WO2022091100A1 - Polynucléotides pour l'édition d'arn et leurs méthodes d'utilisation - Google Patents

Polynucléotides pour l'édition d'arn et leurs méthodes d'utilisation Download PDF

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WO2022091100A1
WO2022091100A1 PCT/IL2021/051281 IL2021051281W WO2022091100A1 WO 2022091100 A1 WO2022091100 A1 WO 2022091100A1 IL 2021051281 W IL2021051281 W IL 2021051281W WO 2022091100 A1 WO2022091100 A1 WO 2022091100A1
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polynucleotide
editing
adenosine
nucleic acid
acid sequence
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PCT/IL2021/051281
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Schraga SCHWARTZ
Ronit NIR
Anna UZONYI
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Yeda Research And Development Co. Ltd.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense

Definitions

  • the present invention in some embodiments thereof, relates to polynucleotides for RNA editing and methods of using same.
  • RNA editing is a natural process through which eukaryotic cells alter the sequence of RNA molecules, often in a site-specific and precise way, thereby increasing the repertoire of genome encoded RNAs by several orders of magnitude.
  • RNA editing enzymes have been described for eukaryotic species throughout the animal and plant kingdoms, and these processes play an important role in managing cellular homeostasis in metazoans from the simplest life forms to humans.
  • RNA editing manipulates genetic information in a reversible and tunable manner making it a promising target for therapeutics enabling manipulations that are either lethal or quickly compensated when done at the genome level. Furthermore, RNA editing could be safer because potential adverse effects and off-target edits should be reversible and dose-dependent.
  • ADAR adenosine deaminase enzyme
  • RNA Adenosine-to-inosine editing in RNA diversifies the transcriptome by recoding of amino acid codons, Start codons and Stop codons, and by alteration of splicing, among other mechanisms [Nishikura et al. Nat. Rev. Mol. Cell Biol. 17, 83-96 (2016)].
  • ADAR is of intense interest from pathophysiological, bioengineering and therapeutic perspectives. Despite the intense interest in this enzyme, the fundamental rules determining which sites within the dsRNAs are to be edited and to what levels remain very poorly understood. To date, studies addressing these questions revealed a degenerate sequence preference, including a bias against a G or a C at the position immediately upstream of the edited site (Eggington et al., 2011), a slight enrichment for ‘G’ at the position following the edited site (Ouyang et al., 2018), and that adenosines in mismatched A-C pairs are preferably edited (Roth et al., 2019; Wong et al., 2001).
  • ADAR1 is known to edit long double- stranded RNAs with a strong secondary structure (Morse et al., 2002).
  • dsRNA with a few mismatches could be a better substrate than perfect double strands (Brummer et al., 2017; Solomon et al., 2017), and that RNA tertiary structure might play a role as well (Reenan, 2005; Tian et al., 2011).
  • Attempts to characterize the effect of structural variations on editing levels have been partially successful for individual sites, but could not provide a comprehensive, globally applicable model (Liu et al., 2019).
  • RNA editing Steering ADAR to specific sites at selected transcripts, a strategy called site-directed RNA editing, holds great promise for the treatment of disease and as a tool to study protein and RNA function.
  • An example comes with ‘A’s in stop codons (UGA, UAA, UAG) which, when edited, allow for read-through during translation; thus, diseases caused by mutations that introduce termination codon (PTCs) can be corrected by RNA editing.
  • a polynucleotide comprising a nucleic acid sequence having at least 70 % complementarity to a target RNA sequence comprising an adenosine, wherein the nucleic acid sequence comprises at least one complementarity mismatch with at least one nucleotide in the target RNA sequence located in at least one of nucleotides 25-40 upstream and/or downstream of the adenosine, such that upon hybridization of the nucleic acid sequence to the target RNA sequence a disruption of a double- stranded structure is formed at the at least one complementarity mismatch and wherein the nucleic acid sequence and the RNA sequence form double- stranded structures upstream and downstream of the disruption.
  • the disruption is a bulge.
  • the at least one complementarity mismatch comprises 1-10 nucleotides.
  • the at least one complementarity mismatch comprises 1-7 nucleotides.
  • the at least one complementarity mismatch comprises 1-4 nucleotides.
  • the nucleic acid sequence further comprises a complementary mismatch with the adenosine.
  • the complementary mismatch with the adenosine is a cytidine.
  • the at least 70 % complementarity is at least 80 % complementarity.
  • the at least 70 % complementarity is at least 90 % complementarity.
  • the nucleic acid sequence exhibits full complementarity to the target RNA sequence other than the complementarity mismatch.
  • the at least one of nucleotides 25-40 upstream is 25-35 upstream.
  • the at least one of nucleotides 25-35 upstream is nucleotide 30 upstream.
  • the at least one of nucleotides 25-40 downstream is 30-40 downstream. According to some embodiments of the invention, the at least one of nucleotides 30-40 downstream is nucleotide 35 downstream.
  • the nucleic acid sequence is at least 100 nucleotides long.
  • the nucleic acid sequence is up to 250 nucleotides long.
  • the nucleic acid sequence is about 150 nucleotides long.
  • the nucleic acid sequence comprises at least 50 nucleotides upstream and downstream of a nucleotide located opposite the adenosine upon the hybridization.
  • the nucleic acid sequence comprises at least 60 nucleotides upstream and downstream of a nucleotide located opposite the adenosine upon the hybridization.
  • the adenosine of the target RNA is comprised in a CAG sequence.
  • the adenosine is not comprised in a GA or GAG sequence.
  • the target RNA sequence is part of a polynucleotide associated with a disease that can benefit from editing the adenosine by ADAR.
  • the adenosine introduces an in-frame premature stop codon.
  • the adenosine introduces or removes a splice site.
  • the adenosine alters a codon.
  • the polynucleotide comprising an additional nucleic acid sequence encoding ADAR.
  • nucleic acid system comprising the polynucleotide and a polynucleotide comprising a nucleic acid sequence encoding ADAR.
  • the ADAR is ADAR1.
  • the polynucleotide is comprised in a nucleic acid construct comprising a cis-acting regulatory element for directing expression of the polynucleotide.
  • the polynucleotide or system being attached to or encapsulated in a delivery vehicle.
  • a cell expressing the polynucleotide or the system.
  • a method of editing a target RNA sequence comprising an adenosine in a cell expressing same comprising contacting the cell with the polynucleotide or the system, thereby editing the target RNA sequence comprising the adenosine.
  • the cell expresses an endogenous ADAR.
  • the cell does not express an endogenous ADAR.
  • the cell expresses an exogenous ADAR.
  • the cell is a eukaryotic cell.
  • a method of treating a disease that can benefit from ADAR RNA editing in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the polynucleotide or the system, thereby treating the disease in the subject.
  • the polynucleotide or the system for use in treating the disease that can benefit from ADAR RNA editing in a subject in need thereof.
  • FIGs. 1A-D demonstrate the design and detection of editing in double- stranded reporter sequences.
  • Figure 1A is a schematic representation of the design of double- stranded inverted repeat and single-stranded repeat constructs.
  • B2 is based on a mouse non-coding B2 element.
  • mNG is based on the mNeonGreen gene.
  • Figure IB shows % editing [defined herein as (A/(A+G))xl00] in each site along the B2 and mNG double-stranded constructs transfected into HEK293T cells.
  • Upper bar charts show editing in the first arm of the double-stranded structure
  • lower bar charts show editing in the second, complementary arm. Base paired sites are aligned.
  • Figure 1C shows the correlation of editing events on opposing nearby sites, using a weighted sliding window. Data for the B2 construct is shown.
  • Figure ID shows analysis of co-occurrence of editing events across single molecules of mNG construct transfected into MCF7 cells. Hierarchically clustered pairwise correlation matrix between occurrences of editing events across all pairs of adenosines within single reads. The level of correlation is color-coded. The % editing at each site is color-coded in a grey scale in the bars on the top and on the left. Only sites edited to at least 10 % are shown.
  • FIGs. 2A-K demonstrate the design and results of a systematic screening of ADAR1 substrates expressed in cells.
  • Figure 2A is a schematic representation of subsets of planned structures and sequences in the oligo library. Numbers indicate different sequences based on B2 (blue) and mNG (red) of the respective subset.
  • Figure 2B is a schematic representation of the preparation and cloning of the oligo library pool. B2 variable second half of the oligo was cloned at the 3’ UTR of the mNG gene in the pzDonor FC plasmid, already containing the invariable first half of the oligo. mNG variable second half was cloned at the mNG 3’ UTR.
  • FIG. 2C is a schematic representation of the expression of the oligo pool in cells, and steps of library preparation.
  • B2 and mNG libraries were expressed in human cell lines [HEK293T with or without perturbation (HEK293T IFN-a or siAdar), MCF7, A549] and a mouse cell line (NIH3T3).
  • RNA was extracted, the invariable first arm of the constructs including the barcode were reverse transcribed, PCR amplified and sequenced on Novaseq 6000 platform with a 300 bp kit.
  • Figure 2D shows the correlation of editing in biological replicates, shown for the B2 construct and the MCF7 cell line.
  • Each dot represents the average editing level of an adenosine in a construct. Pearson correlation coefficient and p-value are indicated.
  • Figure 2E shows the correlation of editing in a subset of the sequences planned with two different barcodes to quantify the impact of barcode choice on editing. Data for the B2 construct from the MCF7 cell line is shown. Each dot represents the average editing level of a single adenosine in a construct. Pearson correlation coefficient and p-value are indicated.
  • Figure 2F shows AD ARI expression levels normalized to GAPDH, as determined by RT-qPCR analysis of HEK293T cells transfected with the oligo pool containing plasmid in basal, IFN-a stimulated or siAdar conditions.
  • Figure 2G shows the correlation of editing in basal and IFN- stimulated HEK293T cells for the B2 construct. Each dot represents the average editing level of an adenosine in a construct.
  • Figure 2H shows % editing in the double- stranded control constructs in human and mouse cell lines. Adenosines are labelled based on their position in the 146 bp long B2 or mNG positive control construct.
  • Figure 21 shows boxplots of number of edited ‘A’s in single molecules of the B2 or mNG perfect double-strands in each cell line used in the study.
  • Figure 2J shows clustering of reads according to the number of edits per molecule from the prefect double- stranded B2 construct based on the number of edited ‘A’s in single molecules. Fine graph depicts frequency of reads with the given number of edits. Data from MCF7 cell line is shown.
  • Figure 2K shows the level of editing in the subset of constructs randomly disrupting the double- stranded structure in 5 % increments, for the B2 construct. Left panel is a graphical presentation of the set. Right panel shows mean % of editing in different cell lines changing with the extent of disruption of the structure.
  • FIGs. 3A-G demonstrate that editing is periodically induced at roughly 30 - 35 bp distances from structural disruptions.
  • Figure 3 A is a heatmap of 3 nt mismatch running through the sequence. Each row represents a construct disrupted at a different position, running from the 5’ end of the construct (top of the heatmap) to the 3’ end (bottom of the heatmap). Black vertical lines indicate location of 3 nt disruption. Dashed lines are placed in a distance of -35 and +30 nt from the location of the disruption. Each column is an adenosine in the construct, and their absolute locations in the construct are indicated. Editing levels are color-coded following scaling across columns using a Z-score transformation. Data for the mNG construct is shown.
  • Figure 3B demonstrates a subset of 3 nt mismatch running through the sequence.
  • Upper panel is a graphical presentation of the subset
  • middle panel shows the mNG construct
  • bottom panel shows the B2 construct.
  • Mismatches placed at different locations in the constructs are centered at position zero on the x-axis.
  • Each dot represents the delta (A) change of editing level of an adenosine, normalized to the non-disrupted construct.
  • Vertical lines in the scatterplots are placed at -70 (mNG only), -35, 0 and +30. Fitted curves show Loess fit of A editing with a span of 0.1. Only values outside of the -5 - +5 range were included, unless less than five such measurements were present for a given location.
  • Figure C demonstrates a subset of 1 nt mismatch running through the sequence.
  • Data is represented as in Figure 3B.
  • Figure 3D demonstrates a subset of TTC bulges running through the sequence.
  • Left panel is a graphical presentation of the subset and right panel shows the mNG construct.
  • Data is represented as Figure 3B.
  • Figure 3E demonstrates a subset of TTCTTCT bulges running through the sequence.
  • Left panel is a graphical presentation of the subset and right panel shows the mNG construct.
  • Data is represented as Figure 3B.
  • Figure 3F demonstrates a subset of double mismatches around selected edited ‘A’s.
  • Upper-left panel is a graphical explanation of the subset.
  • Vertical lines (black) in the heatmap indicate location of the disruption.
  • Dashed lines are placed approximately in a distance of -35 and +30 nt from the location of the disruption.
  • Data is presented as Figure 3A.
  • Each of the four panels on the heatmap indicate a set with a different center of the double mismatch; 91, 64, 58 or 52.
  • Each row is a construct where the disruption is placed in a different distance from the center, from 1 nt (top row) to 19 nt (bottom row), uneven numbers only.
  • Figure 3G demonstrates a subset of double mismatches around selected edited ‘A’s.
  • X and y axes show the distance from the first and second mismatch, respectively.
  • Each dot represents an adenosine in a construct.
  • a editing level normalized to the perfect double-stranded construct is color-coded.
  • Dashed lines mark -35 and 30 bp distance from the disruption. All data is shown for the MCF7 ceil line.
  • FIGs. 4A-D demonstrates that editing is directional and symmetric with a 5 bp offset.
  • Figure 4A shows schematic representations of possible models of symmetric and directional editing. Model 1: Fully symmetric editing; direction is sensed with regards to the beginning of the double strand and the loop. Model 2: Symmetricity with 5 bp offset; direction is sensed with regards to the 5’ - 3’ orientation of the molecule.
  • Figure 4B is a schematic representation demonstrating library preparation steps and characterization of the second, variable arm of the expressed construct. The second, variable arm including the barcode sequence was reverse transcribed and sequenced as single molecules.
  • Figure 4C is a heatmap of the subset of 4 nt running disruption in HEK293T cells along the variable arm.
  • Each row represents a construct disrupted at a different position, running from the 5’ end of the construct (top of the heatmap) to the 3’ end (bottom of the heatmap).
  • Black vertical lines indicate location of 4 nt disruption. Dashed lines are placed in a distance of -35 and +30 nt from the location of the disruption.
  • Each column is an adenosine in the construct, and their absolute locations in the construct are indicated. Editing levels are color-coded following scaling across columns using a Z-score transformation.
  • Figure 4D demonstrates characterization of the subset of 4 nt running disruption in HEK293T cells. First and second arms are placed under each other and base pairing locations are aligned.
  • Mismatches placed at different locations in the constructs are centered at position zero on the x-axis.
  • Each dot represents the change of editing level (A editing) of an adenosine, normalized to the non-disrupted construct.
  • Vertical lines in the scatterplots are placed at 35, 0 and 30. Fitted curves show Loess fit of A editing with a span of 0.1. Only values outside of the - 5 - +5 range were included, unless less than five such measurements were present for a given location.
  • FIGs. 5A-G demonstrate nucleation and termination of editing.
  • Figure 5A shows editing in the subset of constructs opening up the sequence from the beginning (5’) in 10 nt increments. Data for the mNG construct is shown. Left panel is a graphical presentation of the subset. On the heatmap (middle), each column represents an adenosine with the indicated position. Each row is a construct, where the indicated number of bases were disrupted. Editing % is color coded. Vertical black lines indicate how far the disruption reaches. Scatterplots (right) show the effect of the disruption based on the distance for edited ‘A’s. Editing fold change is normalized to the perfect double strand. Lines show Loess fit with a span of 0.6.
  • FIG. 5B shows editing in the subset of constructs opening up the sequence from the 3’ end in 10 nt increments.
  • Data is presented as in Figure 5A.
  • Figure 5C shows editing levels in the MCF7 cell line in a series of constructs increasing the length of the stem of the B2 sequence by 0, 10, 15, 20 or 25 nt.
  • Figure 3D shows a subset of mismatching the T opposite of each A to C one-by-one.
  • Upper panel is a graphical presentation of the subset. On the heatmap, each row is a construct where a C was introduced opposite of the A instead of T.
  • Each column represents an adenosine with the indicated position. Editing % is color coded.
  • FIG. 5E shows A editing as a function of distance from the mismatch (as in Figures 3B-E). Data is shown for two series of constructs: a subset of constructs, in which the T opposite of A is mutated either to A (black) or C (blue). Curve shows Loess fit of A editing as in Figures 3B-E.
  • Figure 5F shows sequence context of sites that change in editing level upon T to C mismatch opposite of ‘A’s. Bars are color coded as indicated based on the identity of the trinucleotide centered around the editing site. Adenosines close to the 3’ end (A33-A41) that never get edited were excluded from this analysis.
  • Figure 5G is a scatterplot of A editing, induced by A-C mismatched (x-axis) or a distant 3 nt mismatch (y-axis, including graphical presentation). Sequence context is color coded. All data is shown for the MCF7 cell line.
  • FIGs. 6A-C demonstrate exemplary application and model of ADAR 1 -mediated A-to-I editing.
  • Figure 6A is a workflow of targeting endogenous SMAD4 mRNA based on (Qu et al., 2019) and additional mismatched targeting oligos. The empty vector contains no targeting oligo. Positive control construct is a 151 bp long complementary oligo, with a T to C mismatch opposite of the targeted A. Mismatch 35 is a construct with an additional 4 bp mismatch 35 bases away from the target A.
  • Figure 6B shows editing level of the SMAD4 mRNA targeting with different lengths, complementary oligos. Based on 1-2 biological replicates.
  • Figure 6C is a schematic representation of a model summarizing directional, symmetric, recursive and periodic editing mechanism.
  • FIGs. 7A-G demonstrate the results of a systematic screening of ADAR1 substrates expressed in cells.
  • Figure 7A shows the correlation of editing in biological replicates of the mNG construct expressed in the MCF7 cell line. Each dot represents the average editing level of an adenosine in a construct. Pearson correlation coefficient and p-value are indicated.
  • Figure 7B shows the correlation of editing in a subset of the sequences planned with two different barcodes to quantify endogenous noise. Data for the mNG construct from the MCF7 cell line is shown. Each dot represents the average editing level of an adenosine in a construct. Pearson correlation coefficient and p-value are indicated.
  • Figure 7C shows the correlation of editing in basal and IFN- stimulated HEK293T cells for the mNG construct. Each dot represents the average editing level of an adenosine in a construct.
  • Figure 7D shows ADAR1 and MDA5 expression levels normalized to GAPDH, as determined by RT-qPCR analysis of HEK293T cells transfected with the oligo pool containing plasmid in basal, IFN-a stimulated or siAdar conditions. Bars are averages of 2 biological replicates normalized to GAPDH housekeeping gene expression and expression level in basal condition, error bars show standard deviation of the mean.
  • Figure 7E shows the correlation of editing in human (MCF7) and mouse (NIH3T3) cell lines.
  • Each dot represents the average editing level of an adenosine in a construct. Pearson correlation coefficient and p-value are indicated.
  • Figure 7D shows clustering of reads according to the number of edits per molecule from the prefect double- stranded mNG construct based on the number of edited ‘A’s in single molecules. Line graph depicts frequency of reads with the given number of edits. Data from MCF7 cell line is shown.
  • Figure 7G shows % editing in the subset of constructs randomly disrupting the double-stranded structure in 5 % increments, for the mNG construct.
  • FIGs. 8A-B demonstrate that disruption of the structure increases editing at defined distances in various human and mouse cell lines.
  • Figure 1A shows a subset of 3 nt mismatch running through the sequence.
  • Top left panel is a graphical presentation of the subset.
  • Vertical lines in the scatterplots are placed at (-70 for mNG only), -35, 0 and +30.
  • Top middle A549 cell line, B2 construct.
  • Top right NIH3T3 cell line, B2 construct.
  • Bottom middle A549 cell line, mNG construct.
  • Bottom right NIH3T3 cell line, mNG construct.
  • Figure 8B shows a subset of 1 nt mismatch running through the sequence.
  • Top left panel is a graphical presentation of the subset.
  • Top middle A549 cell line, B2 construct.
  • Top right NIH3T3 cell line, B2 construct.
  • Bottom middle A549 cell line, mNG construct.
  • Bottom right NIH3T3 cell line, mNG construct.
  • Vertical lines in the scatterplots are placed at -70 (mNG only), -35, 0 and +30.
  • mismatches placed at different locations in the constructs are centered at position zero on the x-axis.
  • Each dot represents the delta (A) change of editing level of an adenosine, normalized to the non-disrupted construct.
  • Fitted curves show Loess fit of A editing with a span of 0.1. Only values outside of the -5 - +5 range were included, unless less than five such measurements were present for a given location.
  • FIGs. 9A-B demonstrate the effect of mismatching nearby bases on the opposite strand.
  • Figure 9A shows a subset of mismatching neighboring bases of edited ‘A’s to all possible other nucleotides.
  • Left panels are graphical presentations of the subsets. From top to bottom, panels show sets mismatching the base opposite of T, G, C and A.
  • the x-axes show the distance from the disruptions.
  • Y-axes show to which base a nucleotide is mismatched.
  • Right panel Individual examples of the sequence context of mismatching each base are shown, together with the respective change of editing in these examples (SEQ ID NOs: 15-30).
  • Data shows MCF7 B2.
  • Figure 9B demonstrates that editing level depends on preceding and following bases. Data is based on B2 and mNG perfect double strands transfected into MCF7 cells. ‘A’s close to the loop are not included in this analysis, since they are not editable, independent of their sequence context.
  • the present invention in some embodiments thereof, relates to polynucleotides for RNA editing and methods of using same.
  • ADAR Adenosine-to-inosine RNA editing effected by the adenosine deaminase enzyme, ADAR, increases the repertoire of genome encoded RNAs.
  • ADAR is a multi-domain protein, comprising a recognition domain and a catalytic domain.
  • site-directed RNA editing holds great promise for the treatment of disease and as a tool to study protein and RNA function.
  • One strategy developed for site-directed RNA editing is to create substrates around a target adenosine that are recognized by ADAR or an engineered ADAR. Essentially, these structures are generated by delivering antisense guide RNA oligos that create editable structures in trans. While the proper design of these guides is critical, the fundamental rules for their construction remain poorly understood.
  • the present inventors exploited the 30-35 bp periodicity to increase the efficiency of mRNA editing by recruitment of endogenous ADAR (Example 2 of the Examples section which follows).
  • the present inventors have designed a polynucleotide targeting SMAD4 mRNA harboring two 75-nt long stretches of complementarity to the targeted adenosine upstream and downstream of it, with a mismatched C opposite of the targeted site and a 4 nucleotides mismatch 35 nucleotides away from the targeted adenosine.
  • This designed polynucleotide achieved a 3 -fold improvement in editing efficiency compared to a control polynucleotide not comprising the mismatch 35 nucleotides away from the targeted adenosine.
  • a polynucleotide comprising a nucleic acid sequence having at least 70 % complementarity to a target RNA sequence comprising an adenosine, wherein said nucleic acid sequence comprises at least one complementarity mismatch with at least one nucleotide in said target RNA sequence located in at least one of nucleotides 25-40 upstream and/or downstream of said adenosine, such that upon hybridization of said nucleic acid sequence to said target RNA sequence a disruption of a double- stranded structure is formed at said at least one complementarity mismatch and wherein said nucleic acid sequence and said RNA sequence form double-stranded structures upstream and downstream of said disruption.
  • polynucleotide refers to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).
  • the polynucleotide is a single stranded nucleic acid sequence.
  • the polynucleotide is an RNA sequence.
  • nucleotide refers to the respective nucleobase-(deoxy)ribosyl-phospholinker, as well as any chemical modifications of the ribose moiety or the phospho group.
  • the nucleotide includes a locked ribosyl moiety (comprising a 2'-4' bridge, comprising a methylene group or any other group, well known in the art), a nucleotide including a linker comprising a phosphodiester, phosphotriester, phosphoro(di)thioate, methylphosphonates, phosphoramidate linkers, or the like.
  • the nucleic acid sequence comprised in the polynucleotide should have sufficient overlap and complementarity to a target RNA sequence comprising an adenosine, to allow for sequence specific hybridization of the complementary nucleic acid sequence with the target RNA sequence.
  • target RNA sequence refers to an RNA sequence at about the same length as the complementary nucleic acid sequence, the target RNA sequence is typically comprised in a polynucleotide which is typically a part of an RNA encoded by a gene of interest.
  • the length and the % complementarity may be routinely determined by a person having ordinary skill in the art. In general, longer sequences provide more specificity - and consequently fewer off-target effects, e.g. through non-specific binding - and stronger binding to the target site.
  • the nucleic acid sequence having the complementarity to the target RNA sequence is at least 75, at least 80, at least 85, at least 90, at least 95, at least 100 nucleotides long.
  • the complementary nucleic acid sequence is at least 100 nucleotides long.
  • the complementary nucleic acid sequence is at least 110, at least 120, at least 130, at least 140, at least 145, at least 150 nucleotides long.
  • the complementary nucleic acid sequence or the polynucleotide is up to 400, up to 350, up to 300, up to 250 or up to 200 nucleotides long.
  • the complementary nucleic acid sequence or the polynucleotide is up to 250 nucleotides long. According to specific embodiments, complementary the nucleic acid sequence or the polynucleotide is 100-400, 100-300, 120-300, 120-250, 120-200 or 140-200 nucleotides long.
  • the complementary nucleic acid sequence or the polynucleotide is 100-250 nucleotides long.
  • the complementary nucleic acid sequence or the polynucleotide is 140-200 nucleotides long.
  • the complementary nucleic acid sequence or the polynucleotide is about 150 nucleotides long.
  • the complementary nucleic acid sequence or the polynucleotide is 151 nucleotides long.
  • the complementary nucleic acid sequence comprises at least 50, at least 55, at least 60, at least 65, at least 70 nucleotides upstream and/or downstream of a nucleotide located opposite said adenosine upon said hybridization.
  • the complementary nucleic acid sequence comprises at least 50, at least 55, at least 60, at least 65, at least 70 nucleotides upstream and downstream of a nucleotide located opposite said adenosine upon said hybridization.
  • the complementary nucleic acid sequence comprises at least 50 nucleotides upstream and downstream of a nucleotide located opposite said adenosine upon said hybridization.
  • the complementary nucleic acid sequence comprises at least 60 nucleotides upstream and downstream of a nucleotide located opposite said adenosine upon said hybridization.
  • complementarity refers to global complementarity, a complementarity over the entire complementary nucleic acid sequence disclosed herein having about the same length as the target RNA sequence comprising an adenosine disclosed herein and not over portions thereof.
  • the nucleic acid sequence has at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 % complementarity to the target RNA sequence comprising the adenosine.
  • the nucleic acid sequence has at least 70 % complementarity to the target RNA sequence comprising the adenosine.
  • the nucleic acid sequence has at least 80 % complementarity to the target RNA sequence comprising the adenosine. According to specific embodiments, the nucleic acid sequence has at least 90 % complementarity to the target RNA sequence comprising the adenosine.
  • the nucleic acid sequence exhibits full complementarity to the target RNA sequence comprising the adenosine other than the complementarity mismatch(es) disclosed herein.
  • the complementary nucleic acid sequence comprises at least one complementarity mismatch with at least one nucleotide in said target RNA sequence such that upon hybridization of the nucleic acid sequence to the target RNA sequence a disruption of a double- stranded structure is formed at the at least one complementarity mismatch.
  • hybridization refers to a reaction in which the nucleic acid sequence and the target RNA sequence react to form a complex that is stabilized via non- covalent bonding between the bases of the nucleotide residues (i.e. double strand structure).
  • the non-covalent bonding may occur by e.g. Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner, including non-naturally occurring/synthetic nucleotides or bonds therebetween as described above.
  • complementarity mismatch refers to at least one nucleotide having no base pair complementation with a given nucleotide.
  • the at least one complementarity mismatch leads to a disruption of the double-stranded structure formed upon hybridization.
  • the complementary nucleic acid sequence and the RNA sequence form double- stranded structures both upstream and downstream of the disruption (e.g. upstream and downstream with respect to the target RNA strand).
  • Non-limiting examples of such a structural disruption may include, but are not limited to, a bulge (a separation of the double- stranded structure on one of the strands of the complex), an internal loop (a separation of the double-stranded structure on both strands of the complex), a hairpin loop (also known as stem loop), a junction or a combination thereof.
  • the disruption is a bulge.
  • the bulge is produced in the complementary nucleic acid sequence strand (i.e. not in the target RNA strand).
  • the bulge is produced in the target RNA strand.
  • the disruption size may vary from a single unpaired residue (caused by a single complementarity mismatch) or a plurality of nucleotides (caused by a plurality of complementarity mismatches).
  • the at least one complementarity mismatch or the disruption comprises 1-30, 1-25, 1-20, 1-15 or 1-10 nucleotides. According to specific embodiments, the at least one complementarity mismatch or the disruption comprises 1-10 nucleotides.
  • the at least one complementarity mismatch or the disruption comprises 1-7 nucleotides.
  • the at least one complementarity mismatch or the disruption comprises 1-4 nucleotides.
  • the at least one complementarity mismatch or the disruption comprises 5 nucleotides.
  • the at least one complementarity mismatch or the disruption comprises 4 nucleotides.
  • the at least one complementarity mismatch or the disruption comprises 3 nucleotides.
  • the at least one complementarity mismatch or the disruption comprises 2 nucleotides.
  • the at least one complementarity mismatch or the disruption comprises 1 nucleotide.
  • the at least one complementarity mismatch is located in at least one of nucleotides 25-40 upstream and/or downstream of the adenosine in the target RNA.
  • the at least one complementarity mismatch is located in at least one of nucleotides 25-40, 25-35 or 30-40 upstream and/or downstream of the adenosine in the target RNA.
  • the at least one complementarity mismatch is located upstream of the adenosine in the target RNA.
  • the at least one complementarity mismatch is located in at least one of nucleotides 25-35, 26-35, 32-35, 28-35, 29-35 upstream of the adenosine in the target RNA.
  • the at least one complementarity mismatch is located in at least one of nucleotides 25-35 upstream of the adenosine in the target RNA.
  • the at least one complementarity mismatch is located in at least one nucleotide about 30 upstream of the adenosine in the target RNA.
  • the at least one complementarity mismatch is located in at least nucleotide 30 upstream of the adenosine in the target RNA. According to specific embodiments, the at least one complementarity mismatch is located in nucleotide 30 upstream of the adenosine in the target RNA.
  • the at least one complementarity mismatch is located downstream of the adenosine in the target RNA.
  • the at least one complementarity mismatch is located in at least one of nucleotides 30-40, 30-39, 30-38, 30-37, 30-36 downstream of the adenosine in the target RNA.
  • the at least one complementarity mismatch is located in at least one of nucleotides 30-40 downstream of the adenosine in the target RNA.
  • the at least one complementarity mismatch is located in at least one nucleotide about 35 downstream of the adenosine in the target RNA.
  • the at least one complementarity mismatch is located in at least nucleotide 35 downstream of the adenosine in the target RNA.
  • the at least one complementarity mismatch is located in nucleotide 35 downstream of the adenosine in the target RNA.
  • ADAR can be increased to only convert a specific target adenosine by providing a nucleic acid sequence that comprises a complementary mismatch opposite the target adenosine.
  • This mismatch can be created by providing a nucleic acid sequence having a cytidine or uridine, according to a specific embodiment a cytidine, opposite the target adenosine in the target RNA sequence.
  • the target RNA sequence Upon deamination of the target adenosine in the target RNA, the target RNA sequence will obtain an inosine which, for most biochemical processes, is "read" by the cell's biochemical machinery as a guanosine.
  • the mismatch is resolved (as inosine is capable of base pairing with the opposite cytidine in the nucleic acid sequence).
  • the complementary nucleic acid sequence further comprises a complementary mismatch with the adenosine in the target RNA.
  • the complementary mismatch with the adenosine is a cytidine.
  • Non-specific editing of adenosines can be further limited by making sure that the adenosines that should not be edited, or at least at a lower frequency, encounter an opposite nucleotide with a 2'-0 modified ribose moiety, such as a 2'-0Me, as the latter is known to reduce the efficiency of editing of the opposite adenosine.
  • the complementary nucleic acid sequence may be chemically modified.
  • the nucleic acid sequence comprises 2'-0 methyl groups in a position(s) which oppose an adenosine(s) when the nucleic acid sequence hybridizes with the target RNA sequence if this adenosine(s) in the target RNA sequence is not a target for editing.
  • an opposing base being a guanine or adenine may be provided, as these nucleobases generally impede deamination of the opposing base.
  • the nucleic acid sequence or the polynucleotide is not chemically modified.
  • the polynucleotide comprises an additional nucleic acid sequence comprising an ADAR-recruiting domain.
  • the polynucleotide does not comprise an additional nucleic acid sequence comprising an ADAR-recruiting domain.
  • ADAR-recruiting domain refers to a nucleic acid sequence or structure that mediates binding of ADAR to the complementary nucleic acid sequence : target RNA sequence complex in a direct or indirect manner.
  • ADAR-recruiting domains include, but are not limited to, GluR-2, GluR-B (R/G), GluR-B (Q/R), GluR-6 (R/G), 5HT2C, and FlnA (Q/R) domain; See for example, Wahlstedt, Helene, and Marie, Wiley Interdisciplinary Reviews: RNA 2.6 (2011): 761-771 and Aquino- Jarquin (2020) Molecular Therapy Nucleic Acids, 19: P1065-1072, which are incorporated herein by reference in their entirety.
  • such an ADAR-recruiting domain comprises a double-strand RNA structure.
  • the recruiting domain comprises or consists of a nucleic acid sequence that is capable of forming a stem-loop structure.
  • the stem structure of the stem- loop structure comprises at least two mismatches.
  • the stem-loop structure comprises a 3-8 nucleotides loop.
  • the stem-loop structure comprises a 5 nucleotides loop.
  • the loop comprises or consists of the nucleic acid sequence GCUAA or GCUCA.
  • the polynucleotide comprises an additional nucleic acid sequence encoding ADAR.
  • Nucleic acid sequences having the complementarity to a target RNA sequence comprising an adenosine described herein and the nucleic acid sequence encoding ADAR can be expressed as a single transcript or as two separate transcripts. Methods of expressing two distinct transcripts from a single polynucleotide are well known in the art and are further provided infra.
  • nucleic acid system comprising the polynucleotide comprising the nucleic acid sequences having the complementarity to a target RNA sequence comprising an adenosine described herein and a separate polynucleotide comprising a nucleic acid sequence encoding ADAR.
  • ADAR adenosine deaminase acting on RNA
  • E.C. No. 3.5.4 refers to an enzyme that catalyzes the conversion of adenosine (A) to inosine (I) by hydrolytic deamination.
  • ADARs share a common modulator organization which consists of a variable N-terminal region, a double stranded RNA binding domain and a zinc containing catalytic domain. Accordingly, the ADAR may be ADAR 1, 2 or 3.
  • the ADAR is ADAR1.
  • the ADAR is ADAR2.
  • the adenosine deaminase is derived from one or more metazoa species, including but not limited to, mammals, birds, frogs, squids, fish, flies and worms.
  • the ADAR is a human ADAR (e.g., hADARl and hADAR2).
  • Non-limiting exemplary sequences of human ADAR1 are provided in the following GenBank Accession Numbers: NP_001020278, NP_001102, NP_001180424, NP_056655 and NP.056656
  • Non-limiting examples of nucleic sequence encoding human AD ARI are provided in the following GenBank Accession Numbers: NM_001025107, NM_001111, NM_001193495, NM_015840 and NM_015841.
  • the nucleic acid sequence encoding human ADAR comprises SEQ ID NO: 31.
  • the nucleic acid sequence encoding human ADAR consists of SEQ ID NO: 31.
  • Non-limiting exemplary sequences of human ADAR2 are provided in the following GenBank Accession Numbers: NP_001103, NP_001153702, NP_001333616, NP_001333617 and NP_056648
  • Non-limiting examples of nucleic sequence encoding human ADAR2 are provided in the following GenBank Accession Numbers: NM_001033049, NM_001112, NM_001160230, NM_015833 and NM_015834.
  • the nucleic acid sequence encoding human ADAR comprises SEQ ID NO: 32.
  • the nucleic acid sequence encoding human ADAR consists of SEQ ID NO: 32.
  • Any coding sequence of ADAR also encompasses functional isoforms, fragments and homologues (naturally occurring or synthetically/recombinantly produced), which exhibit the desired activity as described herein.
  • Such homologues can be, for example, at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identical or homologous to the polypeptide sequences provided herein; or at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least
  • Sequence identity or homology can be determined using any protein or nucleic acid sequence alignment algorithm such as Blast, ClustalW, and MUSCLE.
  • the homolog may also refer to an ortholog, a deletion, insertion, or substitution variant, including a conservative and non-conservative amino acid substitution.
  • the polynucleotides may be ligated into a nucleic acid expression construct, under the transcriptional control of a cis-regulatory sequence (e.g., promoter sequence) suitable for directing constitutive or inducible transcription of the polynucleotide sequence in a cell.
  • a cis-regulatory sequence e.g., promoter sequence
  • the regulatory element is a heterologous regulatory element.
  • the polynucleotide or the system wherein the polynucleotide is comprised in a nucleic acid construct comprising a cis-acting regulatory element for directing expression of said polynucleotide.
  • the nucleic acid construct also referred to herein as an "expression vector" of some embodiments of the invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors).
  • a typical cloning vector may also contain a transcription and translation initiation sequence, transcription and translation terminator and a polyadenylation signal.
  • such constructs will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or a portion thereof.
  • Eukaryotic promoters typically contain two types of recognition sequences, the TATA box and upstream promoter elements.
  • the TATA box located 25-30 base pairs upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase to begin RNA synthesis.
  • the other upstream promoter elements determine the rate at which transcription is initiated.
  • Constitutive promoters suitable for use with some embodiments of the invention are promoter sequences which are active under most environmental conditions and most types of cells such as the cytomegalovirus (CMV) and Rous sarcoma virus (RSV).
  • Inducible promoters suitable for use with some embodiments of the invention include for example the tetracyclineinducible promoter (Zabala M, et al., Cancer Res. 2004, 64(8): 2799-804), the galactose inducible promoter (GALI-1 promoter) or the copper induced promoter (CUP1-1 promoter).
  • the promoter utilized by the nucleic acid construct of some embodiments of the invention is active in the specific cell population transformed.
  • cell type- specific and/or tissue- specific promoters include promoters such as albumin that is liver specific [Pinkert et al., (1987) Genes Dev. 1:268-277], lymphoid specific promoters [Calame et al., (1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cell receptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins; [Banerji et al.
  • neuron-specific promoters such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477], pancreas-specific promoters [Edlunch et al. (1985) Science 230:912-916] or mammary gland-specific promoters such as the milk whey promoter (U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166).
  • Enhancer elements can stimulate transcription up to 1,000 fold from linked homologous or heterologous promoters. Enhancers are active when placed downstream or upstream from the transcription initiation site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer/promoter combinations that are suitable for some embodiments of the invention include those derived from polyoma virus, human or murine cytomegalovirus (CMV), the long term repeat from various retroviruses such as murine leukemia virus, murine or Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1983, which is incorporated herein by reference.
  • CMV cytomegalovirus
  • the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
  • Polyadenylation sequences can also be added to the expression vector in order to increase the efficiency of translation.
  • Two distinct sequence elements are required for accurate and efficient poly adenylation: GU or U rich sequences located downstream from the polyadenylation site and a highly conserved sequence of six nucleotides, AAUAAA, located 11-30 nucleotides upstream.
  • Termination and polyadenylation signals that are suitable for some embodiments of the invention include those derived from SV40.
  • the expression vector of some embodiments of the invention may typically contain other specialized elements intended to increase the level of expression of cloned nucleic acids or to facilitate the identification of cells that carry the recombinant sequence.
  • a number of animal viruses contain DNA sequences that promote the extra chromosomal replication of the viral genome in permissive cell types. Plasmids bearing these viral replicons are replicated episomally as long as the appropriate factors are provided by genes either carried on the plasmid or with the genome of the host cell.
  • the vector may or may not include a eukaryotic replicon. If a eukaryotic replicon is present, then the vector is amplifiable in eukaryotic cells using the appropriate selectable marker. If the vector does not comprise a eukaryotic replicon, no episomal amplification is possible. Instead, the recombinant DNA integrates into the genome of the engineered cell, where the promoter directs expression of the desired nucleic acid.
  • the expression vector of some embodiments of the invention can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.
  • IRS internal ribosome entry site
  • the individual elements comprised in the expression vector can be arranged in a variety of configurations.
  • enhancer elements, promoters and the like, and even the polynucleotide sequence(s) can be arranged in a "head-to-tail" configuration, may be present as an inverted complement, or in a complementary configuration, as an antiparallel strand. While such variety of configuration is more likely to occur with non-coding elements of the expression vector, alternative configurations of the coding sequence within the expression vector are also envisioned.
  • mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+/-), pGL3, pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.
  • Expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses can be also used.
  • SV40 vectors include pSVT7 and pMT2.
  • Vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5.
  • exemplary vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
  • Non-limiting examples of bacterial constructs include the pET series of E. coli expression vectors [Studier et al. (1990) Methods in Enzymol. 185:60-89).
  • yeast a number of vectors containing constitutive or inducible promoters can be used, as disclosed in U.S. Pat. Application No: 5,932,447.
  • vectors can be used which promote integration of foreign DNA sequences into the yeast chromosome.
  • the expression of the coding sequence can be driven by a number of promoters.
  • viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al. (1984) Nature 310:511-514], or the coat protein promoter to TMV [Takamatsu et al. (1987) EMBO J. 3:17-311] can be used.
  • plant promoters such as the small subunit of RUBISCO [Coruzzi et al. (1984) EMBO J.
  • viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms.
  • viruses infect and propagate in specific cell types.
  • the targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell.
  • Recombinant viral vectors are useful for in vivo expression since they offer advantages such as lateral infection and targeting specificity.
  • Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny.
  • Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.
  • the type of vector used by some embodiments of the invention will depend on the cell type transformed.
  • the ability to select suitable vectors according to the cell type transformed is well within the capabilities of the ordinary skilled artisan and as such no general description of selection consideration is provided herein.
  • the cell may be transformed stably or transiently with the nucleic acid constructs disclosed herein.
  • stable transformation the nucleic acid molecule is integrated into the cell genome and as such it represents a stable and inherited trait.
  • transient transformation the nucleic acid molecule is expressed by the cell transformed but it is not integrated into the genome and as such it represents a transient trait.
  • the polynucleotide, system or construct is attached to or encapsulated in a delivery vehicle.
  • delivery vehicles include for example, but not limited to, a viral vector, a lipid-based particle, a liposome, an exosome and a nanoparticle.
  • the present invention also contemplates cells comprising the polynucleotides, systems and constructs.
  • a cell expressing the polynucleotide or the system disclosed herein.
  • the cell may be a prokaryotic or a eukaryotic cell.
  • the cell is a eukaryotic cell.
  • Non-limiting examples of eukaryotic cells which may be used with some embodiments of the invention include but are not limited to, mammalian cells, fungal cells, yeast cells, insect cells, algal cells or plant cells.
  • the cell is not a bacterium.
  • the cell is not E.coli.
  • the cell is a human cell.
  • the cell is a cell like such as, but not limited to HEK293T, HT29, A549, HepG2, RD, SF268, SW13 or HeLa cell.
  • the cell is a primary cell.
  • the cell is a cell in which an endogenous or an exogenous ADAR is capable of editing RNA in.
  • the cell expresses an endogenous ADAR.
  • the cell does not express an endogenous ADAR.
  • the cell expresses an exogenous ADAR.
  • polynucleotides designed according to the present teachings can be used to recruit ADAR to edit the adenosine in the target RNA (i.e. site-directed editing).
  • a method of editing a target RNA sequence comprising an adenosine in a cell expressing same comprising contacting the cell with the polynucleotide or the system disclosed herein, thereby editing the target RNA sequence comprising said adenosine.
  • the contacting is effect in-vivo.
  • the contacting is effect ex-vivo.
  • the adenosine of the target RNA is comprised in a CAG sequence.
  • the adenosine of the target RNA is not comprised in a GA or GAG sequence.
  • a method of selecting a target RNA sequence for ADAR editing comprising identifying in an RNA sequence encoded by a gene of interest a target RNA sequence comprising an adenosine, such that said adenosine is comprised in a CAG sequence and not a GA or GAG sequence, said adenosine being suitable for ADAR editing in the target RNA sequence.
  • the identifying may be effected by sequencing or by using pre-existing databases.
  • the target RNA sequence comprises at least 50, at least 55, at least 60, at least 65, at least 70 nucleotides upstream and/or downstream of the adenosine.
  • Adenosine-to-inosine deamination may introduce or revert, for example, a point mutation, in-frame stop codon, aberrant splice site, alternative splice site and miss-folding of a resulting protein.
  • the adenosine in the target RNA sequence introduces an in-frame premature stop codon.
  • the in-frame premature stop codon is UAG or TAG.
  • the adenosine in the target RNA sequence introduces or removes a splice site.
  • the adenosine in the target RNA sequence alters a codon.
  • the method comprising assessing the editing following the contacting.
  • Assessing editing can be determined using methods well known in the art, including, for example, RNA sequencing, PCR, amino acid sequencing when the RNA editing causes a change to the amino acid sequence encoded by the RNA, assessing the presence of a functional, elongated, truncated, full-length and/or wild-type protein when the RNA editing modifies a stop codon or a splice site.
  • the target RNA sequence is part of a polynucleotide associated with a disease that can benefit from editing the adenosine in the target RNA by ADAR.
  • a method of treating a disease that can benefit from ADAR RNA editing in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the polynucleotide, the system or the construct disclosed herein, thereby treating the disease in the subject.
  • polynucleotide for use in treating a disease that can benefit from ADAR RNA editing in a subject in need thereof.
  • treating refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder, or condition e.g., cancer) and/or causing the reduction, remission, or regression of a pathology.
  • pathology disease, disorder, or condition e.g., cancer
  • Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.
  • the term “subject” includes mammals, e.g. human beings at any age and of any gender which suffer from the pathology. According to specific embodiments, this term encompasses individuals who are at risk to develop the pathology.
  • the phrase “disease that can benefit from ADAR RNA editing” refers to a disease in which a nucleic acid sequence alteration (i.e. mutation) drives onset and/or progression of the disease, wherein this alteration can be repaired by RNA editing.
  • the nucleic acid sequence alteration is a guanosine to adenosine mutation.
  • Non-limiting examples of diseases that can benefit from ADAR RNA editing include, cystic fibrosis, familial hypercholesterolaemia, Haemophilia-B, Tay-Sachs, ataxia telangiectasia, albinism, alpha- 1 -antitrypsin deficiency, Alzheimer disease, Amyotrophic lateral sclerosis, Asthma, P-thalassemia, Cadasil syndrome, Charcot-Marie-Tooth disease, Chronic Obstructive Pulmonary Disease (COPD), Distal Spinal Muscular Atrophy (DSMA), Duchenne/Becker muscular dystrophy, Dystrophic Epidermolysis bullosa, Epidermylosis bullosa, Fabry disease, Factor V Eeiden associated disorders, Familial Adenomatous, Polyposis, Galactosemia, Gaucher's Disease, Glucose-6-phosphate dehydrogenase, Haemophilia, Hereditary Hematochromatosis, Hunter Syndrome
  • Non-limiting examples of specific polynucleotides associated with diseases that can benefit from ADAR RNA editing include CFTR, LDLR, Factor IX, A1AT, LRRK2, hexosaminidase, ATM dystrophin, huntingtin, neurofibromin 1, neurofibromin 2, the P-globin chain of haemoglobin, CEP290 (centrosomal protein 290kDa), the HEXA gene of the P- hexosaminidase A, any one of the Usher genes (e.g. USH2B encoding Usherin), TP53, C0L3A1, BMPR2, AHH, FANCCW506X, MYBPC3, IL2RG.
  • Non-limiting examples of disease-associated mutations that may be restored by the methods disclosed herein include, but are not limited to, mutations in the CFTR gene (e.g. G542X; W1282X; R553X; 1162X; Y122X) associated with cystic fibrosis; W23X mutation in the low-density lipoprotein receptor (LDLR) associated with familial hypercholesterolaemia; mutations in Factor IX (e.g. E27K, G60S, R248Q) associated with Haemophilia-B; G269S mutation in the hexosaminidase A enzyme associated with Tay-Sachs, and mutations in the ATM gene (e.g.
  • mutations in the CFTR gene e.g. G542X; W1282X; R553X; 1162X; Y122X
  • LDLR low-density lipoprotein receptor
  • Factor IX e.g. E27K, G60S, R248Q
  • G2250A, G3676A, R2032K associated with ataxia telangiectasia
  • TP53 X e.g., 158G>A
  • IDUA W402X e.g., TGG>TAG mutation in exon 9
  • MPS I Mucopolysaccharidosis type I
  • C0L3A1 W1278X e.g, 3833G>A mutation
  • BMPR2 W298X e.g., 893G>A
  • AHH W725X e.g., 2174G>A
  • FANCC W506X e.g., 1517G>A
  • MYBPC3 W1098X e.g., 3293G>A
  • IL2RG 27X e.g., 71OG>A
  • compositions, methods or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.
  • the phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.
  • B2 construct was ordered in two parts as gBlocks from IDT (Table 1 herein below). The first half, including part of the loop, was cloned between the Spel (Bcul, Thermo Fisher Scientific) and BstBI (NEB) restriction sites in the pzDonor FC plasmid. The second containing part of the loop, and either the reverse complement of the first half (positive control) or the same sequence (negative control) was cloned using BstBI (NEB) and Asci (Sgsl, Thermo Fisher Scientific). The second half of the mNG construct, repeating or reverse complementing the last 146 bases of the mNG 3’ UTR with a 46 bp long loop inbetween was similarly ordered and cloned.
  • oligo pool - Custom oligo pool designed by the present inventors was ordered from Twist Bioscience and resuspended in 200 pl TE buffer, incubated at 65 °C for 10 minutes and stored at -20 °C.
  • Herculase II Fusion DNA polymerase (Agilent) was used.
  • 5 PCR reactions each with 5 pl DNA pool as starting material was prepared and amplified for 18 PCR cycles. Reactions were pooled and cleaned with QIAQuick PCR purification column (Qiagen). Restriction enzyme cutting of the PCR product was performed in 3 reactions for each subset.
  • Plasmids were prepared and used the same as for the control constructs. The plasmids were digested with Asci and BstBI, treated with alkaline phosphatase FastAP (Thermo Fisher Scientific) and cleaned with QIAQuick PCR purification column. Digested oligo pool was run on a 2.5 % agarose gel and bands were cut from gel.
  • Oligo library and plasmid were ligated in 1 : 1 molar ratio using Lucigen ligase (CloneDirect Rapid Ligation Kits) and transformed into E. Cloni (Lucigen Corporation, cat# LC-60117-2) bacteria by electroporation. Bacteria were seeded on ten 14 cm plates for each sample. Following overnight incubation at 37 °C, bacteria were scraped off the plates and the DNA was extracted with NucleoBond Xtra Maxiprep kit (Macherey Nagel).
  • HEK293T cells were seeded on a 10 cm plate and transfected with the plasmid construct as described hereinabove. At the same time, 650 U / ml IFN-a (pbl assay science) was added.
  • HEK293T cells were subjected to two rounds of Adar siRNA (Ambion #4427038) treatment using the Lipofectamine RNAiMAX (Thermo Fisher Scientific) reagent, and 15 pmol siRNA in a 6-wells format, with 24 hours interval. Cells were reseeded on 10 cm plates and transfected with the plasmid as described hereinabove.
  • RNA extraction, cDNA synthesis and quantitative real-time PCR - RNA was extracted with NucleoZOL (Macherey-Nagel).
  • cDNA was synthesized with MultiScribe Reverse Transcriptase cDNA synthesis kit (Thermo Fisher Scientific).
  • qPCR (see primer sequences in Table 1 hereinabove) was performed with KAPA Sybr Fast qPCR Master Mix (Kapa Biosystems). All gene expression levels were normalized to GAPDH and fold changes were determined with the quantitative Ct method.
  • RNA was poly-A selected with oligo dT -beads (Dynabeads® mRNA DIRECTTM Kit life tech).
  • poly-A selected libraries were prepared and sequenced on Illumina Nextseq platform.
  • Poly-A RNA from oligo-pool transfected cells was reverse transcribed (see primer sequences in Table 1 hereinabove) to obtain the constant or variable fragments of the constructs with the matching 8 nt barcode.
  • PCR amplicons see primer sequences in Table 1 hereinabove
  • of these fragments were generated and sequenced on Illumina Novaseq 6000 platform obtaining 300 bp long reads.
  • Plasmids were transfected into HEK293T cells as described hereinabove. RNA extraction, DNAse treatment and reverse transcription was performed according to standard protocols. Sanger sequencing data was quantified with the EditR tool (Kluesner et al., 2018).
  • the four constructs were expressed from plasmids transfected into HEK293T cells.
  • the perfect double- stranded B2 and mNG constructs contained 14 and 25 sites that were edited over 10 %, respectively, while no such sites were present in the negative controls.
  • a substantial variability of editing levels was observed in the perfect double-strands, with some sites being edited to 80 %, while others were not edited at all (Figure IB).
  • the first 20 nucleotides of the B2 and 10 nucleotides of the mNG element did not have editing levels exceeding 5 %.
  • Adenosines in the last 10 nt of the B2 and 20 nt of the mNG element were edited to less than 1 %.
  • an oligo library was designed on the basis of the B2 and mNG constructs in which their sequences and structures were systematically disrupted (Figure 2A).
  • Each of the 2000 variations was designed to replace the second arm of the double stranded structure of the B2 or mNG construct and were cloned, as a pool, into the plasmid already containing the invariable first arm.
  • Each sequence was designed with a unique 8-nt barcode (Figure 2B).
  • IFN stimulation led to a substantial global increase in editing levels, on average leading to 1.65-fold higher editing levels compared to the basal condition. This increase was consistent with the roughly 2.5-fold increase in total ADAR1 levels achieved following IFN induction ( Figures 2F and 7D).
  • IFN stimulation mainly affected sites with medium (1 - 50 %, average: 1.81 -fold increase) editing levels in basal conditions, while leaving lowly (0 - 1 %) and highly (over 70 %) edited sites almost unchanged, suggestive that AD ARI binding to high affinity sites (modified at high levels under basal conditions) is saturated ( Figures 2G and 7C).
  • ADAR1 knockdown led to nearly complete loss of editing, demonstrating that in these cell lines all the detected editing sites depend on ADAR1 ( Figure 2H). This is consistent with the notion that ADAR2 is expressed in cell lines to very low levels or not at all (Schaffer et al., 2020).
  • editing level at position 24 was 49 % in contrast to 7 % in the undisrupted construct.
  • editing level at position 85 was 44 % compared to 26 % without the disruption.
  • ADAR1 can sense its orientation with respect to the stem-loop structure (e.g. by sensing its relative position with respect to the loop), which determines the offset at which it introduces editing. Under such a scenario a site closer to the loop will be edited at a 30 bp offset, whereby a site closer to the beginning of the stem will be edited at a 35 bp offset ( Figure 4 A, Model 1).
  • ADAR1 senses its relative orientation with respect to the RNA strand, and will introduce editing at a 35 bp distance upstream of the disrupted site, and 30 bp downstream ( Figure 4A, Model 2). It was reasoned that assessing editing levels in the second arm of the construct would allow direct discrimination between these two scenarios. Under the first scenario, editing sites on the second arm should precisely mirror the ones on the top arm (in which case they would occur 30 bp upstream and 35 bp downstream of the disrupted site).
  • the offset is expected to be maintained with respect to the RNA orientation (in which case it would occur 35 bp upstream and 30 bp downstream of the disrupted site), forming a 5 bp gap between the peak edited site on the top arm versus bottom arm.
  • the first question is particularly pertinent in the context of a ‘perfect’ dsRNA structures lacking any disruptions.
  • the second question is crucial to address in the context of a recursive event, which requires an ‘exit clause’ for termination.
  • ADAR1 is essential for the maintenance of hematopoiesis and suppression of interferon signaling. Nat. Immunol. 10, 109- 115.
  • EditR A Method to Quantify Base Editing from Sanger Sequencing. CRISPR J 7, 239-250.
  • RNA editing by ADAR1 prevents MDA5 sensing of endogenous dsRNA as nonself. Science 349, 1115-1120.
  • RNA-editing enzyme ADAR1 controls innate immune responses to RNA. Cell Rep. 9, 1482-1494.
  • RNA hairpins in noncoding regions of human brain and Caenorhabditis elegans mRNA are edited by adenosine deaminases that act on RNA. Proc. Natl. Acad. Sci. U. S. A. 99, 7906-7911.
  • RNA editing level in the mouse is determined by the genomic repeat repertoire. RNA 72, 1802-1809.
  • ADAR 1 -mediated RNA editing is a novel oncogenic process in thyroid cancer and regulates miR-200 activity. Oncogene 39, 3738-3753.
  • ADARs Adenosine deaminases acting on RNA
  • RNA editing by AD ARI leads to context-dependent transcriptome-wide changes in RNA secondary structure. Nat. Commun. 8, 1440.
  • irCLASH reveals RNA substrates recognized by human ADARs. Nat. Struct. Mol. Biol. 27, 351-362.
  • Double-stranded RNAs containing multiple IU pairs are sufficient to suppress interferon induction and apoptosis. Nat. Struct. Mol. Biol. 17, 1043-1050.

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Abstract

L'invention concerne des polynucléotides pour l'édition d'ARN, en conséquence, l'invention concerne un polynucléotide comprenant une séquence d'acide nucléique ayant au moins 70 % de complémentarité par rapport à une séquence d'ARN cible comprenant une adénosine, ladite séquence d'acide nucléique comprenant au moins un mésappariement de complémentarité avec au moins un nucléotide dans ladite séquence d'ARN cible situé dans au moins l'un des nucléotides 25 à 40 en amont et/ou en aval de ladite adénosine, de sorte que lors de l'hybridation de ladite séquence d'acide nucléique à ladite séquence d'ARN cible, une interruption d'une structure à double brin est formée au niveau dudit au moins un mésappariement de complémentarité et ladite séquence d'acide nucléique et ladite séquence d'ARN formant des structures à double brin en amont et en aval de ladite interruption. L'invention concerne également des méthodes d'édition d'une séquence d'ARN cible comprenant une adénosine et des méthodes de traitement d'une maladie qui peut bénéficier d'une édition d'ARN ADAR.
PCT/IL2021/051281 2020-10-29 2021-10-28 Polynucléotides pour l'édition d'arn et leurs méthodes d'utilisation WO2022091100A1 (fr)

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Cited By (2)

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WO2024153694A1 (fr) 2023-01-18 2024-07-25 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Oligonucléotides antisens pour l'édition d'arn et leurs procédés d'utilisation

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