 4 engineered Sm-binding sequences. The engineered nucleic acid may comprise or encode 5 engineered Sm-binding sequences.

[0293] In some embodiments, the engineered Sm-binding sequence may have at least: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides (nt; or base pair / bp). In some embodiments, the engineered Sm-binding sequence may have at most: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 nt / bp.

[0294] In some embodiments, the engineered nucleic acid comprising at least one engineered Sm-binding sequence (or a transcript encoded therefrom) may be expressed at a level that is at least: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart. In some embodiments, the engineered nucleic acid comprising at least one engineered Sm-binding sequence (or a transcript encoded therefrom) may be expressed at a level that is at most: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart. In some embodiments, the engineered nucleic acid comprising at least one engineered Sm-binding sequence (or a transcript encoded therefrom) may have a half-life that is at least: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart. In some embodiments, the engineered nucleic acid comprising at least one engineered Sm-binding sequence (or a transcript encoded therefrom) may have a half-life that is at most: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart. In some embodiments, the amount of the engineered nucleic acid comprising at least one engineered Sm-binding sequence (or a transcript encoded therefrom) that is localized to the nucleus may be at least: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart. In some embodiments, the amount of the engineered nucleic acid comprising at least one engineered Sm-binding sequence (or a transcript encoded therefrom) that is localized to the nucleus may be at most: 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 100 %, 150 %, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, or 1000-fold higher than that of the control counterpart.

[0295] In some embodiments, the engineered Sm-binding sequence may be configured to recruit a splicing regulator to the engineered nucleic acid molecule. In some embodiments, the engineered Sm-binding sequence may be configured to recruit a splicing regulator to the target nucleic or RNA molecule. In some embodiments, the engineered Sm-binding sequence may be configured to recruit at least: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50 or more different splicing regulator proteins to the engineered nucleic acid molecule and / or the target nucleic / RNA molecule. In some embodiments, the engineered Sm-binding sequence may be configured to recruit at most: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50 different splicing regulator proteins to the engineered nucleic acid molecule and / or the target nucleic / RNA molecule. In some cases, the splicing regulator or splicing regulator protein may comprise a Sm ring protein.

[0296] In some embodiments, the engineered Sm-binding site may comprise a Sm-binding site derived from a human, murine, mammalian, plant, eucaryotes, virus, any functional derivatives thereof; or any combinations thereof. In some embodiments, the engineered Sm-binding site may comprise a Sm-binding site derived from a human. In some embodiments, the engineered Sm-binding site may comprise a Sm-binding site derived from a murine. In some embodiments, the engineered Sm-binding site may comprise a Sm-binding site derived from a mammal. In some embodiments, the engineered Sm-binding site may comprise a Sm-binding site derived from a plant. In some embodiments, the engineered Sm-binding site may comprise a Sm-binding site derived from an eucaryote. In some embodiments, the engineered Sm-binding site may comprise a Sm-binding site derived from a virus.

[0297] In some embodiments, the engineered Sm-binding site may comprise a U1 Sm-binding sequence, a U2 Sm-binding sequence, a U4 Sm-binding sequence, a U5 Sm-binding sequence, a sequence derived from major spliceosomal U small nuclear ribonucleoprotein (SmOpt), any functional derivatives thereof; or any combinations thereof. In some embodiments, the engineered Sm-binding site may comprise a U1 Sm-binding sequence. In some embodiments, the engineered Sm-binding site may comprise a U2 Sm-binding sequence. In some embodiments, the engineered Sm-binding site may comprise a U4 Sm-binding sequence. In some embodiments, the engineered Sm-binding site may comprise a U5 Sm-binding sequence. In some embodiments, the engineered Sm-binding site may comprise a sequence derived from major spliceosomal U small nuclear ribonucleoprotein (SmOpt).

[0298] Linkers

[0299] In some embodiments, any two of the various structural and / or sequence elements as described herein; such as the 5’ engineered element, 3’ engineered element, engineered stabilizing sequences / secondary structure sequences, engineered Sm-binding sequences, TTD, engineered promoters, 5’ methyl G cap, antisense sequences, any specific embodiments thereof, or any derivatives thereof; can be separated by at least: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. In some embodiments, any two of the various structural and / or sequence elements as described herein by at most: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides. In some embodiments, any two of the various structural and / or sequence elements as described herein by 0 nucleotides. In some embodiments, the number of nucleotides that separates any two of the various structural and / or sequence elements can be determined by at least in part on whether the number of nucleotides used to separate the any two of the various structural and / or sequence elements would facilitate the engineered nucleic acid to process certain property(ies), as described herein.

[0300] Nucleic Acid Modifications

[0301] In some embodiments, engineered nucleic acid molecules as described herein comprise unmodified forms of nucleotides. In some embodiments, engineered nucleic acid molecules as described herein comprise modified forms of nucleotides. Unmodified nucleotides contain nucleobases that include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleotides are described in more detail below. Modifications may be achieved by incorporating, for example, one or more alternative nucleosides, alternative 2’ sugar moieties, and / or alternative internucleoside linkages. Typically, these types of modifications are introduced to optimize the molecule’s efficacy or biophysical properties (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, reduce immunogenicity, and / or targeting to a particular location or cell type).

[0302] In some embodiments, modified nucleotides comprise nucleoside modifications. In some embodiments, modification of the nucleotides described herein include one or more of the following nucleoside modifications: 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5- trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F- adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and / or 3-deazaguanine and 3-deazaadenine. Further modification of the nucleic acid molecules described herein may include nucleobases disclosed in US 3,687,808; Kroschwitz, J. I., ed. The Concise Encyclopedia of Polymer Science and Engineering, New York, John Wiley & Sons, 1990, pp. 858-859; Englisch et al., Angewandte Chemie, International Edition 30:613, 1991 ; and Sanghvi, Y.S., Chapter 16, Antisense Research and Applications, CRC Press, Gait, M.J. ed., 1993, pp. 289-302.

[0303] In some embodiments, modified nucleotides comprise nucleotide modifications. In some embodiments, modifications of the nucleotides described herein include one or more of the following 2’ sugar modifications: 2’-O-methyl (2’-O-Me), 2’-methoxyethoxy (2’-O-CH2CH2OCH3, also known as 2’-O-(2-methoxyethyl) or 2’-MOE), 2’-dimethylaminooxyethoxy, i.e. , a O(CH2)2ON(CH3)2 group, also known as 2’-DMAOE, and / or 2’-dimethylaminoethoxyethoxy (also known in the art as 2’-0-dimethylamino-ethoxy-ethyl or 2’-DMAEOE), i.e., 2’-O-CH2OCH2N(CH3)2. Other possible 2’-modifications that can modify the modified nucleotides described herein include all possible orientations of OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O- alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Other potential sugar substituent groups include, e.g., aminopropoxy (-OCH2CH2CH2NH2), allyl (-CH2-CH=CH2), -O-allyl (-O-CH2-CH=CH2) and fluoro (F). 2’-sugar substituent groups may be in the arabino (up) position or ribo (down) position. In some embodiments, the 2’-arabino modification is 2’-F.

[0304] In some embodiments, nucleic acid molecules comprise internucleoside linkage modifications. In some embodiments, modifications of the nucleic acid molecules described herein include one or more of the following internucleoside modifications: phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3’-alkylene phosphonates, 5’-alkylene phosphonates, phosphinates, phosphoramidates including 3’-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3’-5’ linkages, 2’-5’ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3’ to 3’, 5’ to 5’ or 2’ to 2’ linkage.

[0305] Nucleic Acid Length and Expression Levels

[0306] In some embodiments, engineered nucleic acid molecules as described herein comprise varying lengths of nucleotides. In some embodiments, the engineered nucleic acid molecules comprising one or more antisense sequences, an engineered Sm-binding site, and a 3’ engineered element; or a 5’ engineered element, one or more antisense sequences, and an engineered Sm-binding site; or a 5’ engineered element, one or more antisense sequences, an engineered Sm-binding site, and a 3’ engineered element may include a nucleotide sequence of 50 to 500 nucleotides in length (e.g., 50, 51 , 52, 53, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 nucleotides in length).

[0307] In some embodiments, an expression cassette encoding an engineered nucleic acid (i.e., a nucleic acid molecule) comprising an engineered promoter, one or more antisense sequences, an engineered Sm-binding site, and a 3’ engineered terminator; or an engineered promoter, a 5’ engineered terminator, one or more antisense sequences, an engineered Sm-binding site, and a 3’ engineered terminator may include a nucleotide sequence of 500 to 5000 nucleotides in length (e.g., 500, 510, 520, 530, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, or 5000 nucleotides in length).

[0308] In some embodiments, the engineered nucleic acid may have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, 250, 251 , 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 , 262, 263, 264, 265, 266, 267, 268, 269, 270, 271 , 272, 273, 274, 275, 276, 277, 278, 279, 280, 281 , 282, 283, 284, 285, 286, 287, 288, 289, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 , 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331 , 332, 333, 334, 335, 336, 337, 338, 339, 340, 341 , 342, 343, 344, 345, 346, 347, 348, 349, 350, 351 , 352, 353, 354, 355, 356, 357, 358, 359, 360, 361 , 362, 363, 364, 365, 366, 367, 368, 369, 370, 371 , 372, 373, 374, 375, 376, 377, 378, 379, 380, 381 , 382, 383, 384, 385, 386, 387, 388, 389, 390, 391 , 392, 393, 394, 395, 396, 397, 398, 399, 400, 401 , 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441 , 442, 443, 444, 445, 446, 447, 448, 449, 450, 451 , 452, 453, 454, 455, 456, 457, 458, 459, 460, 461 , 462, 463, 464, 465, 466, 467, 468, 469, 470, 471 , 472, 473, 474, 475, 476, 477, 478, 479, 480, 481 , 482, 483, 484, 485, 486, 487, 488, 489, 490, 491 , 492, 493, 494, 495, 496, 497, 498, 499, 500, 1000, 2000, 5000 or more nucleotides (nt; or base pair / bp). In some embodiments, the engineered nucleic acid may have at most 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, 250, 251 , 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 , 262, 263, 264, 265, 266, 267, 268, 269, 270, 271 , 272, 273, 274, 275, 276, 277, 278, 279, 280, 281 , 282, 283, 284, 285, 286, 287, 288, 289, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 , 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331 , 332, 333, 334, 335, 336, 337, 338, 339, 340, 341 , 342, 343, 344, 345, 346, 347, 348, 349, 350, 351 , 352, 353, 354, 355, 356, 357, 358, 359, 360, 361 , 362, 363, 364, 365, 366, 367, 368, 369, 370, 371 , 372, 373, 374, 375, 376, 377, 378, 379, 380, 381 , 382, 383, 384, 385, 386, 387, 388, 389, 390, 391 , 392, 393, 394, 395, 396, 397, 398, 399, 400, 401 , 402, 403, 404, 405, 406, 407, 408, 409, 410, 411 , 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441 , 442, 443, 444, 445, 446, 447, 448, 449, 450, 451 , 452, 453, 454, 455, 456, 457, 458, 459, 460, 461 , 462, 463, 464, 465, 466, 467, 468, 469, 470, 471 , 472, 473, 474, 475, 476, 477, 478, 479, 480, 481 , 482, 483, 484, 485, 486, 487, 488, 489, 490, 491 , 492, 493, 494, 495, 496, 497, 498, 499, 500, 1000, 2000, or 5000 nt / bp.

[0309] In some embodiments, the transcribed form of the engineered nucleic acid may have at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, 250, 251 , 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 , 262, 263, 264, 265, 266, 267, 268, 269, 270, 271 , 272, 273, 274, 275, 276, 277, 278, 279, 280, 281 , 282, 283, 284, 285, 286, 287, 288, 289, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331 , 332, 333, 334, 335, 336, 337, 338, 339, 340, 341 , 342, 343, 344, 345, 346, 347, 348, 349, 350, 351 , 352, 353, 354, 355, 356, 357, 358, 359, 360, 361 , 362, 363, 364, 365, 366, 367, 368, 369, 370, 371 , 372, 373, 374, 375, 376, 377, 378, 379, 380, 381 , 382, 383, 384, 385, 386, 387, 388, 389, 390, 391 , 392, 393, 394, 395, 396, 397, 398, 399, 400, 401 , 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431 , 432, 433, 434, 435, 436, 437, 438, 439, 440, 441 , 442, 443, 444, 445, 446, 447, 448, 449, 450, 451 , 452, 453, 454, 455, 456, 457, 458, 459, 460, 461 , 462, 463, 464, 465, 466, 467, 468, 469, 470, 471 , 472, 473, 474, 475, 476, 477, 478, 479, 480, 481 , 482, 483, 484, 485, 486, 487, 488, 489, 490, 491 , 492, 493, 494, 495, 496, 497, 498, 499, 500 or more nucleotides (nt; or base pair / bp). In some embodiments, the transcribed form of the engineered nucleic acid may have at most 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, 250, 251 , 252, 253, 254, 255, 256, 257, 258, 259, 260, 261 , 262, 263, 264, 265, 266, 267, 268, 269, 270, 271 , 272, 273, 274, 275, 276, 277, 278, 279, 280, 281 , 282, 283, 284, 285, 286, 287, 288, 289, 290, 291 , 292, 293, 294, 295, 296, 297, 298, 299, 300, 301 , 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331 , 332, 333, 334, 335, 336, 337, 338, 339, 340, 341 , 342, 343, 344, 345, 346, 347, 348, 349, 350, 351 , 352, 353, 354, 355, 356, 357, 358, 359, 360, 361 , 362, 363, 364, 365, 366, 367, 368, 369, 370, 371 , 372, 373, 374, 375, 376, 377, 378, 379, 380, 381 , 382, 383, 384, 385, 386, 387, 388, 389, 390, 391 , 392, 393, 394, 395, 396, 397, 398, 399, 400, 401 , 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421 , 422, 423, 424, 425, 426, 427, 428, 429, 430, 431 , 432, 433, 434, 435, 436, 437, 438, 439, 440, 441 , 442, 443, 444, 445, 446, 447, 448, 449, 450, 451 , 452, 453, 454, 455, 456, 457, 458, 459, 460, 461 , 462, 463, 464, 465, 466, 467, 468, 469, 470, 471 , 472, 473, 474, 475, 476, 477, 478, 479, 480, 481 , 482, 483, 484, 485, 486, 487, 488, 489, 490, 491 , 492, 493, 494, 495, 496, 497, 498, 499, or 500 nt / bp.

[0310] In some embodiments, at least: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, 250 or more nucleotides of the engineered nucleic acid may be single-stranded. In some embodiments, at most: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 , 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, or 250 nucleotides of the engineered nucleic acid may be single-stranded. In some embodiments, at least: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 156, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 , 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, 250 or more nucleotides of the engineered nucleic acid may be base-paired with another nucleotide(s) of the engineered nucleic acid. In some embodiments, at most: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 156, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171 , 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 , 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249, or 250 nucleotides of the engineered nucleic acid may be base-paired with another nucleotide(s) of the engineered nucleic acid.

[0311] In some embodiments, the engineered nucleic acid may be expressed (or encode a transcript that is expressed therefrom) at a level of at least: 1 picomolar (pM) per 1 x10A6 host cells, 10 pM per 1 x10A6 host cells, 100 pM per 1 x10A6 host cells, 1 nanomolar (nM) per 1 x10A6 host cells, 10 nM per 1 x10A6 host cells, 100 nM per 1 x10A6 host cells, 1 micromolar (pM) per 1 x10A6 host cells, 10 pM per 1 x10A6 host cells, 100 pM per 1 x10A6 host cells, 1 millimolar (mM) per 1 x10A6 host cells, 10 mM per 1 x10A6 host cells, 100 mM per 1 x10A6 host cells or more, wherein the host cells are: (1 ) transfected with a vector that comprises or encodes the engineered nucleic acid; (2) transduced with a virus that comprises or encodes the engineered nucleic acid; or (3) encode the engineered nucleic acid (such as via genome editing or engineering of cells). Detection or measurements of the expression level or quantity of the RNA of the engineered nucleic acid (or a control counterpart) may be carried out via northern blot, quantitative polymerase chain reaction (qPCR), fluorescent hybridization, sequencing, microarray, any functional derivatives thereof, any functional derivatives thereof; or any combinations thereof. Detection or measurements of the expression level or quantity of the engineered nucleic acid (or a control counterpart) may be carried out at least: 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week or more, subsequent the engineered nucleic acid is allowed to be expressed within the host cell. Detection or measurements of the expression level or quantity of the engineered nucleic acid (or a control counterpart) may be carried out at most: 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week, subsequent the engineered nucleic acid is allowed to be expressed within the host cell. Detection or measurements of the expression level or quantity of the engineered nucleic acid (or a control counterpart) may be carried out at least: 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week or more, subsequent the engineered nucleic acid is contacted to the host cell.

[0312] Detection or measurements of the expression level or quantity of the engineered nucleic acid (or a control counterpart) may be carried out at most: 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week, subsequent the engineered nucleic acid is contacted to the host cell. In some embodiments, the engineered nucleic acid may be expressed (or encode a transcript that is expressed therefrom) at a level of at most: 1 picomolar (pM) per 1 x10A6 host cells, 10 pM per 1 x10A6 host cells, 100 pM per 1 x10A6 host cells, 1 nanomolar (nM) per 1 x10A6 host cells, 10 nM per 1 x10A6 host cells, 100 nM per 1 x10A6 host cells, 1 micromolar (pM) per 1 x10A6 host cells, 10 pM per 1 x10A6 host cells, 100 pM per 1 x10A6 host cells, 1 millimolar (mM) per 1 x10A6 host cells, 10 mM per 1 x10A6 host cells, or 100 mM per 1 x10A6 host cells.

[0313] In some embodiments, the engineered nucleic acid may be expressed (or encode a transcript that is expressed therefrom) at a level of at least: 1 picomolar (pM) per culture of host cells, 10 pM per culture of host cells, 100 pM per culture of host cells, 1 nanomolar (nM) per v, 10 nM per culture of host cells, 100 nM per culture of host cells, 1 micromolar (pM) per culture of host cells, 10 pM per culture of host cells, 100 pM per culture of host cells, 1 millimolar (mM) per culture of host cells, 10 mM per culture of host cells, 100 mM per culture of host cells or more. A culture of host cells may have at least: 1 x10A3 host cells, 1 x10A4 host cells, 1 x10A5 host cells, 1 x10A6 host cells, 1 x10A7 host cells, 1 x10A8 host cells or more. A culture of host cells may have at most: 1 x10A3 host cells, 1 x10A4 host cells, 1 x10A5 host cells, 1 x10A6 host cells, 1 x10A7 host cells, or 1 x10A8 host cells. In some embodiments, the engineered nucleic acid may be expressed (or encode a transcript that is expressed therefrom) at a level of at most: 1 picomolar (pM) per culture of host cells, 10 pM per culture of host cells, 100 pM per culture of host cells, 1 nanomolar (nM) per v, 10 nM per culture of host cells, 100 nM per culture of host cells, 1 micromolar (pM) per culture of host cells, 10 pM per culture of host cells, 100 pM per culture of host cells, 1 millimolar (mM) per culture of host cells, 10 mM per culture of host cells, or 100 mM per culture of host cells.

[0314] In some embodiments, at least: 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more engineered nucleic acids (or transcripts expressed by the engineered nucleic acids) are localized to or present within the nucleus. In some embodiments, at most: 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% engineered nucleic acids (or transcripts expressed by the engineered nucleic acids) are localized to or present within the nucleus.

[0315] In some embodiments, the half-life of the engineered nucleic acid (or a transcript expressed therefrom) is at least: 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, or 5 years. In some embodiments, the half-life of the engineered nucleic acid (or a transcript expressed therefrom)is at most: 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, at most: 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, or 5 years.

[0316] Exemplary Engineered Nucleic Acids / Nucleic Acid Molecules

[0317] Engineered nucleic acids as described herein may comprise or encode one or more engineered terminators, engineered Sm-binding sequences, or engineered stabilizing sequences or secondary structure sequences.

[0318] For instance, engineered nucleic acids as described herein may comprise or encode a Triple helix with RNase P cleavage site and t-RNA like structure, mouse Malat1_3’ WT, truncated version of mouse Human Malatl Triple helix with RNase P cleavage site and t-RNA like structure, mouse Malat1_3’ WT+mascRNA, truncated version of mouse MALAT1 triple helix com 14 +mascRNA, truncated version of mouse MALAT1 triple helix comp.1 +mascRNA, truncated version of mouse MALAT1 triple helix comp.6 +mascRNA, truncated version of mouse MALAT1 triple helix comp.7 +mascRNA, truncated version of mouse MALAT1 triple helix comp.9 +mascRNA, truncated version of mouse MALAT1 triple helix comp.10 +mascRNA, truncated version of mouse MALAT1 triple helix comp.11 +mascRNA, truncated version of mouse MALAT1 triple helix comp.12 +mascRNA, truncated version of mouse MALAT1 triple helix comp.14 +mascRNA, Mut U2.5 +mascRNA, Human NEAT1 / MEN p triple helix + mascRNA, Mouse MEN p triple helix + mascRNA, Human MALAT1 mascRNA, Mouse MALAT1 mascRNA, Human MEN beta mascRNA, Mouse MEN beta mascRNA, Human Malatl Triple helix, mouse Malatl Triple helix, truncated version of mouse MALAT 1 triple helix com14, truncated version of mouse MALAT 1 triple helix comp.1 , truncated version of mouse MALAT1 triple helix comp.6, truncated version of mouse MALAT1 triple helix comp.7, truncated version of mouse MALAT1 triple helix comp.9, truncated version of mouse MALAT1 triple helix comp.10, truncated version of mouse MALAT1 triple helix comp.11 , truncated version of mouse MALAT1 triple helix comp.12, truncated version of mouse MALAT1 triple helix comp.14, Mut U2.5 triple helix, Human NEAT1 / MEN p triple helix, Mouse MEN p triple helix, KSHV PAN core Triple Helix, KSHV GCPAN triple helix, KSHV PAN domain triple helix, RRV PAN triple helix, EHV2 triple helix, TYMV pseudoknot, Human MAT2A 3’UTR stem-loop structure Hairpin A, Human MAT2A 3’UTR stem-loop structure Hairpin B, Human MAT2A 3’UTR stem-loop structure Hairpin C, Human MAT2A 3’UTR stemloop structure Hairpin D, Human MAT2A 3’UTR stem-loop structure Hairpin E, Human MAT2A 3’UTR stem-loop structure Hairpin F, IR1 (intergenic region sequence of cipC-cel48F), IR2 (intergenic region sequence of cel48F-cel8C), IR3 (intergenic region sequence of el8C-cel9G), IR4 (intergenic region sequence of cel9G-cel9E), IR5 (intergenic region sequence of cel9E-orfX), IR6 (intergenic region sequence of orfX-cel9H), IR7 (intergenic region sequence of cel9H-cel9J), IR8 (intergenic region sequence of cel9J -Man5K), IR9 (intergenic region sequence of Man5K-cel9M), IR10 (intergenic region sequence of cel9M rgl 11 Y), IR11 (intergenic region sequence of rgl11 Y -cel5N), V. Vulnificus add, B. Subtilis xpt, Cholera Vc2, T. tengcongensis SAM, T. tengcongensis glmS, U4 Sm site, Human U1 Sm site, Human U1 Pseudogene Sm site, Human U1 Pseudogene Sm site, Mouse U7 sm site, Mouse smOPT site, Mouse smOPT site 2, HDV Ribozyme, HDV Ribozyme, CPEB3 Ribozyme, Hammer Head Ribozyme, any fragments thereof, any functional derivatives thereof, or any combinations thereof.

[0319] In some embodiments, the engineered nucleic acid may comprise or encode a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or more sequence identity to any sequence disclosed in Table 6. In some embodiments, the engineered nucleic acid may comprise or encode a sequence having at most 50%, at most 55%, at most 60%, at most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90%, at most 91 %, at most 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, at most 99%, at least 99.5% sequence identity to any sequence disclosed in Table 6. In some embodiments, the engineered nucleic acid may comprise or encode a sequence having 100% sequence identity to any sequence disclosed in Table 6.

[0320] Table 6: Exemplar DNA or RNA Sequences Encoding Engineered Nucleic Acid Sequences Name of Sequence SEQ ID Notes sequence NO

[0321] Human AAGCTGATCTCCAATGCTCTTCAGTAGGGTCATGAAGGTTTTTCTT 1 Engineered 3’ Malatl Triple TTCCTGAGAAAACAACACGTATTGTTTTCTCAGGTTTTGCTTTTTG terminator, helix with GCCTTTTTCTAGCT T AAAAAAAAAAAAAGC AAAAGAT GCTGGTGGT TTD, or RNase P TGGCACTCCTGGTTTCCAGGACGGGGTTCAAATCCCTGCGGCGTCT cleavage cleavage site TTGCTT sequence and t-RNA like

[0322] structure

[0323] mouse GATTCGTCAGTAGGGTTGTAAAGGTTTTTCTTTTCCTGAGAAAACA 2 Engineered 3’ Malatl _3’ ACCTTTTGTTTTCTCAGGTTTTGCTTTTTGGCCTTTCCCTAGCTTT terminator, WT+mascRN AAAAAAAAAAAAGC AAAAGAC GC T GGT GGC T GGC AC T C C T GGT T T C TTD, or A CAGGACGGGGTTCAAGTCCCTGCGGTGTCTTTGCTT cleavage sequence truncated AAAGGTTTTTCTTTTCCTGAGAAATTTCTCAGGTTTTGCTTTTTAA 3 Engineered 3’ version of AAAAAAAGCAAAAGATGCTGGTGGTTGGCACTCCTGGTTTCCAGGA terminator, mouse CGGGGTTCAAAT TTD, or MALAT1 triple cleavage helix com14 sequence +mascRNA

[0324] truncated AAAGGTTTTTCTTTTCCTGATCAGGTTTTGCTTTTTGGCCTTTCCC 4 Engineered 3’ version of TAGC T T T AAAAAAAAAAAAGC AAAAGAC GC T GGT GGC T GGC AC T C C terminator, mouse TGGTTTCCAGGACGGGGTTCAAGTCCCTGCGGTGTCTTTGCTT TTD, or MALAT1 triple cleavage helix comp.1 sequence +mascRNA

[0325] truncated AAAGGTTTTTCTTTTCCTGAGAAAACAACCTTTTGTTTTCTCAGGT 5 Engineered 3’ version of T T T GC T T T T T AAAAAAAAAAAAGC AAAAGAC GC T GGT GGC T GGC AC terminator, mouse TCCTGGTTTCCAGGACGGGGTTCAAGTCCCTGCGGTGTCTTTGCTT TTD, or MALAT1 triple cleavage helix comp.6 sequence +mascRNA

[0326] truncated AAAGGTTTTTCTTTTCCTGAGAAAACAACCTTTTGTTTTCTCAGGT 6 Engineered 3’ version of TTTGCTTTTTGGCCTTTCCCTAGCTTTAAAAAGCAAAAGACGCTGG terminator, mouse TGGCTGGCACTCCTGGTTTCCAGGACGGGGTTCAAGTCCCTGCGGT TTD, or MALAT1 triple GTCTTTGCTT cleavage helix comp.7 sequence

[0327]

[0328] +mascRNA truncated AAAGGTTTTTCTTTTCCTGAGAAAACAATTGTTTTCTCAGGTTTTG 7 Engineered 3’ version of CT T T T Tterminator, mouse GGTTTCCAGGACGGGGTTCAAGTCCCTGCGGTGTCTTTGCTT TTD, or MALAT1 triple cleavage helix comp.9 sequence +mascRNA

[0329] truncated AAAGGTTTTTCTTTTCCTGAGAAAACGTTTTCTCAGGTTTTGCTTT 8 Engineered 3’ version of T T AAAAAAAAAAAAGC AAAAGAC GC T GGT GGC T GGC AC T C C T GGT T terminator, mouse TCCAGGACGGGGTTCAAGTCCCTGCGGTGTCTTTGCTT TTD, or MALAT1 triple cleavage helix comp.10 sequence +mascRNA

[0330] truncated AAAGGTTTTTCTTTTCCTGAGAAAACAACCTTTTGTTTTCTCAGGT 9 Engineered 3’ version of TTTGCTTTTTTAGCTT TAAAAAGC AAAAGAC GCTGGTGGCTGGCAC terminator, mouse TCCTGGTTTCCAGGACGGGGTTCAAGTCCCTGCGGTGTCTTTGCTT TTD, or MALAT1 triple cleavage helix comp.11 sequence +mascRNA

[0331] truncated AAAGGTTTTTCTTTTCCTGAGAAATTTCTCAGGTTTTGCTTTTTAA 10 Engineered 3’ version of AAAAAAAAAAGC AAAAGAC GC T GGT GGC T GGC AC T C C T GGT T T C C A terminator, mouse GGACGGGGTTCAAGTCCCTGCGGTGTCTTTGCTT TTD, or MALAT1 triple cleavage helix comp.12 sequence +mascRNA

[0332] truncated AAAGGTTTTTCTTTTCCTGAGAAATTTCTCAGGTTTTGCTTTTTAA 11 Engineered 3’ version of AAAAAAAGCAAAAGACGCTGGTGGCTGGCACTCCTGGTTTCCAGGA terminator, mouse CGGGGTTCAAGTCCCTGCGGTGTCTTTGCTT TTD, or MALAT1 triple cleavage helix comp.14 sequence +mascRNA

[0333] Mut U2.5 GATTCGTCAGTAGGGTTGTAAAGGTTTTTCTTTTCCTGAGAAAACA 12 Engineered 3’ +mascRNA ACCTTTTGTTTTCTCAGGTTTTGCTTTAAGGCCTTTCCCTAGCTTT terminator, AAAAAAAAAAAAGC AAAAGAC GC T GGT GGC T GGC AC T C C T GGT T T C TTD, or CAGGACGGGGTTCAAGTCCCTGCGGTGTCTTTGCTT cleavage sequence Human CGGAGCCGCCGCAGGTGTTTCTTTTACTGAGTGCAGCCCATGGCCG 13 Engineered 3’ NEAT1 / MEN CACTCAGGTTTTGCTTTTCACCTTCCCATCTGTGAAAGAGTGAGCA terminator, p triple helix + GGAAAAAGCAAAAGGCGCTGGTGGTGGCACGTCCAGCACGGCTGGG TTD, or mascRNA CCGGGGTTCGAGTCCCCGCAGTGTT cleavage sequence Mouse MEN p GGCACGGAGCCGCCGCAGGTGTTTCTTTTCCTGACCGCGGCTCATG 14 Engineered 3’ triple helix + GCCGCGCTCAGGTTTTGCTTTTCACCTTTGTCTGAGAGAACGAACG terminator, mascRNA T GAGC AGGAAAAAGC AAAAGGC AC TGGTGGCGGCACGCCCGCACCT TTD, or CGGGCCAGGGTTCGAGTCCCTGCAGTACCGTGCTTC cleavage sequence Human AAAAAGCAAAAGATGCTGGTGGTTGGCACTCCTGGTTTCCAGGACG 15 Engineered 3’ MALAT1 GGGTTCAAATCCCTGCGGCGTCTTTGCTT terminator, mascRNA TTD, or cleavage sequence Mouse AAAAAGC AAAAGAC GCTGGTGGCTGGCACTCCTGGTTTC C AGGAC G 16 Engineered 3’ MALAT1 GGGTTCAAGTCCCTGCGGTGTCT terminator, mascRNA TTD, or cleavage sequence Human MEN AAAAAGCAAAAGGCGCTGGTGGTGGCACGTCCAGCACGGCTGGGCC 17 Engineered 3’ beta GGGGTTCGAGTCCCCGCAGTGTT terminator, mascRNA TTD, or

[0334] cleavage

[0335]

[0336] sequence Mouse MEN AAAAAGC AAAAGGC AC TGGTGGCGGCACGCCCGCACCTCGGGCCAG 18 Engineered 3’ beta GGTTCGAGTCCCTGCAGTACCGTGCTTC terminator, mascRNA TTD, or cleavage sequence Human Malatl AAGCTGATCTCCAATGCTCTTCAGTAGGGTCATGAAGGTTTTTCTT 19 Engineered Triple helix TTCCTGAGAAAACAACACGTATTGTTTTCTCAGGTTTTGCTTTTTG stabilizing GCCTTTTTCTAGCT T AAAAAAAAAAAAAGC AAAA sequence or secondary structure sequence mouse Malatl GATTCGTCAGTAGGGTTGTAAAGGTTTTTCTTTTCCTGAGAAAACA 20 Engineered Triple helix ACCTTTTGTTTTCTCAGGTTTTGCTTTTTGGCCTTTCCCTAGCTTT stabilizing sequence or secondary structure sequence truncated AAAGGTTTTTCTTTTCCTGAGAAATTTCTCAGGTTTTGCTTTTTAA 21 Engineered version of AAAAAAAGCAAAA stabilizing mouse sequence or MALAT1 triple secondary helix com14 structure sequence truncated AAAGGTTTTTCTTTTCCTGATCAGGTTTTGCTTTTTGGCCTTTCCC 22 Engineered version of T AGC T T T AAAAAAAAAAAAGC AAAA stabilizing mouse sequence or MALAT1 triple secondary helix comp.1 structure sequence truncated AAAGGTTTTTCTTTTCCTGAGAAAACAACCTTTTGTTTTCTCAGGT 23 Engineered version of TTTGCTTTT T AAAAAAAAAAAAGC AAAA stabilizing mouse sequence or MALAT1 triple secondary helix comp.6 structure sequence truncated AAAGGTTTTTCTTTTCCTGAGAAAACAACCTTTTGTTTTCTCAGGT 24 Engineered version of TTTGCTTTTTGGCCTTTCCCTAGCTTTAAAAAGCAAAA stabilizing mouse sequence or MALAT1 triple secondary helix comp.7 structure sequence truncated AAAGGTTTTTCTTTTCCTGAGAAAACAATTGTTTTCTCAGGTTTTG 25 Engineered version of C T T T T T AAAAAAAAAAAAGC AAAA stabilizing mouse sequence or MALAT1 triple secondary helix comp.9 structure sequence truncated AAAGGTTTTTCTTTTCCTGAGAAAACGTTTTCTCAGGTTTTGCTTT 26 Engineered version of T T AAAAAAAAAAAAGC AAAA stabilizing mouse sequence or MALAT1 triple secondary helix comp.10 structure sequence truncated AAAGGTTTTTCTTTTCCTGAGAAAACAACCTTTTGTTTTCTCAGGT 27 Engineered version of T T T GC T T T T T T AGC T T T AAAAAGC AAAA stabilizing mouse sequence or MALAT1 triple secondary helix comp.11 structure sequence truncated AAAGGTTTTTCTTTTCCTGAGAAATTTCTCAGGTTTTGCTTTTTAA 28 Engineered

[0337]

[0338] version of AAAAAAAAAAGCAAAA stabilizing mouse sequence or MALAT1 triple secondary helix comp.12 structure sequence truncated AAAGGTTTTTCTTTTCCTGAGAAATTTCTCAGGTTTTGCTTTTTAA 29 Engineered version of AAAAAAAGCAAAA stabilizing mouse sequence or MALAT1 triple secondary helix comp.14 structure sequence Mut U2.5 triple GATTCGTCAGTAGGGTTGTAAAGGTTTTTCTTTTCCTGAGAAAACA 30 Engineered helix ACCTTTTGTTTTCTCAGGTTTTGCTTTAAGGCCTTTCCCTAGCTTT stabilizing sequence or secondary structure sequence Human CGGAGCCGCCGCAGGTGTTTCTTTTACTGAGTGCAGCCCATGGCCG 31 Engineered NEAT1 / MEN CACTCAGGTTTTGCTTTTCACCTTCCCATCTGTGAAAGAGTGAGCA stabilizing p triple helix GGAAAAAGCAAAA sequence or secondary structure sequence Mouse MEN p GGCACGGAGCCGCCGCAGGTGTTTCTTTTCCTGACCGCGGCTCATG 32 Engineered triple helix GCCGCGCTCAGGTTTTGCTTTTCACCTTTGTCTGAGAGAACGAACG stabilizing T GAGC AGGAAAAAGC AAAA sequence or secondary structure sequence KSHV PAN GGCTGGGTTTTTCCTTCGAAAGAAGGTTTTTATCCCAGTGTATAAA 33 Engineered core Triple AAAAAAAAAAAA stabilizing Helix sequence or secondary structure sequence KSHVGCPAN GGGTTTTTCCTTCGAAAGAAGGTTTTTATCCCTGCCTTCGGGCAAA 34 Engineered triple helix AAAAAA stabilizing sequence or secondary structure sequence KSHV PAN TGTTTTGGCTGGGTTTTTCCTTGTTCGCACCGGACACCTCCAGTGA 35 Engineered domain triple C C AGAC GGC AAGGT T T T T AT C C C AGT GT AT AT T AAAAAAAAAAAAA stabilizing helix AA sequence or secondary structure sequence RRV PAN CGTTTGTGTTGGTTTTTATGACCAGCTTGGTACAAAACCTGCTGGT 36 Engineered triple helix GATTTTTTACC C AAC AAAT AAT AAAT AAAA stabilizing sequence or secondary structure sequence EHV2 triple CTCAAAGAATATTTTTAAAGACTTTTTTCCCCAACCTCTGGGTTGG 37 Engineered helix GT T T T T T T T C T T T AAAAT AT T CAAT AAAA stabilizing sequence or secondary structure sequence TYMV CCUAAGUUCUCGAUCUUUAAAAUCGUUAGCUCGCCAGUUAGCGAGU 38 Engineered pseudoknot CUGUCCCCACACGACAGAUAAUCGGGUGCAACUCCCGCCCCUUUUC stabilizing

[0339]

[0340] CGAGGGUCAUCGGAACCA sequence or secondary structure sequence Human GCCUUUUUUCCCCAGACUUGUUGGCGUAGGCUACAGAGAAGCCUUC 39 Engineered MAT2A 3’UTR AAGCUCUGAGGGAAAGGGC stabilizing stem-loop sequence or structure secondary Hairpin A structure sequence Human ACAGGCACUUGGCAGCCUUGUGAUGUCAUACAGAGAAGUCACAGGG 40 Engineered MAT2A 3’UTR CAGUACCUGAGGGUCUGU stabilizing stem-loop sequence or structure secondary Hairpin B structure sequence Human GCUUUCUGAACAGCUGGUGUAGCUACAGAGAAACCAGCUUCCUUCA 41 Engineered MAT2A 3’UTR GAGAGC stabilizing stem-loop sequence or structure secondary Hairpin C structure sequence Human UCUGGGGUAUGGCGUAAGUACAGAGAAGCCAUCACCUCAGA 42 Engineered MAT2A 3’UTR stabilizing stem-loop sequence or structure secondary Hairpin D structure sequence Human CCAGCAUUCCCAGGUAGGCCAAGGUGUCCUACAGAAAAACCUUGGG 43 Engineered MAT2A 3’UTR UUAGACCUACAGGGGGUCUGG stabilizing stem-loop sequence or structure secondary Hairpin E structure sequence Human CAUGGAGAAAGCUGACUUGGCUGGUGUGGUACAGAGAAGCCAGCUU 44 Engineered MAT2A 3’UTR GUUUACAUGCUUAUUCCAUG stabilizing stem-loop sequence or structure secondary Hairpin F structure sequence IR1 TTATTGAATTTAAATTTTCCATACTTTATGGCATCAAGCCATAAAG 45 Stem loop for (intergenic TATGGAGTAAAAAAACATATGAGCAGATTTTACTCTGCCATAAAAA stability and region processing sequence of TTCAAAGAATAAGGAAGGTGTAAA

[0341] cipC-cel48F)

[0342] IR2 TATTTAATACTATGACGCATATGTAACCTTAAAGTCCGGACAGTAT 46 Stem loop for (intergenic TTGGTTTGATTAAATTACTCATTCTTGTACTGTCCGGGCTTATGAG stability and region T T ACAAAGAAAAAAAGAAAAGGAT TAAGGTAAGAAC processing sequence of

[0343] cel48F-cel8C)

[0344] IR3 AATAAATAAAAATAATTGAGTGAGCATCTCAGGTTAAATTTGTCTT 47 Stem loop for (intergenic AAAAAT GT T T AAAT T T AAT T T T AGGGAGT GAT GGC AAG stability and region processing sequence of

[0345] el8C-cel9G)

[0346] IR4 TAGAAGTTCAGTTTGGAAGTTTAATGAGTTTTTAATGCTTGCATTA 48 Stem loop for (intergenic C T AAAT GT AAGC T T T AAAAAAT AAAAAAT T T T AC T AGGAGGT AAAT stability and region processing sequence of

[0347]

[0348] cel9G-cel9E) IR5 TTTTGAAGTTTTATTTGGAACAATAGGAATGCAACTAGTGCATTCC 49 Stem loop for (intergenic TATTGTTCCTATCCTATTAGTTTCAGATTTATGATTTGAACGAATT stability and region processing sequence of TGTTATTTCCTTTAAT T AAAAT GT T TATAGTAATTTTTTAGTAATA

[0349] cel9E-orfX) AAAT AT AT T GAAT AAAT AAGGAGAAAAGC C

[0350] IR6 GAAC T C AAGAAGAT TAATCCTGTATT C AC AGAGGGT GAGAT AAC AG 50 Stem loop for (intergenic TTAAATAACTGATTCATGCATGGAGTGTGTAAAT stability and region processing sequence of

[0351] orfX-cel9H)

[0352] IR7 CATACCTTTTAACATATCAATATCAATGAAATATACGTAAATCTCT 51 Stem loop for (intergenic GTTTATTGCCCGGCAGACACAAATGGCATAAAGATTGTATTTTGCC stability and region processing sequence of AGTGTATATGCCGGGTAATGACAATACTTAAATTATAAAAATTATT

[0353] cel9H-cel9J) ATTCCAAAGTAGTTGATCAATATATAAAAAATAAAAAGGGGGTAAT

[0354] ATT IRS TGCTCTTGACCTTGCTTTATTAAAGAAGACTCTACTTGGTTAATAC 52 Stem loop for (intergenic AGCCTGTTAATTCGAAAAGTAAGGAGGATTTGCT stability and region processing sequence of

[0355] cel9J -Man5K)

[0356] IR9 ATAATTTATTACAGTGTATTTGACCTGTCCGTTAAAAACGGTTCTT 53 Stem loop for (intergenic T AAGT C GGAC AGAAAT T T T T AAAT AAAAAAGGGAGGGAT AGT T stability and region processing sequence of

[0357] Man5K-cel9M)

[0358] IR10 TATCAATGACTTCCCGGTGGAAGATGATCTTGGGGCGGCTGATGTC 54 Stem loop for (intergenic AACGGAGGTAAGAGTATTGATGCGTTGGACTATGCAGCAGTAAAGA stability and region GCTATTTGCTGCTGCTAATAGCTGAGTTCCCCGGAAGGTAATTTCA processing sequence of GGC AGT T T T AGTAAAAGGAGGGAAAAGT

[0359] cel9Mrgl11Y)

[0360] IR11 CACTAGACTTGGCATTACTAAAAGCAAGTTTGCTTTCGTAGTGTTT 55 Stem loop for (intergenic AGGAAAGGT T AT T AAAC AAAAAGGAGAAGAAC C T stability and region processing sequence of

[0361] rgl11Y-cel5N)

[0362] V. GGCTTCATATAATCCTAATGATATGGTTTGGGAGTTTCTACCAAGA 56 Riboswitch Vulnificus add GCCTTAAACTCTTGATTATGAAGT

[0363] B. GGATCATATAATCGCGTGGATATGGCACGCAAGTTTCTACCGGGCA 57 Riboswitch Subtilis xpt CCGTAAATGTCCGACTATGGTC

[0364] V. GGAAAAATGTCACGCACAGGGCAAACCATTCGAAAGAGTGGGACGC 58 Riboswitch Cholera Vc2 AAAGC C T C C GGC C T AAAC C AGAAGAC AT GGT AGGT AGC GGGGT T AC

[0365] CGATGG

[0366] T. T T AAAAT C T C T T AT C AAGAGAGGT GGAGGGAC TGGCCCGAT GAAAC 59 Riboswitch tengcongensis CCGGCAACCAGCCTTAGGGCATGGTGCCAATTCCTGCAGCGGTTTC

[0367] SAM GCTGAAAGATGAGAGATTCTTGTAGTCTCTTCTTTTAGCG

[0368] T. CCGGCTTTAAGTTGACGAGGGCAGGGTTTATCGAGACATCGGCGGG 60 Riboswitch tengcongensis TGCCCTGCGGTCTTCCTGCGACCGTTAGAGGACTGGTAAAACCACA

[0369] glmS

[0370] GGCGACTGTGGCATAGAGCAGTCCGGGCAGGAA

[0371] U4 Sm site AAUUUUUGA 61 Natural Sm-

[0372]

[0373] binding sites Human U1 AAUUUGUGG 62 Natural Sm-Sm site binding sites Human U1 AAUUUUUGGUAG 63 Natural Sm-Pseudogene binding sites Sm site

[0374] Human U1 AAUUUGUGGUAG 64 Natural Sm-Pseudogene binding sites Sm site

[0375] Mouse U7 sm AAUUUGUCUAG 65 Natural Sm-site binding sites Mouse AAUUUUUGGAG 66 Engineered smOPT site Sm-binding sites Mouse AAUUUUUGGAGCA 67 Engineered smOPT site 2 Sm-binding sites HDV UGGGCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACA 68 Engineered 3’ Ribozyme UUCCGAGGGGACCGUCCCCUCGGUAAUGGCGAAUGGGAC terminator, TTD, or cleavage sequence HDV GGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAU 69 Engineered 3’ Ribozyme GCUUCGGCAUGGCGAAUGGGAC terminator, short TTD, or cleavage sequence CPEB3 AGGGGGCCACAGCAGAAGCGUUCACGUCGCAGCCCCUGUCAGAUUC 70 Engineered 3’ Ribozyme UGGUGAAUCUGCGAAUUCUGCUG terminator, TTD, or cleavage sequence Hammer Head CUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUC 71 Engineered 3’ Ribozyme terminator, TTD, or cleavage sequence 5’ transcription C T C T GGC T AAC T AGAGAAC 125

[0376] start site

[0377] stabilizer

[0378] 2x proximal TAACAACATAGGAGCTGTGATTGGCTGTTTTCAGCCAATCAGCACT 126

[0379] sequence GAC T C AT T T GC AT AGC C T T T AC AAGC GGT C AC AAAC T C AAGAAAC G

[0380] element

[0381] AGCGGTTTTAATAGTCTTTTAGAATATTGTTTATCGAACCGAATAA GGAACTGTGCTTTGTGATTCACATATCAGTGGAGGGGTGTGGAAAT GGCACCTTGATCTCACCCTCATCGAAAGTGGCTCACCCTCATCGAA AGTGGAGTTGATGTCCTTCCCTGGCTCGCTACAGACGCACTTCCGC AA

[0382] 3x distal TAACAACATAGGAGCTGTGATTGGCTGTTTTCAGCCAATCAGCACT 127

[0383] sequence GACTCATTTGCATATCATTTGCATATCATTTGCATAGCCTTTACAA

[0384] element

[0385] GCGGTCACAAACTCAAGAAACGAGCGGTTTTAATAGTCTTTTAGAA TATTGTTTATCGAACCGAATAAGGAACTGTGCTTTGTGATTCACAT ATCAGTGGAGGGGTGTGGAAATGGCACCTTGATCTCACCCTCATCG AAAGTGGAGTTGATGTCCTTCCCTGGCTCGCTACAGACGCACTTCC GCAA

[0386]

[0387] 3’ Sl4 U1 GTAGTGGGGACTGCGTTCGCGCTTTCCCCTG 128

[0388] stem-loop

[0389] 2x smOPT AAT T T T T GGAGC AAAT T T T T GGAGC A 129

[0390] 2x truncated CCCAATTTCACTGGTCTACAATGAAAGCAAAACAGTTCTCTTCCCC 130

[0391] U7 terminator GCTCCCCGGTGTGTCCCAATTTCACTGGTCTACAATGAAAGCAAAA

[0392] CAGTTCTCTTCCCCGCTCCCCGGTGTGT

[0393]

[0394] “Homology” or “identity” or “similarity” can refer to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which can be aligned for purposes of comparison. When a position in the compared sequence can be occupied by the same base or amino acid, then the molecules can be homologous at that position. A degree of homology between sequences can be a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the disclosure. Sequence homology can refer to a % identity of a sequence to a reference sequence. As a practical matter, whether any particular sequence can be at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to any sequence described herein (which can correspond with a particular nucleic acid sequence described herein), such particular polypeptide or polynucleotide sequence can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for embodiment, 95% identical to a reference sequence, the parameters can be set such that the percentage of identity can be calculated over the full length of the reference sequence and that gaps in sequence homology of up to 5% of the total reference sequence can be facilitate.

[0395] In some cases, the identity between a reference sequence (query sequence, i.e., a sequence of the disclosure) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In some embodiments, parameters for a particular embodiment in which identity can be narrowly construed, used in a FASTDB nucleotide alignment, can include: Scoring Scheme=PAM (Percent Accepted Mutations) 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=O, Cutoff Score=1 , Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject sequence, whichever can be shorter. According to this embodiment, if the subject sequence can be shorter than the query sequence due to 5’ or 3’terminal deletions, not because of internal deletions, a manual correction can be made to the results to take into consideration the fact that the FASTDB program does not account for 5’ and 3’terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the 5’ and 3’termini, relative to the query sequence, the percent identity can be corrected by calculating the number of residues of the query sequence that can be lateral to the 5’ and 3’terminal of the subject sequence, which can be not matched or aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. A determination of whether a residue can be matched or aligned can be determined by results of the FASTDB sequence alignment. This percentage can be then subtracted from the percent identity, calculated by the FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score can be used for the purposes of this embodiment. In some cases, only residues to the 5’ and 3’termini of the subject sequence, which can be not matched / aligned with the query sequence, can be considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest 5’ and 3’terminal residues of the subject sequence can be considered for this manual correction. For example, a 90-residue subject sequence can be aligned with a 100-residue query sequence to determine percent identity. The deletion occurs at the 5’terminus of the subject sequence, and therefore, the FASTDB alignment does not show a matching / alignment of the first 10 residues at the 5’terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the 5’ and 3’termini not matched / total number of residues in the query sequence) so 10% can be subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched, the final percent identity can be 90%. In another example, a 90-residue subject sequence can be compared with a 100-residue query sequence. This time the deletions can be internal deletions, so there can be no residues at the 5’ or 3’termini of the subject sequence which can be not matched / aligned with the query. In this case, the percent identity calculated by FASTDB can be not manually corrected. Once again, only residue positions outside the 5’ and 3’terminal ends of the subject sequence, as displayed in the FASTDB alignment, which can be not matched / aligned with the query sequence can be manually corrected for. “Complementary sequence” can refer to two sequences that can hybridize to each other via canonical base-pairing.

[0396] In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or more sequence identity to any one of SEQ ID NOs: 1-18, 68-71, and 130. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, at least about 99.5% sequence identity to any one of SEQ ID NOs: 1-18 68-71, and 130. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having 100% sequence identity to any one of SEQ ID NOs: 1-18, 68-71, and 130. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or more sequence identity to SEQ ID NO: 1. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, at least about 99.5% sequence identity to SEQ ID NO: 1. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having 100% sequence identity to SEQ ID NO: 1. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or more sequence identity to SEQ ID NO: 2. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, at least about 99.5% sequence identity to SEQ ID NO: 2. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having 100% sequence identity to SEQ ID NO: 2. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or more sequence identity to SEQ ID NO: 3. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, at least about 99.5% sequence identity to SEQ ID NO: 3. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having 100% sequence identity to SEQ ID NO: 3. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or more sequence identity to SEQ ID NO: 4. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, at least about 99.5% sequence identity to SEQ ID NO: 4. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having 100% sequence identity to SEQ ID NO: 4. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or more sequence identity to SEQ ID NO: 5. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, at least about 99.5% sequence identity to SEQ ID NO: 5. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having 100% sequence identity to SEQ ID NO: 5. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or more sequence identity to SEQ ID NO: 6. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, at least about 99.5% sequence identity to SEQ ID NO: 6. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having 100% sequence identity to SEQ ID NO: 6. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or more sequence identity to SEQ ID NO: 7. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, at least about 99.5% sequence identity to SEQ ID NO: 7. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having 100% sequence identity to SEQ ID NO: 7. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or more sequence identity to SEQ ID NO: 8. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, at least about 99.5% sequence identity to SEQ ID NO: 8. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having 100% sequence identity to SEQ ID NO: 8. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or more sequence identity to SEQ ID NO: 9. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, at least about 99.5% sequence identity to SEQ ID NO: 9. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having 100% sequence identity to SEQ ID NO: 9. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or more sequence identity to SEQ ID NO: 10. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, at least about 99.5% sequence identity to SEQ ID NO: 10. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having 100% sequence identity to SEQ ID NO: 10. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or more sequence identity to SEQ ID NO: 11. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, at least about 99.5% sequence identity to SEQ ID NO: 11. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having 100% sequence identity to SEQ ID NO: 11. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or more sequence identity to SEQ ID NO: 12. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, at least about 99.5% sequence identity to SEQ ID NO: 12. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having 100% sequence identity to SEQ ID NO: 12. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or more sequence identity to SEQ ID NO: 13. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, at least about 99.5% sequence identity to SEQ ID NO: 13. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having 100% sequence identity to SEQ ID NO: 13. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or more sequence identity to SEQ ID NO: 14. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, at least about 99.5% sequence identity to SEQ ID NO: 14. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having 100% sequence identity to SEQ ID NO: 14. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or more sequence identity to SEQ ID NO: 15. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, at least about 99.5% sequence identity to SEQ ID NO: 15. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having 100% sequence identity to SEQ ID NO: 15. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or more sequence identity to SEQ ID NO: 16. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, at least about 99.5% sequence identity to SEQ ID NO: 16. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having 100% sequence identity to SEQ ID NO: 16. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or more sequence identity to SEQ ID NO: 17. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, at least about 99.5% sequence identity to SEQ ID NO: 17. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having 100% sequence identity to SEQ ID NO: 17. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or more sequence identity to SEQ ID NO: 18. In some embodiments, the engineered 3’ terminator, TTD, or cleavage sequence may comprise a DNA sequence having at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 96%, at most about 97%, at most about 98%, at most about 99%, at least about 99.5% sequence identity to SEQ ID NO: 18. In some embodim...

Claims

CLAIMS1. A nucleic acid molecule comprising:i) one or more antisense sequences, an engineered Sm-binding site, and a 3’ engineered element;ii) a 5’ engineered element, one or more antisense sequences, and an engineered Sm- binding site; oriii) a 5’ engineered element, one or more antisense sequences, an engineered Sm- binding site, and a 3’ engineered element.

2. The nucleic acid molecule of claim 1 , wherein the engineered Sm-binding site is configured to recruit a splicing regulator to a ribonucleic acid (RNA) molecule encoded by the nucleic acid molecule.

3. The nucleic acid molecule of claim 2, wherein the splicing regulator comprises a Sm ring protein.

4. The nucleic acid molecule of any one of claims 1-3, wherein the engineered Sm-binding site comprises a U1 Sm-binding sequence, a U2 Sm-binding sequence, a U4 Sm-binding sequence, a U5 Sm-binding sequence, a sequence from a major spliceosomal U small nuclear ribonucleoprotein (SmOpt), or any combination thereof.

5. The nucleic acid molecule of any one of claims 1-4, wherein the engineered Sm-binding site comprises a Sm-binding site from a human, a murine, a plant, a virus, or any combination thereof.

6. The nucleic acid molecule of any one of claims 1-5, wherein the engineered Sm-binding site comprises any one of SEQ ID NOs: 61-67 and 129.

7. The nucleic acid molecule of any one of claims 1-6, wherein the 3’ engineered element comprises one or more of an engineered stabilizing sequence or a secondary structure sequence and a terminator.

8. The nucleic acid molecule of any one of claims 1-7, wherein the 3’ engineered element is configured to form at least one secondary structure.

9. The nucleic acid molecule of claim 7 or claim 8, wherein the terminator is configured to facilitate: (1) 3’ end processing of the artificial nucleic acid molecule and / or (2) stabilization of the nucleic acid molecule.

10. The nucleic acid molecule of any one of claims 1-9, wherein the 5’ engineered element comprises one or more of a methylation element, an engineered stabilizing sequence or a secondary structure, and a recruitment site.

11. The nucleic acid molecule of claim 10, wherein the recruitment site comprises a binding site for RNA binding proteins (RBPs) or a protein recruitment domain.

12. The nucleic acid molecule of claim 10, wherein the methylation element comprises a 5’ methyl G cap.

13. The nucleic acid molecule of claim 12, wherein the 5’ methyl G cap comprises 2,2,7 trimethyl G (m3G) cap.

14. The nucleic acid molecule of any one of claims 7-13, wherein the engineered stabilizing sequence or the secondary structure sequence comprises a stem-loop sequence, a triple helix sequence, a quadruplex sequence, a tRNA-like sequence, a pseudoknot sequence, or any combinations thereof.

15. The nucleic acid molecule of claim 14, wherein the stem-loop sequence comprises a small nucleolar RNA snoRNA (H / ACA and C / D box) derived stem-loop sequence, a viral snRNA derived stem-loop sequence, a plant snRNA derived stem-loop sequence, a non-coding or viral RNA derived triple helix sequence, a clustered regularly interspaced short palindromic repeats (CRISPR) single-guide (sg) RNA derived stem-loop sequence, an intergenic region forming a stem loop structure, a long non-coding RNA (LncRNA) derived stem-loop sequence, a PlWI-interacting RNA (piRNA) derived stem-loop sequence, a stem-bulge RNA (sbRNA) derived stem-loop sequence, a transfer RNA (tRNA) derived stem-loop sequence, or any combinations thereof.

16. The nucleic acid molecule of any one of claims 7-15, wherein the engineered stabilizing sequence or the secondary structure sequence comprises any one of SEQ ID NO: 19-60, 72-74, and 105-118.

17. The nucleic acid molecule of any one of claims 1 -16, wherein the one or more antisense sequences have a length of 15 to 50 nucleotides.

18. The nucleic acid molecule of any one of claims 1-17, wherein the one or more antisense sequences are complementary to a region of one or more target RNA molecules.

19. The nucleic acid molecule of claim 18, wherein the one or more antisense sequences are complementary to an intron of the one or more target RNA molecules, an exon of the one or more target RNA molecules, an intron-exon junction of the one or more target RNA molecules, an untranslated region (UTR) of one or more the target RNA molecules, or any combinations thereof.

20. The nucleic acid molecule of claim 19, wherein the UTR is a 5’ UTR and / or a 3’ UTR.

21. The nucleic acid molecule of any one of claims 1 -20, wherein the one or more antisense sequences comprise a steric-blocking antisense oligonucleotide, a small interfering RNA, a splice-switching oligonucleotide, or an RNA binding protein interacting decoy sequence.

22. The nucleic acid molecule of any one of claims 1 -21 , wherein the engineered Sm-binding site is located 3’ to the one or more antisense sequences.

23. The nucleic acid of any one of claims 1-22, wherein the engineered Sm-binding site is located 5’ to the 3’ engineered element.

24. The nucleic acid molecule of any one of claims 1-23, wherein the engineered Sm-binding site is located 3’ to the 5’ engineered element.

25. The nucleic acid molecule of any one of claims 1-24, wherein the nucleic acid is less than 500, less than 400, less than 300, less than 250, less than 200, less than 150 nucleotides, or less than 100 nucleotides in length.

26. The nucleic acid molecule of any one of claims 1-25, wherein the nucleic acid molecule does not comprise a native U7 stem-loop sequence.

27. The nucleic acid molecule of any one of claims 1-26, wherein the nucleic acid molecule does not comprise a native U7 Sm-binding sequence.

28. The nucleic acid molecule of any one of claims 1-27, wherein the nucleic acid molecule is RNA.

29. The nucleic acid molecule of claim 28, wherein the nucleic acid RNA molecule comprises one or more synthetic or modified nucleobases or nucleotides.

30. The nucleic acid molecule of claim 29, wherein the synthetic or modified nucleobase comprises hypoxanthine, xanthine, 7-methylguanine, 5, 6- dihydrouracil, 5-methylcytosine, or 5-hydroxymethoyl cytosine.

31. The nucleic acid molecule of claim 29, wherein the synthetic or modified nucleotide comprises a 2’-O-methyl (2’-OMe) modification, a 2'-0-methoxyethyl (2'-O-MOE) modification, or a 2'-fluoro (2’-F) modification.

32. An expression cassette encoding a nucleic acid molecule comprising:i) an engineered promoter, one or more antisense sequences, an engineered Sm-binding site, and a 3’ engineered terminator; orii) an engineered promoter, a 5’ engineered element, one or more antisense sequences, an engineered Sm-binding site, and a 3’ engineered terminator.

33. The expression cassette of claim 32, wherein the 3’ engineered terminator comprises an engineered stabilizing sequence or a secondary structure sequence, an insulator, an engineered transcription terminator domain (TTD), an engineered cleavage / processing site, or any combinations thereof.

34. The expression cassette of claim 32 or claim 33, wherein the 3’ engineered terminator is from a LncRNA, an animal, a plant, a virus, a CRISPR sequence, a small nucleolar RNAs (snoRNA), a histone messenger RNA (mRNA), or any combinations thereof.

35. The expression cassette of any one of claims 32-34, wherein the 3’ engineered terminator comprises any one SEQ ID NOs: 1-18, 119-124, and 130.

36. The expression cassette of any one of claims 33-35, wherein the engineered stabilizing sequence or the secondary structure sequence comprises a stem-loop sequence, a triple helix sequence, a quadruplex sequence, a tRNA-like sequence, a pseudoknot sequence, or any combinations thereof.

37. The expression cassette of claim 36, wherein the stem-loop sequence comprises a small nucleolar RNA snoRNA (H / ACA and C / D box) derived stem-loop sequence, a viral snRNA derived stem-loop sequence, a plant snRNA derived stem-loop sequence, a non-coding or viral RNA derived triple helix sequence, a CRISPR sg RNA derived stem-loop sequence, an intergenic region forming a stem loop structure, a LncRNA derived stem-loop sequence, a piRNA derived stem-loop sequence, a sbRNA derived stem-loop sequence, a tRNA derived stem-loop sequence, or any combinations thereof.

38. The expression cassette of any one of claims 33-37, wherein the engineered stabilizing sequence or the secondary structure sequence comprises any one of SEQ ID NO: 19-60, 72-74, 105-118, and 125.

39. The expression cassette of any one of claims 33-38, wherein the engineered cleavage / processing site comprises a RNAse P recognition sequence, a RNAse Z recognition sequence, a yeast U1 or U2 snRNA maturation sequence, a CCA-adding enzyme recognition sequence, a ribozyme sequence, or any combinations thereof.

40. The expression cassette of claim 39, wherein the ribozyme comprises: a hammerhead ribozyme, a twister ribozyme, a hepatitis delta virus (HDV) ribozyme, the mammalian CPEB3 ribozyme, a pistol ribozyme, or a combination thereof.

41. The expression cassette of claim 39 or claim 40, wherein the ribozyme sequence comprises any one of SEQ ID NO: 68-71.

42. The expression cassette of any one of claims 33-41 , wherein the TTD comprises one or more of distinct 3’ box domains, tRNA derived stem-loop sequence, MALAT1 -associated small cytoplasmic RNAs (mascRNAs), or other transcription termination domains.

43. The expression cassette of any one of claims 33-42, wherein the TTD comprises any one SEQ ID NOs: 1-18, 119-124, and 130.

44. The expression cassette of any one of claims 32-43, wherein the 3’ engineered terminator is configured to facilitate: (1) transcriptional termination of the nucleic acid molecule, (2) 3’ end processing of the nucleic acid molecule, and / or (3) stabilization the nucleic acid molecule.

45. The expression cassette of any one of claims 33-44, wherein the engineered stabilizing sequence is configured to form at least one secondary structure.

46. The expression cassette of any one of claims 32-45, wherein the 5’ engineered element comprises one or more of a methylation element, an engineered stabilizing sequence or a secondary structure, and a recruitment site.

47. The expression cassette of claim 46, wherein the recruitment site comprises a binding site for RNA binding proteins (RBPs) or protein recruitment domain.

48. The expression cassette of claim 46, wherein the methylation element comprises a 5’ methyl G cap.

49. The expression cassette of claim 48, wherein the 5’ methyl G cap comprises a m3G cap.

50. The expression cassette of any one of claims 46-49, wherein the engineered stabilizing sequence or the secondary structure sequence comprises a stem-loop sequence, a triple helix sequence, a quadruplex sequence, a tRNA-like sequence, a pseudoknot sequence, or any combinations thereof.

51. The expression cassette of claim 50, wherein the stem-loop sequence comprises a small nucleolar RNA snoRNA (H / ACA and C / D box) derived stem-loop sequence, a viral snRNA derived stem-loop sequence, a plant snRNA derived stem-loop sequence, a non-coding or viral RNA derived triple helix sequence, a CRISPR sg RNA derived stem-loop sequence, an intergenic region forming a stem loop structure, a LncRNA derived stem-loop sequence, a piRNA derived stem-loop sequence, a sbRNA derived stem-loop sequence, a tRNA derived stem-loop sequence, or any combinations thereof.

52. The expression cassette of any one of claims 46-51 , wherein the engineered stabilizing sequence or the secondary structure sequence comprises any one of SEQ ID NO: 19-60, 72-74, 105-118, and 125.

53. The expression cassette of any one of claims 32-52, wherein the engineered promoter comprises a U1 promoter, a U2 promoter, a U4 promoter, or a U7 promoter.

54. The expression cassette of any one of claims 32-52, wherein the engineered promoter comprises a tissue-specific promoter.

55. The expression cassette of claim 54, wherein the tissue-specific promoter comprises an eye-specific promoter, a brain-specific promoter, a muscle-specific promoter, or any combinations thereof.

56. The expression cassette of any one of claims 32-52, wherein the promoter comprises a constitutively active promoter.

57. The expression cassette of any one of claims 32-56, wherein the promoter comprises a CMV, RSV, SV40, CBA, CAG, truncated CAG, Cbh, EF-1a, EFS, PGK, UBC, GUSB, UCOE, hAAT, TBG, GRM6, 770En_454P, HSA, Desmin, SkCRM4 / Des, MCK, CK6, MHCK7, dMCK, tMCK, CK8, CK8e, C5-12, NSE, CMV-MyoD, SynM, Synapsin, aMHC, PDGF, MLC2v, cTnT, MecP2, CaMKII, mGluR2, NFL, NFH, np2, PPE, ENK, EAAT2, GFAP, Myo, AUSEx3, SPcA5-12, unc45b, SPc5-12, MBP, Cox-2, PCP2, CLDN5, NR2E1, PITX3, PDE6H, RHO, RHOK, CNGA1, CNGB1, PDE6B, GRK1, SAG, ARR3, S-opsin, L / M-opsin, mopsin-500, CNGA3, CNGB3, RPGR, RPGRIP1, IRBP, NHPH5, PRPH2, CRX, STK38L, RPE65, BEST1, PR1.7, PRO.5, 3LCR-PRO.5, PR2.1 , CAR, IRBP, Pou4f3, Ocp1 , Mathl , Prestin, Myo7a, Opto-mGluR6, H1 , 7SK, U1 , U2, U4, U5, U6, or U7 promoter.

58. The expression cassette of any one of claims 32-57, wherein the promoter comprises any one of SEQ ID NOS: 77-92 and 132-139.

59. The expression cassette of any one of claims 32-58, wherein the engineered promoter further comprises regulatory elements.

60. The expression cassette of claim 59, wherein the regulatory elements comprise enhancers or internal ribosomal entry sites (IRES).

61. The expression cassette of claim 60, wherein the enhancer comprises any one of SEQ ID NOS: 93-104.

62. The expression cassette of any one of claims 32-61 , wherein the engineered Sm-binding site is configured to recruit a splicing regulator to an RNA molecule encoded by the nucleic acid molecule.

63. The expression cassette of claim 62, wherein the splicing regulator comprises a Sm ring protein.

64. The expression cassette of any one of claims 32-63, wherein the engineered Sm-binding site comprises a U1 Sm-binding sequence, a U2 Sm-binding sequence, a U4 Sm-binding sequence, a U5 Sm-binding sequence, a sequence from SmOpt, or any combination thereof.

65. The expression cassette of any one of claims 32-64, wherein the engineered Sm-binding site comprises a Sm-binding site from a human, a murine, a plant, a virus, or any combination thereof.

66. The expression cassette of any one of claims 32-65, wherein the engineered Sm-binding site comprises any one of SEQ ID NOs: 61-67 and 129.

67. The expression cassette of any one of claims 32-66, wherein the one or more antisense sequences have a length of 15 to 50 nucleotides.

68. The expression cassette of any one of claims 32-67, wherein the one or more antisense sequences are complementary to a region of one or more target RNA molecules.

69. The expression cassette of any one of claims 32-68, wherein the one or more antisense sequences are complementary to an intron of the one or more target RNA molecules, an exon of the one or more target RNA molecules, an intron-exon junction of the one or more target RNA molecules, a UTR region of one or more the target RNA molecules, or any combinations thereof.

70. The expression cassette of claim 69, wherein the UTR is a 5’ UTR and / or a 3’ UTR.

71. The expression cassette of any one of claims 32-70, wherein the one or more antisense sequences comprise a steric-blocking antisense oligonucleotide, a small interfering RNA, a splice-switching oligonucleotide, or an RNA binding protein interacting decoy sequence.

72. The expression cassette of any one of claims 32-71 , wherein the engineered Sm-binding site is located 3’ to the one or more antisense sequences.

73. The expression cassette of any one of claims 32-72, wherein the engineered Sm-binding site is located 5’ to the 3’ engineered element.

74. The expression cassette of any one of claims 32-73, wherein the engineered Sm-binding site is located 3’ to the 5’ engineered element.

75. The expression cassette of any one of claims 32-74, wherein the expression cassette is less than 5000, less than 4000, less than 3000, less than 2500, less than 2000, less than 1500 nucleotides, or less than 1000 nucleotides in length.

76. The expression cassette of any one of claims 32-75, wherein the nucleic acid is less than 500, less than 400, less than 300, less than 250, less than 200, less than 150 nucleotides, or less than 100 nucleotides in length.

77. The expression cassette of any one of claims 32-76, wherein the nucleic acid molecule does not comprise a native U7 stem-loop sequence.

78. The expression cassette of any one of claims 32-77, wherein the nucleic acid molecule does not comprise a native U7 Sm-binding sequence.

79. The expression cassette of any one of claims 32-78, wherein the encoded nucleic acid molecule is RNA.

80. A vector comprising (1 ) the artificial nucleic acid of any one of claims 1-31 or (2) the expression cassette of any one of claims 32-79.81 . The vector of claim 80, wherein the vector is a viral vector.

82. A method of targeting a splicing-regulatory element or a protein binding site of a target nucleic acid sequence, the method comprising contacting the target nucleic acid sequence with (1) the nucleic acid molecule of any one of claims 1 -31 , (2) the expression cassette of any one of claims 32-79, or (3) the vector of any one of claims 80-81 .

83. A method of modulating expression of a protein, the method comprising contacting a target nucleic acid sequence encoding the protein with (1 ) the nucleic acid molecule of any one of claims 1 -31 , (2) or a nucleic acid molecule transcribed from the expression cassette of any one of claims 32-79, (3) or a nucleic acid molecule transcribed from the vector of any one of claims 80-81 .

84. A method of modulating levels of a protein, the method comprising contacting a target nucleic acid sequence encoding the protein with (1 ) the nucleic acid molecule of any one of claims 1 -31 , (2) or a nucleic acid molecule transcribed from the expression cassette of any one of claims 32-79, (3) or a nucleic acid molecule transcribed from the vector of any one of claims 80-81 .

85. The method of claim 84, wherein the target nucleic acid sequence is a mature mRNA.

Images