WO2026006151A2 - Fonctionnalisation d'ace-arnt codant pour des picovecteurs linéaires synthétiques - Google Patents

Fonctionnalisation d'ace-arnt codant pour des picovecteurs linéaires synthétiques

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WO2026006151A2
WO2026006151A2 PCT/US2025/034736 US2025034736W WO2026006151A2 WO 2026006151 A2 WO2026006151 A2 WO 2026006151A2 US 2025034736 W US2025034736 W US 2025034736W WO 2026006151 A2 WO2026006151 A2 WO 2026006151A2
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trna
oligonucleotide
sense strand
oligonucleotide set
ace
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WO2026006151A3 (fr
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John LUECK
Joseph Porter
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University of Rochester
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University of Rochester
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
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    • C12N2310/3513—Protein; Peptide
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3519—Fusion with another nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/532—Closed or circular
    • C—CHEMISTRY; METALLURGY
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    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • This invention relates to vectors, such as synthetic linear picovectors, that encode anticodon-edited transfer RNAs (ACE-tRNAs), as well as to agents and methods for producing such vectors.
  • ACE-tRNAs anticodon-edited transfer RNAs
  • This invention also relates to ACE-tRNAs and related agents and methods for treating disorders associated with premature termination codons (PTCs).
  • the genetic code is comprised of nucleotide triplets, referred to as codons, each of which corresponds to a specific amino acid incorporated during the process of protein synthesis.
  • codons each of which corresponds to a specific amino acid incorporated during the process of protein synthesis.
  • 61 codons specify the insertion of an amino acid into a nascent polypeptide chain during translation by the ribosome.
  • the remaining three codons (TAA, TAG, and TGA) function as stop codons, signaling the termination of translation.
  • a point mutation particularly a single-nucleotide substitution that converts an amino acid-encoding codon into one of the three stop codons, is referred to as a nonsense mutation.
  • Such mutations introduce a premature termination codon (PTC) into the protein-coding sequence of a gene.
  • PTC premature termination codon
  • the presence of a PTC frequently results in the production of a truncated protein product, which is typically nonfunctional or exhibits significantly impaired biological activity.
  • the presence of a PTC also triggers nonsense-mediated mRNA decay (NMD), thereby reducing the overall level of the mutated transcript.
  • NMD nonsense-mediated mRNA decay
  • Nonsense mutations represent a substantial proportion of pathogenic genetic alterations, accounting for approximately 10% to 15% of all mutations associated with inherited diseases. These mutations have been implicated in nearly 1,000 severe genetic disorders, including but not limited to cystic fibrosis, Duchenne muscular dystrophy, and certain cancers.
  • compositions, compounds, and therapeutic methods capable of suppressing PTCs or otherwise restoring the expression and function of full-length proteins encoded by genes harboring nonsense mutations.
  • present disclosure addresses this need by providing agents and methods for the treatment or prevention of diseases or disorders associated with, or resulting from, nonsense mutations.
  • the disclosure provides an oligonucleotide set comprising: (a) a first hairpin oligonucleotide comprising from 5’ end to 3’ end: a first antisense strand, a first loop, and a first sense strand that is complementary to the first antisense strand; and (b) a second hairpin oligonucleotide comprising from 5’ end to 3’ end: a second sense strand, a second loop, and a second antisense strand that is complementary to the second sense strand.
  • the first sense strand and the second sense strand are adapted to be joined together to form a nucleic acid sequence encoding a RNA molecule.
  • the first loop or the second loop or another part of the oligonucleotide is directly or indirectly linked with a nuclear targeting moiety.
  • the first hairpin oligonucleotide and the second hairpin oligonucleotide form a closed- end DNA thread (CEDT) molecule, which is also called a picovector in some cases.
  • CEDT closed- end DNA thread
  • the first hairpin oligonucleotide comprises a nucleic acid sequence encoding a tRNA leader.
  • the second hairpin oligonucleotide comprises a nucleic acid sequence encoding a RNA polymerase III termination signal.
  • the oligonucleotide set further comprises a third sense strand and a third antisense strand having a sequence complementary to the third sense strand.
  • the first sense strand, the third sense strand, and the second sense strand are adapted to be joined together in order to form a second nucleic acid sequence encoding the RNA molecule.
  • the disclosure provides an oligonucleotide set comprising: (a) a first hairpin oligonucleotide comprising from 5’ end to 3’ end: a first antisense strand, a first loop, and a first sense strand that is complementary to the first antisense strand; (b) a second hairpin oligonucleotide comprising from 5’ end to 3’ end: a second sense strand, a second loop, and a second antisense strand that is complementary to the second sense strand; and (c) a third sense strand and a third antisense strand having a sequence complementary to the third sense strand.
  • the first sense strand, the third sense strand, and the second sense strand are adapted to be joined together in an order to form a second nucleic acid sequence encoding a RNA molecule.
  • the first loop or the second loop or another part of the oligonucleotide is directly or indirectly linked with a nuclear targeting moiety.
  • the first hairpin oligonucleotide, the third sense and antisense strands, and the second hairpin oligonucleotide form a CEDT molecule.
  • the third sense strand comprises a nucleic sequence encoding a tRNA leader. In some embodiments, the third sense strand comprises a nucleic sequence encoding a RNA polymerase III termination signal.
  • the RNA molecule comprises tRNA.
  • the tRNA comprises an anti-codon edited-tRNA (ACE-tRNA).
  • the ACE-tRNA causes a ribosome to read through one or more stop codons during translation.
  • the one or more stop codons comprise a premature termination codon (PTC). Examples of the PTC include PTCs that result in disease or PTCs that result in nonsense- associated diseases.
  • the PTC is present in a nucleic acid sequence encoding cystic fibrosis transmembrane conductance regulator (CFTR).
  • CFTR cystic fibrosis transmembrane conductance regulator
  • the tRNA is selected from the group consisting of Arg-tRNA- UGA, Gln-tRNA-UAA, Gln-tRNA-UAG, Trp-tRNA-UGA, Trp-tRNA-UAG, Glu-tRNA- UAA, Glu-tRNA-UAG, Cys-tRNA-UGA, Tyr-tRNA-UAG, Tyr-tRNA-UAA, Leu-tRNA- UGA, Leu-tRNA-UAG, Leu-tRNA-UAA, Lys-tRNA-UAG, Lys-tRNA-UGA, Ser-tRNA- UGA, Ser-tRNA-UAG, and Ser-tRNA-UAA.
  • the nucleic acid sequence has a size of from 200 nucleotides to 1,000 nucleotides.
  • the first loop or the second loop or another part of the oligonucleotide is linked with an agent such as the nuclear targeting moiety.
  • the agent or the nuclear targeting moiety comprises a labeling agent, a peptide, a bioactive agent, or a combination thereof.
  • the labeling agent comprises any one of N-hydroxysuccinimide (NHS), thiol-maleimide, and azidedibenzocyclooctyne (DBCO).
  • NHS N-hydroxysuccinimide
  • DBCO azidedibenzocyclooctyne
  • Such an agent can be linked to the oligonucleotide via any suitable methods known in the art, such as bioorthogonal chemistry and click chemistry.
  • Exemplary reactions may include native chemical ligation and the Staudinger ligation, copper- catalyzed azide-alkyne cycloaddition, strain-promoted [3 + 2] reactions, tetrazine ligation, metal-catalyzed coupling reactions, oxime and hydrazone ligations as well as photoinducible bioorthogonal reactions.
  • the first hairpin oligonucleotide or the second hairpin oligonucleotide comprises one or more chemically modified nucleotides.
  • the one or more chemically modified nucleotides comprise a 2’-O-methyl- modified sugar moiety.
  • the one or more chemically modified nucleotides comprise a modified intemucleoside linkage.
  • the nuclear targeting moiety comprises a peptide or a protein having a nuclear localization signal or sequence (NLS).
  • the peptide or protein is linked to the first loop or the second loop or another part of the oligonucleotide via a linker.
  • the protein is a transcription factor or a nuclear protein.
  • the linker is a small molecule compound, a polypeptide, or a oligonucleotide.
  • the nuclear targeting moiety comprises a nucleic acid having a DNA targeting sequence (DTS).
  • DTS DNA targeting sequence
  • the nuclear targeting moiety comprises a binding pair having (i) a first member that is linked to the first loop or the second loop or another part of the oligonucleotide, and (ii) a second member that contains to a NLS or a DTS or is linked to the NLS or DTS.
  • the first member and the second member are designed to bind to each other.
  • a composition comprising the oligonucleotide set described herein. In one embodiment, the composition is formulated as a nanoparticle formulation.
  • this disclosure also provides a kit comprising the oligonucleotide set described herein and, optionally, a ligase.
  • the ligase is a T4 DNA ligase.
  • this disclosure further provides a method for making a CEDT molecule.
  • the method comprises: providing an oligonucleotide set described herein; and ligating components of the oligonucleotide set, thereby obtaining the CEDT molecule.
  • the oligonucleotide set is synthesized chemically. In some embodiments, the oligonucleotide set is synthesized with chemically modified nucleotides.
  • the CEDT molecule is further linked to a labeling agent, a peptide, a bioactive agent, or a combination thereof.
  • this disclosure provides a CEDT molecule comprising the components described above.
  • a CEDT molecule can be made according to the method described herein.
  • this disclosure additionally provides a method of treating a disease associated with a PTC in a subject in need thereof.
  • the method comprises administering to the subject the CEDT molecule described herein or a pharmaceutical composition thereof.
  • the disease is selected from the group consisting of cystic fibrosis, Duchenne and Becker muscular dystrophies, retinoblastoma, neurofibromatosis, ataxia- telangiectasia, Tay-Sachs disease, Wilm’s tumor, hemophilia A, hemophilia B, Menkes disease, Ullrich’s disease, b-Thalassemia, type 2A and type 3 von Willebrand disease, Robinow syndrome, brachydactyly type B (shortening of digits and metacarpals), inherited susceptibility to mycobacterial infection, inherited retinal disease, inherited bleeding tendency, inherited blindness, congenital neurosensory deafness and colonic agangliosis and inherited
  • FIG. 1 shows a representative schematic of a process for ACE-tRNA targeting.
  • the diagram depicts key steps in the preparation of ACE-tRNA constructs for site-specific incorporation of materials with chemi cal/biologi cal functionalities.
  • FIG. 2A shows an alternate labeling strategy referred to as Alternate Labeling Method 1.
  • This method facilitates combinatorial screening of nuclear localization signal (NLS) sequences using a single ACE-tRNA picovector scaffold. Instead of synthesizing a large variety of NLS peptides, NLS sequences were appended to the ALFA tag nanobody through high-throughput cloning and expression in Escherichia coli. While strain-promoted azidealkyne cycloaddition (SPAAC) chemistry was previously used for labeling due to its biocompatibility in aqueous systems, it required extended reaction times exceeding 48 hours at the concentrations employed.
  • SPAAC strain-promoted azidealkyne cycloaddition
  • FIG. 2B shows ACE-tRNA LPV (linear picovector; equivalent to synthetic linear picovector, sLPV) constructs used in experiments involving ALFA-Nb-NLS fusions. These constructs were tested for their capacity to restore expression in systems bearing premature termination codons (PTCs).
  • PTCs premature termination codons
  • FIG. 3 shows ACE-tRNA LPV functionalized with nuclear localization signal peptides (NLS) through strain-promoted azide-alkyne cycloaddition (SPAAC) coupling chemistry, which were employed in functional rescue assays.
  • HEK293T cells harboring a stably integrated nanoluciferase reporter containing a PTC were cultured in black 96-well plates and treated with either vehicle control (0.1% DMSO) or aphidicolin (to a final concentration of 5 pg/mL, dissolved in DMSO), a compound known to arrest the cell cycle in early S-phase without disrupting transcriptional or translational machinery. This approach simulated the conditions of non-dividing cells encountered in vivo.
  • FIG. 4 shows that the experiment shown in FIG. 3 can be extended to multiple ACE- tRNAs encoded as sLPV (ArgUGA and OptLeuUGA) with NLS conjugates to enhance rescue in functional PTC suppression assays. Further, this is demonstrated in two cell models (HEK293T a.k.a. 293 T, and 16HBE14o- a.k.a. HBE) harboring a stably integrated nanoluciferase reporter containing a PTC. As demonstrated in FIG. 3, while constructs lacking NLS showed modest PTC suppression in proliferating cells (vehicle), significant enhancement was observed in arrested cells (aphidicolin) cells when NLS sequences were appended. Taken together, the results shown here extend the findings demonstrated in FIG. 3.
  • sLPV ArgUGA and OptLeuUGA
  • FIG. 5 shows results obtained from HeLa cells transfected with the ACE-tRNA LPV constructs described above, using Lipofectamine 2000 as the transfection reagent.
  • a custom buffer (20 mM HEPES, pH 7.2, 150 mM NaCl, and 10 mM EDTA), followed by sonication and heat treatment at 95 °C for 5 minutes. Lysates were then centrifuged at 20,000 x g for 5 minutes to remove debris. The resulting supernatant was analyzed using a 2’,3’-cyclic GMP-AMP (cGAMP) ELISA Kit (INVITROGEN) to quantify intracellular levels of cGAMP.
  • cGAMP 2’,3’-cyclic GMP-AMP
  • the cGAMP-activated cGAS-STING innate immunity pathway is triggered by the presence of DNA in the cytoplasm, providing a host defense against microbial pathogens. This pathway has impacts on autophagy, cellular senescence, and antitumor immunity, while overactivity results in autoimmune and inflammatory diseases. Given the intracellular route of nonviral delivery of DNA vectors, this pathway may be triggered spuriously, resulting in unintended cellular damage. Larger plasmid DNA results in the highest cGAMP levels demonstrated here, with the smaller LPVs displaying significantly lower amounts of cGAMP production.
  • FIG. 6 shows a second alternate labeling strategy (Alternate Labeling Method 2), wherein an all-DNA ACE-tRNA LPV construct is engineered to include a DNA targeting sequence (DTS).
  • DTS DNA targeting sequence
  • the DTS is recognized by transcription factors or other DNA-binding proteins that are actively transported into the nucleus.
  • FIG. 7 shows the ACE-tRNA LPV constructs utilized in conjunction with DTS elements, further demonstrating modular design flexibility for nuclear targeting applications.
  • FIG. 8 shows a third alternate labeling strategy (Alternate Labeling Method 3), which involves the site-specific incorporation of para-azido-phenylalanine (pAzF) into proteins via genetic code expansion in cell culture.
  • This method enables conjugation of full-length proteins, such as transcription factors, which inherently contain nuclear targeting domains that may not be fully functional when isolated as peptides.
  • the use of intact proteins preserves subnuclear localization properties that are critical for biological activity and specificity in nuclear compartments.
  • FIG. 9 shows a comparison of LPV functionalized with nuclear localization signal peptides (NLS) through strain-promoted azide-alkyne cycloaddition (SPAAC) coupling chemistry, to that of LPV functionalized with NbALFA-NLS fusion proteins.
  • NLS nuclear localization signal peptides
  • SPAAC strain-promoted azide-alkyne cycloaddition
  • FIG. 10 shows exemplary ACE-tRNA LPV (linear picovector) constructs utilized in experiments evaluating NLS functionality through through strain-promoted azide-alkyne cycloaddition (SPAAC) coupling chemistry to proteins containing para-azido-phenylalanine (pAzF). These constructs were designed to assess intracellular delivery and nuclear targeting efficiency.
  • SPAAC strain-promoted azide-alkyne cycloaddition
  • FIG. 11 shows a representative chemical synthesis scheme for the generation of DTS- labeled ACE-tRNA synthetic LPV (sLPV) wherein an all-DNA ACE-tRNA LPV construct is engineered to include a DNA targeting sequence (DTS).
  • DTS in this approach is composed of a covalently-closed-end linear DNA fragment, in contrast to the scheme presented in FIG. 6.
  • the covalently-closed-end DTS moiety increases ease of production and in vivo stability of the sLPV conjugate.
  • FIG. 12 shows a synthetic scheme for the preparation of sLPV constructs labeled with NbALFA-NLS. This conjugation enables targeted delivery of the ACE-tRNA sLPV construct to the nucleus via nanobody-mediated localization.
  • FIGS. 13A, 13B, and 13C shows that NbALFA-NLS-labeled sLPV constructs exhibit biological behavior comparable to those directly conjugated to synthetic NLS peptides.
  • FIG. 13A shows a Coomassie Blue-stained SDS-PAGE gel confirming expression and purification of the NbALFA-NLS fusion proteins.
  • FIG. 13B shows results from a gel mobility shift assay, indicating that the NbALFA-NLS effectively binds to and alters the electrophoretic mobility of ptLeu-sLPV-ALFA peptide.
  • FIG. 13A shows a Coomassie Blue-stained SDS-PAGE gel confirming expression and purification of the NbALFA-NLS fusion proteins.
  • FIG. 13B shows results from a gel mobility shift assay, indicating that the NbALFA-NLS effectively binds to and alters the electrophoretic mobility of ptLeu-sLPV-ALFA peptide.
  • 13C shows data demonstrating successful suppression of premature termination codons (PTCs) in non-dividing 16HBE14o- cells harboring a stably integrated nanoluciferase reporter containing a PTC, following transfection with the [ ptLeu-sLPV-ALFA-peptide]-[NbALFA-hLa-NLS] conjugate.
  • FIGS. 14A and 14B depict functional measurements of cystic fibrosis transmembrane conductance regulator (CFTR) activity in ex vivo wild-type mouse trachea.
  • FIG. 14A shows a representative transepithelial short-circuit current (Lc) trace demonstrating CFTR-mediated chloride transport.
  • FIG. 14B quantifies the peak forskolin and IB MX (F&I) responses as mean ⁇ standard error of the mean (SEM), confirming CFTR functionality.
  • FIG. 15 shows that nucleotide optimization of ACE-tRNA Leu resulted in a significant enhancement of PTC suppression efficiency in 16HBE14o- cells, with observed translational readthrough activity increasing by approximately 6.5-fold relative to the unoptimized construct.
  • FIG. 16 shows that the inclusion of appended NLS sequences significantly increased the efficiency of ptLeu-sLPV-mediated PTC suppression in non-dividing 16HBE14o- reporter cells.
  • FIG. 17 shows that conjugation of the ptLeu-sLPV with human La (hLa) NLS peptide led to a substantial reduction in cyclic GMP-AMP (cGAMP) production in HeLa cells via lessened activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway.
  • cGAMP cyclic GMP-AMP synthase
  • the present disclosure relates to the field of nucleic acid therapeutics and delivery systems. More specifically, it concerns engineered transfer RNA constructs and their use in gene therapy applications, including compositions and methods for efficient delivery to target cells.
  • ACE-tRNA DNA-based anticodon-engineered transfer RNA
  • PTCs premature termination codons
  • NPs poly(amine-co-ester)
  • PACE NP formulations have been evaluated for in vivo biocompatibility and safety, including assessments in murine models to determine toxicity, biodistribution, and therapeutic efficacy.
  • sLPVs functionalized ACE-tRNA Picovectors
  • NLSs nuclear localization signals
  • NoLSs nucleolar localization sequences
  • DTSs DNA nuclear targeting sequences
  • this disclosure provides an advanced platform for targeted gene therapy using ACE-tRNA constructs, addressing challenges associated with delivery, expression, and therapeutic performance in the treatment of genetic diseases caused by nonsense mutations.
  • this disclosure provides an oligonucleotide set.
  • the oligonucleotide set comprises: (a) a first hairpin oligonucleotide comprising from 5’ end to 3’ end: a first antisense strand, a first loop, and a first sense strand that is complementary to the first antisense strand; and (b) a second hairpin oligonucleotide comprising from 5’ end to 3’ end: a second sense strand, a second loop, and a second antisense strand that is complementary to the second sense strand, wherein the first sense strand and the second sense strand are adapted to be joined together to form a nucleic acid sequence encoding a RNA molecule, and wherein the first loop or the second loop or another part of the oligonucleotide is directly or indirectly linked with a nuclear targeting moiety.
  • this disclosure provides an oligonucleotide set comprising: (i) a first hairpin oligonucleotide comprising from 5’ end to 3’ end: a first antisense strand, a first loop, and a first sense strand that is complementary to the first antisense strand; (ii) a second hairpin oligonucleotide comprising from 5’ end to 3’ end: a second sense strand, a second loop, and a second antisense strand that is complementary to the second sense strand; and (iii) a third sense strand and a third antisense strand having a sequence complementary to the third sense strand, wherein the first sense strand, the third sense strand, and the second sense strand are adapted to be joined together in an order to form a second nucleic acid sequence encoding a RNA molecule, and wherein the first loop or the second loop or another part of the oligonucleotide is directly or indirectly linked with a nuclear targeting
  • nuclear targeting moiety refers to a molecule, structure, domain, or complex that is capable of directing a substance (e.g., a nucleic acid or polypeptide) associated with it to the nucleus of a host cell.
  • a nuclear targeting moiety enhances nuclear binding and/or uptake and facilitates the entry of the substance through the nuclear membrane into the nucleus of the host cell.
  • nuclear targeting moi eties include, but are not limited to, peptides, polypeptides, proteins, nucleic acids, complexes of peptides/polypeptides/proteins, complexes of nucleic acids, and complexes comprising both peptides/polypeptides/proteins and nucleic acids.
  • the nuclear targeting moiety comprises a peptide or a protein having a nuclear localization signal or sequence (NLS).
  • NLS nuclear localization signal or sequence
  • nuclear targeting is achieved by incorporating a nuclear membrane transport peptide, a NLS peptide, or a small molecule that provides equivalent NLS functionality.
  • suitable NLS peptides include those described herein, such as hLa NLS (GGPVKRAREETDKEEPASKQQKTENGAGDQ) (SEQ ID NO: 1), Tyl NLS (GGPNSKKRSLEDNETEIKVSRDTWNTKNMRSLEPPRSKKRIH) (SEQ ID NO: 2), and SV40 NLS (GGGPKKKRKVED) (SEQ ID NO: 3).
  • nuclear targeting may alternatively be achieved by incorporating a DNA nuclear targeting sequence (DTS).
  • DTS DNA nuclear targeting sequence
  • Non-limiting examples include SV40 DTS (e.g., tggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacc) (SEQ ID NO: 4); TGT SV40 DTS (e.g., TGTtggttgctgactaattgagatgcatgctttgcatac ttctgctgctggggagcctggggactggac tttccacaccGAACG) (SEQ ID NO: 5); and NFKB DTS and 3XNFKB DTS (e.g., TACGGGAAATTCCACCTCGGGAAATTCCTGATCGGGA AATTCCGAACG) (SEQ ID NO: 6).
  • an NLS consists of one or more short sequences of positively charged amino acids, such as lysines or arginines, that are exposed on the surface of the protein.
  • the best- characterized transport signal is the classical NLS (cNLS) for nuclear protein import, which consists of either one (monopartite) or two (bipartite) stretches of basic amino acids.
  • Monopartite cNLSs are exemplified by the SV40 large T antigen NLS (PKKKRRV) (SEQ ID NO: 7) or (KKKRKVE) (SEQ ID NO: 8), while bipartite cNLSs are exemplified by the nucleoplasmin NLS (KRPAATKKAGQAKKKK) (SEQ ID NO: 9).
  • the nuclear targeting moiety is selected from the group consisting of a nuclear localization signal peptide, a nuclear membrane transport peptide, and a steroid receptor-binding moiety.
  • the nuclear targeting moiety may be a protein interaction domain that mediates signaling to an NLS-containing protein.
  • the nuclear targeting moiety may be covalently or noncovalently attached to the substance (e.g. , a vector as described herein), or may be recombinantly expressed as a fusion with the substance.
  • nuclear targeting moieties include Lamin A/C, nucleoporins (e.g, NUP), ASHL2, ESET, histones, LSD1, DNA repair enzymes such as PARP, and P84/THOC1, as well as the respective NLS sequences thereof.
  • Suitable peptides are described, for example, in U.S. Pat. Nos. 5,795,587 and 5,670,347, and in WO 98/58955, each of which is incorporated herein by reference in its entirety. (See also Aronsohn et al.. J. Drug Targeting 1 : 163 (1997); Zanta et al., Proc. Nat’l Acad. Sci. USA 96:91-96 (1999); Ciolina et al. Cold Spring Harbor Laboratory Meeting Abstracts (1999), p. 20; and Saphire et al., J. Biol. Chem. 273:29764 (1999)).
  • a nuclear targeting peptide may be a NLS peptide or a nuclear membrane transport peptide, and may comprise natural or non-natural amino acids, including D-amino acids and chemical analogues such as peptoids.
  • the NLS may be composed of amino acids or their analogues arranged in a natural or reverse sequence.
  • the peptide or protein is operably linked to the first loop, the second loop, or an alternative region of the oligonucleotide through a suitable linker.
  • the linker may be selected to provide appropriate spatial orientation, flexibility, or functional separation between the peptide or protein and the oligonucleotide component.
  • the protein comprises a transcription factor or another nuclear- localized protein, such as a chromatin-modifying enzyme, a nuclear receptor, or a DNA- binding regulatory protein.
  • a transcription factor or another nuclear- localized protein, such as a chromatin-modifying enzyme, a nuclear receptor, or a DNA- binding regulatory protein.
  • the linker comprises a small molecule compound, a polypeptide (e.g, a flexible or rigid peptide linker), or an oligonucleotide segment.
  • the linker may be chemically or enzymatically cleavable, or otherwise designed to permit controlled release or separation under specific cellular conditions.
  • the nuclear targeting moiety includes a nucleic acid comprising a DNA targeting sequence (DTS), which facilitates the localization of the oligonucleotide construct to the nucleus and promotes interaction with chromosomal DNA.
  • DTS DNA targeting sequence
  • the DTS may be derived from a naturally occurring sequence known to mediate nuclear localization or may be a synthetic sequence optimized for enhanced nuclear delivery.
  • the nuclear targeting moiety comprises a binding pair comprising: (i) a first member that is linked to the first loop, the second loop, or another region of the oligonucleotide; and (ii) a second member that contains or is linked to an NLS or a DTS, wherein the first member and the second member are designed to bind specifically to each other.
  • oligonucleotide refers to a compound comprising a plurality of linked nucleosides joined together by phosphodiester or other intemucleosidic linkages.
  • the term encompasses naturally occurring oligonucleotides (e.g., unmodified RNA or DNA) as well as synthetic or chemically modified oligonucleotides, including but not limited to those comprising modified sugars, bases, or backbone structures.
  • an oligonucleotide may include one or more unmodified RNA and/or DNA nucleosides and/or one or more modified nucleosides.
  • hairpin refers to a secondary structure formed within a single nucleic acid strand when two regions of the strand, typically complementary when read in reverse orientation, undergo intramolecular base pairing to form a double-stranded stem ending in a single-stranded loop.
  • Such structures are commonly observed in both natural and synthetic nucleic acid sequences and may play structural or functional roles, including in gene regulation and nucleic acid stability.
  • the term “antisense strand” refers to a nucleic acid strand that is complementary to the corresponding “sense” strand.
  • the antisense strand typically hybridizes to the sense strand according to Watson-Crick base pairing.
  • the antisense strand may be referred to by the symbol (-), whereas the sense strand may be referred to by the symbol (+).
  • the antisense strand may be used in various applications, including antisense therapeutics, RNA interference (RNAi), and hybridization assays.
  • complementarity refers to the relationship between two nucleic acid sequences that can form a stable duplex through Watson- Crick or other recognized base-pairing interactions.
  • DNA sequence 5’-C-A- G-T-3’ is complementary to 5’-A-C-T-G-3’.
  • Complementarity may be complete (z.e., each nucleotide in a first strand pairs with its corresponding complementary base in a second strand) or partial (z.e., one or more mismatches are present).
  • the degree of complementarity influences hybridization stability and specificity, which are critical factors in techniques such as polymerase chain reaction (PCR), molecular probes, and nucleic acid-based therapeutics.
  • self-complementary sequence refers to a nucleic acid sequence that is capable of forming intramolecular or intermolecular base pairs with a complementary sequence that is its reverse complement.
  • a self-complementary sequence on a first oligonucleotide may be designed such that a second oligonucleotide includes the reverse complement, enabling hybridization between the two.
  • Such self- complementary sequences are useful for generating duplexes, hairpins, or other structured motifs relevant in structural biology, nanotechnology, and nucleic acid therapeutics.
  • the first hairpin oligonucleotide and the second hairpin oligonucleotide form a closed- end DNA thread (CEDT) molecule.
  • a CEDT molecule comprises a linear double-stranded DNA segment in which the terminal ends are covalently linked to form hairpin loop structures, thereby eliminating free ends. Due to these covalently closed termini, the structure may functionally and topologically resemble a circular single-stranded DNA molecule upon denaturation.
  • Each CEDT molecule includes two covalently closed hairpin termini that physically join the 5’ and 3’ ends of each complementary strand.
  • These hairpins are typically formed through self-annealing or enzymatic ligation of palindromic or complementary sequences and may lack complete base pairing at the apex due to torsional or conformational strain inherent in the loop structure.
  • the unpaired bases at the apex typically 1-4 nucleotides
  • the hairpin sequences are entirely self-complementary; in others, one or more base mismatches (“wobbles”) are tolerated without compromising structural integrity.
  • a CEDT molecule When denatured, a CEDT molecule can be visualized as a circular single-stranded DNA composed of forward (sense or plus) and reverse (antisense or minus) sequences positioned adjacently within the same strand. This contrasts with traditional plasmid or minicircle DNA vectors, in which complementary sequences are distributed between separate circular strands.
  • the CEDT molecule is preferably at least 75% complementary over its length, and may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary in certain embodiments.
  • complementary refers to the conventional base pairing of nucleotides (adenine (A) with thymine (T) or uracil (U), and cytosine (C) with guanine (G)) and applies to both inter- and intramolecular interactions in either antiparallel or palindromic arrangements.
  • the CEDT molecule may comprise a wide variety of nucleotide sequences, including naturally derived, synthetic, or chimeric segments.
  • the CEDT molecule includes one or more processing enzyme target sites that facilitate post-synthetic modification or functionalization of the vector.
  • target sites may be recognized by: restriction endonucleases, which bind and cleave DNA at specific palindromic recognition sequences; site-specific recombinases (e.g., Cre, FLP), which mediate directional DNA exchange at their respective recognition sites such as loxP or FRT; integrases (e.g., phiC31), which catalyze insertion of genetic material at specific attP/attB sequences; or RNA polymerases, wherein the target sequence corresponds to a promoter element, enabling transcription initiation.
  • Suitable promoters may include eukaryotic, viral, or synthetic promoters (e.g., CMV, EFla, SV40, or T7 promoters), optionally in combination with a eukaryotic transcriptional terminator.
  • the CEDT comprises an expression cassette, which may consist of a promoter operably linked to a coding sequence (e.g., for a therapeutic RNA such as tRNA or mRNA, or for a polypeptide), and optionally a polyadenylation or transcription termination sequence.
  • a promoter operably linked to a coding sequence
  • a coding sequence e.g., for a therapeutic RNA such as tRNA or mRNA, or for a polypeptide
  • a polyadenylation or transcription termination sequence e.g., for a therapeutic RNA such as tRNA or mRNA, or for a polypeptide
  • operably linked refers to the functional arrangement of a regulatory element (e.g., a promoter) and a coding sequence such that transcription of the coding sequence is initiated under appropriate biological conditions.
  • the length of a CEDT molecule may vary depending on the application. In some embodiments, the total size of the molecule ranges from about 100 base pairs (bp) to about 4 kilobases (kb), more typically from 200 bp to 2 kb, or 200 bp to 800 bp.
  • the modularity of the CEDT structure permits the inclusion of multiple coding or regulatory elements. For example, CEDTs of 200 bp or greater may accommodate multiple ACE-tRNA cassettes, thereby facilitating enhanced expression of engineered tRNAs from a single vector.
  • a CEDT comprises both a Leucine ACE-tRNA and a Tryptophan ACE-tRNA, designed to suppress the W1282X nonsense mutation in the CFTR gene associated with cystic fibrosis.
  • the dual inclusion of ACE-tRNAs that utilize distinct aminoacyl tRNA synthetases is anticipated to provide synergistic or additive effects in nonsense suppression efficiency, offering a novel therapeutic strategy for diseases characterized by PTCs.
  • the CEDT molecule described herein represents a structurally distinct and functionally versatile DNA construct comprising a linear, double-stranded sequence flanked by covalently closed ends in the form of hairpin loops.
  • the CEDT molecule may include internal coding and regulatory sequences, enzyme recognition sites, or other modular genetic elements, and offers a robust platform for use in gene therapy, RNA expression, genome editing, and other nucleic acid-based applications.
  • the first hairpin oligonucleotide comprises a nucleic acid sequence encoding a tRNA leader sequence.
  • the tRNA leader may facilitate transcription initiation or enhance transcript stability when expressed in a eukaryotic or prokaryotic host cell.
  • the second hairpin oligonucleotide comprises a nucleic acid sequence encoding a transcriptional termination signal recognized by RNA polymerase III.
  • termination signals typically include a stretch of thymidine residues (e.g., TTTTT) and are effective in terminating RNA polymerase Ill-mediated transcription in mammalian and other eukaryotic cells.
  • the oligonucleotide set further comprises a third sense strand and a third antisense strand, wherein the third antisense strand is at least partially complementary to the third sense strand.
  • the third sense and antisense strands may hybridize to form a double-stranded region, which may encode a functional RNA sequence, such as an siRNA, shRNA, or other regulatory RNA molecule.
  • the first sense strand, third sense strand, and second sense strand are adapted to be sequentially ligated, assembled, or otherwise joined together in a defined order to generate a composite or contiguous nucleic acid molecule.
  • the resulting sequence may encode a full-length RNA molecule comprising functional domains derived from the first, third, and second sense strands.
  • the resulting construct forms a CEDT molecule.
  • the CEDT molecule may adopt a dumbbell-like structure and may be resistant to exonuclease degradation, making it particularly suitable for use in therapeutic or diagnostic applications.
  • the third sense strand comprises a nucleic acid sequence encoding a tRNA leader sequence, which may facilitate efficient transcription or translation of the associated RNA product.
  • the third sense strand comprises a nucleic acid sequence encoding an RNA polymerase III transcription termination signal, thereby contributing to efficient and precise termination of transcription when the CEDT molecule is transcribed in a host cell.
  • the RNA molecule comprises tRNA.
  • the tRNA comprises an ACE-tRNA.
  • An ACE-tRNA is an engineered tRNA molecule capable of reverting a PTC into the originally lost amino acid or a different amino acid.
  • Such engineered tRNAs allow for “re- editing” of a disease-causing nonsense codon to a specific amino acid.
  • the small size of these tRNA molecules makes them amenable to ready expression, as the tRNA and the promoter together can be only about 300 bp.
  • an oligonucleotide can be synthesized to include the structural component of a tRNA gene that is functional in human cells.
  • the sequence of this oligonucleotide can be designed based on a known sequence with substitutions made in the anticodon region of the tRNA, causing the specific tRNA to recognize nonsense or other specific mutations.
  • ACE-tRNAs include those described in WO2019090154, WO 2019090169, WO2021252354A1, and Lueck, J. D. et al. Nature communications 10, 822 (2019), the contents of which are incorporated herein by reference.
  • an ACE-tRNA has a four-arm structure comprising a T-arm, a D-arm, an anticodon-arm, and an acceptor arm (see, e.g., Figure 2 of WO2019090169).
  • the T-arm is made up of a “T-stem” and a “TYE loop.”
  • the T-stem is modified to increase the stability of the tRNA.
  • the ACE-tRNA has a modified T- stem that increases the biological activity to suppress stop sites relative to the endogenous T- stem sequence.
  • ACE-tRNAs can be used for suppression of PTCs.
  • This ACE-tRNA approach offers several significant benefits over other readthrough strategies, including (1) codon specificity; (2) ACE-tRNAs suppression of PTCs resulting in seamless rescue, thus negating spurious effects on protein stability, folding, trafficking, and function; and (3) in vitro delivery of these of ACE-tRNA resulting in significant functional rescue of affected protein, such as CFTR channels with p.G542X or p.W1282X CF mutations.
  • the ACE-tRNAs have shown to be efficient at PTC suppression in several cDNA genes with varied PTC positions in multiple cell types. Because ACE-tRNAs exhibit high efficiency in PTC suppression with no known detrimental effects, they can be used as therapeutics.
  • ACE-tRNAs can be made according to the methods described in WO2019090154, W02019090169, WO2021252354A1, and Lueck, J. D. et al.. Nature communications 10, 822 (2019). Using the described methods, an extensive library of ACE-tRNAs for effective rescue of PTCs in cell culture can be generated.
  • Other engineered human tRNA sequences to suppress disease-causing PTCs include those described in W02019090154, W02019090169, WO2021252354A1, and Lueck, J. D. et al., Nature communications 10, 822 (2019), the contents of which are incorporated herein by reference.
  • the tRNA is selected from the group consisting of Arg-tRNA- UGA, Gln-tRNA-UAA, Gln-tRNA-UAG, Trp-tRNA-UGA, Trp-tRNA-UAG, Glu-tRNA- UAA, Glu-tRNA-UAG, Cys-tRNA-UGA, Tyr-tRNA-UAG, Tyr-tRNA-UAA, Leu-tRNA- UGA, Leu-tRNA-UAG, Leu-tRNA-UAA, Lys-tRNA-UAG, Lys-tRNA-UGA, Ser-tRNA- UGA, Ser-tRNA-UAG, and Ser-tRNA-UAA.
  • the ACE-tRNA causes a ribosome to read through one or more stop codons during translation by the ribosome. Such readthrough may occur during protein synthesis and results in continued elongation of the polypeptide chain beyond the canonical stop site.
  • the one or more stop codons comprise a premature termination codon (PTC), which may arise from a genetic mutation.
  • the ACE-tRNA may be specifically designed to recognize and suppress the PTC, thereby restoring expression of a full-length functional protein.
  • the PTC is located within a nucleic acid sequence encoding the CFTR protein. Restoration of full-length CFTR protein expression by suppression of the PTC may be beneficial in treating cystic fibrosis or related disorders.
  • the nucleic acid sequence harboring the PTC may have a length ranging from approximately 200 nucleotides to approximately 1,000 nucleotides (e.g., 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000 nucleotides or any intermediate number therebetween), although sequences outside of this range may also be suitable depending on the context and intended application.
  • the ACE-tRNA molecule comprises one or more structural elements, such as a first loop, a second loop, or another region of the oligonucleotide, which is linked to an agent.
  • the agent comprises a labeling moiety, a peptide, a bioactive agent, or any combination thereof.
  • agents may be used, for example, to facilitate detection, purification, cellular targeting, or therapeutic modulation of the ACE-tRNA molecule.
  • the labeling moiety includes, but is not limited to, N- hydroxysuccinimide (NHS) esters, thiol-maleimide conjugates, or azide-dibenzocyclooctyne (DBCO) click chemistry reagents. Combinations of such labeling moieties may also be employed to provide multifunctionality or enhanced detection capabilities.
  • NHS N- hydroxysuccinimide
  • DBCO azide-dibenzocyclooctyne
  • the nucleic acid molecules may include one or more chemically modified nucleotides, such as a 2’-O-methyl modified sugar moiety.
  • chemically modified nucleotides may include a modified internucleoside linkage.
  • a “modified oligonucleotide” refers to an oligonucleotide comprising at least one modified nucleoside and/or at least one modified internucleoside linkage. Examples of modified oligonucleotides include single-stranded and double-stranded compounds, such as antisense compounds, siRNAs, shRNAs, ssRNAs, and occupancy-based compounds.
  • nucleoside refers to a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA) and modified nucleosides. Nucleosides may be linked to a phosphate moiety.
  • a “chemical modification” refers to a chemical difference in a compound when compared to a naturally occurring counterpart. Chemical modifications of oligonucleotides may include nucleoside modifications (such as sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications. As used herein, an “internucleoside linkage” refers to a covalent linkage between adjacent nucleosides in an oligonucleotide. In reference to an oligonucleotide, a chemical modification does not include differences only in nucleobase sequence.
  • a “sugar moiety” refers to a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside.
  • a “modified sugar moiety” refers to a substituted sugar moiety or a sugar surrogate.
  • a “substituted sugar moiety” refers to a furanosyl that is not a naturally occurring sugar moiety. Substituted sugar moieties include, but are not limited to furanosyls comprising substituents at the 2’ -position, the 3’- position, the 5’-position and/or the deposition. Certain substituted sugar moieties are bicyclic sugar moieties.
  • a “2’-substituted sugar moiety” refers to a furanosyl comprising a substituent at the 2’-position other than H or OH. Unless otherwise indicated, a 2’-substituted sugar moiety is not a bicyclic sugar moiety (z.e., the 2 ’-substituent of a 2 ’-substituted sugar moiety does not form a bridge to another atom of the furanosyl ring.
  • modified sugar moieties are substituted sugar moieties.
  • modified sugar moieties are bicyclic or tricyclic sugar moieties.
  • modified sugar moieties are sugar surrogates. Such sugar surrogates may include one or more substitutions corresponding to those of substituted sugar moieties.
  • modified sugar moieties are substituted sugar moieties comprising one or more substituents, including but not limited to substituents at the 2’ and/or 5’ positions.
  • sugar substituents suitable for the 2’-position include but are not limited to: 2’-F, 2’-OCH 3 (“OMe” or “O-methyl”), and 2’-O(CH 2 ) 2 OCH3 (“MOE”).
  • sugar substituents at the 5 ’-position include but are not limited to: 5’-methyl (R or S), 5’-vinyl, and 5’-methoxy.
  • substituted sugars may include more than one non-bridging sugar substituent, for example, 2 ’-F-5’ -methyl sugar moi eties see, e.g., PCT International Application WO 2008/101157, for additional 5’,2’- bis substituted sugar moieties and nucleosides).
  • Nucleosides comprising 2 ’-substituted sugar moieties are herein referred to as 2’- substituted nucleosides.
  • a 2 ’-substituted nucleoside may include a 2’- substituent group selected from halo, allyl, amino, azido, O — C1-C10 alkoxy; O — C1-C10 substituted alkoxy, SH, CN, OCN, CF3, OCF3, O-alkyl, S-alkyl, N(R m )-alkyl; O-alkenyl, S- alkenyl, or N(R m )-alkenyl; O-alkynyl, S-alkynyl, N(R m )-alkynyl; O-alkynyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH
  • These 2 ’-substituent groups can be further substituted with one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl, and alkynyl.
  • a 2’-substituted nucleoside may include a sugar moiety comprising a 2 ’-substituent group selected from F, O — CEE, and OCEECEEOCEE.
  • modified sugar moieties may include a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety.
  • the bicyclic sugar moiety may include a bridge between the 4’ and the 2’ furanose ring atoms.
  • Examples of such 4’ to 2’ sugar substituents include, but are not limited to: — [C(R a )(Rb)]n — , — [C(Ra)(Rb)]n— O— — C(RaRb)— N(R)— O— or, — C(RaRb)— O— N(R)— ; 4’-CH 2 -2’, 4’- (CH 2 ) 2 -2’, 4’-(CH 2 ) 3 -2’, 4’-(CH 2 )— 0-2’ (LNA); 4’-(CH 2 )— S-2; 4’-(CH 2 ) 2 — 0-2’ (ENA); 4’- CH(CH 3 ) — 0-2’ (cEt) and 4’-CH(CH 2 OCH 3 ) — 0-2’, and analogs thereof (see, e.g., U.S.
  • this disclosure provides a method for making a CEDT molecule.
  • the method comprises: providing an oligonucleotide set as described herein; and ligating components of the oligonucleotide set to obtain the CEDT molecule.
  • ligation of components of the oligonucleotide set may be facilitated by an enzyme, such as a ligase.
  • the ligase may be a eukaryotic ligase, a prokaryotic ligase, a single-stranded DNA ligase, or a double-stranded DNA ligase.
  • Suitable examples of DNA ligases include, but are not limited to, T4 DNA ligase, Taq DNA ligase, T7 DNA ligase, T3 DNA ligase, 9°NTM DNA ligase, and E. coli DNA ligase.
  • the oligonucleotide set is chemically synthesized.
  • the oligonucleotide set comprises chemically modified nucleotides.
  • the first hairpin oligonucleotide, the second hairpin oligonucleotide, or the resulting CEDT molecules may each include one or more chemically modified nucleotides, such as a 2’- O-methyl modified sugar moiety.
  • the chemical modification includes a modified internucleoside linkage.
  • labeling agent refers to any composition that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means.
  • labeling agents include, without limitation, biotin (for staining with labeled streptavidin conjugates), magnetic beads (e.g., Dynabeads®), fluorescent dyes (e.g., fluorescein, Texas Red, rhodamine, green fluorescent protein, and similar), radiolabels (e.g., tritium ( 3 H), iodine-125 ( 125 I), sulfur-35 ( 35 S), carbon-14 ( 14 C), or phosphorus-32 ( 32 P)), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, late
  • Radiolabels can be detected using photographic film or scintillation counters; fluorescent labels can be detected using photodetectors to monitor emitted light; enzymatic labels may be detected by providing the enzyme with a substrate and measuring the resulting product; and colorimetric labels may be detected by direct visual inspection.
  • labeling or chemical modification of the hairpin, CEDT, or picovector does not adversely affect PTC (premature termination codon) suppression. Accordingly, labeled hairpins or labeled CEDT/picovectors may be employed in various applications, including the identification of NLS sequences and/or cell-penetrating peptides (CPPs) that enhance picovector delivery and intracellular localization.
  • CPPs cell-penetrating peptides
  • bioactive agent refers to any substance suitable for use in therapeutic or diagnostic applications. Such agents may be used, for example, in methods for diagnosing the presence or absence of disease in a subject, or in methods for treating disease in a subject.
  • this disclosure provides a CEDT molecule produced according to the methods described herein.
  • the nucleic acid molecules such as oligonucleotide sets, hairpin oligonucleotides, or CEDT molecules generated from the disclosed oligonucleotide sets, can be provided in a composition (e.g., a pharmaceutical composition) or in a kit.
  • the composition may include an oligonucleotide set as described herein.
  • the composition may include the first hairpin oligonucleotide and/or the second hairpin oligonucleotide, as described herein.
  • the composition may include a CEDT molecule prepared from an oligonucleotide set as described herein.
  • this disclosure provides a kit comprising the oligonucleotide set described herein and, optionally, a ligase.
  • the ligase is T4 DNA ligase.
  • nucleic acids e.g., DNA
  • Any pharmaceutically acceptable carrier or excipient may be used.
  • Auxiliary substances such as wetting or emulsifying agents, pH- buffering substances, and the like, may be included in the excipient or vehicle.
  • These excipients, vehicles, and auxiliary substances are generally pharmaceutical agents that may be administered without undue toxicity and that, in the case of vaccine compositions, do not induce an immune response in the individual receiving the composition.
  • a suitable carrier may include, for example, a liposome.
  • Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, and ethanol.
  • compositions may also be included, such as mineral acid salts (e.g., hydrochlorides, hydrobromides, phosphates, sulfates) and salts of organic acids (e.g., acetates, propionates, malonates, benzoates).
  • the preparation may also include excipients that serve as stabilizers, particularly for peptides, proteins, or similar molecules.
  • suitable carriers that act as stabilizers for peptides include, without limitation, pharmaceutical grades of dextrose, sucrose, lactose, trehalose, mannitol, sorbitol, inositol, dextran, and the like.
  • Suitable carriers include, without limitation, starch, cellulose, sodium or calcium phosphates, citric acid, tartaric acid, glycine, high molecular weight polyethylene glycols (PEGs), or combinations thereof.
  • PEGs polyethylene glycols
  • compositions containing active ingredients can be prepared using known procedures and readily available components.
  • active ingredients such as oligonucleotides or CEDT molecules
  • the compositions can also be formulated as solutions suitable for parenteral administration, such as intramuscular, subcutaneous, or intravenous injection.
  • the compositions can be in the form of an aqueous or anhydrous solution or dispersion, or as an emulsion or suspension.
  • the compositions may be in powder form, obtained via aseptic isolation of sterile solids or by lyophilization from solution, for constitution with a suitable vehicle e.g., sterile, pyrogen-free water) before use.
  • the compositions disclosed herein may be formulated as lipid nanoparticles (LNPs), such as those described in WO2020263883, WO2013123523, W02012170930, WO2011127255, W02008103276, and US20130171646, each of which is incorporated herein by reference in its entirety.
  • LNPs lipid nanoparticles
  • the present disclosure provides nanoparticle compositions comprising a lipid composition and at least one nucleic acid, such as a CEDT molecule, along with a delivery agent.
  • the lipid composition may encapsulate the nucleic acid.
  • Nanoparticle compositions are typically sized on the order of micrometers or smaller and may include a lipid bilayer. Nanoparticle compositions include, for example, LNPs, liposomes (e.g., lipid vesicles), and lipoplexes. In some embodiments, the nanoparticle is a liposome with a lipid bilayer and a diameter of 500 nm or less.
  • the nanoparticle composition includes one or more lipid bilayers, optionally arranged as concentric bilayers separated by aqueous compartments.
  • the lipid bilayers may be functionalized and/or crosslinked, and may include one or more ligands, proteins, or channels.
  • a lipid nanoparticle may include an ionizable lipid, a structural lipid, a phospholipid, and a nucleic acid of interest.
  • the lipid nanoparticle includes an ionizable lipid, a PEG-modified lipid, a sterol, and a structural lipid.
  • the molar ratio of the components may range from about 20-60% ionizable lipid, about 5- 25% structural lipid, about 25-55% sterol, and about 0.5-15% PEG-modified lipid.
  • the lipid nanoparticle has a poly dispersity index of less than 0.4 and exhibits a net neutral charge at physiological pH.
  • the mean diameter of the lipid nanoparticle may range from 50-150 nm, or from 80-100 nm in certain embodiments.
  • lipid refers to a small molecule with hydrophobic or amphiphilic properties. Lipids may be naturally occurring or synthetic. Classes of lipids include, but are not limited to, fats, waxes, sterol -containing metabolites, vitamins, fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides, and prenol lipids. Due to their amphiphilic nature, some lipids can self-assemble into liposomes, vesicles, or membranes in aqueous media.
  • nucleic acids are formulated into lipid nanoparticles with diameters from about 10 to 100 nm, or alternatively, from about 10 to 500 nm. In some embodiments, the nanoparticle diameter exceeds 100 nm.
  • the largest dimension of a nanoparticle composition may be 1 pm or smaller (e.g., 1 pm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, or 50 nm).
  • Nanoparticle compositions can be relatively homogeneous.
  • the polydispersity index which reflects particle size distribution, may be used to characterize homogeneity. A value below 0.3 generally indicates a narrow size distribution.
  • the poly dispersity index is from about 0 to 0.25, for example, 0.01 to 0.25, or more specifically, from about 0.10 to 0.20.
  • the nucleic acids described herein are formulated for controlled release and/or targeted delivery.
  • controlled release refers to the release of a pharmaceutical composition or compound in a predefined pattern over time to achieve a desired therapeutic effect.
  • the nucleic acids may be encapsulated using delivery agents known in the art for such applications.
  • the term “encapsulate” means to enclose, surround, or encase. Encapsulation may be complete, substantial, or partial.
  • substantially encapsulated means that more than 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the composition is enclosed within the delivery agent. “Partially encapsulated” means that less than 10%, 20%, 30%, 40%, or 50% of the composition is enclosed.
  • the composition is formulated for sustained release.
  • sustained release refers to a formulation that releases its active ingredient over a prolonged period, such as hours, days, weeks, months, or years.
  • sustained release nanoparticle compositions may be formulated as disclosed in W02010075072, US20100216804, US20110217377, US20120201859, and US20130150295, each of which is incorporated herein by reference in its entirety.
  • the nanoparticle compositions are formulated for tissue- or cellspecific targeting, as described in WO2008121949, W02010005726, W02010005725, WO201 1084521, WO2011084518, US20100069426, US20120004293, and US20100104655, each of which is incorporated herein by reference in its entirety.
  • the nucleic acid molecules e.g., oligonucleotide sets, hairpin oligonucleotides, CEDT molecules
  • the kit includes a container comprising at least one nucleic acid molecule or a composition thereof, along with optional informational material.
  • the informational material may include descriptive, instructional, or marketing content related to the methods and uses described herein, such as instructions for manufacturing, recommended dosage, or administration schedules.
  • the kit includes an additional therapeutic agent.
  • Multiple containers may be provided, for example, one containing the nucleic acid composition and another containing a separate therapeutic agent.
  • the containers may hold unit dosages of the pharmaceutical composition.
  • the kit may include other components such as solvents, buffers, adjuvants, stabilizers, preservatives, or combinations thereof.
  • a delivery device suitable for administration e.g., a syringe
  • the device may be pre-loaded or provided empty for loading prior to use.
  • this disclosure provides a method for treating a disease associated with a PTC in a subject in need thereof.
  • the method comprises administering to the subject a CEDT molecule as described herein, or a pharmaceutical composition thereof.
  • the disclosed method offers several advantages. First, it provides improved stop codon suppression specificity.
  • the therapeutic ACE-tRNAs of the present disclosure can selectively target a specific stop codon, such as TGA, thereby reducing off-target effects at stop codons unrelated to the disease.
  • the method enables amino acid specificity.
  • the expressed tRNA is engineered to specifically restore the amino acid lost due to insertion of the disease- associated stop codon, thereby minimizing undesired effects on protein stability, folding, and trafficking.
  • the method is adaptable to “personalized” therapy to correct any disease- related PTC.
  • the human genome encodes nine distinct tryptophan (Trp) tRNAs that are recognized by Trp-tRNA synthetase and suppress the UGG codon. Each of these nine Trp tRNAs may be exploited for codon re-editing tolerance (e.g., for UGG to UGA).
  • Trp tryptophan
  • arginine codons frequently mutate to PTCs in PTC-associated diseases due to their proximity to stop codons in the genetic code.
  • Arg tRNAs available for use, and ACE-tRNAs encoding arginine may serve as therapeutic agents for Arg ⁇ PTC mutations irrespective of the affected gene. For instance, approximately 35% of Leber congenital amaurosis (LCA) cases are caused by nonsense mutations, the majority of which involve arginine to stop codon transitions.
  • LCA Leber congenital amaurosis
  • Another advantage of the disclosed method is its compact and modular design, which enables facile expression and cell-specific delivery.
  • the complete expression system, including the tRNA and promoter sequence, is compact enough to facilitate delivery by various gene therapy vectors.
  • Diseases or disorders caused by or associated with PTCs include, but are not limited to, variants of Duchenne muscular dystrophy and Becker muscular dystrophy due to a PTC in dystrophin; retinoblastoma due to a PTC in RBI; neurofibromatosis due to a PTC in NF1 or NF2; ataxia-telangiectasia due to a PTC in ATM; Tay-Sachs disease due to a PTC in HEXA; cystic fibrosis due to a PTC in CFTR; Wilms’ tumor due to a PTC in WT1; hemophilia A due to a PTC in factor VIII; hemophilia B due to a PTC in factor IX; p53 -associated cancers due to a PTC in TP53; Menkes disease; Ullrich’s disease; P-thalassemia due to a PTC in P-globin; type 2A and type 3 von Willebrand disease due to a PTC in
  • Additional disorders that may be treated using the compositions and methods described herein include, but are not limited to, Liddle’s syndrome, xeroderma pigmentosum, Fanconi anemia, various forms of anemia, hypothyroidism, and cancers associated with mutations in the TP53 gene (e.g., squamous cell carcinoma, hepatocellular carcinoma, ovarian carcinoma).
  • Other treatable conditions may include esophageal carcinoma, osteosarcoma, breast cancer, malignant fibrous histiocytoma, SRY-associated sex reversal, triosephosphate isomerase deficiency-related anemia, diabetes mellitus, and various forms of rickets, among others.
  • the method comprises treating a disease or disorder associated with a nonsense mutation by reversing or mitigating the effects of such mutation through administration of a CEDT molecule as described in this disclosure.
  • the disease or disorder may be cystic fibrosis, wherein the CEDT molecule targets a specific nonsense mutation within the CFTR gene to restore functional protein expression.
  • Other diseases or disorders that may be addressed include, for example, Hurler syndrome, Dravet syndrome, spinal muscular atrophy, Usher syndrome, aniridia, choroideremia, ocular coloboma, retinitis pigmentosa, dystrophic epidermolysis bullosa, pseudoxanthoma elasticum, Alagille syndrome, Waardenburg-Shah syndrome, infantile neuronal ceroid lipofuscinosis, cystinosis, X-linked nephrogenic diabetes insipidus, and polycystic kidney disease.
  • PTCs premature termination codons
  • ocular diseases include various ocular diseases. These eye diseases may involve one or more nonsense mutations in genes associated with inherited retinal or visual disorders.
  • cone dystrophies including Stargardt’s disease (STGD1), cone-rod dystrophy, retinitis pigmentosa (RP), and increased susceptibility to age-related macular degeneration (AMD) — may be associated with PTCs in genes such as KCNV2 (e.g., Glu43X, Glu306X, Gln76X, Glul48X), CACNA2D4 (Tyr802X, Arg628X), RP2 (Argl20X), RHO (Ser334X), RPE65 (Arg44X), and PDE6A (Lys455X).
  • STGD1 Stargardt’s disease
  • RP retinitis pigmentosa
  • AMD age-related macular degeneration
  • KCNV2 e.g., Glu43
  • Congenital stationary night blindness may also be amenable to treatment, with type 2 (CSNB2) linked to mutations in CACNA1F (Arg958X, Arg830X), and type 1 (CSNB1) associated with mutations in TRPM1 (Glnl lX, Lys294X, Arg977X, Ser882X) and NYX (Trp350X).
  • Best disease involves mutations in BEST1 (Tyr29X, Arg200X, Ser517X).
  • Leber congenital amaurosis may involve mutations in KCNJ13 (Trp53X, Argl66X), CEP290 (Argl51X, Glyl890X, Lysl575X, Argl271X, Argl782X), CRB1 (Cysl332X), GUCY2D (Ser448X, Arg4091X), LCA5 (Gln279X), RDH12 (Tyrl94X, Glu275X), SPATA7 (ArglO8X), and TULP1 (Gln301X).
  • Usher syndrome type 1 may involve nonsense mutations in USH1C (Arg31X), PCDH15 (Arg3X, Arg245X, Arg643X, Arg929X), IQCB1 (Arg461X, Arg489X), PDE6A (Gln69X), and ALMS1 (Ser999X, Arg3804X). Additional treatable conditions may include aniridia (PAX6, Glyl94X), ocular coloboma (PAX2, Argl39X; IAMBI, Arg524X), and choroideremia (REP 1, CHM, Gln32X). These disorders are characterized by the presence of nonsense mutations that result in truncated, non-functional proteins, and may benefit from therapeutic methods that promote readthrough of PTCs or otherwise restore expression of full-length functional proteins.
  • compositions described herein can be administered in one or more doses using techniques well known to those skilled in the medical arts, taking into account factors such as the age, sex, weight, and general health condition of the subject, as well as the desired route of administration.
  • the dosage of the composition may range from about 1 picogram (pg) to about 10 milligrams (mg) of active component per kilogram (kg) of body weight per administration. In some embodiments, the dosage may be from about 20 pg to about 10 mg of active component per kg of body weight per administration.
  • the compositions may be administered at intervals of every 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, or 31 days, depending on the therapeutic protocol and the patient’s response.
  • the total number of doses for an effective treatment regimen may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses, or more.
  • the agent or composition may be administered either prophylactically to prevent disease or therapeutically to treat an existing condition.
  • the compositions are administered to a subject in need thereof in an amount sufficient to elicit a desired therapeutic response. Such an amount is referred to herein as a “therapeutically effective dose.”
  • a therapeutically effective dose Such an amount is referred to herein as a “therapeutically effective dose.”
  • the precise dosage required will vary depending on factors such as the specific composition used, the formulation and route of administration, the stage and severity of the disease being treated, the overall health status of the subject, and the clinical judgment of the treating physician.
  • compositions may be administered by any method known in the art. Examples of suitable methods are described, for instance, in Donnelly et al., Annual Review of Immunology 15:617-648 (1997), U.S. Patent Nos. 5,580,859, 5,703,055, and 5,679,647, the contents of each of which are incorporated herein by reference in their entirety.
  • the DNA component of the composition may be complexed with particles or beads and delivered to a subject, for example, via a needle-free injection device such as a vaccine gun.
  • a needle-free injection device such as a vaccine gun.
  • a pharmaceutically acceptable carrier including a physiologically acceptable compound, may depend on the route of administration.
  • compositions may be administered by a variety of routes, including but not limited to parenteral administration, such as intradermal, intramuscular, or subcutaneous injection. Additional administration routes include oral, intranasal, intravaginal, transdermal (e.g., via iontophoresis), or topical application to the skin or mucosal tissues.
  • parenteral administration such as intradermal, intramuscular, or subcutaneous injection. Additional administration routes include oral, intranasal, intravaginal, transdermal (e.g., via iontophoresis), or topical application to the skin or mucosal tissues.
  • the composition may be administered to interstitial spaces of tissues, as described in U.S. Patent Nos. 5,580,859 and 5,703,055. Epidermal administration may also be employed, which can involve mechanical or chemical irritation of the outermost layer of the skin to stimulate an immune response (see, e.g., U.S. Patent No. 5,679,647).
  • the compositions may be formulated for nasal administration.
  • suitable nasal formulations may include coarse powders having particle sizes ranging from about 10 to about 500 microns, administered by rapid inhalation (e.g., as snuff).
  • the formulation may be delivered as a nasal spray, nasal drops, or as an aerosol using a nebulizer.
  • Nasal formulations may include aqueous or oily solutions containing the active component.
  • compositions may also be prepared as liquid formulations, such as suspensions, syrups, or elixirs.
  • the compositions are formulated for injectable administration, including subcutaneous, intradermal, intramuscular, or intravenous injection.
  • Such formulations may be provided as sterile solutions, suspensions, or emulsions.
  • composition described herein may be incorporated into liposomes, microspheres, or other polymeric matrices.
  • examples of such formulations are disclosed in U.S. Patent No. 5,703,055 and in Liposome Technology, Volumes I to III (2nd ed. 1993) by Gregoriadis, the contents of which are hereby incorporated by reference in their entirety.
  • Liposomes may be composed of phospholipids or other biocompatible lipids and can serve as nontoxic, physiologically acceptable, and metabolizable carriers. Such carriers are generally straightforward to prepare and are amenable to various routes of administration.
  • composition may be administered by any of a variety of routes, including, but not limited to, oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhalational, buccal, intrapleural, intravenous, intra-arterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intrathecal, intra-articular, or any combination thereof.
  • routes including, but not limited to, oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhalational, buccal, intrapleural, intravenous, intra-arterial, intraperitoneal, subcutaneous, intramuscular, intranasal, intrathecal, intra-articular, or any combination thereof.
  • routes including, but not limited to, oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, inhalational, buccal, intrapleural, intravenous, intra-arterial, intraperitoneal, sub
  • the composition may also be delivered using a variety of physical delivery systems, including but not limited to traditional syringes, needleless injection devices, microprojectile bombardment (e.g., “gene guns”), electroporation (EP), hydrodynamic injection, or ultrasound- mediated delivery.
  • the composition is administered to a mammal using one or more established nucleic acid delivery technologies. These may include direct DNA injection with or without in vivo electroporation, liposome-mediated delivery, nanoparticle-facilitated delivery, or delivery using recombinant vectors, such as recombinant adenovirus, recombinant adeno-associated virus (AAV), or recombinant vaccinia virus.
  • the ACE-tRNA, or a nucleic acid molecule encoding the ACE-tRNA may be introduced via direct DNA injection, optionally in conjunction with in vivo electroporation to enhance cellular uptake and expression.
  • nucleic acid or “polynucleotide” refers to a DNA molecule (e.g. , cDNA or genomic DNA), an RNA molecule (e.g, mRNA), or a DNA or RNA analog. DNA or RNA analogs may be synthesized from nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double-stranded.
  • isolated nucleic acid refers to a nucleic acid whose structure is not identical to that of any naturally occurring nucleic acid or fragment thereof.
  • This term includes, for example: (a) a DNA molecule having a sequence identical to a portion of a naturally occurring genomic DNA but not flanked by both coding sequences that naturally flank that portion; (b) a nucleic acid incorporated into a vector or a genome in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, genomic fragment, PCR product, or restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene encoding a fusion protein.
  • the above-described nucleic acids may be used to express a tRNA according to this invention. For such expression, the nucleic acid can be operatively linked to appropriate regulatory sequences to form an expression vector.
  • translation refers to the process by which a polypeptide (e.g, a protein) is synthesized from an mRNA template.
  • an increase in translation refers to an increase in the number of polypeptide molecules produced per mRNA molecule encoding said polypeptide.
  • non-complementary in reference to nucleobases means a pair of nucleobases that do not form hydrogen bonds with each other.
  • mismatch means a nucleobase of a first oligomeric compound that cannot base pair with a corresponding nucleobase of a second oligomeric compound when aligned.
  • One or both of the oligomeric compounds may be oligonucleotides.
  • a “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked.
  • the vector may or may not be capable of autonomous replication or genomic integration. Examples include plasmids, cosmids, and viral vectors.
  • the vector typically comprises a nucleic acid in a form suitable for expression in a host cell and may include one or more regulatory sequences operatively linked to a nucleic acid of interest.
  • a “regulatory sequence,” as used herein, includes promoters, enhancers, and other expression control elements such as polyadenylation signals. Regulatory sequences may direct constitutive, tissue-specific, or inducible expression. The design of an expression vector may depend on factors such as host cell type and the desired expression level.
  • a promoter is a DNA sequence that directs RNA polymerase binding and initiation of RNA synthesis.
  • a “strong promoter” is one that initiates transcription at a high frequency.
  • promoter refers to a nucleotide sequence that initiates and regulates transcription of a polynucleotide. Promoters may be inducible, repressible, or constitutive. The term includes full-length promoters and functional fragments that direct transcription. The terms “promoter” and “control element” are used interchangeably.
  • operably linked refers to an arrangement wherein a regulatory element such as a promoter is positioned relative to a nucleic acid sequence to enable expression.
  • the promoter need not be immediately adjacent to the sequence, as long as it functions effectively. Thus, untranslated sequences may be present between the promoter and coding region.
  • An “expression cassette” refers to a nucleic acid sequence capable of directing expression of a nucleotide sequence in a host cell. It typically includes a promoter operably linked to a coding region and may further include termination signals and translation control sequences.
  • the cassette may be chimeric or recombinant and may be under the control of a constitutive or regulatable promoter.
  • suitable promoters include PGK, CMV, RSV, Hl, or U6 (Pol II and Pol III promoters).
  • nucleic acid fragment refers to a portion of a given nucleic acid molecule.
  • “Substantial identity” in the context of nucleic acid sequences means at least 70-99% sequence identity compared to a reference sequence, as determined using standard alignment algorithms. “Substantial identity” between nucleic acids or polypeptides indicates at least 90%-99% identity using alignment tools such as FASTA, BLAST, or GAP. “Substantial similarity” in polypeptides may include conservative amino acid substitutions, which do not significantly alter the protein’s function. Sequence comparisons can be conducted using tools such as GAP, BESTFIT, FASTA, or BLAST, with default parameters. Conservative substitutions are defined by physicochemical properties or substitution matrices such as PAM250. These algorithms help determine whether two sequences encode proteins with similar or identical function.
  • a “minivector” refers to a compact circular DNA molecule (e.g., mini circle or closed linear DNA such as CEDT), lacking a bacterial origin of replication and antibiotic resistance gene, and ranging from about 100 bp to about 5 kbp in size. Minivectors may be generated by site-specific recombination of parental plasmids and typically include only a transgene expression cassette, such as one expressing an ACE-tRNA for suppressing premature termination codons (PTCs), without bacterial-derived sequences.
  • PTCs premature termination codons
  • disease is generally synonymous with “disorder” and “condition” and refers to any abnormal physiological state that impairs function and reduces quality or duration of life.
  • a “subject” or “subject in need thereof’ includes humans and non-human animals (e.g., mammals, birds, amphibians, and reptiles).
  • the subject is an experimental model or a human patient.
  • a “PTC-associated disease” refers to any condition caused by a nonsense mutation that introduces a premature termination codon, leading to a truncated and typically nonfunctional protein.
  • Treating” or “treatment” refers to administration of an agent to cure, prevent, delay, or ameliorate a disease, its symptoms, or predisposition thereto. “Preventing” includes full or partial inhibition of disease onset or progression.
  • a “pharmaceutical composition” refers to a combination of an active agent with a carrier suitable for diagnostic or therapeutic use in vivo or ex vivo.
  • a “pharmaceutically acceptable carrier” is one that does not cause undesirable effects and is compatible with the active ingredient.
  • Carriers may include solubilizing agents, biocompatible vehicles, excipients, and stabilizers, among others.
  • agent refers broadly to chemical compounds, biological macromolecules (e.g., nucleic acids, antibodies), or extracts from biological materials.
  • a “therapeutic agent” is an agent capable of producing a beneficial biological effect, such as ameliorating a disease or condition.
  • Doses are often expressed in relation to body weight. Accordingly, a dose expressed as [g, mg, or other unit]/kg (or g, mg, etc.) typically refers to [g, mg, or other unit] per kilogram (or g, mg, etc.) of body weight, even if the term “body weight” is not explicitly stated.
  • zzz vitro refers to events that occur in an artificial environment, such as in a test tube, reaction vessel, or cell culture, rather than within a multicellular organism.
  • zzz vivo refers to events that occur within a multicellular organism, such as a non-human animal.
  • compositions that is “substantially free” of component Y may, in some instances, be entirely free of Y. Where context permits, the term “substantially” may be omitted from the definition of the invention.
  • each when referring to a collection of items, is intended to identify an individual item within the collection, but does not necessarily refer to every item, unless clearly dictated by the context.
  • This disclosure is directed to: (1) the development and optimization of highly functional DNA-based anticodon-engineered transfer RNA (ACE-tRNA) therapeutic constructs; (2) the formulation and co-delivery of these ACE-tRNA constructs with poly(amine-co-ester) (PACE) polymeric nanoparticle (NP) delivery systems to facilitate efficient transfection and gene delivery to airway epithelial cells; and (3) the evaluation of the biocompatibility and safety profile of the PACE NP formulations in vivo, including in murine models.
  • the experimental methods described herein were designed to overcome deficiencies in the prior art and to provide improved approaches for the effective delivery of therapeutic genetic material to airway epithelial cells, while maintaining a favorable safety profile.
  • ACE-tRNA-sLPVs Active nuclear import of ACE-tRNA encoding DNA in non-dividing cells is essential for successful PTC correction therapy. It was demonstrated that the ACE-tRNA-sLPVs can be functionalized with SV40, Tyl and hLa NLSs to support significant PTC suppression in nondividing cells without marked activation of the cGAS-STING pathway. It was hypothesized that efficient ACE-tRNA-sLPV trafficking to the nucleus will support robust PTC suppression and minimize toxicity. The goal is to identify NLSs, nucleolar localization sequences (NoLSs) and DNA nuclear targeting sequences (DTSs) that provide superior ACE-tRNA-sLPV localization for maximized PTC suppression in non-dividing cells.
  • NoLSs nucleolar localization sequences
  • DTSs DNA nuclear targeting sequences
  • TREs transcription response elements
  • SV40 enhancer sequence Transcription factors that are actively trafficked to the nucleus bind TREs of cytoplasmic DNA and drag them into the nucleus through nuclear pore complexes (NPCs).
  • NPCs nuclear pore complexes
  • SV40, NFkB and 325 unique TREs identified by SELEX and ChIP sequencing (Jolma, A. et al. Cell 152, 327-339 (2013) and recently used for generation of synthetic promoters, are screen for DTS function with ACE- tRNAs.
  • Identified TREs are short sequences ranging from 6-23 nucleotides that were concatenated with 3x end-to-end and ordered as complimentary oligos from IDT (below the 90nt cutoff for standard oligos).
  • the dsDNA was Golden-Gate cloned into a positive/negative Type IIS restriction enzyme MCS in the backbone of the mini pUC57 plasmid that encodes ptACE-tRNA Leu uGA. It was demonstrated this cloning strategy to result in near 100% cloning efficiency of thousands of oligos, with errors arising solely from oligo synthesis.
  • 3xTRE-ACE-tRNA plasmids were transfected into dividing and non-dividing (aphi dicolin, 5mg/ml) SGG I6HBE0- cells as previously described in 96-well plates.
  • Rescued Nluc-PTC expression was quantified by plate-reader luminometry 24 later, with higher luminescence indicating active DNA trafficking to the nucleus.
  • the TREs that support the highest PTC suppression in non-dividing cells were repeated with a dose-response (0.75 to 40ng) in non-dividing SGG 16HBEo-cells.
  • the top 10 performing TREs that support maximum PTC suppression at the lowest DNA cone were generated as closed-end dsDNA by ligated oligos with a single azide modification on the 5’ end and clicked to the ptLeu-sLPV as outlined in Figs. 11-12.
  • ptLeu-sLPV-DTSs were transfected into dividing and non-dividing SGG I6HBE0- cells with a dose-response (0.75 to 40ng) in 96 well plates to identify the DTS that supports the highest maximal PTC suppression at the lowest DNA cone.
  • a cGAMP ELISA was performed to determine if DTS assisted nuclear targeting reduces the GAS-STING cellular response, as observed with hLa NLS (Fig. 17).
  • SV40-, Tyl- and hLa-NLSs enhance ptLeu-sLPV- dependent PTC suppression.
  • These three NLSs were the first sequences that were investigated, and it was hypothesize that other NLS (and/or NoLS) exist that support more potent ptLeu- sLPV nuclear trafficking.
  • the hLa-NLS was highly potent in supporting qptLeu-sLPV PTC suppression in non-dividing cells.
  • hLa protein binds to the 3’ ends of precursor tRNAs to protect from exonuclease digestion, and the hLa-NLS localizes to RNAs in both the nucleus and nucleolus.
  • NoLSs sequences that may exhibit further improved qptLeu-sLPV function
  • the Nucleolar localization sequence Detector prediction software (NoD) was used to identify 27 “strong” NoLSs in tRNA transcription protein complexes (PolIII, TFIIIB, TFIIIC) (Scott, M. S., et al. BMC Bioinformatics 12, 317 (2011); Scott, M. S., et al. Nucleic Acids Research 38, 7388-7399 (2010)).
  • NoLSs Nucleolar localization sequence Detector prediction software
  • NbALFA-NLS protein in a high throughput manner
  • a high throughput cloning MCS at the 3 ’ end of the NbALFA was generated to clone in NLS DNA sequence with high efficiency.
  • the NbALFA-NLS are expressed in E. coll with a N-terminal PelB signal sequence to co-translationally translocate the NbALFA-NLS to the periplasm, where it is subsequently removed by a peptidase.
  • Nbs fold well in the periplasm and allow for rapid purification with limited steps using a N-terminal 6xHIS tag.
  • Fig. 13 shows CB stain of NbALFA protein with no NLS, scrambled and hLa NLS (Fig.
  • the top performing sequences (—20) were used to synthesize peptides for direct conjugation to qptLeu-sLPV, where a dose response study in 16HBE14o- cells that express a stably integrated nanoluciferase reporter containing a PTC, will be performed, and also tested for reduced generation of cGAMP. It was found that NoLS/NLS peptides from the screen support efficient nuclear transport of ptLeu-sLPV that is superior to hLa. A head-to-head comparison of top performing DTSs, NoLSs and NLSs following electroporation was performed in lungs of Stop-Go-Gio PTC reporter mice and PTC reporter 16HBE14o- cells.
  • PTC suppression efficiency in Stop-Go-Glow PTC reporter mice was used as the main readout for function by developing electroporation-mediated gene transfer technologies for alveoli in the deep lung to enhance treatment for acute lung injury/acute respiratory distress syndrome (ALLARDS).
  • ALLARDS acute lung injury/acute respiratory distress syndrome
  • This approach is also a highly effective for airway gene delivery.
  • the method is safe, rapid, and highly effective for gene delivery throughout all cell types in the lung of the mouse, rat, and pig.
  • Safety is the major concern for lung electroporation. The idea of passing an electric field across the chest may be fine for a mouse, but some feel that a person is different.
  • Electroporation-mediated gene transfer technology was adopted here to test how the different nuclear targeting moieties in Stop-Go-Glow PTC reporter mice. The top 2 performing DTS, NoLS and NLS sequences were compared head-to-head with ptLeu-sLPV-hLa.
  • IVIS IVIS imaging of the chest of intact Stop-Go-Glow PTC reporter mice.
  • excised lungs and lung lysate was analyzed by IVIS and the left or right lobe was inflated and fixed for ptLeu-sLPVs-AF647 imaging, and mNeonGreen expression was recovered with HA antibodies and with DAPI.
  • Hematoxylin and eosin (H&E) histological analyses are performed to detect changes in lung morphology. The other lobe is homogenized for analysis of Nluc luminescence. A more comprehensive analysis of cell-type delivery is conducted using the top-performing constructs.
  • PTC premature termination codon
  • the top-performing construct is used for PACE NP delivery to SGG and W1282X-CFTR mouse lungs upon its identification.
  • Timepoints shorter or longer
  • DNA concentrations typically lower are adjusted to distinguish differences in PTC suppression efficacy among various nuclear localization moieties.
  • ACE-tRNA dependent suppression of NTCs using Ribo-seq was previously described.
  • total cellular RNA is isolated and subjected to RNAse treatment.
  • the ribosomes remain bound to the RNA, and protect 26 to 34nt footprints that can then be gel isolated and sequenced to determine their location on each transcript across the entire transcriptome.
  • cDNA plasmids encoding ACE-tRNA Gln uAA, - tRNA Glu uAG, -tRNA ⁇ uGA, -tRNA Gly uGA and -tRNA Trp uGA were transfected into HEK293 cells, and after 48hrs, the cellular RNA was subjected to Ribo-seq to determine if ribosome occupancy on the 3’ untranslated region (UTR) was higher in the presence of ACE-tRNAs compared to scrambled control.
  • the amount of 3’UTR occupancy of ribosomes was nominal, indicating that ACE-tRNAs do not significantly suppress NTCs under conditions that significantly suppress PTCs, indicating a therapeutic window.
  • nonsense codon (NTC) readthrough of all transcripts is assessed.
  • Freshly dissected sections from the left lung lobe ( ⁇ 30 mg) of wild-type (WT) mice are collected at 4-7 days post-treatment to generate ribosomal footprint mRNA libraries. While lung tissue permits isolation of total RNA via trituration in lysis buffer, obtaining high-quality RNA remains challenging.
  • Sequencing reads are aligned to the longest transcript variant of each mouse gene/transcript, as annotated in the UCSC RefSeq GRCm38 reference genome.
  • the disclosed picovector technology is expected to exhibit durability that correlates with the half-life of the target cells.
  • airway epithelial cells are known to have extended turnover times, with reported half-lives of over six months in the trachea and approximately 17 months in the bronchioles in murine models, and approximately 50 days in humans.
  • the picovector platform is anticipated to require infrequent redosing, potentially less than once per month in human patients, thereby enhancing patient compliance and reducing treatment burden.
  • Nanoparticles (NPs) formulated using poly(amine-co-ester) (PACE) polymers represent advantageous vehicles for the delivery of nucleic acids. These polymers exhibit several favorable characteristics, including biocompatibility, biodegradability, and structural versatility, which allow for chemical customization to optimize delivery performance. Additionally, PACE polymers possess a mild cationic charge, which facilitates electrostatic interaction with negatively charged nucleic acid cargo, thereby enhancing encapsulation and delivery efficiency.
  • PACE polymers were synthesized in small batches via enzymatic copolymerization of diesters with amino-substituted diols. Terpolymers have three component monomers: a lactone that confers hydrophobicity, NP stability and reduces cytotoxicity, an amino-diol that confers a mildly cationic charge, and an ester that enables additional hydrophobic control. Higher MW polymers are efficient in condensing nucleic acids, whereas hydrophobic domains enhance vehicle stability and facilitate cargo release. A set of principles that guide optimization for nucleic acid payloads (DNA and RNA) was already identified by variation of molecular weight (MW) and hydrophobicity to focus the screening process. Further, an advantage of PACE compared to other cationic polymers is that the mild cationic charge improves association with cells and promotes endosomal escape without causing toxicity.
  • MW molecular weight
  • the present disclosure provides formulations that efficiently encapsulate and deliver ptLeu-sLPV-hLa to the airways in vivo.
  • These polymer-based delivery systems are optimized for targeted pulmonary administration in murine models, with the ultimate objective of developing a viable therapeutic for cystic fibrosis (CF) and other airway diseases caused by nonsense mutations.
  • CF cystic fibrosis
  • Cre mRNA was delivered directly to the airway via IT instillation using a PACE formulation optimized for airway delivery (PACE-Lung).
  • PACE-Lung a PACE formulation optimized for airway delivery
  • IT delivery of Cre mRNA resulted in -10% expression in bulk lung and -30% expression in cells from bronchoalveolar lavage fluid (BALF) of treated animals.
  • BALF bronchoalveolar lavage fluid
  • Efficient delivery of firefly luciferase (Flue) mRNA to the lung was achieved using PACE polymers, which they assessed by Flue luminescence.
  • ptLeu-sLPV-hLa (w/ AF647) is encapsulated into 20 unique polymeric NPs composed of PACE polymers designed for IT delivery.
  • NPs are formulated with ptLeu-sLPV-hLa- AF647 and characterized (size, morphology, surface charge, loading, release kinetics). Unloaded NPs are included as negative controls.
  • Antibodies (Abs) used for co-staining include keratin 5 (basal cells in submucosal glands), acetylated-tubulin and FoxJl (ciliated or non-ciliated airway epithelial cells depending on staining), nerve growth factor receptor, keratin 14, p63 (basal cells in airways), and Muc5AC (goblet cells), Foxll lonocytes, EpCAM+ epithelial cells, CD31+ endothelial cells, and CD45+ leukocytes.
  • NP encapsulated ptLeu-sLPV- hLa and scrambled control are tested for safety and efficacy in SGG mice. Safety is assessed by standard serum chemistry analysis (including AST, ALT, Aik Phos, T.
  • Bilirubin, BUN, Creatinine), serum and bronchoalveolar lavage (BAL) cytokine analyses including IL-6, IL- la, MIP-1 a, GM-CSF, RANTES, KC, G-CSF
  • cytokine analyses including IL-6, IL- la, MIP-1 a, GM-CSF, RANTES, KC, G-CSF
  • histopathological examination of organs brain, heart, lung, liver, spleen, kidney
  • Transfection and PTC suppression efficiency of ptLeu-sLPV-hLa in live mice, ex vivo lungs, and homogenized lung tissue is assessed for Nluc luminescence by IVIS luminometry.
  • NP delivery is assessed by flow cytometry and microscopy as described above in E2.1, and transfection efficiency and ACE- tRNA PTC suppression activity is and quantified by luminescence of homogenized lung tissue. Safety is assessed as described above.
  • NP delivery is assessed by flow cytometry and microscopy as described above, with transfection efficiency and ACE-tRNA activity assessed and quantified by luminescence of homogenized lung tissue.
  • Safety studies are performed as described above, 24 hours after the 2 nd dose.
  • W1282X-CFTR mouse models are valuable tools for evaluating premature termination codon (PTC)-targeted therapeutics, as the observed alterations in CFTR transcripts closely mirror those in human patients.
  • PTC premature termination codon
  • the strategy is further evaluated in the G542X-CF rat model or in a genetically engineered CF ferret model harboring a relevant nonsense mutation.
  • intratracheal (IT) administration of PACE nanoparticle (NP) formulations has been shown to exhibit similar delivery profiles in both murine and large animal models, supporting translational relevance of the proposed delivery system.
  • the optimal PACE NP formulation and dosing regimen identified above are utilized to administer optLeu-sLPV-hLa-AF647 and a scrambled control formulation to the lungs of 3- week-old homozygous CFTR W1282X mice.
  • Post-administration evaluations are conducted at 7, 14, and 21 days. Given that only approximately 40% of homozygous CFTR W1282X mice survive beyond 40 days of age due to intestinal obstruction, the study is initiated with sufficiently large cohorts to ensure statistical robustness at each time point. Specifically, 16 mice are included in the 21-day cohort, and 12 mice each is included in the 7-day and 14-day cohorts, in order to achieve a minimum of six surviving mice per endpoint.
  • Group sizes are calculated to ensure statistical confidence in detecting a 10% rescue of endpoints, including CFTR mRNA expression and function.
  • the proposed sample sizes are determined to achieve 80% power to detect differences in the range of 10 to 16, depending on the number of mice available at each time point. Both male and female mice are utilized to satisfy the required sample numbers.
  • tracheas are excised, longitudinally bisected, and subjected to Ussing chamber measurements according to established protocols.
  • the change in CFTR short-circuit current is determined following the sequential basolateral administration of forskolin and IBMX (F&I), followed by CFTR inhibition using Inhl72 (Fig. 14).
  • CFTR activity is also measured in intestinal segments (duodenum, ileum, jejunum, colon) harvested from the same animals, serving as negative controls, wherein CFTR function is not expected.
  • a correction exceeding 10% in short-circuit current is predicted in airway tissues obtained from mice administered ptLeu-sLPV-hLa, whereas no such correction is anticipated in mice receiving a scramble control.
  • the right lung lobe is perfused, inflated with agarose, and embedded in OCT compound for preparation of frozen sections.
  • the left lung lobe is divided into upper, middle, and lower segments. These segments are allocated across the cohort for one of the following purposes: (1) flash freezing for subsequent Western blot protein analysis, or (2) immediate RNA extraction for quantitative reverse transcription PCR (qRT-PCR) or digital droplet PCR (ddPCR) analysis of CFTR mRNA levels.
  • qRT-PCR quantitative reverse transcription PCR
  • ddPCR digital droplet PCR
  • CFTR mRNA is quantified using quantitative reverse transcription PCR (qRT-PCR) or digital droplet PCR (ddPCR).
  • qRT-PCR quantitative reverse transcription PCR
  • ddPCR digital droplet PCR
  • Tissue sections from the right lung lobe are excised at three anatomical levels — top, middle, and bottom — and imaged by confocal microscopy to detect optLeu-sLPV-hLa-AF647 and DAPI fluorescence. Quantification is conducted by determining the number of AF647-positive cells relative to the total number of cells across 10 sections per anatomical level. Automated image analysis is performed using MATLAB-based software developed by the Piotrowski- Daspti laboratory.
  • H&E hematoxylin and eosin
  • the therapeutic platform combining ACE-tRNAs and PACE nanoparticles (NPs) resulted in the restoration of greater than 10% of normal CFTR function in W1282X CF mice.
  • Picovectors containing NLS signals for enhanced nuclear entry
  • NLS sequences Three previously characterized, but structurally distinct, nuclear localization signal (NLS) sequences — SV40, Tyl, and hLa — were evaluated alongside a scrambled hLa peptide (Scram) as a negative control. Notably, all tested NLS sequences were found to significantly enhance PTC suppression in aphidicolin-treated PTC reporter 16HBE14o- cells. Among the sequences tested, hLa demonstrated the highest level of enhancement, exhibiting a 3.5-fold increase in suppression activity relative to the Scram control.
  • the 5 ’-conjugated nuclear localization signal (NLS) sequences were subsequently paired with the ptACE-tRNA Leu uGA-sLPVs (also referred to as “optLeu-sLPVs”), and this pairing was found to significantly enhance premature termination codon (PTC) suppression in aphidicolin-treated PTC reporter 16HBE14o- cells (Fig. 16).
  • PTC premature termination codon
  • cytoplasmic double-stranded DNA (dsDNA) is known to activate the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) innate immune pathway — a recognized concern for therapeutic DNA delivery to airway cells — it was necessary to evaluate whether the disclosed technologies induced such activation.
  • cyclic GMP- AMP (cGAMP) levels were assessed via enzyme-linked immunosorbent assays (ELISA; ThermoFisher) in HeLa cells following transfection with various DNA platforms, including CFTR and ACE-tRNA plasmid cDNA (pcDNA), ACE-tRNA- sLPV, and optLeu-sLPV-hLa constructs (Fig. 17).
  • Oligonucleotides were synthesized by Integrated DNA Technologies (IDT) with 5’ phosphorylation modifications. Each oligonucleotide comprising a hairpin end was synthesized with an internal PEG4-DBCO modification in the loop and a 5’ phosphorylation modification. All oligonucleotides were resuspended to a concentration of 100 pM in TE buffer (Fisher Scientific).
  • Azide-containing NLS peptides (Genscript) or Alexa Fluor fluorescent dyes (Thermo Fisher Scientific) were resuspended to 1 mM in either molecular biology-grade water (for peptides) or DMSO (for Alexa Fluor dyes). Labeling reactions were conducted individually for each oligonucleotide hairpin end. Atypical labeling reaction consisted of 15 pL DBCO-hairpin (200 pmol hairpin oligonucleotide at a final concentration of 1.33 pM in the reaction), 30 pL azide-label (4 nmol azide-label at a final concentration of 26.7 pM in the reaction), and 105 pL water. Reactions were incubated at room temperature in the dark for 48 hours.
  • Oligonucleotides comprising the ACE-tRNA cargo were annealed in 1 * IDT annealing buffer (100 mM potassium acetate, 30 mM HEPES, pH 7.5), with each oligonucleotide present at a final concentration of 10 pM in the annealing mixture. The mixture was heated to 95 °C for 5 minutes and then cooled to 4 °C over 30 minutes using a thermocycler.
  • IDT annealing buffer 100 mM potassium acetate, 30 mM HEPES, pH 7.5
  • the labeled hairpin ends were annealed to the core ACE-tRNA assembly by mixing 30 pL of each labeled hairpin with 30 pL of the annealed core ACE-tRNA.
  • the complete linear picovector annealing mixture was heated to 65 °C for 5 minutes and then cooled to 4 °C over 20 minutes in a thermocycler.
  • a typical linear picovector assembly reaction consisted of 90 pL of the annealed oligonucleotide mixture [30 pL of each labeled hairpin (133 nM each, final concentration in the ligation reaction) and 30 pL of annealed core ACE-tRNA (1 pM, final concentration in the ligation reaction)], 30 pL T4 DNA ligase buffer (New England Biolabs), 6 pL T4 DNA ligase (2400 units; NEB), and 174 pL water. The ligation reaction was incubated at room temperature in the dark for 16 hours.
  • GGGPKKKRKVED (SEQ ID NO: 3)
  • GGPSEVANKEPQQTAEGKEGKTRAKRDEDQ (SEQ ID NO: 10)

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Abstract

La présente divulgation concerne des compositions et des méthodes de traitement de troubles génétiques causés par des mutations non-sens à l'aide de constituants d'ARN de transfert à modification d'anticodon (ACE-ARNt). Ces constituants d'ACE-ARNt à base d'ADN sont conçues pour supprimer des codons de terminaison prématurés (PTC) et pour restaurer l'expression de protéines fonctionnelles pleine longueur. La divulgation concerne en outre des formulations de constituants d'ACE-ARNt comportant des nanoparticules polymères de poly(amine-co-ester) (PACE) pour améliorer la stabilité, pour protéger des acides nucléiques et pour améliorer l'administration aux cellules épithéliales des voies respiratoires. La divulgation concerne également des picovecteurs d'ACE-ARNt fonctionnalisés (sLPV) incorporant des éléments de ciblage tels que des signaux de localisation nucléaire (NLS), des séquences de localisation nucléolaire (NoLS), et des séquences de ciblage nucléaire d'ADN (DTS) pour améliorer l'importation et la localisation nucléaires.
PCT/US2025/034736 2024-06-24 2025-06-23 Fonctionnalisation d'ace-arnt codant pour des picovecteurs linéaires synthétiques Pending WO2026006151A2 (fr)

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