WO2020051555A1 - Rna and dna base editing via engneered adar recruitment - Google Patents

Rna and dna base editing via engneered adar recruitment Download PDF

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Publication number
WO2020051555A1
WO2020051555A1 PCT/US2019/050095 US2019050095W WO2020051555A1 WO 2020051555 A1 WO2020051555 A1 WO 2020051555A1 US 2019050095 W US2019050095 W US 2019050095W WO 2020051555 A1 WO2020051555 A1 WO 2020051555A1
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Prior art keywords
domain
rna
nucleic acid
vector
sequence
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PCT/US2019/050095
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English (en)
French (fr)
Inventor
Prashant MALI
Dhruva KATREKAR
Dario Meluzzi
Genghao CHEN
Kyle M. FORD
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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Priority to EP19857915.3A priority Critical patent/EP3847255A4/en
Priority to CN201980072798.1A priority patent/CN112996912A/zh
Priority to AU2019336793A priority patent/AU2019336793B2/en
Priority to GB2104889.7A priority patent/GB2590880A/en
Priority to CA3110998A priority patent/CA3110998A1/en
Priority to CN202410647165.1A priority patent/CN119193702A/zh
Application filed by University of California Berkeley, University of California San Diego UCSD filed Critical University of California Berkeley
Priority to EP23214540.9A priority patent/EP4332224A3/en
Priority to US17/273,885 priority patent/US20220010333A1/en
Priority to JP2021511542A priority patent/JP7720623B6/ja
Publication of WO2020051555A1 publication Critical patent/WO2020051555A1/en
Anticipated expiration legal-status Critical
Priority to US19/261,672 priority patent/US20260092292A1/en
Ceased legal-status Critical Current

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Definitions

  • the third domain sequence can comprise an RNA editing entity recruiting domain encoding sequence that forms a secondary structure having a two dimensional shape that can be substantially a cruciform.
  • at least one base of the non-naturally occurring RNA can comprise a chemical modification.
  • at least one sugar of the non-naturally occurring RNA can comprise a chemical modification.
  • a kit can comprise the non-naturally occurring RNA in a container.
  • the kit can further comprise a syringe.
  • the container can be the syringe.
  • an isolated cell can comprise the non-naturally occurring RNA.
  • a pharmaceutical composition can comprise the non-naturally occurring RNA in unit dose form.
  • the ADAR protein can be a human ADAR protein. In some cases, the ADAR protein can be a recombinant ADAR protein. In some cases, the ADAR protein can be a modified ADAR protein.
  • the nucleic acid can be chemically synthesized. In some cases, the nucleic acid can be genetically encoded. In some cases, a kit can comprise the nucleic acid in a container. In some cases, the kit can further comprise a syringe. In some cases, the container can be the syringe. In some cases, an isolated cell can comprise the nucleic acid. In some cases, a pharmaceutical composition can comprise the nucleic acid in unit dose form. In some cases, the
  • FIG. 1 is a schematic showing endogenous recruitment of ADARs.
  • FIG. 3 shows the results of in vitro and in vivo screening of exemplary adRNAs.
  • FIG. 10 shows engineering next-gen adRNAs with enhanced ADAR1 and ADAR2 recruitment potential.
  • the first series of columns show relative activity employing no adRNA.
  • the second series of columns show relative activity employing a construct comprising a GluR2 domain.
  • the third series of columns show relative activity employing a construct comprising an Alu domain associated with two cruciform structures.
  • FIG. 11A-D shows different construct designs.
  • FIG. 11A exemplifies an antisense domain linked to two GluR2 domains.
  • FIG. 11B exemplifies an anti-sense domain only.
  • FIG. 36C Analysis of RNA editing yields across a spectrum of endogenous targets chosen to cover a range of expression levels.
  • FIG. 47 is a schematic showing exon skipping via creation of a splice acceptor and/or branch point mutation.
  • the vector can comprise a nucleic acid with a polynucleotide sequence encoding for at least one RNA editing entity recruiting domain.
  • the polynucleotide sequence may not form a secondary structure comprising a stem-loop.
  • the polynucleotide sequence can form one or more stem-loops.
  • the polynucleotide sequence can form a secondary structure comprising a cruciform.
  • the polynucleotide sequence can form a secondary structure that can be substantially linear.
  • a polynucleotide sequence can encode for more than one RNA editing recruiting domains.
  • a polynucleotide sequence can encode for a plurality of recruiting domains.
  • a polynucleotide sequence can encode for 2, 3, 4, 5, 6 or more recruiting domains.
  • a recruiting domain of a plurality can include an Alu domain, an APOBEC domain, a GluR2 domain, Casl3 domain, or any combination thereof.
  • the Alu domain, APOBEC domain, Casl3 domain, or GluR2 domain can be a naturally occurring recruiting domain.
  • the term“about,” as used herein can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which can depend in part on how the value is measured or determined, e.g., the limitations of the measurement system.
  • “about” can mean plus or minus 10%, per the practice in the art.
  • “about” can mean a range of plus or minus 20%, plus or minus 10%, plus or minus 5%, or plus or minus 1% of a given value.
  • the term can mean within an order of magnitude, within 5-fold, or within 2-fold, of a value.
  • adenosine deaminases acting on RNA can refer to an adenosine deaminase that can convert adenosines (A) to inosines (I) in an RNA sequence.
  • ADAR1 and ADAR2 are two exemplary species of ADAR that are involved in mRNA editing in vivo. Non-limiting exemplary sequences for ADAR1 can be found under the following reference numbers: HGNC: 225; Entrez Gene: 103; Ensembl: ENSG
  • Glur2 mRNA as used herein can refer to the mRNA encoding ionotropic AMPA glutamate receptor 2 (“Glur2”) which undergoes adenosine to inosine (A -> I) editing. This mRNA recruits ADARs in a site specific manner.
  • Glur2 ionotropic AMPA glutamate receptor 2
  • oligonucleotide refers to a structure formed in single stranded oligonucleotide when sequences within the single strand which are complementary when read in opposite directions base pair to form a region whose conformation resembles a hairpin or loop.
  • “Hybridization” can refer to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding can occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner.
  • the complex can comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self- hybridizing strand, or any combination of these.
  • a hybridization reaction can constitute a step in a more extensive process, such as the initiation of a PC reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.
  • a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
  • the term also can refer to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
  • the term“vector” can refer to a nucleic acid construct deigned for transfer between different hosts, including but not limited to a plasmid, a virus, a cosmid, a phage, a BAC, a YAC, etc.
  • A“viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
  • plasmid vectors can be prepared from commercially available vectors.
  • viral vectors can be produced from baculoviruses, retroviruses, adenoviruses, AAVs, etc.
  • the pharmaceutical compositions for the administration of the AdRNA can be conveniently presented in dosage unit form and can be prepared by any of the methods well known in the art of pharmacy.
  • the pharmaceutical compositions can be, for example, prepared by uniformly and intimately bringing the compounds provided herein into association with a liquid carrier, a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation.
  • the compound provided herein is included in an amount sufficient to produce the desired therapeutic effect.
  • the recruiting domain may not comprise an APOBEC domain.
  • the recruiting domain may not comprise a C as 13 domain.
  • a nucleic acid can comprise between 20 and several hundred nucleotides.
  • longer targeting portions provide more specificity for the target site of the RNA sequence to be edited, less off-target effects due to unintentional (off-target) binding as well as more room to create secondary structures, such as stem-loop structures, cruciforms, toe hold structures, within the targeting portion itself, mismatches or wobble-bases (due to mismatches with one or more of the complementary base(s) in the targeted RNA sequence at or near the site to be edited), and so forth.
  • targeting portions can be complementary to the target RNA sequence over the entire length of the targeting portion except for the mismatch opposite the nucleotide to be edited, and optionally one or two wobble bases.
  • An RNA editing entity recruiting domain can comprise a structure that can be substantially a toehold.
  • An RNA editing entity can comprise one or more mismatch bulges.
  • an excipient can comprise a sweetener.
  • suitable sweeteners can include glucose (com syrup), dextrose, invert sugar, fructose, and mixtures thereof (when not used as a carrier); saccharin and its various salts such as a sodium salt; dipeptide sweeteners such as aspartame; dihydrochalcone compounds, glycyrrhizin; Stevia Rebaudiana (Stevioside); chloro derivatives of sucrose such as sucralose; and sugar alcohols such as sorbitol, mannitol, sylitol, and the like.
  • the engineered adRNA of this disclosure further comprises, or alternatively consists essentially of, or yet further consists of an ADAR2 recruiting domain derived from GluR2 mRNA.
  • the engineered adRNA of this disclosure further comprises, or alternatively consists essentially of, or yet further consists of ADAR1 recruiting domain derived from Alu repeats.
  • the engineered adRNA of this disclosure further comprises, or alternatively consists essentially of, or yet further consists of two MS2 hairpins flanking the sequence complementary to a target RNA.
  • an engineered adRNA wherein the ADAR2 recruiting domain of the engineered adRNA derived from GluR2 mRNA is located at the 5’ end or the 3’ end of the engineered adRNA.
  • the GluR2 mRNA is located at both the 5’end and the 3’ end of the engineered adRNA.
  • Example 4 in vivo RNA editing of point mutations via RNA-guided adenosine deaminases.
  • a similar contingency matrix was used for each selected reference T-site, except that G was replaced with C in the above definitions.
  • the p-values calculated for all selected reference sites and for a given comparison of samples were adjusted for multiple testing using the Benjamini-Hochberg method.
  • A-sites and T-sites with adjusted p-values less than a false discovery rate (FDR) of 1% and with a fold change of at least 1.1 in editing yield were deemed to have a significant change in A-to-G editing yield on forward and reverse transcripts, respectively.
  • the counts of these sites for each comparison of samples are shown as /V S ig in FIG. 38 - FIG. 42, and are reported under the column“changed sites” in Table 5.
  • ADAR2 e.g . Stafforst, Zhang, and Rosenthal labs
  • Casl3d Casl3d based inhibition of splicing
  • genes involved in cancer pathways harbor single amino acid substitution. Creation of dominant negative mutants, constitutionally active mutants and catalytically inactive mutants is possible by creating A->G substitutions in the mRNA sequences of these genes. Some of these genes include KRAS, HRAS, JAK2, GSK3 , b-catenin, SmoM2, Caspase3, Caspase 8, TGF-b, p53.
  • ADARs have been shown to edit double stranded RNA as well as both strands of a DNA-RNA hybrid, it is possible to recruit ADARs via single stranded DNA or DNA- RNA hybrids to edit both DNA and RNA. This can be used to modify the current adenine base editing approach to Cas9 (or Cpfl)-ADAR-deaminase domain fusions (ADAR1,
  • compositions, methods and systems are disclosed herein. Specific exemplary embodiments of these compositions, methods and systems are disclosed below.
  • Embodiment 1 An engineered AD AR1 or ADAR2 guide RNA (“adRNA”) comprising: a sequence complementary to a target RNA.
  • adRNA engineered AD AR1 or ADAR2 guide RNA
  • Embodiment 16 The vector of embodiment 15, wherein the vector is selected from a group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno- associated viral vector.
  • Embodiment 25 The method of embodiment 23 or 24, wherein the subject is an animal.
  • Embodiment 6 An engineered ADAR2 guide RNA (“adRNA”) encoded by a sequence selected from the group of sequences provided in TABLE 1 or FIG. 2.
  • adRNA engineered ADAR2 guide RNA
  • Embodiment 1 An ADAR system for exon skipping comprising an adRNA targeting a splice acceptor and/or a branch point in an intron and, optionally, an ADAR enzyme.
  • Embodiment 2. The ADAR system of claim 1, wherein the ADAR enzyme is ADAR1, ADAR2, or a mutant or variant each thereof.
  • Embodiment 7 The method of claim 6, wherein the disease, disorder, or condition is selected from Duchenne muscular dystrophy or ornithine transcarbamylase deficiency.

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  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Nitrogen And Oxygen As The Only Ring Hetero Atoms (AREA)
  • Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)
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AU2019336793A AU2019336793B2 (en) 2018-09-06 2019-09-06 RNA and DNA base editing via engineered ADAR recruitment
GB2104889.7A GB2590880A (en) 2018-09-06 2019-09-06 RNA and DNA base editing via engineered ADAR recruitment
CA3110998A CA3110998A1 (en) 2018-09-06 2019-09-06 Rna and dna base editing via engneered adar recruitment
EP19857915.3A EP3847255A4 (en) 2018-09-06 2019-09-06 Rna and dna base editing via engineered adar recruitment
JP2021511542A JP7720623B6 (ja) 2018-09-06 2019-09-06 遺伝子操作されたａｄａｒのリクルートを介したｒｎａ及びｄｎａ塩基の編集
CN202410647165.1A CN119193702A (zh) 2018-09-06 2019-09-06 经由改造的adar募集的rna和dna碱基编辑
US17/273,885 US20220010333A1 (en) 2018-09-06 2019-09-06 Rna and dna base editing via engineered adar recruitment
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WO2022078995A1 (en) * 2020-10-12 2022-04-21 Eberhard Karls Universität Tübingen Artificial nucleic acids for rna editing
WO2022103852A1 (en) * 2020-11-11 2022-05-19 Shape Therapeutics Inc. Rna-editing compositions and methods of use
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JP2023040852A (ja) * 2021-09-10 2023-03-23 隆光 矢野 －１フレームシフトを誘導するための１本鎖核酸分子及び組成物
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WO2023150784A3 (en) * 2022-02-07 2023-08-31 The Regents Of The University Of California Method of interfering with repetitive rna
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EP4074825A4 (en) * 2019-12-09 2025-01-22 Astellas Pharma Inc. ANTISENSE GUIDE RNA WITH ADDED FUNCTIONAL REGION FOR TARGET RNA EDITING
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WO2021113264A1 (en) * 2019-12-02 2021-06-10 The Regents Of The University Of California Engineering circular guide rnas
US11827880B2 (en) 2019-12-02 2023-11-28 Shape Therapeutics Inc. Therapeutic editing
EP4074825A4 (en) * 2019-12-09 2025-01-22 Astellas Pharma Inc. ANTISENSE GUIDE RNA WITH ADDED FUNCTIONAL REGION FOR TARGET RNA EDITING
WO2021216853A1 (en) * 2020-04-22 2021-10-28 Shape Therapeutics Inc. Compositions and methods using snrna components
WO2021242870A1 (en) * 2020-05-26 2021-12-02 Shape Therapeutics Inc. Compositions and methods for genome editing
WO2021242778A1 (en) * 2020-05-26 2021-12-02 Shape Therapeutics Inc. Methods and compositions relating to engineered guide systems for adenosine deaminase acting on rna editing
WO2021242889A1 (en) * 2020-05-26 2021-12-02 Shape Therapeutics Inc. Engineered circular polynucleotides
JP2023528039A (ja) * 2020-05-26 2023-07-03 シェイプ セラピューティクス インコーポレイテッド 改変環状ポリヌクレオチド
WO2022007803A1 (zh) * 2020-07-06 2022-01-13 博雅辑因（北京）生物科技有限公司 一种改善的rna编辑方法
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WO2022046667A1 (en) * 2020-08-24 2022-03-03 Wave Life Sciences Ltd. Cells and non-human animals engineered to express adar1 and uses thereof
WO2022078995A1 (en) * 2020-10-12 2022-04-21 Eberhard Karls Universität Tübingen Artificial nucleic acids for rna editing
WO2022078569A1 (en) * 2020-10-12 2022-04-21 Eberhard Karls Universität Tübingen Artificial nucleic acids for rna editing
JP2023552037A (ja) * 2020-10-12 2023-12-14 エバーハルト カール ウニヴェルジテート テュービンゲン Ｒｎａ編集のための人工核酸
WO2022103852A1 (en) * 2020-11-11 2022-05-19 Shape Therapeutics Inc. Rna-editing compositions and methods of use
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WO2022138929A1 (ja) * 2020-12-25 2022-06-30 アステラス製薬株式会社 標的ｒｎａのポリアデニル化シグナル配列を編集するためのガイドｒｎａ
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US12152239B2 (en) 2021-09-03 2024-11-26 Tacit Therapeutics, Inc. RNA editing via recruitment of spliceosome components
JP2023040852A (ja) * 2021-09-10 2023-03-23 隆光 矢野 －１フレームシフトを誘導するための１本鎖核酸分子及び組成物
WO2023101993A3 (en) * 2021-11-30 2023-07-27 Minghong Zhong Segmented nucleic acids
WO2023150784A3 (en) * 2022-02-07 2023-08-31 The Regents Of The University Of California Method of interfering with repetitive rna
WO2023196853A1 (en) * 2022-04-05 2023-10-12 Astellas Gene Therapies, Inc. Compositions and methods for the treatment of muscular dystrophies
WO2026080897A1 (en) * 2024-10-11 2026-04-16 Proqr Therapeutics Ii B.V. Antisense oligonucleotides for the treatment of chronic pain

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JP7720623B2 (ja) 2025-08-08
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EP3847255A1 (en) 2021-07-14
AU2019336793A1 (en) 2021-04-08
CN119193702A (zh) 2024-12-27
AU2019336793B2 (en) 2024-11-21
US20260092292A1 (en) 2026-04-02
GB202104889D0 (en) 2021-05-19
US20220010333A1 (en) 2022-01-13
EP4332224A3 (en) 2024-06-05
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GB2590880A (en) 2021-07-07
JP7720623B6 (ja) 2025-09-18
CN112996912A (zh) 2021-06-18

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