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EP4532715A1 - Molécule d'acide nucléique fonctionnelle - Google Patents

Molécule d'acide nucléique fonctionnelle

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Publication number
EP4532715A1
EP4532715A1 EP23729102.6A EP23729102A EP4532715A1 EP 4532715 A1 EP4532715 A1 EP 4532715A1 EP 23729102 A EP23729102 A EP 23729102A EP 4532715 A1 EP4532715 A1 EP 4532715A1
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
acid molecule
functional nucleic
sequence
ires
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23729102.6A
Other languages
German (de)
English (en)
Inventor
Stefano GUSTINCICH
Sabrina D'AGOSTINO
Bianca PIERATTINI
Massimiliano VOLPE
Remo SANGES
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fondazione Istituto Italiano di Tecnologia
Original Assignee
Fondazione Istituto Italiano di Tecnologia
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fondazione Istituto Italiano di Tecnologia filed Critical Fondazione Istituto Italiano di Tecnologia
Publication of EP4532715A1 publication Critical patent/EP4532715A1/fr
Pending legal-status Critical Current

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    • 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
    • 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
    • C12N15/67—General methods for enhancing the expression
    • 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
    • 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
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/532—Closed or circular
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/10—Vectors comprising a special translation-regulating system regulates levels of translation
    • C12N2840/105—Vectors comprising a special translation-regulating system regulates levels of translation enhancing translation
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • the present invention relates to a circularfunctional nucleic acid molecule comprising one or more target binding sequences and a regulatory sequence comprising an internal ribosome entry site (IRES).
  • the invention also encompasses a linear functional nucleic acid molecule comprising one or more target binding sequences and a regulatory sequence comprising an IRES. Also included are methods of enhancing protein translation efficiency, and methods of treating gene defects using the circular and linear functional nucleic acid molecules of the invention.
  • Natural SINEUPs are a functional class of antisense IncRNAs that promote translation of their sense protein encoding genes. They are generated from genomic loci with partially overlapping sense/antisense transcript pairs organized in a head-to-head conformation.
  • SINEUP activity depends on the combination of two RNA domains: an overlapping antisense region, defined as the Binding Domain (BD), which drives SINEUP specificity, and a SINEB2 element, which is often in inverted orientation (invSINEB2), and acts as an Effector Domain (ED), determining the up-regulation of target m RNA translation.
  • a representative member of natural SINEUPs is AS Uchl1 , a IncRNA antisense to the mouse orthologue of the human ubiquitin C-terminal hydrolase L1 (Uchl1) gene. When overexpressed, AS Uchl1 increases UchL1 protein expression without affecting its m RNA levels. This activity requires the concomitant presence of ED and BD RNA sequences.
  • AS Uchl1 promotes the association of Uchl1 mRNA to heavy polysomes and consequent increased UCHL1 protein expression.
  • SINEUPs can increase expression of the protein encoded by a target mRNA by around 1.5 to 3 fold, making SINEUPs an ideal tool to increase target protein levels in vivo within a normal physiological range.
  • the present invention seeks to optimise the role of IRES containing functional nucleic acids.
  • CircRNAs are single-stranded, covalently closed RNA molecules produced from pre-m RNAs through a process called back-splicing. They were first observed in several viroid genomes and then visualized in the cytoplasm of HeLa cells with electron microscopy. With the advent of nextgeneration sequencing, thousands of circRNAs have been identified. They are regulated separately from their linear counterparts and are mostly conserved. CircRNAs can regulate protein expression through several mechanisms, such as binding to RBPs or by miRNA sponging. Without wishing to be bound by theory, it is envisaged that circularfunctional nucleic acids may be more resistant to degradation than their linear counterparts.
  • the inventors provide herein a circular functional nucleic acid molecule in which the molecular functionality of IRES sequences is retained when utilised as a trans- acting effector domain (ED), such that targeted translational upregulation of specific proteins is achieved when utilising IRES sequence EDs in conjunction with antisense target binding domains.
  • ED trans- acting effector domain
  • nucleic acid molecule wherein the nucleic acid molecule is linear and may act as a precursorto the circularfunctional nucleic acids.
  • a functional nucleic acid molecule comprising:
  • a regulatory sequence comprising an internal ribosome entry site (IRES), wherein the functional nucleic acid molecule is circular.
  • IRS internal ribosome entry site
  • a functional nucleic acid molecule comprising:
  • a DNA molecule encoding the functional nucleic acid molecule as defined herein.
  • an expression vector comprising the functional nucleic acid molecule or the DNA molecule, as defined herein.
  • a composition comprising the functional nucleic acid molecule, the DNA molecule or the expression vector, as defined herein.
  • nucleic acid molecule for enhancing translation of one or more target mR NA sequences.
  • an in vitro method for enhancing protein translation comprising administering to a cell the functional nucleic acid molecule, the DNA molecule, the expression vector, the composition or the pharmaceutical composition, as defined herein, to a subject.
  • Circ5533 comprises an IRES sequence (light grey) as ED and an upstream region potentially acting as mono- or multi BD (yellow) to one or more endogenous target mRNAs.
  • Circ5533 acts as a circUP increasing target protein levels.
  • BDs putative Binding Domains
  • Putative BD1 (pink) and BD2 (blue) are located on the region upstream of the IRES sequence of circ5533 (dark yellow).
  • BDs were identified by looking for the longest sequence pairing, in antisense orientation, between circ5533 RNA and Pxk mRNA; a minimal perfect match of 8nts was considered.
  • BD1 can potentially target 3 different regions on the 5’UTR of Pxk mRNA, identified as target sites (TSs) 1 , 2 and 3 (pink).
  • BD2 could pair with TS4 and/or TS5 (blue) on the 3’UTR of Pxk mRNA.
  • Pxk CDS is highlighted in green.
  • Circ5533 activity on PXK-FI_AG mutant fold inductions Identified TSs were individually deleted from Pxk-flag RNA; each derived construct was co-transfected with circ5533 in HEK293T cells (1 :12). After 48 h, cells were harvested and proteins and RNA extracted. Empty pcDNA3.1 (+)-Laccase2 vector, co-transfected with different PXK-mutants, was used as negative control; PXK-FI-AG WT + circ5533was the positive control. Deletion of TS1 from Pxk 5’UTR or of TS4 from Pxk 3’UTR inhibited the circ5533-mediated increase in PXK- FI-AG protein.
  • Plots report PXK-FLAG fold induction mean ⁇ SD; they represent 4 biological replicates; *p ⁇ 0.05 and **p ⁇ 0.01 were calculated with one- sample t-test.
  • the results provided herein demonstrate that IRESs can act as trans- acting translational up- regulators of protein expression within a circular functional nucleic acid.
  • the functional nucleic acid molecule provided may be utilised for the targeted upregulation of proteins of interest without affecting mRNA levels.
  • the functional nucleic acid molecule described herein may be used to enhance translation of a target mRNA sequence, such as a therapeutic target mRNA sequence which encodes a therapeutic target protein, without inducing negative side-effects associated with increasing expression of the target above physiological levels.
  • the functional nucleic acid molecule of the invention is preferably an RNA molecule.
  • the term “functional RNA molecule” refers to instances wherein the functional nucleic acid molecule is formed of RNA.
  • the functional RNA molecule is generally capable of enhancing the translation of a target m RNA.
  • a circular functional nucleic acid molecule according to the invention may be more stable than a linear nucleic acid molecule as it may be less susceptible to degradation, e.g., since exonucleases cannot degrade circular molecules as they lack ‘free ends’.
  • a circular functional nucleic acid molecule of the invention may therefore remain active for a longer time than a corresponding linear functional nucleic acid molecule.
  • a circular functional nucleic acid molecule may exhibit prolonged activity due to a prolonged lifetime and therefore be advantageous over a conventional non-circular (i.e. linear) nucleic acid molecule which does not or cannot circularise.
  • a functional nucleic acid molecule of the present invention comprises one or more target binding sequences comprising one or more sequences reverse complementary to one or more target mRNA sequences; and a regulator sequence comprising an internal ribosome entry site (IRES), wherein the functional nucleic acid molecule is linear and wherein the one or more target binding sequences and the regulatory sequence are positioned between two complementary intronic repeats.
  • IRS internal ribosome entry site
  • linear functional nucleic acid molecule refers to a nucleic acid molecule that possesses two termini: a 5’- and 3’ -terminus. Such nucleic acids may generally be considered to adopt a linear structure.
  • the linear functional nucleic acid molecule of the invention comprises two complementary intronic repeats, within which the regulatory sequences (binding domain(s) and effector domain) are positioned.
  • Intronic repeats are known to promote circRNA formation in cis, and are further regulated (in trans) by RNA binding proteins, such as splicing factors.
  • the intronic repeats disclosed herein may promote the formation of the circularfunctional nucleic acid molecule of the invention.
  • RNA ‘circ5533’ is endogenously expressed in eukaryotic cells.
  • Complementary intronic repeats such as those disclosed herein, are used for the over-expression of exogenous circ5533 through circularizing plasmids such as pcDNA3.1 (+)ZKSCAN or pcDNA3.1 (+) Laccase2 MCS Exon Vectors.
  • the intronic repeats are derived from the pCDNA3.1 (+) ZKSCAN1 MCS Exon Vector and comprise or consist of SEQ ID NO: 21 (‘upstream 5533 insert’), or an RNA sequence encoded thereby.
  • the intronic repeats are derived from the pCDNA3.1 (+) ZKSCAN1 MCS Exon Vector and comprise orconsist of SEQ ID NO: 22 (‘downstream 5533 insert’) or an RNA sequence encoded thereby. In one embodiment, the intronic repeats are derived from the pcDNA3.1 (+) Laccase2 MCS Exon Vector and comprise orconsist of SEQ ID NO: 23 (‘upstream 5533 insert’), or an RNA sequence encoded thereby.
  • the intronic repeats are derived from the pcDNA3.1 (+) Laccase2 MCS Exon Vector and comprise or consist of SEQ ID NO: 24 (‘downstream 5533 insert’), or an RNA sequence encoded thereby.
  • the intronic repeats comprise or consist of RNA sequences encoded by the DNA sequences of any one or more of SEQ ID NOs: 21 - 24.
  • the linear functional nucleic acid molecule of the invention may constitute a precursor of the circularfunctional nucleic acid molecule of the invention.
  • the linear functional nucleic acid molecule of the invention may circularise or be circularised, e.g., in vitro prior to use; in vitro within a cell, or in vivo (e.g., within a cell); by a natural process, such as endogenous back- splicing; or by synthetic methods, such as in vitro ligation.
  • linear and “circular” nucleic acid molecule refer to a nucleic acid molecule having a generally or overall linear (i.e., possessing 5’ and 3’ termini) or circular (i.e., covalently ‘closed’) configuration. These terms do not preclude, for example, the existence of regions within said functional nucleic acid molecule that possess some structural characteristics themselves, e.g., RNA secondary structure.
  • the functional nucleic acid molecule of the present invention is preferably an RNA molecule or modified RNA molecule as described herein.
  • the functional nucleic acid molecule of the invention is preferably a circRNA or linear precursor thereof.
  • the functional nucleic acid molecule of the invention comprises or consists of a sequence from the circRNAs identified in Table 1.
  • the functional nucleic acid molecule provided herein is trans- acting such that it functionally modulates sequences present on another RNA molecule.
  • the functional nucleic acid molecule further comprises at least one spacer sequence between the target determinant sequence and the regulatory sequence.
  • SEQ ID NO: 1 is a non-limiting example of a spacer/linker sequence which may be used in the functional nucleic acid molecule of the present invention.
  • the spacer/linker sequence may comprise SEQ ID NO: 1 .
  • the spacer/linker sequence may consist of SEQ ID NO: 1.
  • the functional nucleic acid molecule is single stranded.
  • the functional nucleic acid molecule consists of RNA nucleotides.
  • the functional nucleic acid molecule consists of DNA nucleotides.
  • the functional nucleic acid molecule is DNA.
  • the functional nucleic acid molecule comprises one or more modifications or chemical modifications.
  • modification refers to a structural change in, or on, the most com mon, natural ribonucleotides: adenosine, guanosine, cytidine, thymidine, or uridine ribonucleotides.
  • the chemical modifications described herein may be changes in or on a nucleobase (i.e. a chemical base modification), or in or on a sugar (i.e. a chemical sugar modification).
  • the chemical modifications may be introduced co-transcriptionally (e.g. by substitution of one or more nucleotides with a modified nucleotide during synthesis), or post-transcriptionally (e.g. by the action of an enzyme). Chemical modifications are known in the art, for exam pie as described in The RNA
  • the chemical modification is a chemical base modification.
  • the chemical base modification may be selected from a modification of an adenine, cytosine, thymine and/or uracil base.
  • the chemical sugar modification is a 2’ modification, such as a 2'-O- Methyl modification.
  • the functional nucleic acid molecule comprises a 3’ -polyadenylation (polyA) tail.
  • a “3’-polyA tail” refers to a long chain of adenine nucleotides added to the 3’-end of the nucleic acid which provides stability to the RNA molecule and can promote translation.
  • the functional nucleic acid molecule comprises a 5’ -cap.
  • a “5’-cap” refers to an altered nucleotide at the 5’ -end of the transcript which provides stability to the molecule, particularly from degradation from exonucleases, and can promote translation.
  • the 5’-cap may be a 7-m ethylguanylate cap (m7G), i.e. a guanine nucleotide connected to the RNA via a 5' to 5' triphosphate linkage and methylated on the 7 position.
  • m7G 7-m ethylguanylate cap
  • the term “functional nucleic acid” as it applies to the linear functional nucleic acid molecule disclosed herein may refer to functionality of the linear functional nucleic acid and/or functionality, e.g., translational regulatory activity, of the circular functional nucleic acids for which the linear nucleic acid molecule serves as a precursor.
  • the linear functional nucleic acid molecule when acting as a precursor, may not necessarily exhibit the functionality of the circular functional nucleic acid molecule, which it generates.
  • the at least one target determinant sequence comprises a sequence reverse complementary to a target m RNA sequence for which protein translation is to be enhanced.
  • target or “target m RNA sequence” refers to an endogenous or exogenous mRNA within a cell in vitro, an endogenous or exogenous mRNA /n vivo, e.g., within a cell; or any mRNA/n vitro, with which the functional nucleic acid molecule of the invention is reverse complementary, such that their translation is enhanced when in the presence of the functional nucleic acid molecule of the invention.
  • the at least one target determinant sequence comprises a sequence reverse complementary to a therapeutic target mRNA sequence for which protein translation is to be enhanced.
  • therapeutic target or “therapeutic target mRNA sequence” refers to a target which may be used to treat a disease or condition in said subject when its translation is enhanced, such as enhanced by using a functional nucleic acid molecule according to the present invention.
  • a therapeutic target when expressed in a subject (such as in a cell of a subject), may: restore or otherwise increase levels of a protein that are deficient or abnormal in a cell; augment an existing pathway in a cell; and/or provide a novel function or activity in a cell; thereby treating a disease or condition of said subject.
  • the therapeutic target comprises at least one gene defect that results in abnormal levels of a protein of interest.
  • the gene defect may be haploinsufficiency.
  • a target binding sequence needs to have only about 60% similarity with a sequence reverse complementary to the target mRNA in order to increase protein translation. In fact, the target binding sequence can even display a large number of mismatches and retain activity.
  • the target binding sequences of the functional nucleic acid molecule of the invention may each display about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% similarity with a sequence reverse complementary to the target mRNA.
  • polypeptide or polynucleotide sequences are said to be the same as or “identical” to other polypeptide or polynucleotide sequences, if they share 100% sequence identity over their entire length.
  • Residues in sequences are numbered from left to right, i.e. from N- to C- terminus for polypeptides; from 5’ to 3’ terminus for polynucleotides. If closely related sequences are not identical they may be similar, i.e., they may possess a certain, quantifiable, degree of sequence identity, e.g., a sequence may have 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence identity to another sequence.
  • any quoted sequence identity will be understood as being calculated across the residue range over which the two sequences are aligned.
  • the aligned residue range may represent the entirety of one or more of the input sequences or a contiguous section of sequence of one or more of the input sequences, and is typically determined by standard tools known in the art, e.g., NCBI BLAST.
  • the “% sequence identity” between a first nucleotide sequence and a second nucleotide sequence may be calculated using NCBI BLAST, using standard settings for nucleotide sequences (BLASTN).
  • the “% sequence identity” between a first polypeptide sequence and a second polypeptide sequence may be calculated using NCBI BLAST, using standard settings for polypeptide sequences (BLASTP).
  • a “difference” between sequences refers to an insertion, deletion or substitution of a single nucleotide in a position of the second sequence, compared to the first sequence. Insertions, deletions or substitutions in a second sequence which is otherwise identical (100% sequence identity) to a first sequence result in reduced % sequence identity.
  • “Complementarity” relates to the Watson-Crick base pairing principle that ‘A nucleotides will hydrogen bond with T (or ‘U’) nucleotides, and ‘G’ nucleotides with ‘C’ nucleotides to form double stranded structures that associate via said “complementary” nucleotides.
  • a “complementary” sequence is a sequence closely-related to another sequence such that such base pairing can occur.
  • Complementary sequences may be 100% complementary such that they may base pair across their entire length, or they may be e.g., 99%, 90%, 80%, 70%, or 60% complementary etc., such that they base pair across portions of their sequence.
  • a complementary sequence may also be called a “reverse complementary” sequence.
  • the target binding sequence may be less than about 250 nucleotides long, preferably less than about 200 nucleotides long, less than about 150 nucleotides long, less than about 140 nucleotides long, less than about 130 nucleotides long, less than about 120 nucleotides long, less than about 110 nucleotides long, less than about 100 nucleotides long, less than about 90 nucleotides long, less than about 80 nucleotides long, less than about 70 nucleotides long, less than about 60 nucleotides long or less than about 50 nucleotides long.
  • the target binding sequence is between about 4 and about 50 nucleotides in length, such as between about 18 and about 44 nucleotides in length.
  • the target binding sequence may be designed to hybridise with the 5’-untranslated region (5’ UTR) of the target mRNA sequence.
  • the sequence is reverse complementary to 0 to 50 nucleotides, such as 0 to 40, 0 to 39, 0 to 38, 0 to 37, 0 to 36, 0 to 35, 0 to 34, 0 to 33, 0 to 32, 0 to 31 , 0 to 30, 0 to 29, 0 to 28, 0 to 27, 0 to 26, 0 to 25, 0 to 24, 0 to 23, 0 to 22, 0 to 21 0 to 20, 0 to 19, 0 to 18, 0 to 17, 0 to 16, 0 to 15, 0 to 14, 0 to 13, 0 to 12, 0 to 11 , 0 to 10, 0 to 9, 0 to 8, 0 to 7, or 0 to 6 nucleotides of the 5’ UTR.
  • the target binding sequence may be designed to hybridise to a region upstream of an AUG site (start codon), such as a start codon within the CDS, of the target mRNA sequence.
  • the sequence is reverse complementary to 0 to 80 nucleotides, such as 0 to 70, 0 to 60, 0 to 50, 0 to 40, 0 to 39, 0 to 38, 0 to 37, 0 to 36, 0 to 35, 0 to 34, 0 to 33, 0 to 32, 0 to 31 , 0 to 30, 0 to 29, 0 to 28, 0 to 27, 0 to 26, 0 to 25, 0 to 24, 0 to 23, 0 to 22, 0 to 21 , 0 to 20, 0 to 19, 0 to 18, 0 to 17, 0 to 16, 0 to 15, 0 to 14, 0 to 13, 0 to 12, 0 to 11 , 0 to 10, or 0 to 9 nucleotides upstream of the AUG site.
  • the target binding sequence may be designed to hybridise to the target mRNA sequence downstream of said AUG site.
  • the sequence is reverse complementary to 0 to 40 nucleotides, such as 0 to 39, 0 to 38, 0 to 37, 0 to 36, 0 to 35, 0 to 34, 0 to 33, 0 to 32, 0 to 31 , 0 to 30, 0 to 29, 0 to 28, 0 to 27, 0 to 26, 0 to 25, 0 to 24, 0 to 23, 0 to 22, 0 to 21 , 0 to 20, 0 to 19, 0 to 18, 0 to 17, 0 to 16, 0 to 15, 0 to 14, 0 to 13, 0 to 12, 0 to 11 , 0 to 10, 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, or 0 to 4 nucleotides of the target mRNA sequence downstream of said AUG site.
  • the target determinant sequence is at least 10 nucleotides long and comprises,
  • the target determinant sequence is at least 14 nucleotides long and comprises, from 3’ to 5’:
  • the coding sequence starts on the first AUG site (M1) of the mRNA.
  • the nucleotides of the 5’UTR sequence are numbered sequentially using decreasing negative numbers approaching the AUG site on the target mRNA (e.g. -3, -2, -1).
  • the nucleotides of the CDS sequence are numbered sequentially using increasing positive numbers (e.g. +1 , +2, +3) from the AUG site, such that the A of the AUG site is numbered +1.
  • the region bridging the 5’UTR and the CDS will therefore be numbered -3, -2, -1 , +1 , +2, +3, with the A of the AUG site numbered +1 .
  • the functional nucleic acid of the invention may be designed to target any suitable mRNA in order to increase protein translation of said mRNA.
  • Particularly suitable targets are mRNAs for which their natural abundance is reduced e.g., due to a haploinsufficiency or microdeletion.
  • Suitable targets may include, but are not limited to, OPA1 , FXN, GRN, GBA, PRPF31 , GDNF, BDNF, NGF, TRKA, TRKB, and/or RET.
  • the functional nucleic acid molecule of the invention may be designed to target a given mRNA using the principles disclosed in the foregoing section, “Target determinant sequences”.
  • a functional nucleic acid molecule according to the invention, wherein the one or more target binding sequence comprises a sequence reverse complementary to a PXK mRNA sequence.
  • the one or more target binding sequence consists of a sequence reverse complementary to a sequence within SEQ ID NO: 25.
  • the target binding sequence comprises a sequence reverse complementary to a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence within SEQ ID NO: 25.
  • the target binding sequence consists of a sequence reverse complementary to a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence within SEQ ID NO: 25.
  • the target sequence consists of a sequence encoded by SEQ ID NO: 25.
  • the one or more target binding sequence comprises or consists of a sequence as selected from SEQ ID NOs: 26 and SEQ ID NO: 27.
  • the target sequence comprises a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence encoded by SEQ ID NOs: 28 and/or 29.
  • Checkpoint kinase 1 (CHK1 or Chk1), is a serine/threonine protein kinase that is encoded by the CHEK1 gene. CHK1 haploinsufficiency is associated with anaemia and defective erythropoiesis. Since CHK1 overexpression is implicated in deleterious processes such as tumorigenesis, the use of the functional nucleic acid of the present invention to restore protein levels (i.e. , without exceeding wild-type physiological levels) is advantageous in avoiding such unwanted effects.
  • the one or more target binding sequence comprises a sequence reverse complementary to a sequence within SEQ ID NO: 30.
  • the one or more target binding sequence consists of a sequence reverse complementary to a sequence within SEQ ID NO: 30.
  • the target binding sequence comprises a sequence reverse complementary to a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence within SEQ ID NO: 30.
  • the target sequence comprises a sequence encoded by SEQ ID NO: 30.
  • the target sequence comprises a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence encoded by SEQ ID NO: 30.
  • SEQ ID NO: 30 is a CHK1 transcript with the accession numbers ENST00000438015 (ensemble) and NM_001114122.3 (NCBI).
  • Mitofusin-2 is a mitochondrial outer membrane GTPase that is encoded by the MFN2 gene.
  • Mitofusin-2 protein may be referred to as MFN2, as may the corresponding mRNA that encodes it.
  • Mutations in MFN2 are associated with Charcot-Marie-Tooth (CMT) disease-2A, which is a neurological disorder that presents neuropathy-related features and systemic impairment of the central nervous system.
  • CMT disease-2A Charcot-Marie-Tooth
  • Up-regulation of MFN2 triggers apoptotic cell death of vascular smooth muscle cells and cardiomyocytes, indeed MFN2 protein levels are associated with several heart diseases.
  • Down-regulation of MFN2 leads to vascular proliferative disorders and cardiac dysfunction.
  • the one or more target binding sequence comprises a sequence reverse complementary to a MFN2 mR NA sequence.
  • the one or more target binding sequence comprises a sequence reverse complementary to a target mRNA sequence selected from the group consisting of: human MFN2 and mouse MFN2.
  • the one or more target binding sequence comprises a sequence reverse complementary to a sequence within SEQ ID NO: 31 .
  • the target binding sequence comprises a sequence reverse complementary to a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence within SEQ ID NO: 31.
  • the target binding sequence consists of a sequence reverse complementary to a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence within SEQ ID NO: 31.
  • the target sequence comprises a sequence encoded by SEQ ID NO: 31.
  • the target sequence consists of a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence encoded by SEQ ID NO: 31.
  • SEQ ID NO: 31 is a MFN2 transcript with the accession numbers ENST00000235329 (ensemble) and NM_014874.4 (NCBI).
  • a functional nucleic acid molecule according to the invention, wherein the one or more target binding sequence comprises a sequence reverse complementary to a GUCY1 B1 mRNA sequence.
  • the one or more target binding sequence comprises a sequence reverse complementary to a target mRNA sequence selected from the group consisting of: human GUCY1 B1 and mouse GUCY1 B1.
  • the one or more target binding sequence consists of a sequence reverse complementary to a sequence within SEQ ID NO: 32.
  • the target binding sequence comprises a sequence reverse complementary to a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence within SEQ ID NO: 32.
  • the target binding sequence consists of a sequence reverse complementary to a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence within SEQ ID NO: 32.
  • the target sequence comprises a sequence encoded by SEQ ID NO: 32.
  • the target sequence consists of a sequence encoded by SEQ ID NO: 32.
  • the target sequence comprises a sequence with about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identity to a sequence encoded by SEQ ID NO: 32.
  • SEQ ID NO: 32 is a GUCY1 B1 transcript with the accession numbers ENST00000264424 (ensemble) and NM_000857.5 (NCBI).
  • the functional nucleic acid molecule of the invention comprises a regulatory sequence comprising an internal ribosome entry site (IRES).
  • IRES internal ribosome entry site
  • a dual luciferase (Firefly luciferase [Flue], Renilla Luciferase [Rluc]) encoding plasmid is used for experimental tests.
  • Said test may be considered “The Standard Bicistronic Plasmid Test for Cellular mRNA IRESs” used to test putative IRES sequences.
  • the foregoing is a functional test wherein the putative IRES sequence is inserted between RLuc and FLuc, e.g., as described in Jasckson, Cold Spring Harb Perspect Biol 2013; 5:a011569, wherein the translational function of the putative IRES sequence is determined by the Fluc/RLuc value, thus measuring c/s-acting activity.
  • a web-based tool is available to search for sequence-based and structure-based similarities between a query sequence of interest and the entirety of annotated and experimentally validated IRES sequences within the database.
  • the output of the program is a probability score for any nucleotide sequence to be able to act as IRES in a validation experiment with bicistronic constructs.
  • Additional sequence-based and structurebased web-based browsing tools are available to suggest, with a numerical predicting value, the IRES activity potentials of any given nucleotide sequence (http://rna.informatik.uni- frburg.de/; http://regrna.mbc.nctu.edu.tw/index1.php).
  • the regulatory sequence has protein translation enhancing activity.
  • the regulatory sequence increases or enhances translation of the target m RNA sequence.
  • expression of the protein encoded by the target mRNA is increased by at least 1 .2 fold, such as at least 1 .5 fold, in particular at least 2 fold.
  • expression of the protein encoded by the target mRNA is increased between 1 .5 to 3 fold, such as between 1 .6 and 2.2 fold.
  • These increases in protein expression are within physiological ranges. It is envisaged that increasing protein expression within these ranges will allow the treatment of diseases associated with one or more gene defects, such as cancer or neurodegenerative diseases, without leading to negative side effects associated with increasing expression of the target above non-disease state or ‘wildtype’ physiological levels.
  • the expression of the protein encoded by the target mRNA is increased by at least about 1.1 fold, at least about 1 .2 fold, at least about 1 .3 fold, at least about 1 .4 fold, at least about 1 .5 fold, at least about 1 .6 fold, at least about 1 .7 fold, at least about 1 .8 fold, at least about 1 .9 fold, at least about 2.0 fold, at least about 2.1 fold, at least about 2.2 fold, at least about 2.3 fold, at least about 2.4 fold, at least about 2.5 fold, at least about 2.6 fold, at least about 2.7 fold, at least about 2.8 fold, at least about 2.9 fold, or at least about
  • the expression of the protein encoded by the target mRNA is increased about 1.1 fold, about 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, about 1.6 fold, about 1.7 fold, about 1.8 fold, about 1.9 fold, about 2.0 fold, about 2.1 fold, about 2.2 fold, about 2.3 fold, about 2.4 fold, about 2.5 fold, about 2.6 fold, about 2.7 fold, about 2.8 fold, about 2.9 fold, or about 3.0 fold.
  • the expression of the protein encoded by the target mRNA is increased by less than about 1 .2 fold, less than about 1 .3 fold, less than about 1 .4 fold, less than about
  • the regulatory sequence is in a direct orientation relative to the 5’ to 3’ orientation of the functional nucleic acid molecule, i.e., the regulatory sequence is embedded (inserted) with the same 5’ to 3’ orientation as the functional nucleic acid molecule.
  • the regulatory sequence com prises an IRES sequence, or a fragment thereof. Said sequence enhances translation of the target mRNA sequence.
  • IRESs having sequences ranging from 48 to 576 nucleotides have been tested with success, e.g. human Hepatitis C Virus (HCV) IRESs (e.g. SEQ ID NO: 2 and 3), human poliovirus IRESs (e.g. SEQ ID NO: 4 and 5), human encephalomyocarditis (EMCV) virus (e.g. SEQ ID NO: 6 and 7), human cricket paralysis (CrPV) virus (e.g. SEQ ID NO: 8 and 9), human Apaf-1 (e.g.
  • HCV Hepatitis C Virus
  • poliovirus IRESs e.g. SEQ ID NO: 4 and 5
  • EMCV human encephalomyocarditis
  • CrPV human cricket paralysis virus
  • Apaf-1 e.g.
  • SEQ ID NO: 10 and 11 human ELG-1 (e.g. SEQ ID NO: W and 13), human c-MYC (e.g. SEQ ID NO: 14-17) and human dystrophin (DMD) (e.g. SEQ ID NO: 18 and 19).
  • ELG-1 e.g. SEQ ID NO: W and 13
  • human c-MYC e.g. SEQ ID NO: 14-17
  • human dystrophin e.g. SEQ ID NO: 18 and 19
  • the regulatory sequence comprises a sequence selected from the group consisting of SEQ ID NOs 2 - 20, or a fragment thereof.
  • the regulatory sequence consists of a sequence selected from the group consisting of SEQ ID NOs 2 - 20, or a fragment thereof.
  • the regulatory element has at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 86% sequence identity, at least about 87% sequence identity, at least about 88% sequence identity, at least about 89% sequence identity, at least about 90% sequence identity, at least about 91 % sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, or 100% sequence identity to any one of SEQ ID NOs 2 - 20.
  • the regulatory sequence is a sequence comprising or consisting of a sequence selected from the circRNAs identified in Table 1.
  • the regulatory sequence comprises or consists of a fragment of any one of SEQ ID NOs 2 - 20, wherein the fragment is about is about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about
  • the fragment is a functionally active fragment that retains IRES activity within the definition provided above.
  • the fragment is a functionally active fragment that retains protein translation enhancing activity.
  • a “functionally active fragment” of an IRES might also be considered an IRES perse.
  • “functionally active fragment” of an IRES is utilised to delineate IRES sequences that are shorter in length as compared with ‘parental’ IRES sequences from which they are designed or derived.
  • an expression vector comprising said DNA molecule.
  • the mammalian expression plasmid is a pCDNA3.1 (+) ZKSCAN1 MCS Exon Vector.
  • the mammalian expression plasmid is a pcDNA3.1 (+) Laccase2 MCS Exon Vector
  • the viral vector is rcLV -TetOne-Puro.
  • the viral vector is pLPCX-link.
  • Vectors of the invention may comprise any one of more features selected from the list comprising: a CAG promoter, a CMV enhancer, SV40 late poly(A) terminator, a LTR-TREt (Tre-Tight) promoter, and/or a BGH poly(A) terminator.
  • any promoter may be used in the vector. Since the activity of the functional nucleic acids of the invention is independent of the promoter it is envisaged that these will work just as well as those exemplified above.
  • the present invention also relates to com positions com prising the functional nucleic acid molecule, the DNA molecule or the expression vector described herein.
  • composition may comprise components which enable delivery of said functional nucleic acid molecule by viral vectors (AAV, lentivirus and the like) and non-viral vectors (nanoparticles, lipid particles and the like).
  • viral vectors AAV, lentivirus and the like
  • non-viral vectors nanoparticles, lipid particles and the like.
  • the functional nucleic acid molecule of the invention may be administered as naked or unpackaged RNA
  • the functional nucleic acid molecule may be administered as part of a composition, for example a composition comprising a suitable carrier.
  • the carrier is selected based upon its ability to facilitate the transfection of a target cell with one or more functional nucleic acid molecules.
  • composition comprising the functional nucleic acid molecule described herein.
  • a pharmaceutical composition comprising at least one functional nucleic acid molecule, at least one DNA molecule, or at least one expression vector according to the present invention.
  • a pharmaceutical composition may com prise at least one functional nucleic acid molecule, at least one DNA molecule, or at least one expression vector according to the present invention with a suitable pharmaceutical excipient, diluent or carrier.
  • the suitable pharmaceutical excipient, diluent or carrier may depend on the intended route of administration and standard pharmaceutical practice.
  • a suitable carrier may include any of the standard pharmaceutical carriers, vehicles, diluents or excipients known in the art and which are generally intended for use in facilitating the delivery of nucleic acids, such as RNA.
  • Liposomes, exosomes, lipidic particles or nanoparticles are examples of suitable carriers that may be used for the delivery of RNA.
  • the carrier or vehicle delivers its contents to the target cell such that the functional nucleic acid molecule is delivered to the appropriate subcellular compartment, such as the cytoplasm.
  • a method for enhancing translation of a target mRNA comprising administering the functional nucleic acid molecule, DNA molecule, expression vector or composition as defined herein to the cell.
  • a target mRNA such as a therapeutic target mRNA
  • the cell is a mammalian cell, such as a human or a mouse cell.
  • an in vitro method for increasing the protein synthesis of a target in a cell or cell-free system comprising administering the functional nucleic acid molecule, DNA molecule, expression vector or the composition described herein, to the cell or cell-free system.
  • a method for increasing the protein synthesis of a target in a cell comprising administering the functional nucleic acid molecule, DNA molecule, expression vector or the composition described herein, to the cell.
  • the cell is a mammalian cell, such as a human or a mouse cell.
  • a method for increasing the protein synthesis efficiency of a target in a cell comprising administering the functional nucleic acid molecule, DNA molecule, expression vector or the composition described herein, to the cell.
  • the cell is a mammalian cell, such as a human or a mouse cell.
  • Methods of the invention result in increased levels of target protein in a cell and therefore find use, for example, in methods of treatment of diseases which are associated with gene defects (e.g. one or more gene defects which result in reduced protein levels and/or loss-of- function mutations of the encoding gene).
  • Methods of the invention find particular use in diseases caused by a quantitative decrease in the predetermined, normal protein level, such as haploinsufficiency.
  • Methods of the invention can be performed in vitro, ex vivo or in vivo.
  • the methods described herein may com prise transfecting into a cell the functional nucleic acid molecule, DNA molecule, expression vector or composition as defined herein.
  • the functional nucleic acid molecule, DNA molecule, expression vector or composition may be administered to target cells using methods known in the art and include, for exam pie, microinjection, lipofection, electroporation, using calcium phosphate, self-infection by the vector or transduction of a virus.
  • nucleic acid molecule DNA molecule
  • expression vector or the composition such as pharmaceutical composition, as defined herein for use in therapy.
  • nucleic acid molecule DNA molecule
  • expression vector or the composition such as pharmaceutical composition, as defined herein for use as a medicament.
  • the functional nucleic acid molecule of the invention find use in increasing the level of a target protein, such as a therapeutic target within a cell.
  • nucleic acid molecule DNA molecule
  • expression vector or composition such as pharmaceutical composition, for use in the treatment of a disease-associated with one or more gene defects.
  • a disease associated with one or more gene defects may be a cancer or a neurodegenerative disease.
  • the functional nucleic acid molecule, DNA molecule, expression vector or composition such as pharmaceutical composition, for use in the treatment of cancer.
  • the functional nucleic acid molecule, DNA molecule, expression vector or composition, such as pharmaceutical composition for use in the treatment of a neurodegenerative disease.
  • a method of treating a disease associated with one or more gene defects comprising administering a therapeutically effective amount of the functional nucleic acid molecule, the DNA molecule, the expression vector, or the composition, such as pharmaceutical composition, as defined herein to a subject in need thereof.
  • a method of treating a disease associated with one or more gene defects comprising administering a therapeutically effective amount of the functional nucleic acid molecule, the DNA molecule, the expression vector, or the composition, such as the pharmaceutical composition, as defined herein to a subject in need thereof, wherein the disease is a cancer or a neurodegenerative disease.
  • Example 1 Circularized SINEUP(IRES) RN A maintains its activities in trans
  • the Virtual Ribosome tool (Dna2pep v1 .1) was used to predict the longest complete ORF for each circRNA by searching across all positive reading frames with methionine as start codon and a canonical stop codon.
  • circRNAs from 32 genomic loci presented a predicted ORF of 100 or fewer aa in length (Table 1 , below).
  • hsa_circ_0085533 showed complete absence of ORF according to the used constraints.
  • Circ5533 is transcribed from the c-myc locus, is 555-nts-long, and contains an IRES sequence of a length of 380 nts, usually included in the 5’UTR of c- myc mRNA, with well-established evidence of cis activity.
  • c- myc IRES acts in trans as ED in both linear synthetic SINEUP and in circlIP ( Figure 1 ).
  • circ5533 also presents additional 175 nts of unknown function that could contain BD sequences ( Figure 2a).
  • Table 1 List of circRNAs containing an IRES sequence with a predicted ORF of 100 aa or fewer in length
  • the table shows identified circRNAs by reporting their chromosome coordinates (Chromosome, Chrom Start and ChromEnd), their ID reference numbers in circBase (circBase ID), the gene strand on which they are encoded (Strand), the number of exons contained in the circRNA (Exon number), their predicted ORF length (ORF length), the best transcript RefSeq ID (Best Transcript) and the gene name.
  • Example 3 - ci rc 5533 positively regulates PXK protein levels through two defined BDs in trans
  • Pxk as a representative mRNA target of the circlIP activity of circ5533.
  • circ5533 was not dependent on the type of circularizing plasmid.
  • circ5533’s ability to increase endogenous PXK protein levels with no change in mRNA levels Figure 4a-c).
  • TS4 and TS5 were identified in the 3’UTR of Pxk mRNA, that could pair with a second antisense region (BD2) in the non-IRES sequence of circ5533, which spans position 104 to 112.
  • BD2 second antisense region
  • deletions of TS1 and TS4 inhibited the circ5533-mediated increase of PXK-FLAG protein, demonstrating that TS1 and TS4 are essential for circ5533 activity and suggesting that both BD1 and BD2 are functionally active.
  • query sequence (circ5533) was divided in two main regions identified as non-IRES and IRES sequences, and then aligned against Pxk 5’UTR, CDS and 3’UTR, respectively. For each region, overlapping matches are indicated with 1 (Yes) and no matches with 0. SEQUENCES

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Abstract

La présente invention concerne des molécules d'acide nucléique fonctionnelles circulaires comprenant une ou plusieurs séquences de liaison cibles et une séquence régulatrice comprenant un site d'entrée interne des ribosomes (IRES). L'invention concerne également des molécules d'acide nucléique fonctionnelles linéaires comprenant une ou plusieurs séquences de liaison cibles et une séquence régulatrice comprenant un IRES. L'invention concerne également des procédés d'amélioration de l'efficacité de traduction de protéine, et des procédés de traitement de défauts de gène à l'aide des molécules d'acide nucléique fonctionnelles circulaires et des molécules d'acide nucléique linéaires de l'invention.
EP23729102.6A 2022-05-26 2023-05-26 Molécule d'acide nucléique fonctionnelle Pending EP4532715A1 (fr)

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