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WO2010084371A1 - Nouvelles molécules d'arn interférent circulaire - Google Patents

Nouvelles molécules d'arn interférent circulaire Download PDF

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WO2010084371A1
WO2010084371A1 PCT/IB2009/000305 IB2009000305W WO2010084371A1 WO 2010084371 A1 WO2010084371 A1 WO 2010084371A1 IB 2009000305 W IB2009000305 W IB 2009000305W WO 2010084371 A1 WO2010084371 A1 WO 2010084371A1
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rna
intron
sequence
gene
rna molecule
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Guillaume Plane
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MITOPROD
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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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/50—Methods for regulating/modulating their activity
    • C12N2320/51—Methods for regulating/modulating their activity modulating the chemical stability, e.g. nuclease-resistance

Definitions

  • the invention relates to novel circular interfering RNA molecules (ciRNA) which can be produced by in vitro or in vivo transcription.
  • ciRNA circular interfering RNA molecules
  • the invention relates to genetic constructs and transcription vectors useful for the production of said circular interfering RNAs.
  • the invention relates also to a method for producing large amounts of said circular interfering RNAs in yeast in a form which is stable and easy to purify.
  • RNA interference is a post-transcriptional gene-silencing mechanism in which double-stranded RNA (dsRNA) triggers degradation of homologous messenger RNA in the cytoplasm (reviewed in Shuey et al, Drug Discovery Today, 2002, 7, 1040-1046 and Tuschl T. and Borkhardt A., MoI. Interv., 2002, 2, 158-167).
  • dsRNA double-stranded RNA
  • long dsRNAs are cleaved by the RNase III class endoribonuclease Dicer into 21-23 base duplexes having 2-base 3 '-overhangs and 5'- terminal phosphate groups.
  • RNA-induced silencing complex RNA-induced silencing complex
  • RNAi is an important mechanism for the regulation of gene expression in a broad range of eukaryotic organisms, including both plants (Van Der Krol et al, Plant Cell., 1990, 2, 291-299) and animals (Fire et al, Nature, 1998, 391, 806-81 1).
  • siRNAs for use as therapeutic agents to reduce activity of specific gene products are also receiving considerable attention.
  • Silencing can be induced in target cells by directly introducing synthetically produced siRNAs or short hairpin RNAs (shRNAs), or alternatively by transfecting cells with engineered plasmid or viral vectors expressing siRNAs or shRNAs under the transcriptional control of RNA polymerase II or III promoter (U6 and Hl).
  • Short hairpin RNAs are siRNA derived molecules that contain the sense strand and the antisense strand of siRNA molecules and a short loop sequence between the sense and antisense strands.
  • shRNAs are formed of one RNA molecule that folds into a hairpin.
  • shRNAs are converted into small interfering RNA (siRNA) by the cellular ribonuclease III, Dicer (Dykxhoorn et al, Nat. Rev. MoI. Cell. Biol., 2003, 4, 457-467; Hammond, S.M., FEBS Lett., 2005, 579, 5822-5829; Hannon, G.J. and Rossi, J.J., Nature, 2004, 431, 371-378; Rossi J.J., Hum. Gene Ther., 2008, 19, 313- 317).
  • plasmids or viral vectors are highly effective at expressing siRNAs or shRNAs, in vitro in cell culture as well as in vivo in laboratory animals, they cause safety and ethic problems in clinical applications.
  • the vector DNA may be inserted in the chromosomal DNA and thus induce alteration of this DNA which may lead to cancer by activation of cellular oncogenes by insertional mutagenesis. Therefore, synthetic siRNAs or shRNAs are considered as the RNAi molecules of choice for clinical purposes.
  • RNA polymerases from their cognate promoters (T7, T3 or SP6; Seyhan et al., oligonucleotides, 2006, 16, 353-363).
  • RNAP bacteriophage RNA polymerases
  • RNA polymerase from their natural promoters results in 5'-termini triphosphate that can trigger an interferon response in vivo.
  • shRNA transcripts may have extra 5 '-nucleotides that can constrain the sequences that can be targeted.
  • the 3' ends may have an additional n+1 nucleotide not encoded by the template.
  • efficient transcription of siRNAs requires two separate dsDNA templates and four oligodeoxynucleotides must be synthesized for each siRNA duplex.
  • the European Patent EP 1646724 describes a method for producing a heterologous RNA of interest including a siRNA, which uses yeast lacking mitochondrial DNA ⁇ rho ) whose mitochondria are transformed with a DNA encoding the heterologous RNA of interest (synthetic rho ) for producing the RNA of interest in their mitochondria.
  • This RNA is readily isolated in a stable form and in large amounts, from the mitochondria of the synthetic rho ' strain, insofar as the only RNAs produced in the mitochondria of said synthetic rho ' strain are those which are encoded by the DNA used for the transformation.
  • the synthetic RNA molecules made of natural (unmodified) nucleotides which are produced by the preceding methods are not stable in biological fluids since their extremities are rapidly degraded by 3'-exonuclease, one of the major enzymes involved in the degradation of nucleic acids in vivo. Therefore, the production of stable synthetic siRNAs is required for therapeutic applications.
  • siRNA can be stabilized by the modification of the terminal ribose moieties with a 2'-deoxy, 2'-O-methyl or 2'-fluoro group or replacement of the terminal phosphodiester bonds with phosphorotioates.
  • the chemically modified siRNAs are costly, they are often less active than their unmodified counterpart and they may be toxic to human.
  • RNA dumbbell-shaped circular RNA that contains a 15 to 23 bp stem sequence encoding the firefly luciferase gene (siRNA sequence) and two 9- mer loops was synthesized from two RNA strands that were closed at both ends with the loop sequences, using T4 RNA ligase (Abe et al, J. Am. Chem. Soc, 2007, 129, 15108-15109).
  • the RNA dumbbell is resistant to degradation in serum, because of the shape of the molecule, an endless structure that cannot be degraded by 3'-exonuclease.
  • RNA splicing reactions can generate circular exon sequences when the 3' end of the exon is joined to a splice site at an upstream rather than a downstream position (donor (5') splice site is 3' of the acceptor (3') splice site ; figure 1). Circular RNAs generated by splicing have been demonstrated with in vitro manipulated Group I and Group II intron sequences.
  • Group I catalytic introns are self-splicing ribozymes. Their size is variable (from 68 over 300 nucleotides; most are over 400 nucleotides). They catalyze their own excision from mRNA, tRNA and rRNA precursors in bacteria, lower eukaryotes and higher plants. However, their occurrence in bacteria seems to be more sporadic than in lower eukaryotes, and they have become prevalent in higher plants.
  • the genes that group I introns interrupt differ significantly: they interrupt rRNA, mRNA and tRNA genes in bacterial genomes, as well as in mitochondrial and chloroplast genomes of lower eukaryotes, but only invade rRNA genes in the nuclear genome of lower eukaryotes.
  • RNA forms the active site and directs the cleavage and ligation reaction at the 5' and 3' splice sites. Splicing is initiated with an external G nucleotide (cofactor) and processed by two sequential transesterification steps catalysed by RNA in vitro.
  • the catalytic core of Group I introns consists of two structural domains, P4-P6 and P3-P9.
  • P4-P6 has been proposed to interact with the substrate helix Pl which contains the 5' splice site
  • P3-P9 contains the binding site for the guanosine nucleophile.
  • the P4-P6 and P3-P9 domains of the Tetrahymena Group I intron can self-assemble into an active structure (Tanner et al, Science, 1997, 275, 847-849).
  • Most Group I introns are able to splice themselves in the absence of proteins, i.e. the RNA itself is catalytic (ribozyme). However, not all group I introns are truly catalytic.
  • Splicing of some group I introns in vivo is modulated by a number of proteins which play a role in folding and 3D structure (maturases) and are encoded by various genes independent from the introns or by the introns themselves.
  • Group I catalytic intron and flanking 5' and 3' exons sequences are available in the sequence data base.
  • the cyanobacteria Anabaena sp. pre-tRNA-Leu gene sequence including the self-splicing Group I intron sequence corresponds to the accession numbers GenBank M38962 and M38961.
  • the Tetrahymena ribosomal Group I intron sequence (399 bases in its circular form) is described for example in figure 1 of G. Dinter-Gott Kunststoff, Proc. Natl. Acad. Sd., USA, 1986, 83, 6250-6254.
  • PIE Group I self-splicing permuted intron-exon
  • the RNA molecule to be made circular is prepared by inserting a DNA sequence encoding the RNA of interest in a DNA construct comprising the following sequences from a Group I self-splicing intron (figure 1) : (1) a sequence encoding a 3' portion of the intron, (2) a sequence encoding the 3' splice site, (3) a sequence encoding the RNA of interest inserted at the fusion point of the 3 'exon fused end-to-end to the 5 'exon or of a fragment of the fused exons having few nucleotides (less than 50, preferably less than 20 nucleotides) of the 3' exon (Exon 2) sequence flanking the 3' splice site and few nucleotides of the 5' exon (Exon 1) sequence flanking the 5' splice and eventually a cloning site inserted at
  • the DNA construct is cloned in an expression plasmid under the transcriptional control of an appropriate promoter. Transcription from the promoter, results in the production of an autocatalytic messenger RNA (mRNA circular RNA precursor) which generates a circular form of the RNA of interest by self-splicing.
  • mRNA circular RNA precursor autocatalytic messenger RNA
  • a permuted bacteriophage T4-derived Group I intron was also used to generate circular messenger RNA sequences in vitro, in E.coli and in yeast (Ford E. and Ares M., Proc. Natl. Sci. USA, 1994, 91, 3117-3121; US Patent 5,773,244 by Ares M. and Ford, E.E.) as well as a circular form of streptavidin RNA aptamer in vitro (Umekage, S. and Kikuchi, Y., Nucleic Acids Symposium Series N°50, 2006, 323-324).
  • the circular RNA molecules generated by self-splicing of Group I permuted intron-exon (PIE) sequences comprise exon sequences flanking the splice junction; for example in the case of Anabaena-derived Group I intron the circular RNA comprises at least a 7 bp stem terminated by a 7 nucleotide loop (see for example, figure 1 of Puttaraju et al, Nucleic Acids Res., 1993, 21, 4253-4258; figure 1 of Bohjanen et al, Nucleic Acids Res., 1996, 24, 3733-3738 or figure 2 of Puttaraju, M. and Been, M.D., The Journal of Biological Chemistry, 1996, 42, 26081-26087).
  • PIE permuted intron-exon
  • the circular RNA molecules which have been generated are enzymatic molecules (ribozyme, RNase P) or protein ligands (sptreptavidine, Tat).
  • ribozyme ribozyme, RNase P
  • protein ligands sptreptavidine, Tat.
  • the secondary structures from the Group I PIE splice- junction sequences were added at the end of a stem comprising the 5' and 3' ends of the RNA molecules.
  • the structure and the function of the RNA molecule were not altered substantially since the secondary structures from the Group I PIE splice-junction sequences were added in a region distant from the active site (cleavage or binding site).
  • the inventor has designed a circular siRNA (ciRNA) molecule that contains a siRNA sequence targeting the luciferase gene closed at one end by a small loop and at the other end by the stem and the loop derived from Anabaena Group I PIE splice-junction sequences (figure 2A).
  • the ciRNA molecule was generated by in vitro or in vivo transcription (bacteria, yeast) from a transcription plasmid comprising a T7 promoter operatively linked to a DNA construct derived from the Anabaena Group I PIE constructs previously described by Puttaraju et al. (Puttaraju, M.
  • the ciRNA molecule is stable (resistant to 3'- Exonuclease digestion; figure 6) and has an RNAi activity which is at least equivalent to that of the corresponding shRNA molecule (figure 9).
  • the invention relates to a circular RNA molecule (ciRNA) which comprises the sense and the antisense strands of an interfering RNA molecule (RNAi) targeting a gene of interest, wherein said sense and antisense strands are closed at one end by a loop structure (first loop) and at the other end by a splice junction sequence generated by splicing activity of permuted intron-exon sequences, and wherein the circular RNA molecule inhibits the expression of the gene of interest in cells expressing said gene of interest.
  • ciRNA circular RNA molecule
  • RNAi interfering RNA molecule
  • interfering RNA molecule or RNAi molecule refers to a double- stranded RNA molecule of at least 19 bp, comprising complementary sense and antisense strands, wherein the antisense strand is complementary to the sequence of a target gene and the introduction of the RNAi molecule in cells expressing the target gene inhibits the expression of said target gene.
  • the interfering RNA molecule may be long ( > 100 pb) or short (small; ⁇ 100 pb); small interfering RNA (siRNA) molecules consist preferably of 19 to 30 bp.
  • nucleic acid - “complementary” refers to the ability of a nucleic acid to form hydrogen bond(s) by either traditional Watson-Crick base-pairing or other non- traditional type base-pairing.
  • the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies for nucleic acid molecules is well-known in the art (see, e.g., Turner et al, 1987, CSH Symp. Quant.
  • a percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base-pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides, in the first oligonucleotide being base- paired to a second nucleic acid sequence having 10 nucleotides represents 50 %, 60 %, 70 %, 80 %, 90 % and 100 % complementarity, respectively).
  • Perfectly complementary means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • “few nucleotides” refers to less than 50 nucleotides, preferably less than 20 nucleotides, more preferably around 15 nucleotides.
  • target gene refers to a gene whose expression is to be down- regulated.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the inhibition of the target gene expression by the circular RNA molecule of the invention is assayed at the RNA or protein level, by methods well- known in the art, for example by real time quantitative RT-PCR, Northern-blot, FACS or Western-blot.
  • the portion of the ciRNA molecule comprising the sense strand of the RNAi molecule, the first loop and the antisense strand of the RNAi forms a shRNA. Therefore, this portion of the ciRNA molecule is also referred to as shRNA.
  • the sense and antisense strands of the RNAi molecule comprise at least 19 ribonucleotides, for example 21 to 27 (e.g. 21, 22, 23, 25, 26 or 27) ribonucleotides.
  • the RNAi molecule may have overhanging ribonucleotide(s) at one or both end(s), preferably, 1 to about 5 (e.g. about 1, 2, 3, 4, 5) overhanging ribonucleotides.
  • the overhanging ribonucleotides which are advantageously at the 3 'end of the antisense strand, are preferably uridines.
  • the splice junction sequence may be generated by splicing activity of any permuted intron-exon sequence consisting of : (1) a portion of the 3' half of an intron sequence including the 3' splice site, (2) exon sequence (sequences flanking said intron or an exogenous sequence) and (3) a portion of the 5' half of said intron sequence including the 5' splice.
  • the splice junction sequence is generated by splicing activity of permuted intron- sequences derived from an intron of a yeast mitochondrial gene.
  • the splice junction sequence is generated by self-splicing activity of permuted intron-exon sequences.
  • the permuted intron-exon sequences are derived from a Group I catalytic intron, such as for example the Group I catalytic intron of the cyanobacteria Anabaena sp. pre-tRNA-Leu gene or the Tetrahymena ribosomal gene.
  • the splice junction sequence may comprise or consist of a loop structure (second loop) which is similar or identical to that of the first loop.
  • the splice junction sequence may form a loop structure (Figure 2A) or it may comprise a stem-loop structure, derived for example from Anabaena sp. Group I PIE splice-junction sequence (figure 2B).
  • the strands of the RNAi molecule are connected to the strands of the stem-loop structure, either directly or by 3 to 10 non- complementary nucleotides forming a bulge (figure 2B and SEQ ID NO: 2).
  • the loop(s) (first and/or second loop) which may be identical or different comprise at least 3 ribonucleotides, for example about 3, 4, 5, 6, 7, 8, 9, or 10 ribonucleotides.
  • the loop(s) comprise at least 5 ribonucleotides.
  • the loop(s) comprise the sequence SEQ ID NO: 1.
  • the invention relates also to a genetic construct (DNA construct; figure 3) designed for the production of the ciRNA molecule of the invention, said genetic construct comprising: (1) a transcription initiation region (promoter), (2) a sequence encoding a 3' portion of a self-splicing Group I intron (Intron 2), (3) a sequence encoding the 3' splice site of said self-splicing Group I intron, (4) a sequence encoding the shRNA of the invention inserted at the junction (fusion point) of the 3'exon (Exon 2) of said self-splicing Group I intron fused end-to-end to the 5'exon (Exon 1) of said self-splicing Group I intron or of a fragment of the fused exons having few nucleotides of the 3' exon sequence flanking the 3' splice site and few nucleotides of the 5' exon sequence flanking the 5' splice and eventually a cloning site inserted at the junction of the
  • the sequences (2) to (6) of the construct are operatively linked to the transcription initiation (1) and termination (7) regions.
  • In vitro or in vivo transcription from the promoter produces an autocatalytic unspliced RNA (ciRNA RNA precursor or ciRNA precursor) which generates a circular form of the siRNA of interest by self-splicing.
  • the genetic construct comprises advantageously restriction sites 5' and 3' of the promoter and transcription termination regions (figure 3).
  • the DNA construct of the invention may be derived from the Group I PIE constructs previously described (Puttaraju, M.
  • Group I PIE constructs may be derived from other Group I autocatalytic introns, using the same strategy.
  • the self-splicing Group I intron is from the cyanobacteria Anabaena sp. pre-tRNA-Leu gene.
  • the portion of the genetic construct which starts immediately downstream of the promoter and terminates immediately upstream of the sequence encoding the shRNA of the invention comprises the sequence SEQ ID NO: 3 and the portion of the genetic construct which starts immediately downstream of the sequence encoding the shRNA of the invention and terminates immediately upstream of the transcription termination region comprises the sequence SEQ ID NO: 4. Therefore, the sequence encoding the shRNA of the invention is inserted between the sequences SEQ ID NO: 3 and SEQ ID NO: 4.
  • the transcription initiation region may be from RNA polymerase promoters from the host-cell that is used for producing the RNA, including for example prokaryotic RNA polymerase promoters and eukaryotic RNA polymerase I, II or III (pol I, II or III) promoters. Transcripts from pol II or pol III promoters are expressed at high levels in all cells. Transcription units derived from genes encoding U6 small nuclear transfer RNA and adenovirus VA RNA are useful in generating high concentrations of desired ciRNA in cells.
  • the promoter may be constitutive or inducible.
  • inducible promoters are: eukaryotic metal lothionine promoter which is induced by increased levels of heavy metals, prokaryotic lacZ promoter which is induced in response to isopropyl- ⁇ -D-thiogalacto-pyranoside (IPTG) and eukaryotic heat shock promoter which is induced by increased temperature.
  • the transcription initiation region may be from an exogenous RNA polymerase (T7, T3 or SP6) promoter, providing that said exogenous RNA polymerase enzyme is expressed in the host cell in which the expression vector is introduced.
  • the ciRNA RNA precursor is under the transcriptional control of the bacteriophage T7 RNA polymerase promoter and terminator sequences. Examples of said sequence are SEQ ID NO: 5 and SEQ ID NO: 6, respectively for the T7 RNA polymerase promoter and the T7 terminator. According to another preferred embodiment of said genetic construct, the ciRNA RNA precursor is under the transcriptional control of transcription initiation and termination regions which are functional in yeast mitochondria.
  • said genetic construct comprises the COX2 3'UTR sequence (SEQ ID NO: 7) which includes the dodecameric motif (aataatattctt ; SEQ ID NO : 8) that protects the mitochondrial RNA from the RNA degradosome.
  • the C0X2 3'UTR sequence is inserted 3' to the sequence encoding the shRNA and 5' to the transcription terminator.
  • a preferred genetic construct derived from Anabaena sp. Group I PIE sequences and in which the ciRNA RNA precursor is expressed under the transcriptional control of the T7 RNA polymerase promoter and terminator sequences comprises the sequence encoding the shRNA of the invention inserted between the sequences SEQ ID NO: 9 and SEQ ID NO: 10.
  • This construct is flanked by EcoRl and Smal sites at both ends and comprises Pst ⁇ and Pmel sites 3' of the T7 promoter as well as Xbal, Nrul, Spel and EcoRV sites 5'of the T7 terminator.
  • the COX2 3'UTR sequence flanked at both ends by Spel and EcoRV sites is inserted 5' to the T7 terminator.
  • a similar strategy may be used to generate genetic constructs from other permuted intron-exon sequences, as described above, For example, from PIE sequences derived from an intron of a yeast mitochondrial gene, such as Saccharomyces cerevisiae mitochondrial 21 S rRNA gene, rl intron encoding a putative transposase (GenBank accession number Ml 1280 and SEQ ID NO: 11) or S. cerevisiae COXl gene Group I al 4 intron.
  • the invention concerns also a vector comprising the genetic construct as defined above.
  • a vector which can be used in the present invention includes, but is not limited to, a viral vector, a plasmid, a RNA vector or a linear or circular DNA or RNA molecule which may consists of a chromosomal, non chromosomal, semisynthetic or synthetic nucleic acids.
  • Preferred vectors are those capable of autonomous replication (episomal vector) and/or expression of nucleic acids to which they are linked (transcription vectors).
  • Vectors can also comprise selectable markers, for example: neomycin phosphotransferase, histidinol dehydrogenase, dihydro folate reductase, hygromycin phosphotransferase, herpes simplex virus thymidine kinase, adenosine deaminase, glutamine synthetase, and hypoxanthine-guanine phosphoribosyl transferase for eukaryotic cell culture; TRPl for S. cerevisiae; tetracycline, rifampicin or ampicillin resistance in E. coli.
  • selectable markers for example: neomycin phosphotransferase, histidinol dehydrogenase, dihydro folate reductase, hygromycin phosphotransferase, herpes simplex virus thymidine kinase, adenosine deaminase,
  • the transcription vector is a plasmid containing a replication origin which is active in the host cells, and a selection marker.
  • the host cells are bacteria or yeasts.
  • Yeast expression vectors comprise advantageously the Ori5 mitochondrial replication origin sequence (SEQ ID NO: 12) for maintenance of the plasmid vector in the yeast cell progeny and eventually a COX2 gene fragment (SEQ ID NO: 13) for the selection of mitochondrial transformants.
  • the invention concerns also eukaryotic or prokaryotic cells which are modified by a vector as defined above.
  • the invention concerns also a method for producing the ciRNA of the invention derived from the method described in the EP Patent 1646724, which method is characterized in that it comprises at least the following steps: (1) transforming the mitochondria of yeast cells (in particular
  • S. cerevisiae cells lacking mitochondrial DNA (rho° strain) with a mitochondrial transcription vector comprising the genetic construct as defined above under the control of a promoter and a transcription terminator that are functional in yeast mitochondria, and a mitochondrial transformation reporter gene or a fragment of said reporter gene; a mitochondrial transformant or a synthetic rho strain is thus obtained;
  • step (3) culturing the yeast mitochondrial transformants selected in step (2), preferably in the exponential growth phase;
  • step (3) (4) isolating the mitochondria from the yeast mitochondrial transformants cultured according to step (3), and (5) extracting and purifying the ciRNA of interest from said mitochondria.
  • the ciRNA of the invention may be used as a therapeutic agent to reduce activity of specific gene products.
  • the ciRNA of the invention may also be used in functional genomics to knock down single genes for detailed study or hundreds to thousands of genes in high-throughput functional genomic surveys.
  • the invention also concerns a ciRNA molecule or a vector as defined above, as a medicament.
  • the invention concerns also a pharmaceutical composition comprising at least a ciRNA of the invention in an acceptable carrier, such as stabilizer, buffer and the like.
  • a pharmaceutical composition or formulation refers to a form suitable for administration, e.g., systemic or local administration, into a cell or subject, including for example a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, inhalation, or by injection. These compositions or formulations are prepared according to any method known in the art for the manufacture of pharmaceutical compositions.
  • the invention features a composition wherein the ciRNA molecule or vector is associated to a compound that allows the delivery of the ciRNA/vector into target cells.
  • the compound may be a membrane peptide, transporter, lipid, hydrophobic moiety, cationic polymer, PEL
  • the ciRNA and the compound are formulated in microspheres, nanoparticules or liposomes.
  • the ciRNA molecule or vector may be associated with a compound that allows a specific targeting of the target cell, such as a ligand of a cell-surface antigen or receptor, for example a peptide or an antibody specific for said antigen/receptor .
  • a pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence or treat (alleviate a symptom to some extent, preferably all the symptoms) of a disease or state.
  • the pharmaceutically effective dose of the ciRNA depends upon the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors, that those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered.
  • the ciRNA of the invention may be administered by a single or multiple route(s) chosen from: local (intratumoral, for example intracerebral (intrathecal, intraventricular)), parenteral (percutaneous, subcutaneous, intravenous, intramuscular, intraperitoneal), oral, sub-lingual, or inhalation.
  • local intraarticular, for example intracerebral (intrathecal, intraventricular)
  • parenteral percutaneous, subcutaneous, intravenous, intramuscular, intraperitoneal
  • oral sub-lingual, or inhalation.
  • the invention further comprises other features which will emerge from the description which follows, which refers to examples illustrating the ciRNA molecules of the invention and their use according to the invention, as well as to the appended drawings in which: - figure 1 illustrates the production of circular RNA molecules using permuted intron-exon sequences.
  • FP fusion point.
  • 5'ss 5' splice site.
  • 3'ss 3'splice site.
  • SJ splice junction.
  • - figure 2 represents the structure of circular RNA molecules according to the invention.
  • - figure 3 represents the structure of a genetic construct designed for the production of the circular molecule according to the invention.
  • Intron 2 intron 3' half.
  • Exon 2 3'exon sequence flanking the 3' splice site.
  • Intron 1 intron 5' half.
  • Exon 1 5'exon sequence flanking the 5' splice site.
  • FIG. 4 represents the sequence of a genetic construct designed for the production of the circular molecule according to the invention.
  • FIG. 5 represents the pVciLuc (A) and pVmutciLuc (B) plasmid map.
  • - figure 6 illustrates the resistance of the circular RNA ciLUC to exonuclease R treatment.
  • the linear precursor of ciLUC produced by in vitro transcription was circularized by incubation with GTP in Hepes buffer for 18 h at 32 °C.
  • An aliquot of the reaction mixture was incubated with exonuclease R (IUI per microgram of RNA) for Ih at 37 °C and analyzed by electrophoresis on denaturing 1 % agarose gel (ciLuc (+)).
  • An aliquot of the reaction mixture, not treated with exonuclease R (ciLuc (-)) and a linear RNA (LIN) were used as controls.
  • - figure 7 represents the pucMod (A) and pPT24 (B) vector map.
  • - figure 8 represents the T7 vector map.
  • - figure 9 illustrates the RNA interference activity of the ciLUC RNA.
  • Huh-7 cells (10 5 cells) were transfected with 900 ng of RNA. The expression of the two luciferase genes was assayed at 48 h. The values correspond to the percentage of luminescence inhibition by the RNA (ratio of RLUs in the cells transfected with RNA versus control cells (no RNA) x 100).
  • the RNA interference activity of the circular siRNA, ciLuc was compared to that of the corresponding shRNA (siLUC) and a non-relevant shRNA (siNCE).
  • the plotted data are the means ⁇ standard deviation of 4 i experiments.
  • Example 1 Design of a ciRNA targeting the luciferase gene (ciLUC) shLUC is a known shRNA targeting the luciferase gene
  • ciLUC The structure of ciLUC is presented in figure 2B; it contains the shLUC sequences mentioned above and additional sequences (underlined) consisting of the tRNA Leu anticodon stem-loop of Anabaena group I PIE (in bold) and sequences from a HDV ribozyme (AGGCG and CUGGGCU).
  • the tRNA Leu anticodon stem-loop of Anabaena group I PIE contains a 7-nucleotide loop (AAAA TUUC) corresponding to the splice junction (arrow) and a 5 base-pair stem (UCGCU and AGCGA).
  • RNA non-circular RNA (mutant ci-LUC) which differs from ciLUC by the introduction of two mutations (AA to UG and UU to AC; marked with asterisks), respectively in the 3' and 5' splice sites, was also produced: 5'
  • a genetic construct was designed for the transcription of ciLUC in vitro and in vivo in bacteria and yeast (figures 3 and 4).
  • the ciRNA precursor is under the transcriptional control of the T7 RNA polymerase promoter and T7 terminator sequences (SEQ ID NO: 5 and SEQ ID NO: 6, respectively).
  • This construct contains sequences from the Anabaena Group I PIE plasmid pRIOO (Puttaraju, M. and M.D. Been, Nucleic Acids Res., 1992, 20, 5357- 5364; see in particular figure 1) and from the derived plasmids, pR120, containing a shorter fused-exon sequence and pRCl, containing an HDV ribozyme into the exon (Puttaraju et al, Nucleic Acids Res., 1993, 21, 4253-4258; see in particular figure Ic and 2).
  • sequences consist of two fragments: a first fragment including (A) plasmid sequences, (B) a 3' portion of the intron, (C) the 3' splice site, the first nucleotides of the 3' exon and HDV ribozyme sequences (AAAATCGCTAGGCG) and a second fragment including: (D) HDV ribozyme sequences, the last nucleotides of the 5' exon and the 5 1 splice site (CTGGGCT AGCGACTT), (E) a 3' portion of the intron and (F) other plasmid sequences.
  • the shLUC coding sequence is inserted between the first and the second fragments.
  • the construct contains also the COX2 3'UTR sequence (SEQ ID NO: 1
  • the COX2 3'UTR sequence is inserted 3' to the sequence encoding the ciRNA precursor and 5' to the T7 transcription terminator.
  • the construct contains restriction sites. It is flanked by EcoRl and Smal sites at both ends and comprises Pstl and Pmel sites 3' to the T7 promoter as well as Xbal, Nrul, Spel and EcoRV sites 5 'to the T7 terminator.
  • the COX2 3'UTR sequence (SEQ ID NO: 7) is flanked at both ends by Spel and EcoRV sites as well as by Xbal and Nrul sites, at its 3' end only. The sequence of this construct corresponds to SEQ ID NO: 17.
  • Two control constructs were also generated: a first control (SEQ ID NO: 18) in which the sequence encoding ciLUC was replaced with the sequence encoding mutant ciLUC, and a second control encoding shLUC only (deletion of the intron and exon sequences from Anabaena Group I PIE).
  • the genetic constructs encoding the ciLUC or shLUC RNAs were generated by synthesis of two complementary oligonucleotides corresponding to each strand of the DNA construct and annealing of the oligonucleotides.
  • Each construct was then inserted into the EcoRV site of the pUC57 cloning vector (# SDl 176; GENSCRIPT) to give the recombinant plasmids pVciLuc (figure 5A) and pVshLuc, using standard recombinant DNA techniques.
  • the plasmid pVmutciLuc (figure 5B) containing the genetic construct encoding mutant-ciLUC was derived from pVciLuc by site-directed mutagenesis of the ciLuc insert (SEQ ID NO: 17) at positions 204-205 (AA to TG mutation) and 286-287 (TT to AC mutation).
  • the constructs were also cloned into appropriate vectors for in vivo transcription in yeast or bacteria using T7 RNA polymerase.
  • RNA pellets were then washed in ethanol (70 %) and resuspended in sterile water (100 ⁇ L).
  • RNA produced by in vitro transcription was incubated in Hepes buffer (40 raM Hepes, pH 7.5; 200 mM NaCl; 20 mM MgCl 2 ) for 5 min at 50 °C.
  • RNA was digested with exonucleases having a 3'-5'(RnaseR, RnaseT, PNPase), or 5 '-3' (Exoribonuclease I and II) activity, endonucleases specific for single-strand (Rnase A or RNAse Tl) or double-strand (Rnase V) RNA, according to the manufacturer's instructions.
  • Exonuclease treatment was performed in the presence of 1 UI of Exonuclease R (TEBU-BIO) per microgram of RNA, for Ih at 37 °C.
  • TEBU-BIO 1 UI of Exonuclease R
  • Denaturing agarose gel (1 g agarose, 10 mL 1OX MOPS buffer (0.4 MOPS, 0.1 M sodium acetate, 10 mM EDTA), 18 mL formaldehyde, 72 mL water, 1 ⁇ L ethidium bromide (10 mg/mL)) were used with running buffer containing IX MOPS buffer (0.04 MOPS, 0.01 M sodium acetate, 1 mM EDTA).
  • Native agarose gel (1 g agarose, 10 mL 1OX TAE buffer, 90 mL water, 1 ⁇ L ethidium bromide (10 mg/mL) were used with IX TAE buffer.
  • a RNA ladder (# LAD-DT-25, MITOPROD) was used as a molecular weight marker. Bands on gel were visualized using a UV (302 nm) transilluminator. e) Northern Blot Analysis
  • RNA samples were separated by gel electrophoresis as described above, transferred to a PVDF membrane and detected by standard hybridization technique with a 32 P labeled DNA probe specific for the intron or exon portion of the Anabaena Group I PIE sequences, or for the shLUC, prepared using the
  • RNA sample eventually diluted in loading buffer A (30 mM lithium perchlorate, 20 mM sodium acetate, pH 6.5), was denatured at 65-70°C for 10 min. Then, it was loaded at a low flow-rate on a anion exchange analytic column (DNApac200, DIONEX) equilibrated with the loading buffer A. The chromatography was performed at a low flow-rate (lmL/min) with a gradient of elution buffer B (30 to 300 mM lithium perchlorate). The column was maintained at the desired temperature (usually 65-70°C); the buffers were at room temperature. Alternatively, the column was at room temperature and the buffers were at 65-70°C.
  • RNA quantification was carried out on an AKTA Purifier 10 (GE Healthcare). Data analysis and reporting were performed on computers using the Unicorn 5.0 control system software. The different peaks were identified and the area of each peak was measured to evaluate the efficiency of production of the circular RNA. Purified circular RNA was recovered from the corresponding peak. g) RNA quantification
  • a sample containing RNA was diluted in a neutral buffer (usually water) and the amount of RNA present in the sample was determined by measuring the absorbance (OD) of the diluted sample at 260 nm using a spectrophotometer. The level of contaminants was evaluated by measuring the absorbance at 230 nm and 280 nm and calculating the ratios OD 260 /OD 28 o and OD 260 /OD 230 . Salts (EDTA, acetate), solvents (trizol) and protein (peptid bond) absorb at 230 nm; a good sample has a OD 260 /OD 23 o ratio > 1.8.
  • RNA stability assay Polysaccharides, glycogen, fats and lipids absorb at 280 nm; a good sample has a OD 260 /OD 28 o ratio > 1.6 and ⁇ 2.1. If the ratio is below, the RNA is not completely solubilized or the sample contains many proteins. If the ratio is greater than 2.1 , the RNA is degraded, h) RNA stability assay
  • RNAs ciLUC, mutant-ciLUC, shLUC
  • the stability of the RNAs was assayed by 3'exonuclease digestion as described above. It was also assayed by incubating the ciLUC and shLUC RNAs at 22 °C and 37 °C for different time periods and compairing the ciLUC and shLUC chromatographic profiles by anion echange HPLC, as described above. 2) Results
  • RNA products obtained by in vitro transcription from the vectors encoding the ciLUC, mutant-ciLUC or shLUC RNAs, and circularization or not, of the linear transcripts in the presence of GTP were treated with various exonucleases and endonucleases, and analysed by anion exchange HPLC, native or denaturing gel electrophoresis, and eventually by Northern-blot analysis, to identify the different RNA molecules produced (linear precursor (unspliced), intermediates and products (circular (spliced), 5' and 3' linear fragments; see figure Ic of Puttaraju M. and M.D. Been, Nucleic Acids Res., 1992, 20, 5357-5364 or figure 1 of the present application).
  • the proportion of circular RNA was measured to determine the efficiency of production of this circular RNA by in vitro transcription of a linear unspliced precursor and circularization by self-splicing in the presence of GTP.
  • the proportion of circular RNA may be compared with that of shRNA (shLUC) produced in the same conditions.
  • Circular RNA (at least 80 % pure) was purified by HPLC. The stability of the circular RNA (ciLUC) was tested and compared with that of mutant- ciLUC produced in the same conditions.
  • the transcription vectors comprising the genetic constructs encoding ciLUC, mutant-ciLUC or shLUC (example 2) were transformed into an E.coli strain expressing the T7 RNA polymerase from a lac promoter (T7 express competent (OZYME), BL21(DE3)(NOVAGEN or AGILENT TECHNOLOGIES), using the heat shock method.
  • BL21(DE3), F ompT gal dcm Ion hsdS B (r B " m B " ) ⁇ (DE3 [lad lacUV5- T7 gene 1 indl sam7 nin5]) is an E.
  • Transformed bacteria were cultivated in fresh medium complemented with the appropriate antibiotic to reach 2 OD/ml. IPTG (2mM) was added to the cultures which were then incubated for 3 hours at 37 °C under agitation. After OD measurement, the culture was centrifugated and the RNA was extracted from the pellet using trizol® (INVITROGEN) or trireagent® (EUROMEDEX). The cell pellet was resuspended in Trireagent (1 ml solution for each 20 OD; sample volume should not exceed 10 % of the volume of Trireagent).
  • Chloroform (0.2 mL per ImL Trireagent) was then added and the mixture was vortexed for 15 sec (2 times), stored few minutes (2 to 15 min) at room temperature and centrifugated at 8500g to 1200Og for 15-20 min at 4 °C.
  • the aqueous phase was transferred to a fresh tube and isopropanol was added (0.5 mL for each 20 OD).
  • the mixture was stored 5 to 10 min at room temperature and centrifugated for 15 to 20 min at 4 to 25 °C at 9000g to 1200Og.
  • the supernatant was removed and the RNA pellet was washed with 75 % ethanol (0.5 to ImL for each 20 OD) and centrifugated at 750Og for 5 min at 4 to 25°C.
  • RNA was dissolved in water or in buffer A (for chromatography) by passing the solution a few times through a pipette tip and incubating if necessary for 10-15 min at 55-60 0 C, to improve the solubilization.
  • RNA products obtained by in vivo transcription from bacteria expressing the T7 RNA polymerase under the control of a lac promoter, transformed with vectors encoding the ciLUC, mutant-ciLUC or shLUC RNAs (example 2), induced or not by IPTG, were treated with various exonucleases and endonucleases, and analysed by anion exchange HPLC, native or denaturing gel electrophoresis, and eventually by Northern-blot analysis, as described in example 3, to identify the different RNA molecules produced (linear precursor (unspliced), intermediates and products (circular (spliced), 5' and 3' linear fragments; see figure Ic of Puttaraju M. and M.D.
  • ciLUC circular RNA
  • the proportion of circular RNA (ciLUC) was measured to determine the efficiency of production of this circular RNA by in vivo transcription and in vivo circularization by self-splicing.
  • the proportion of circular RNA (ciLUC) may be compared with that of mutant-ciLUC produced in the same conditions.
  • Circular RNA (at least 80 % pure) was purified by HPLC.
  • the stability of the circular RNA (ciLUC) was tested and compared with that of mutant-ciLUC produced in the same conditions.
  • Example 5 Production of ciLUC by in vivo transcription in yeast mitochondria 1) Material and methods The production of heterologous RNA by transcription in yeast mitochondria is described in the EP Patent 1646724.
  • the genetic constructs encoding ciLUC, mutant-ciLUC or shLUC described in example 2 were cloned individually into the EcoRl site of pucMod or pPT24 (figure 7) mitochondrial transcription vectors.
  • the pucMod vector is a pUC57 derived plasmid containing the Ori5 sequence which allows the maintenance of the plasmid in the mitochondria of the yeast progeny and a COX2 gene fragment (SEQ ID NO: 13) for the selection of the mitochondrial transformants.
  • the plasmid pPT24 is described in Thorness, P.E. and T.D. Fox, Genetics, 1993, 134, 21-28.
  • W303-1B strain Mus ⁇ , ade2, trpl, his3, Ieu2, ura3
  • W303- IB ATCC No. 201238
  • A/50 was transformed with a vector (figure 8) containing the a T7 RNA polymerase gene operatively linked to a mitochondrial targeting sequence (MTS), COX4 leader sequence or ATP9 MTS, under the control of the galactose inducible promoter, GALlO, and an auxotrophic marker (LEU2 or ADEI).
  • MTS mitochondrial targeting sequence
  • COX4 leader sequence COX4 leader sequence
  • ATP9 auxotrophic marker
  • the biolistic method was used to transform the recombinant mitochondrial transcription vectors into the mitochondria of rho 0 (lacking mitochondrial DNA) derivatives of W303- IB strain nuclear transformants (expressing the T7 RNA polymerase).
  • the mitochondrial transformants were isolated by crossing with a tester strain (rho + , C OX2 ' ) and isolation of cells capable of growing on a non- fermentable medium.
  • Mitochondrial transformants were cultivated in YPGA medium supplemented with glucose and galactose to induce T7 RNA polymerase expression to reach 5 to 10 OD/ml.
  • the culture was centrifugated and the mitochondria were isolated as described in the EP Patent 164 and the mitochondrial RNA was extracted using the RN AXEL® and RNABIND® reagents according to the manufacturer's instructions (EUROMEDEX). 2) Results
  • RNA products obtained by transcription in the mitochondria of yeast expressing the T7 RNA polymerase under the control of a galactose inducible promoter transformed with mitochondrial transcription vectors encoding the ciLUC, mutant-ciLUC or shLUC RNAs (example 2), induced or not by galactose, were treated with various exonucleases and endonucleases, and analysed by anion exchange HPLC, native or denaturing gel electrophoresis, and eventually by Northern-blot analysis, as described in example 3, to identify the different RNA molecules produced (linear precursor (unspliced), intermediates and products (circular (spliced), 5' and 3' linear fragments; see figure Ic of Puttaraju M.
  • RNA interference activity of ciLUC 1 Material and methods
  • Huh-7 human hepatocarcinoma derived cells expressing constitutively both the Firefly and Renilla luciferase genes were grown in 24 well plates (10 5 cells/well) in DMEM medium supplemented with 20 % fetal calf serum (complete medium), for 16 h at 37 °C with 5 % CO 2 .
  • the transfection was performed by incubating 0.9 ⁇ g of RNA and 2.1 ⁇ L of DMRIE-C liposome (INVITROGEN) in 500 ⁇ L OptiMEM® (GIBCO), for 30 min at room temperature.
  • the 500 ⁇ L transfection mixture was added to the cells rinsed with PBS and the cells were incubated for 4 h at 37 °C with 5 % CO 2 .
  • the light emitted by the luciferase-catalyzed chemoluminescent reaction was measured in the cells transfected with RNA and in the control cells (no RNA), using a luminometer.
  • the silencing of the luciferase gene was calculated from the ratio of RLUs in the transfected cells versus control cells.
  • RNA interference activity of the ciLUC RNA was compared with that of shLUC RNA (siLuc) and a non-relevant shRNA (siNCE) produced in vitro as described in example 3.
  • shLUC RNA shLUC RNA
  • siNCE non-relevant shRNA

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Abstract

La présente invention porte sur une molécule d'ARN interférent circulaire (ARNic) comprenant les brins sens et antisens d'une molécule d'ARN interférent ciblant un gène d'intérêt, lesdits brins sens et antisens étant fermés à une extrémité par une structure de boucle et à l'autre extrémité par une séquence de jonction d'épissage produite par une activité d'épissage de séquences d'intron-exon permutées, et la molécule d'ARN circulaire inhibant l'expression du gène d'intérêt dans des cellules exprimant ledit gène d'intérêt. L'invention porte également sur des produits de recombinaison génétique et des vecteurs de transcription pour la production dudit ARNic. L'invention porte en outre sur un procédé permettant la production de grandes quantités dudit ARNic dans des mitochondries de levure sous une forme qui est stable et facile à purifier.
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