WO2024023580A2 - Stabilisation d'arn avec des motifs de liaison au miarn - Google Patents

Stabilisation d'arn avec des motifs de liaison au miarn Download PDF

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WO2024023580A2
WO2024023580A2 PCT/IB2023/000449 IB2023000449W WO2024023580A2 WO 2024023580 A2 WO2024023580 A2 WO 2024023580A2 IB 2023000449 W IB2023000449 W IB 2023000449W WO 2024023580 A2 WO2024023580 A2 WO 2024023580A2
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cell
mirna
mir
target rna
rna molecule
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Luca GEBERT
Ian MACRAE
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The Scripps Research Institute
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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—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 against viruses
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
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    • C12N2310/141—MicroRNAs, miRNAs
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    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24221—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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    • C12N2830/00—Vector systems having a special element relevant for transcription

Definitions

  • MicroRNAs form regulatory networks in metazoans. Viruses engage miRNA networks in numerous ways, with Flaviviridae members exploiting direct interactions of their RNA genomes with host miRNAs.
  • the present invention is directed to applications of miRNA binding to stabilize RNA sequences of interest and promote their expression in specified mammalian cell types.
  • the invention provides modified RNA molecules that contain a stabilizing motif or stabilizing element at the 5’ of an RNA sequence of interest (or “target RNA”).
  • the stabilizing motif in the modified RNA molecules is capable of binding via base paring to a miRNA sequence that is specifically or predominantly expressed in one or several specific tissue or cell types.
  • the target RNA sequence that is modified with the stabilizing motif is an mRNA encoding a polypeptide of interest (e.g., a therapeutic polypeptide).
  • the stabilizing motif is complementary to the seed region of the miRNA.
  • the stabilizing motif is additionally complementary to the supplementary region of the miRNA.
  • the stabilizing motif contains a sequence that is substantially identical to the first 29 nucleotides at the 5’ of human HCV genome (SEQ ID NO: 1).
  • the stabilizing motif contains from 5’ to 3’ (a) nucleotides 1-4 that are complementary to nucleotides 17-14 of the miRNA, wherein nucleotide 1 of the stabilizing motif is complementary to nucleotide 17 of the miRNA, (b) a hairpin of about 16 nucleotides, (c) a G nucleotide, (d) a sequence of about 7 nucleotides that are complementary to nucleotides 8-2 of the miRNA, and (e) an A nucleotide.
  • the hairpin contains the sequence 5’ - gccgccugauggcggc - 3’ (SEQ ID NO:2).
  • the RNA sequence of interest in the modified RNA molecules encodes a therapeutic polypeptide, an antibody or an antibody fragment.
  • the invention provides methods for stabilizing a target RNA molecule in a specific tissue or cell. These methods involve (a) appending or fusing at the 5’ end of the target RNA molecule a stabilizing motif to generate a modified target RNA molecule, whereas the stabilizing motif can bind via base pairing to a miRNA that is specifically or abundantly expressed in the specific tissue or cell; and (b) introducing into the cell the modified target RNA molecule or a DNA sequence (e.g., via an expression vector) encoding the modified target RNA molecule. Typically, the target RNA molecule is uncapped or non-canonically capped prior to appending the stabilizing motif. In some preferred embodiments, the target RNA molecule is an mRNA.
  • the stabilizing motif appended to the target RNA sequence is complementary to the seed region of the miRNA. In some embodiments, the stabilizing motif is further complementary to the supplementary region of the miRNA. In some embodiments, the stabilizing motif contains a sequence that is substantially identical to the first 29 nucleotides at the 5’ of human HCV genome (SEQ ID NO: 1), and the specific cell is a liver cell.
  • the stabilizing motif appended to the target RNA sequence contains from 5’ to 3’ (a) nucleotides 1-4 that are complementary to nucleotides 17-14 of the miRNA (with nucleotide 1 of the target RNA being complementary to nucleotide 17 of the miRNA), (b) a hairpin structure of about 16 nucleotides, (c) a G nucleotide, (d) a sequence of about 7 nucleotides that are complementary to nucleotides 8-2 of the miRNA, and (e) an A nucleotide.
  • the hairpin contains the sequence 5’- gccgccugauggcggc - 3’ (SEQ ID NO:2).
  • the specific tissue or cell into which the modified target RNA molecule is to be introduced is a cancer cell, a cardiac cell or a neuron.
  • the target RNA molecule encodes a therapeutic polypeptide, an antibody or an antibody fragment.
  • the specific tissue or cell is brain, and the miRNA is miR-9, miR-124 or miR-128a/b.
  • the specific tissue or cell is pituitary gland, and the miRNA is miR-7, miR-375, miR-141 or miR200a.
  • the specific tissue or cell is thyroid or hematopoietic cell, and the miRNA is miR-142, miR-144, miR150, miR-155 or miR-223.
  • the specific tissue or cell is myocardial or muscle, and the miRNA is miR-l-3p, miR-133a-3p, miR-133b or miR-206.
  • the specific tissue or cell is melanocyte, and the miRNA is miR205-5p.
  • the specific tissue or cell is skin, testis and colon, and the miRNA is respectively miR-205-5p, miR-514a-3p and miR-192-5p.
  • the specific tissue or cell is spleen tissue, and the miRNA is miR-449c-3p or miR-449b-3p. In some other methods, the specific tissue or cell is testis tissue, and the miRNA is miR-514a-3p. In some other methods, the specific tissue or cell is kidney or small intestine, and the miRNA is miR-449c-5p or miR-449b-5p. In still some other methods, the specific tissue or cell is lung, kidney and brain, and the miRNA is miR-449a.
  • the invention provides methods for expressing, or enhancing the expression of, a target RNA molecule in a specific tissue or cell. These methods entail (a) appending at the 5’ end of the target RNA molecule a stabilizing motif to generate a modified target RNA molecule, whereas the stabilizing motif is complementary to a miRNA that is specifically or abundantly expressed in the specific tissue or cell; and (b) introducing the modified target RNA molecule or a DNA sequence encoding the modified target RNA into the specific cell type.
  • the target RNA molecule is exogenous to the specific tissue or cell.
  • the specific tissue or cell is present in a human subject.
  • the specific tissue or cell is a cancer cell, a cardiac cell or a neuron.
  • the target RNA molecule encodes a therapeutic polypeptide, an antibody or an antibody fragment.
  • Figure 1 shows the structure of the Ago2:miR-122:HCV RNA complex.
  • A Cartoon ribbon representation of the atomic model. Shown in the panel are Ago2 protein, its globular domains (N, PAZ MID, and PIWI), miR-122, and HCV RNA. Locations of the HCV RNA 5' terminus, SL1 and G21 are indicated.
  • B Cartoon schematic of miR-122:HCV RNA three-way junction.
  • C Detailed schematic of base-pairing within the miR-122 (SEQ ID NO:3):HCV RNA (SEQ ID NO: 1) three-way junction.
  • Figure 2 shows that HCV RNA stability is modulated by specific interactions with the Ago2-miR122 complex.
  • FIG. 3 shows examples of tissue-specific expression patterns of abundant human miRNAs. Bar graphs showing the relative abundance of six different miRNAs in diverse adult human tissues. Highly expressed miRNAs are often restricted to a small set of specific tissue types. Data obtained from PMID: 26921406.
  • Figure 4 illustrates a general strategy for stabilizing RNAs of interest via interactions with the Ago-miRNA complex. Shown in the figure are the stabilizing element (SEQ ID NO:4) to be added to the 5' end of the RNA of interest, and the complementary stabilizing miRNA (SEQ ID NO: 5). N indicates any nucleotide, provided the specified Watson-Crick base-pairing interactions (indicated by gray lines) are established. RNAs are numbered counting from their 5' ends. A indicates adenine nucleotide, G indicates guanine.
  • Figure 5 shows enhanced expression of mRNAs appended with an miR-binding motif in HEK293 cells using exogenously supplied miRNAs.
  • Figure 6 shows enhanced expression of an mRNA appended with a miR-122 binding motif in Huh-7 cells that endogenously expresses miR-122.
  • the present invention provides compositions and methods for stabilizing RNA sequences and promoting their expression in specific tissue or cell types.
  • the invention is predicated in part on the studies undertaken by the inventors to stabilize RNAs in specified mammalian cell types.
  • HCV hepatitis C virus
  • binding of liver-abundant miR-122 stabilizes the viral RNA and regulates viral translation.
  • the inventors investigated the structural basis for these activities, taking into consideration that miRNAs function in complex with Argonaute (Ago) proteins.
  • the crystal structure of the Ago2:miR-122:HCV complex reveals a structured RNA motif that traps Ago2 on the viral RNA, masking its 5’ end from enzymatic attack
  • the trapped Ago2 can recruit host factor PCBP2, implicated in viral translation, while binding of a second Ago2:miR-122 competes with PCBP2, creating a potential molecular switch for translational control.
  • Combined results revealed a viral RNA structure that modulates Ago2:miR-122 dynamics and repurposes host proteins to generate a functional analog of the mRNA cap-binding complex.
  • RNA of interest cellular Argonaute (Ago) proteins
  • miRNAs cellular microRNAs
  • Ago proteins form a stable complex with miRNAs in most human cells.
  • the inventors identified a set of parameters that, when appended to the 5' end of the RNA of interest, can recruit and trap Ago-miRNA complexes and thereby shield the 5' end of the RNA of interest from degradation by cellular exonucleases and recognition by the innate immune response.
  • RNA of interest i.e., an uncapped or non-canonically capped target mRNA molecule
  • RNA of interest i.e., an uncapped or non-canonically capped target mRNA molecule
  • the inventors demonstrated that expressions of RNAs modified with a 5’ appended stabilizing motif in live mammalian cells are upregulated by several folds when the corresponding miRNA is either exogeneously supplied or endogenously expressed.
  • the invention provides modified RNA molecules with enhanced stability in one or more specific tissue or cell types.
  • the modified RNA molecules contain at its 5’ an appended stabilizing motif that can bind via base pairing to a miRNA that is specifically or abundantly expressed in the specific tissue or cell types.
  • Related methods for stabilizing a target RNA sequence and for promoting its expression in one or more specific tissue or cell types are also provided in the invention.
  • RNA molecules of the invention and related methods of the invention can all be generated or performed in accordance with the procedures exemplified herein or routinely practiced methods well known in the art. Unless otherwise indicated, the practice of the present invention employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art Such techniques are explained fully in the literature.
  • agent includes any substance, molecule, element, compound, entity, or a combination thereof. It includes, but is not limited to, e.g., protein, polypeptide, small organic molecule, polysaccharide, polynucleotide, and the like. It can be a natural product, a synthetic compound, or a chemical compound, or a combination of two or more substances. Unless otherwise specified, the terms “agent”, “substance”, and “compound” are used interchangeably herein.
  • complementary refers to a nucleotide or nucleotide sequence that hybridizes to a given nucleotide or nucleotide sequence.
  • the nucleotide A is complementary to T and vice versa
  • the nucleotide C is complementary to G and vice versa.
  • the nucleotide A is complementary to the nucleotide U and vice versa
  • the nucleotide C is complementary to the nucleotide G and vice versa.
  • Complementary nucleotides include those that undergo Watson and Crick base pairing and those that base pair in alternative modes (non-Watson and Crick base pairing).
  • the nucleotide G is complementary to the nucleotide U and vice versa
  • the nucleotide A is complementary to the nucleotide G and vice versa. Therefore, in an RNA molecule, the complementary base pairs are A and U, G and C, G and U, and A and G. Other combinations, e.g., A and C or C and U, are considered to be non- complementary base pairs.
  • a complementary sequence is comprised of individual nucleotides that are complementary to the individual nucleotides of a given sequence, where the complementary nucleotides are ordered such that they will pair sequentially with the nucleotides of the given sequence. Such a complementary sequence is said to be the "complement" of the given sequence.
  • RNA capping refers to a highly methylated modification of the 5’ end of RNA Pol Il-transcribed RNA. It protects RNA from degradation, recruits complexes involved in RNA processing, export and translation initiation, and marks cellular mRNA as “self’ to avoid recognition by the innate immune system.
  • eukaryotic mRNAs contain a cap structure - an N7-methylated guanosine linked to the first nucleotide of the RNA via a reverse 5’ to 5’ triphosphate linkage.
  • the mRNA cap In addition to its essential role of cap-dependent initiation of protein synthesis, the mRNA cap also functions as a protective group from 5’ to 3’ exonuclease cleavage and a unique identifier for recruiting protein factors for pre-mRNA splicing, polyadenylation and nuclear export. It also acts as the anchor for the recruitment of initiation factors that initiate protein synthesis and the 5’ to 3’ looping of mRNA during translation.
  • a great number of uncapped or non-canonically capped RNA species have been found to exist in plants and in mammals. See, e.g., Gregory et al., Dev Cell. 14: 854-66, 2008; Jiao et al., Plant Cell.
  • RNA decapping refers to the removal of the cap from a capped mRNA by decapping enzymes. This usually directs mRNA to be degraded Decapping is also regulated by RNA binding proteins which promote or antagonize the recruitment of decapping complexes to the mRNA.
  • miRNAs encompass a family of ⁇ 22 nucleotide (nt) noncoding RNAs that are used by the Argonaute (Ago) proteins as guides for identifying complementary sites in the messenger RNAs (mRNAs) that are targeted for repression.
  • RNAs have been identified in organisms ranging from nematodes to humans. Many miRNAs are evolutionarily conserved widely across phyla, regulating gene expression by post-transcriptional gene repression. It has been estimated that the expression of more than 50% of the protein-coding genes in humans are under miRNA control.
  • the long primary transcripts (pri-miRNAs) are transcribed by RNA polymerase II; processed by a nuclear enzyme Drosha; and released as a ⁇ 60bp hairpin precursor (pre-miRNAs). Pre-miRNAs are processed by RNase in enzymes, Dicer, to ⁇ 22 nt (mature miRNAs) and then incorporated into RISC (RNA-induced silencing complex). The complex of miRNAs-RISC binds the 3’ UTR of the target mRNAs and conducts translational repression or degradation of mRNAs.
  • RISC RNA-induced silencing complex
  • MiRNAs are categorized into families that are based on the sequence similarity, and each miRNA is able to target multiple mRNAs. The most conserved part of miRNA is located at 5'-end and is called the seed region. Guide (g) nucleotides from the seed region (g2-g7 or g2-g8) play a primary role in target recognition because more than 80% of the interactions between miRNAs and targets occur via seed pairing. However, for 20% of the miRNA-target interactions, other regions such as the supplementary region (g13-g16) of miRNA might be involved in targeting. Although the miRNAs that belong to the same family share the same seed sequence, they contain divergent 3' regions.
  • miRNA nucleic acid is defined as RNA or DNA that encodes a miR as defined above, or is complementary to a nucleic acid sequence encoding a miR, or hybridizes to such RNA or DNA and remains stably bound to it under appropriate stringency conditions. Specifically included are genomic DNA, cDNA, mRNA, miRNA and antisense molecules, pri-miRNA, pre-miRNA, mature miRNA, miRNA seed sequence, as well as nucleic acids based on alternative backbones or including alternative bases. MiRNA nucleic acids can be derived from natural sources or synthesized.
  • MicroRNA seed sequence "miRNA seed sequence,” “seed region” and “seed portion” are used to refer to nucleotides 2-7 or 2-8 of the mature miRNA sequence.
  • the miRNA seed sequence is typically located at the 5' end of the miRNA.
  • RNA-induced silencing complex RISC is responsible for the gene silencing phenomenon known as RNA interference (RNAi).
  • RNAi RNA interference
  • Argonaute proteins bind different classes of small non-coding RNAs, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). Small RNAs guide Argonaute proteins to their specific targets through sequence complementarity (base pairing), which then leads to mRNA cleavage, translation inhibition, and/or the initiation of mRNA decay.
  • the Argonaute (AGO) gene family encodes for six characteristic domains: N- terminal (N), Linker-1 (L1), PAZ, Linker-2 (L2), Mid, and a C-terminal PIWI domain.
  • a “host cell” refers to a living cell into which a heterologous polynucleotide sequence is to be or has been introduced.
  • the living cell includes both a cultured cell and a cell within a living organism.
  • Means for introducing the heterologous polynucleotide sequence into the cell are well known, e.g., transfection, electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, and/or the like.
  • the heterologous polynucleotide sequence to be introduced into the cell is a replicable expression vector or cloning vector.
  • host cells can be engineered to incorporate a desired gene on its chromosome or in its genome.
  • CHO cells e.g., CHO cells
  • host cells e.g., CHO cells
  • the host cell is a mammalian cell.
  • operably linked or “operably associated” refers to functional linkage between genetic elements that are joined in a manner that enables them to carry out their normal functions.
  • a DNA sequence encoding a modified RNA molecule described herein is operably linked to a promoter when its transcription is under the control of the promoter and the transcript produced is correctly translated into a protein of interest that is normally encoded by the DNA gene.
  • a “substantially identical” nucleic acid or amino acid sequence refers to a polynucleotide or amino acid sequence which comprises a sequence that has at least 75%, 80% or 90% sequence identity to a reference sequence as measured by one of the well-known programs described herein (e.g., BLAST) using standard parameters.
  • the sequence identity is preferably at least 95%, more preferably at least 98%, and most preferably at least 99%.
  • the subject sequence is of about the same length as compared to the reference sequence, i.e., consisting of about the same number of contiguous amino acid residues (for polypeptide sequences) or nucleotide residues (for polynucleotide sequences).
  • Sequence identity can be readily determined with various methods known in the art.
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
  • Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • a cell has been “transformed” or “transfected” by exogenous or heterologous polynucleotide when such polynucleotide has been introduced inside the cell
  • the transforming polynucleotide may or may not be integrated (covalently linked) into the genome of the cell.
  • the transforming polynucleotide may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming polynucleotide has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication.
  • a “clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • vector refers to polynucleotide sequence elements arranged in a definite pattern of organization such that the expression of genes/gene products that are operably linked to these elements can be predictably controlled. Typically, they are transmissible polynucleotide sequences (e.g., plasmid or virus) into which a segment of foreign polynucleotide sequence can be spliced in order to introduce the foreign DNA into host cells to promote its replication and/or transcription.
  • vectors capable of directing the expression of heterologous polynucleotide or target gene sequences encoding for one or more polypeptides are referred to as "expression vectors" or "expression constructs”.
  • the invention provides modified or stabilized RNA molecules that can be expressed in specific tissue or cell types.
  • a general strategy for obtaining such modified RNAs of the invention is shown in Figure 4.
  • the modified RNAs contain an RNA of interest or “target RNA” (e.g., an mRNA encoding a therapeutic protein) and a sequence motif (or “stabilizing motif’ or “stabilizing element”) that is appended at the 5’ end of the RNA of interest.
  • target RNA e.g., an mRNA encoding a therapeutic protein
  • sequence motif or “stabilizing motif’ or “stabilizing element”
  • the target RNA of interest is an mRNA.
  • modified target RNA molecule or “modified RNA” is able to bind via base pairing to a miRNA molecule (e.g., miR-122) that is specifically or abundantly expressed in one or more target tissue or cell types (e.g., liver cells).
  • miRNA molecule e.g., miR-122
  • the target RNA of interest to be modified encodes a therapeutic polypeptide, an antibody or an antibody fragment.
  • the miRNA to which the stabilizing motif binds can regulate gene expressions that are associated with a cellular process or disease development, e.g., tumor development.
  • the sequence motif amended to the target RNA is complementary to a tissue- or cell-specific miRNA.
  • the stabilizing motif is complementary to the seed region of the miRNA. In some of these embodiments, the stabilizing motif is further complementary to the supplementary region of the miRNA.
  • RNA element found near the 5' terminus of the RNA genome of Hepatitis C Virus can be employed as the stabilizing motif in the invention.
  • this sequence motif is able to base pair with miRNA-122, which is highly expressed in liver, associated with cholesterol metabolism and hepatocellular carcinoma, and plays an important role in HCV replication.
  • HCV is a positive-strand RNA virus and human pathogen that can persist in liver cells for decades. Interactions between the HCV genomic RNA and the liver-specific miRNA miR-122 have been shown to be essential for maintaining HCV RNA abundance during viral infection (PMID: 16141076, 18621012, 19965718).
  • the stabilizing motif in the modified RNA molecules of the invention contains a sequence that is substantially identical (e.g., at least 75%, 85%, 90%, 95% or 99% identical) to the first 29 nucleotides at the 5’ of human HCV genome, 5’- gcca gccg ccug augg cggc gaca cucca - 3’ (SEQ ID NO: 1).
  • SEQ ID NO: 1 the first 29 nucleotides at the 5’ of human HCV genome
  • nucleotides 1-4 (counting from the 5' end) of the stabilizing motif correspond to the reverse complement of nucleotides 14-17 of miR-122 (where nucleotide 1 of the modified target RNA is complementary to nucleotide 17 of the miRNA).
  • the modified target RNA further has its sequence immediately after nucleotide 4 encoding an RNA hairpin structure. For example, starting at residue 5, the modified target RNA can contain the sequence: gccgccugauggcggc (SEQ ID NO:2).
  • the modified target RNA has immediately following the hairpin sequence a single G nucleotide (corresponding to t9G).
  • the sequence after the G nucleotide in the modified target RNA should be the reverse complement of nucleotides 2-8 of the reference miRNA.
  • the final nucleotide in the appended motif of the modified target RNA is an A residue (corresponding to tlA).
  • the stabilizing motif contains a sequence that is conservatively modified variant of SEQ ID NO: 1.
  • the sequence of the stabilizing motif is identical to SEQ ID NO:1.
  • the stabilizing motif appended at the 5’ of a target RNA is modified from the 29-nt HCV 5’ sequence (SEQ ID NO:1).
  • This modified stabilizing motif is made to be complementary to the target RNA pairing site of another tissue-specific miRNA (“reference miRNA”) other than miR-122.
  • sequence of the HCV derived stabilizing motif can be altered so that it is complementary to the seed and/or supplementary regions of other miRNAs.
  • RNAs modified at the 5’ end with such altered stabilizing motifs are stabilized in cell types in which a given miRNA is specifically or highly expressed.
  • nucleotides 1 ⁇ 4 (counting from the 5' end) of the altered stabilizing motif RNA of interest are the reverse complement of the nucleotide sequence of 14-17 of the given miRNA (where nucleotide 1 of the altered stabilizing motif is complementary to nucleotide 17 of the miRNA).
  • the sequence immediately after nucleotide 4 in the altered stabilizing motif encodes an RNA hairpin structure of about 16 nucleotides.
  • the hairpin sequence can immediately be followed by a single G nucleotide (corresponding to t9G).
  • the sequence after the G nucleotide should be the reverse complement of nucleotides 2-8 of the given miRNA.
  • the final nucleotide in the altered stabilizing element appended to the RNA of interest can be an A (corresponding to tl A).
  • RNAs of interest By appending to a target RNA of interest a stabilizing motif that is complementary to various miRNAs that are specifically or abundantly expressed in different tissue or cell types, the modified RNAs of interest are stabilized in the different target tissue or cell types.
  • each human cell type typically expresses one or two abundant miRNAs, which can be used as signatures for each cell type. Studies of miRNA expression patterns have shown that abundant miRNAs are often restricted to discrete human tissues.
  • Notable miRNA families with high tissue specificity include, e.g., miR-378, miR-506, miR-8, miR-28, miR-15, miR-320, miR-10, miR181-, let-7, miR-17, miR-379, miR-29, miR-154, miR-30, miR-368, miR-188, miR-743, miR-34, miR-1273, miR-374, miR-130, miR-500, miR-515, miR-449, and 548. See, e.g., Lugwig et al., Nucleic Acids Res. 44(8):3865-77, 2016.
  • a stabilizing motif corresponding to a miRNA that is specifically or abundantly expressed in a given cell type a target RNA can be stabilized and/or specifically expressed in many different types of tissues and cells.
  • miRNAs and corresponding tissue/cell specificities include, e.g., miR-9, miR-124 and miR 128a/b in brain; miR-7, miR-375, miR-141 and miR200a in pituitary gland; miR-142, miR-144, miR150, miR-155 and miR-223 in thyroid and hematopoietic cells (vein and spleen); miR-l-3p, miR-133a-3p, miR-133b and miR-206 in myocardial tissues and muscle; miR205-5p in melanocytes, and miR-514a-3p and miR-192-5p in skin, testis and colon, respectively; miR-514a-3p in testis tissue; miR-449c-3p in spleen tissue; miR-449c-5p and miR-449b-5p in kidney and small intestine; miR-9, miR-124 and miR 128a/b in brain; miR-7, miR-375, mi
  • tissue specificities of various human miRNAs can be found in, e.g., Lugwig et al., Nucleic Acids Res. 44(8):3865-77, 2016; Guo et al., Sci Rep 4, 5150, 2014; Panwar et al., Bioinformatics 33: 1554-60, 2017; McCall et al., Genome Res. 27: 1769-1781, 2017; Juzenas et al., Nucleic Acids Res. 45: 9290-9301, 2017; Pomper et al., Scientific Reports 10: 4921, 2020; Moreau et al., Arterioscler Thromb Vase Biol. 41 :2149-2167, 2021.
  • Appending a target RNA sequence with a 5’ stabilizing motif can be readily performed with standard techniques of molecular biology.
  • the target RNA sequence can be modified directly before being introduced into a specific cell type.
  • fusing of the stabilizing motif to the target RNA sequence can be accomplished at the DNA level, i.e., by manipulating a DNA sequence that encodes the target RNA sequence.
  • a modified DNA sequence that encodes the target RNA with an appended 5’ stabilizing motif can then be introduced into the desired tissue or cell type, typically via an expression construct, to promote expression of an encoded polypeptide of interest.
  • the invention accordingly also provides expression constructs (RNA or DNA) that harbor or encode the modified RNAs described herein.
  • the expression constructs of the invention contain or encode a modified RNA described herein, and are suitable for introducing into a target tissue or cell. They are typically circular vectors and, in addition to the stabilizing motif bearing sequence (DNA or RNA) that encodes the polypeptide of interest, can also contain selectable markers, an origin of replication, and other elements.
  • the vector can contain a selection marker.
  • the selection marker allows one to select for cells into which the vector has been introduced and/or stably integrated.
  • the selection marker can be a polynucleotide encoding a protein or enzyme that confers to the cells visually identifiable characteristics.
  • the vector can harbor a selection marker encoding Renilla luciferase reporter enzyme.
  • the selection marker for identifying host cells into which the vector was introduced and/or stably integrated can be an antibiotic resistance gene.
  • antibiotic resistance genes include antibiotic resistance genes for neomycin, chloramphenicol, blasticidin, hygromycin, and zeocin.
  • the expression vectors of the invention can also bear other DNA sequences that may be necessary for proper RNA transcription and processing, as well as proper ribosome assembly and function.
  • some vectors of the invention additionally harbor sequences corresponding to the 5’-ETS and ITS elements of the precursor RNA sequence.
  • the expression constructs can be recombinantly produced with many vectors well known in the art. These include viral vectors such as recombinant adenovirus, retrovirus, lentivirus, herpesvirus, poxvirus, papilloma virus, or adeno-associated virus.
  • the vectors can be present in liposomes, e.g., neutral or cationic liposomes, such as DOSPA/DOPE, DOGS/DOPE or DMRIE/DOPE liposomes, and/or associated with other molecules such as DNA-anti-DNA antibody-cationic lipid (DOTMA/DOPE) complexes.
  • DOTMA/DOPE DNA-anti-DNA antibody-cationic lipid
  • the expression vectors may be administered to a subject via any route including, but not limited to, intramuscular, buccal, rectal, intravenous or intracoronary routes.
  • Target proteins encoded by modified RNAs containing stabilizing motif can encode any exogenous proteins or polypeptides that are desired for the specific cell type.
  • the target RNA molecule encodes a therapeutic polypeptide.
  • These methods can be employed in therapeutic applications for delivering and expressing the stabilizing motif-appended RNA molecules in human tissues and cells that have abundant expression of a corresponding miRNA complementary to the stabilizing motif.
  • the methods of the invention can be used in the prevention and/or cure of many important human diseases, including many diseases which are not amenable to treatment by other therapies.
  • a stabilized mRNA molecule of the invention can be transferred, for example to treat cancer cells, to express immunomodulatory genes to fight viral infections, or to replace a gene’s function as a result of a genetic defect.
  • the exogenous mRNA (or corresponding DNA sequence) can also encode an antigen of interest for the production of antibodies.
  • the exogenous sequence to be transferred with the methods of the present invention is a polynucleotide that encodes an enzyme.
  • the gene can encode a cyclin-dependent kinase (CDK).
  • Additional embodiments of the invention encompass transferring into target cells exogenous genes that encode cell adhesion molecules or therapeutic antibodies, other tumor suppressors such as p21 and BRCA2, inducers of apoptosis such as Bax and Bak, other enzymes such as cytosine deaminases and thymidine kinases, hormones such as growth hormone and insulin, and interleukins and cytokines.
  • RNA molecules of the invention can encode an antibody or antibody fragment.
  • These include many known antibody-related molecules that are well characterized in the art, e.g., CD4-Ig, eCD4-Ig, PG9, PG16, PGT121, PGT128, 10-1074, PGT145, PGT151, CAP256, 2F5, 4E10, 10E8, 3BNC117, VRC01, VRC07, VRC13, PGDM1400, PGV04, 2G12, bl2, N6, TR66, etanercept, abatacept, rilonacept, aflibercept, belatacept, romiplostim, efmoroctocog, eftrenonacog, asfotase alpha, muromonab-CD3, edrecolomab, capromab, ibritumomab, blinatumomab, abciximab
  • the transgene in the expression vectors of the invention can encode at least a chain or functional fragment derived from any of the other known cellular proteins such as cellular receptors, other cell surface molecules, enzymes, cytokines, chemokines, costimulatory molecules, interleukins, and physiologically active polypeptide factors.
  • these known cellular proteins include, e.g., CD4, TPST1, TPST2, TNFR II, CD28, CTLA-4, PD-1, PD-L1, PD-L2, 4-1BBL, 4-1BB, EPO, Factor VIII, Factor IX, alkaline phosphatase, hemoglobin, fetal hemoglobin, and RPE65.
  • the polypeptide expressed from the rAAV vectors of the invention is at least part of a chimeric antigen receptor (CAR).
  • RNA molecules of the invention can encode one or more other therapeutic polypeptide agents that are well-known known in the art. These include factor VIII, factor IX, ⁇ -globin, low-density lipoprotein receptor, adenosine deaminase, purine nucleoside phosphorylase, sphingomyelinase, glucocerebrosidase, cystic fibrosis transmembrane conductance regulator, a-antitrypsin, CD-18, ornithine transcarbamylase, argininosuccinate synthetase, phenylalanine hydroxylase, branched-chain a-ketoacid dehydrogenase, fumarylacetoacetate hydrolase, glucose 6-phosphatase, a-L-fucosidase, ⁇ - glucuronidase, a-L-iduronidase, galactose 1 -phosphate uridyltransferase
  • pathogens e.g., E. coli, P. aeruginosa, S. aureus, malaria, HIV, rabies virus, HBV, and cytomegalovirus
  • proteins such as lactoferrin, thioredoxin and beta-casein.
  • therapeutic agents or proteins of interest include, but are not limited to, insulin, erythropoietin, tissue plasminogen activator (tPA), urokinase, streptokinase, neutropoiesis stimulating protein (also known as filgastim or granulocyte colony stimulating factor (G-CSF)), thrombopoietin (TPO), growth hormone, emoglobin, insulinotropin, imiglucerase, sarbramostim, endothelian, soluble CD4, and antibodies and/or antigen-binding fragments (e g., FAbs) thereof (e g., orthoclone OKT-e (anti-CD3), GPIIb/IIa monoclonal antibody), ciliary neurite transforming factor (CNTF), granulocyte macrophage colony stimulating factor (GM-CSF), brain-derived neurite factor (BDNF), parathyroid hormone(PTH)-like hormone, insulinotrophic hormone, insulin-like
  • the invention provides methods of stabilizing or enhancing stability of an RNA molecule in selected tissue or cell types.
  • the methods utilize modified RNAs that contain an appended stabilizing motif as described above.
  • the stabilizing motif can enable the target RNA to bind to a miRNA molecule specifically or predominantly expressed in one or several specific tissue or cell types.
  • Binding by the miRNA to the modified target RNA molecule protects it from degradation by cellular exonucleases (primarily XRN-1 and XRN-2), thereby stabilizing the target RNAs in the specific tissue- or cell- types.
  • RNAs lacking a canonical 5' cap structure are subject to degradation by the cellular exonucleases.
  • cells or tissues lacking the specified miRNA will be unable to accumulate the target RNA of interest.
  • the invention also provides methods of promoting or enhancing expression of a target RNA (e g , an mRNA) in a specific or specified cell type.
  • a target RNA e g , an mRNA
  • the target RNA is modified by fusing or appending at its 5’ a stabilizing motif that specifically binds to a miRNA.
  • the target RNA molecule thus modified (or its encoding DNA sequence) is introduced into a specific cell type (e g., liver cell) in which the corresponding miRNA (e.g., miR-122) is differentially or abundantly expressed.
  • the stabilizing element may be appended to the 5' end of any uncapped or non-canonically capped RNA for which accumulation in specific cell types is desired. If placed upstream of an appropriate Internal Ribosomal Entry Site (IRES), the stabilizing element may enable exogenous mRNAs to be expressed in specified cell types (e g., cancer cells, cardiac cells, neurons, etc.).
  • IRS Internal Ribosomal Entry Site
  • a stabilizing motif bearing RNA of interest can be directly introduced into a corresponding specific cell type via standard techniques of molecule biology.
  • delivering a modified exogenous RNA sequence into the specific cell type is achieved by first generating a DNA sequence encoding the stabilized RNA and then introducing the DNA sequence (e.g., via suitable expression vector) into the desired specific cell type.
  • the modified RNA molecules of the invention or DNA sequences encoding the same can be delivered to specific tissues or cells with various methods well known in the art.
  • a transfection agent is used.
  • a transfection agent, or transfection reagent or delivery vehicle is a compound that binds to or complexes with oligonucleotides and polynucleotides, and enhances their entry into cells.
  • transfection reagents include, but are not limited to, cationic liposomes and lipids, polyamines, calcium phosphate precipitates, polycations, histone proteins, polyethylenimine, polylysine, and polyampholyte complexes.
  • Transfection reagents are well known in the art. Examples of transfection reagents suitable for delivery of mRNA molecules include MagicTM mRNA Transfection Reagent (Creative Biolabs) and in vivo-jetRNA® (Polyplus).
  • Reagents for delivery of modified target RNA molecules of the invention or their encoding DNA sequences into specific tissue or cells include, but are not limited to protein and polymer complexes (polyplexes), lipids and liposomes (lipoplexes), combinations of polymers and lipids (lipopolyplexes), and multilayered and recharged particles.
  • Transfection agents may also condense nucleic acids. Transfection agents may also be used to associate functional groups with a polynucleotide.
  • Functional groups can include cell targeting moieties, cell receptor ligands, nuclear localization signals, compounds that enhance release of contents from endosomes or other intracellular vesicles (such as membrane active compounds), and other compounds that alter the behavior or interactions of the compound or complex to which they are attached (interaction modifiers).
  • complexes made with sub-neutralizing amounts of cationic transfection agent may be preferred.
  • polycations are mixed with polynucleotides for delivery to a cell.
  • Poly cations are a very convenient linker for attaching specific receptors to DNA and as result, DNA/polycation complexes can be targeted to specific cell types.
  • An endocytic step in the intracellular uptake of DNA/polycation complexes is suggested by results in which functional DNA delivery is increased by incorporating endosome disruptive capability into the polycation transfection vehicle.
  • Polycations also cause DNA condensation.
  • the volume which one DNA molecule occupies in complex with polycations is drastically lower than the volume of a free DNA molecule.
  • the size of DNA/polymer complex may be important for gene delivery in vivo.
  • Polymer reagents for delivery of a nucleic acid molecule may incorporate compounds that increase their utility. These groups can be incorporated into monomers prior to polymer formation or attached to polymers after their formation.
  • An anti-miRNA transfer enhancing moiety is typically a molecule that modifies a nucleic acid complex and can direct it to a cell location (such as tissue cells) or location in a cell (such as the nucleus) either in culture or in a whole organism. By modifying the cellular or tissue location of the complex, the desired localization and activity of the polynucleotide can be enhanced.
  • the transfer enhancing moiety can be, for example, a protein, peptide, lipid, steroid, sugar, carbohydrate, nucleic acid, cell receptor ligand, or synthetic compound.
  • the transfer enhancing moi eties can enhance cellular binding to receptors, cytoplasmic transport to the nucleus and nuclear entry or release from endosomes or other intracellular vesicles.
  • Nuclear localizing signals can also be used to enhance the targeting of the nucleic acid molecule into proximity of the nucleus and/or its entry into the nucleus.
  • Such nuclear transport signals can be a protein or a peptide such as the SV40 large Tag NLS or the nucleoplasmin NLS.
  • These nuclear localizing signals interact with a variety of nuclear transport factors such as the NLS receptor (karyopherin alpha) which then interacts with karyopherin beta.
  • the nuclear transport proteins themselves could also function as NLS's since they are targeted to the nuclear pore and nucleus.
  • Compounds that enhance release from intracellular compartments can cause DNA release from intracellular compartments such as endosomes (early and late), lysosomes, phagosomes, vesicle, endoplasmic reticulum, Golgi apparatus, trans Golgi network (TGN), and sarcoplasmic reticulum and could be used to aid delivery of the anti-miRNA molecule.
  • Endosomes early and late
  • lysosomes lysosomes
  • phagosomes phagosomes
  • vesicle endoplasmic reticulum
  • Golgi apparatus Golgi apparatus
  • TGN trans Golgi network
  • sarcoplasmic reticulum sarcoplasmic reticulum
  • Such compounds include chemicals such as chloroquine, bafilomycin or Brefeldin Al and the ER-retaining signal (KDEL sequence), viral components such as influenza virus hemagglutinin subunit HA-2 peptides and other types of amphipathic peptides.
  • chemicals such as chloroquine, bafilomycin or Brefeldin Al and the ER-retaining signal (KDEL sequence)
  • viral components such as influenza virus hemagglutinin subunit HA-2 peptides and other types of amphipathic peptides.
  • Cellular receptor moieties are any signal that enhances the association of the nucleic acid molecule with a cell. Enhanced cellular association can be accomplished by either increasing the binding of the polynucleotide or polynucleotide complex to the cell surface and/or its association with an intracellular compartment, for example: ligands that enhance endocytosis by enhancing binding the cell surface.
  • Cellular receptor moieties include agents that target to asialoglycoprotein receptors by using asialoglycoproteins or galactose residues. Other proteins such as insulin, EGF, or transferrin can be used for targeting. Peptides that include the RGD sequence can also be used to target many cells.
  • Chemical groups that react with sulfhydryl or disulfide groups on cells can also be used to target many types of cells.
  • Folate and other vitamins can also be used for targeting.
  • Other targeting groups include molecules that interact with membranes such as lipids fatty acids, cholesterol, dansyl compounds, and amphotericin derivatives.
  • viral proteins could be used to target cells.
  • the invention further provides engineered mammalian cells which express a modified RNA sequence described herein.
  • Various mammalian cells can be employed for introducing an expression construct of the invention or by stably integrating the rDNA described herein into the host genome.
  • Polynucleotides containing or encoding the modified RNA can be introduced into an appropriate host cell (e.g., a mammalian cell such as 293T cell, N2a cell or CHO cell, or PBMC, or primary immune cells) by any means known in the art.
  • the cells can transiently or stably express the introduced vectors containing or encoding the target RNA.
  • mammalian cells are used in these embodiments of the invention.
  • Mammalian expression systems allow for proper post-translational modifications of expressed mammalian proteins to occur, e.g., proper processing of the primary transcript, glycosylation, phosphorylation and advantageously secretion of expressed product.
  • Suitable cells include cells rodent, cow, goat, rabbit, sheep, non-human primate, human, and the like).
  • Specific examples of cell lines include CHO, BHK, HEK293, N2a, VERO, HeLa, COS, MDCK, and W138. Any convenient protocol may be employed for in vitro or in vivo introduction of the expression vector into the host cell, depending on the location of the host cell.
  • the expression construct may be introduced directly into the cell under cell culture conditions permissive of viability of the host cell, e.g., by using standard transformation techniques.
  • the crystal structure shows base-pairing interactions between the HCV RNA and miR-122. Specifically, nucleotides 2-8 and 14-17 of miR-122 (counting from the 5' end of miR-122) pair with the HCV RNA, forming two RNA duplex regions termed ‘seed duplex’ and ‘supplementary duplex’, respectively ( Figure 1). Seed and supplementary duplexes are separated by a stem -loop structure (SL1) in the HCV RNA, thereby forming a three-way RNA junction.
  • SL1 stem -loop structure
  • the crystal structure also shows contacts between Ago2 and the HCV RNA.
  • SL1 of the HCV RNA inserts between the Ago2 PIWI and PAZ domains, locking Ago2 in an opened conformation.
  • HCV nucleotide G21 (corresponding to the ‘t9’ position in miRNA- targeting nomenclature) inserts into a surface pocket in the Ago2 PIWI domain, termed the t9G-binding pocket.
  • HCV nucleotide A29 inserts into a binding pocket in the Ago2 MID domain termed the tlA-binding pocket (PMID: 26359634).
  • Example 2 Defined contacts to Ago2-miR122 stabilize the HCV RNA
  • Example 3 Human miRNAs display discrete and diverse expression patterns [0070] Studies of miRNA expression patterns have shown that abundant miRNAs are often restricted to discrete human tissues (PMID: 26921406). Tumors and cancer cells also often display unique miRNA expression patterns. Thus, in many cases, expression of a particular miRNA may be used as a molecular signature for a specific cell type ( Figure 3).
  • Example 4 Expressing stabilized exogenous mRNAs in specific cell types [0071] This example describes expression of exogenous mRNAs appended with a stabilizing motif in a specific cell type in the presence of corresponding miRNAs. [0072] We first tested expression of modified mRNAs in HEK293 cells with exogeneously introduced miRNAs that are recognized by the 5’ stabilizing motif in the modified mRNAs. Specifically, uncapped mRNAs, encoding a luciferase reporter gene, were produced with stabilizing elements specific to either miRNA-A or miRNA-B (two artificial miRNAs, not naturally present in HEK293 cells).
  • mRNAs were transfected into HEK293 cells with miRNA-A, or miRNA-B, or no exogenous miRNA. Expression levels of the luciferase reporter were subsequently measured in each sample ( Figure 5, A). The results indicate that co-transfection of the mRNA containing a stabilizing element recognized by miRNA-A with miRNA-A led to approximately 10 times more luciferase activity than transfection of the same mRNA without a miRNA or with miRNA-B, demonstrating upregulation dependent on recognition by miRNA-A (( Figure 5, B, right). Similarly, the presence of miRNA-B promoted the expression of the mRNA containing a stabilizing element specifically recognized by miRNA-B ( Figure 5, B, left). These results indicate that the miRNA-specific stabilizing effects observed in vitro are recapitulated in living cells.

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Abstract

La présente invention concerne des molécules d'ARN modifiées ayant une stabilité améliorée dans un ou plusieurs types tissulaires ou cellulaires spécifiques. Les molécules d'ARN modifiées contiennent à l'extrémité 5' un motif de stabilisation ajouté qui peut se lier par un appariement de bases à un miARN qui est exprimé de manière spécifique ou abondante dans les types tissulaires ou cellulaires spécifiques. L'invention concerne également des procédés associés destinés à stabiliser une séquence d'ARN cible et à favoriser son expression dans un ou plusieurs types tissulaires ou cellulaires spécifiques.
PCT/IB2023/000449 2022-07-26 2023-07-25 Stabilisation d'arn avec des motifs de liaison au miarn WO2024023580A2 (fr)

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