WO2005001039A2 - Production de petits arn interferents (arnsi) regulee par les ribozymes et procedes d'utilisation de ceux-ci - Google Patents

Production de petits arn interferents (arnsi) regulee par les ribozymes et procedes d'utilisation de ceux-ci Download PDF

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WO2005001039A2
WO2005001039A2 PCT/US2004/017034 US2004017034W WO2005001039A2 WO 2005001039 A2 WO2005001039 A2 WO 2005001039A2 US 2004017034 W US2004017034 W US 2004017034W WO 2005001039 A2 WO2005001039 A2 WO 2005001039A2
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ribozyme
sirna
rna
kit
allosteric
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WO2005001039A3 (fr
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Garrett A. Soukup
Alexis Kertsburg
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Creighton University
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/123—Hepatitis delta
    • 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
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/127—DNAzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3519—Fusion with another nucleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2330/00—Production
    • C12N2330/30—Production chemically synthesised

Definitions

  • This invention relates to the fields of molecular biology and modulation of gene expression. Specifically, the invention provides materials and methods for the controlled production of small inhibitory RNA (siR ⁇ A) and methods of use thereof.
  • RNA interference is a biological mechanism for double stranded RNA (dsRNA) induced post- transcriptional gene silencing that has become a valuable component of strategies for investigating gene function and achieving potential therapeutic outcomes (McManus, M.T. and P. A. Sharp (2002) Nat. Rev. Genet. 3 : 737-747 ;Paddison, P.J. and G.J. Hannon (2002) Cancer Cell. 2:17-23; Shuey, D.J., et al . (2002) Drug Discov. Today.
  • dsRNA double stranded RNA
  • RNAi is believed to be dependent upon the action of Dicer (Bernstein, E., et al . (2001) Nature. 409 ; 363-366; Retting, R.F., et al . (2001) Genes & Dev. 15:2654-2659; Knight, S.W. and B.L. Bass (2001) Science. 293:2269-2271).
  • Dicer is a ribonuclease III family member that processes dsRNA into small interfering RNA duplexes ( siRNAs ) comprised of -21 nucleotide (nt) strands and having 2 nt 3 ' overhangs (Zamore, P.D., et al.
  • siRNAs are subsequently manipulated by the RNA-induced silencing complex (RISC) , where siRNA strands are utilized as guide sequences to mediate mRNA recognition and cleavage (Tuschl, T., et al . (1999) Genes & Dev. 13:3191-3197; Hammond, S.M., et al . (2000) Nature. 404:293-296) .
  • RISC RNA-induced silencing complex
  • RNAi can be effected by exogenous delivery of siKNAs that are produced by chemical synthesis and hybridization of complementary strands (Elbashir, S.M., et al . (2001) Nature. 411:494-498; Elbashir, S.M., et al . (2001) Genes & Dev. 15:188-200), by in vitro transcription (Donze, 0. and D. Picard (2002) Nucleic Acids Res. 30:e46; Paddison, P.J., et al.
  • RNAi can be effected by siRNAs that are intracellularly transcribed following transfection (Paddison, P.J., et al .
  • siRNA species expression of siRNA species is commonly achieved by transcription from RNA polymerase III (pol III) promoters derived from genes encoding U6 or Hi RNA (Paddison, P.J., et al.
  • RNA pol III initiates and terminates transcription at distinct sites.
  • RNA pol III promoters are ideal for intracellular transcription of small RNAs, as pol III initiates and terminates transcription at distinct sites.
  • the hepatitis delta virus (HDV) ribozyme is a self-cleaving ribozyme that may be modified (e.g. through the addition of a stem-loop structure) so that the cleavage activity of the ribozyme is ligand-dependent (Kertsburg, A. and G.A. Soukup (2002) Nucleic Acids Res. 30:4599-4606).
  • Such allosteric ribozymes perform catalysis upon recognition and binding of specific effector molecules (Soukup, G.A. and R.R. Breaker (2000) Curr. Opin. Struct. Biol . 10:318-325; Silverman, S.K. (2003) RNA. 9:377-383).
  • the HDV ribozyme is particularly suited to the task of ribozyme-mediated and allosteric ribozyme-regulated cleavage.
  • the ribozyme catalyzes self-cleavage and production of a 5' terminal fragment that is not an integral structural component of the ribozyme (Ferre-D'Amare, A.R. , et al . (1998) Nature. 395:567-574; Shih, I.H. and M.D. Been (2002) Annu. Rev. Biochem. 71:887-917). Consequently, the 5'-cleavage product can represent virtually any RNA sequence.
  • a method for producing a small, interfering RNA comprises 1) providing an RNA construct comprising a ribozyme and the sense strand of an siRNA wherein the cleavage activity of the ribozyme causes the cleavage of the sense strand of the siRNA from the RNA construct, 2) providing an RNA construct comprising a ribozyme and the antisense strand of the siRNA wherein the cleavage activity of the ribozyme causes the cleavage of the antisense strand of the siRNA from the RNA construct, and 3) placing the RNA constructs in conditions wherein the cleavage activity of the ribozymes is allowed and the sense and antisense strands of the siRNA are allowed to hybridize to generate the desired siRNA.
  • the method of producing siRNA can be performed in vivo or in vi tro .
  • the ribozyme is an allosteric ribozyme whose activity is modulated by an effector such as, without limitation, the theophylline modulated HDV ribozyme described hereinbelow.
  • the ribozymes linked to the sense strand of the siRNA and to the antisense strand of the siRNA may be the same or different.
  • pharmaceutical preparations for modulating the expression of a gene by ribozyme mediated siRNA production is provided.
  • the pharmaceutical preparation comprises an RNA construct comprising a ribozyme and a sense stand of an siRNA targeted to the desired gene and an RNA construct comprising a ribozyme and an antisense stand of the siRNA in a biologically acceptable medium.
  • the RNA constructs of the pharmaceutical preparation are encoded by DNA and optionally placed in a suitable vector.
  • the ribozyme of the RNA constructs is an allosteric enzyme and a second pharmaceutical preparation comprising the effector of the allosteric enzyme is employed to modulate the production of the siRNA.
  • a method for controlling the expression of a gene in a cell is provided.
  • the method comprises 1) providing an RNA construct comprising a ribozyme and the sense strand of an siRNA targeted to the desired gene wherein the cleavage activity of the ribozyme causes the cleavage of the sense strand of the siRNA from the RNA construct, 2) providing an RNA construct comprising a ribozyme and an antisense strand of the siRNA wherein the cleavage activity of the ribozyme causes the cleavage of the antisense strand of the siRNA from the RNA construct, and 3) administering the RNA constructs to the cells in an amount sufficient to control expression of the desired gene, whereby the RNA constructs enter the cells expressing the desired gene and ribozyme-mediated cleavage occurs to allow for siRNA formation and inhibition of the expression of the desired gene.
  • the RNA constructs are encoded by DNA, optionally in a vector, and administered to the cells in DNA form whereby the RNA constructs are transcribed.
  • the ribozymes are allosteric enzymes and the effector of the allosteric enzymes is administered to the cells to modulate the ribozyme-mediated cleavage of the RNA constructs.
  • an RNA construct comprising a ribozyme operably linked to a small hairpin RNA (shRNA) comprising the sequence of an siRNA may be employed in the instant invention in place of the RNA constructs comprising a ribozyme and the sense and antisense strands of the siRNA.
  • kits comprising a recombinant vector containing a nucleic acid sequence encoding a ribozyme operably linked to a promoter suitable for expression in a desired host cell or in vi tro transcription and a multiple cloning site suitable for cloning a nucleic acid encoding a strand of the siRNA of interest or an shRNA encoding the siRNA of interest so that the cleavage activity of the ribozyme produces the strand of the siRNA or the siRNA as an shRNA.
  • the ribozyme is an allosteric ribozyme and the kit further comprises the effector of the allosteric enzyme in a suitable buffer.
  • the kits of the invention may further comprise host cells suitable for expression of the recombinant vector; at least one reagent for in vi tro transcription of said recombinant vector; at least one reagent suitable for introducing the recombinant vector into test samples, cells, or subjects; at least one reagent suitable for introducing the transcribed RNA product into test samples, cells, or subjects; and/or instruction material .
  • Figure 1 depicts a strategy for allosteric-ribozyme- regulated siRNA production. Specifically, the wild-type sense ribozyme-siRNA-1 construct (sense (S) ribozymes;
  • SEQ ID NO: 1 SEQ ID NO: 1 and the wild-type antisense ribozyme-siRNA- 1 construct (antisense (A) ribozymes; SEQ ID NO: 2) are depicted. Arrowheads indicate cleavage sites which yield siRNA-1 (sense strand SEQ ID NO: 4; antisense strand SEQ ID NO: 5) and wild-type ribozyme (SEQ ID NO: 3).
  • the P4 stem-loop sequence (P4 labeled box; SEQ ID NO: 15) , the stem-loop sequence comprising a theophylline-binding domain and communication module (labeled boxes; SEQ ID NO: 16) , and the sequence of siKNA-2 (sense strand SEQ ID NO: 10; antisense strand SEQ ID NO: 11) are also provided. Additionally, the location of the C75U point mutation is indicated.
  • Figures 2A through 2D are pictures of gels of transcribed ribozyme-siRNA constructs.
  • Figures 2A and 2B are transcription products of various ribozyme-siRNA-1 constructs separated on denaturing PAGE (Fig. 2A) or native PAGE (Fig. 2B) .
  • Figures 2C and 2D are transcription products of various ribozyme-siRNA-2 constructs separated on denaturing PAGE (Fig. 2C) or native PAGE (Fig. 2D) .
  • Grey arrowheads indicate precursor riboyme-siRNAs
  • black arrowheads indicate large 3' cleavage products
  • open arrowheads indicate small 5' cleavage products
  • the asterisks indicate the siRNA.
  • Control lanes X, Y, X+Y, and Z of Figures 2B and 2D represent siRNA strands isolated from the denaturing gels (Figs. 2A and 2C) as indicated by corresponding labeled boxes and allowed to anneal prior to separation by native PAGE.
  • Figures 3A and 3B are pictures of gels of transcribed ribozyme-siRNA constructs.
  • Figures 3A and 3B are transcription products of various theophylline (theo) responsive ribozyme ⁇ siRNA-l (lanes 1-6) and ribozyme- siRNA-2 (lanes 7-12) constructs separated on denaturing
  • FIG. 3A PAGE
  • Fig. 3B native PAGE
  • Grey arrowheads indicate precursor ribozyme-siRNAs
  • black arrowheads indicate large 3' cleavage products
  • open arrowheads indicate small 5' cleavage products
  • the asterisks indicate the siRNA.
  • the control lanes in Figure 3B were prepared as in Figures 2B and 2D.
  • Figure 4A provides the sequences of shRNA-1 (SEQ ID NO: 20) and shRNA-2 (SEQ ID NO: 21) .
  • Figures 4B and 4C are pictures of gels depicting Dicer cleavage products. Radiolabeled RNAs were incubated in the presence or absence of Dicer. dsRNA is a 317-bp double stranded RNA containing nucleotides 12-328 of the open reading frame of EGFP.
  • Pre-siRNA-1 and pre-siRNA-2 are purified ribozyme-siR ⁇ A complexes of A mut and S mut containing siR ⁇ A-1 and siRNA-2, respectively.
  • the control lane (Z) was prepared as in Figures 2B and 2D. Grey arrowheads indicate precursor ribozyme-siRNA constructs, open arrowheads indicate siRNA strands, and asterisks indicate siRNA.
  • siRNAs can be controlled by employing fusion constructs consisting of a strand (sense or antisense) of an siRNA and a ribozyme.
  • two ribozyme-siRNA constructs may be employed to generate the desired siRNA wherein one comprises a ribozyme with the sense strand of the siRNA following the ribozyme cleavage site and the other construct comprises a ribozyme with the antisense strand of the siRNA following the ribozyme cleavage site.
  • the siRNA may then be generated by cleavage by the ribozyme in vi tro or in vivo .
  • the sense ribozyme-siRNA and antisense ribozyme-siRNA constructs are preferably comprised of the same ribozyme, but may alternatively be comprised of different ribozymes (for example, one ribozyme may be allosteric and the other may not) .
  • a single RNA construct comprising a ribozyme operably linked to an shRNA may be employed to generate the desired siRNA, wherein the shRNA contains the sequence of the siRNA and is cleaved from the RNA construct by the catalytic activity of the ribozyme.
  • the ribozymes employed in the instant invention may be any RNA capable of cleaving a target RNA.
  • Such ribozymes include, without limitation, naturally occurring ribozymes such as the hepatitis delta virus (HDV) ribozyme, the hammerhead, hairpin, and VS ribozymes, the group I self-splicing intron, and RNase P in addition to artificial (i.e. man-made or altered) ribozymes such as the X-motif ribozyme (Tang and Breaker (2000) Proc. Natl. Acad. Sci. USA 97:5784-5789).
  • the ribozyme is an allosteric ribozyme, i.e. a ribozyme whose activity is modulated by an effector (e.g.
  • ribozymes examples include, without limitation, the HDV, hammerhead, VS, and X-motif ribozymes and the group I introns which may be modified to be allosteric by methods describe in, for example, Soukup and Breaker
  • an HDV ribozyme (SEQ ID NO: 25) modified to be modulated by theophylline is employed.
  • the siRNAs of the instant invention may be directed to any target desired by the user.
  • the siRNA may target, without limitation, cellular genes, viral genes, bacterial genes, exogenous genes, and reporter genes (e.g. green fluorescent protein).
  • reporter genes e.g. green fluorescent protein
  • nucleic acid or a “nucleic acid molecule” as used herein refers to any DNA or RNA molecule, either single or double stranded and, if single stranded, the molecule of its complementary sequence in either linear or circular form.
  • nucleic acid molecules a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5 ' to 3 ' direction. With reference to nucleic acids of the invention, the term “isolated nucleic acid” is sometimes used.
  • an "isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plas id or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism.
  • a vector such as a plas id or virus vector
  • isolated nucleic acid may refer to an RNA molecule encoded by an isolated DNA molecule as defined above.
  • the term may refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it would be associated in its natural state (i.e., in cells or tissues) .
  • An isolated nucleic acid may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.
  • an isolated nucleic acid may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.
  • the term “specifically hybridizing” refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed “substantially complementary”).
  • the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.
  • Appropriate conditions enabling specific hybridization of single stranded nucleic acid molecules of varying complementarity are well known in the art. For instance, one common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology is set forth below (Sambrook et al . , 1989) :
  • T m 81.5°C + 16.6 og [Na+] + 0.41 (% G+C) - 0.63 (% formamide) - 600/#bp in duplex
  • the T m is 57°C.
  • the T m of a DNA duplex decreases by 1 - 1.5°C with every 1% decrease in homology.
  • targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42°C.
  • the stringency of the hybridization and wash depend primarily on the salt concentration and temperature of the solutions. In general, to maximize the rate of annealing of the probe with its target, the hybridization is usually carried out at salt and temperature conditions that are 20-25°C below the calculated T m of the hybrid.
  • Wash conditions should be as stringent as possible for the degree of identity of the probe for the target . In general, wash conditions are selected to be approximately 12-20°C below the T m of the hybrid. In regards to the nucleic acids of the current invention, a moderate stringency hybridization is defined as hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 ⁇ g/ml denatured salmon sperm DNA at 42°C, and washed in 2X SSC and 0.5% SDS at 55°C for 15 minutes.
  • a high stringency hybridization is defined as hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 ⁇ g/ml denatured salmon sperm DNA at 42°C, and washed in IX SSC and 0.5% SDS at 65°C for 15 minutes.
  • a very high stringency hybridization is defined as hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 ⁇ g/ml denatured salmon sperm DNA at 42°C, and washed in 0. IX SSC and 0.5% SDS at 65°C for 15 minutes.
  • probe refers to an oligonucleotide, polynucleotide or DNA molecule, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe.
  • a probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
  • the probes herein are selected to be complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to "specifically hybridize” or anneal with their respective target strands under a set of pre-determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment may be attached to the 5' or 3 ' end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically.
  • primer refers to a DNA oligonucleotide, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to functionally act as an initiator of template-dependent nucleic acid synthesis .
  • suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as a suitable temperature and pH
  • the primer may be extended at its 3 ' terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield a primer extension product.
  • the primer may vary in length depending on the particular conditions and requirement of the application.
  • the oligonucleotide primer is typically 15-25 or more nucleotides in length.
  • the primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able anneal with the desired template strand in a manner sufficient to provide the 3 ' hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme. It is not required that the primer sequence represent an exact complement of the desired template.
  • a non-complementary nucleotide sequence may be attached to the 5 ' end of an otherwise complementary primer.
  • non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product.
  • Polymerase chain reaction (PCR) has been described in U.S. Patent Nos: 4,683,195, 4,800,195, and 4,965,188, the entire disclosures of which are incorporated by reference herein.
  • the terms “percent similarity” , “percent identity” and “percent homology” when referring to a particular sequence are used as set forth in the University of Wisconsin GCG software program.
  • the term “functional” as used herein implies that the nucleic or amino acid sequence is functional for the recited assay or purpose.
  • a “replicon” is any genetic element, for example, a plasmid, cosmid, bacmid, phage or virus, that is capable of replication largely under its own control.
  • a replicon may be either RNA or DNA and may be single or double stranded.
  • a “vector” is a replicon, such as a plasmid, cosmid, bacmid, phage or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication of the attached sequence or element .
  • An "expression operon” refers to a nucleic acid segment that may possess transcriptional and translational control sequences, such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons) , polyadenylation signals, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.
  • oligonucleotide refers to sequences, primers and probes of the present invention, and is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application and use of the oligonucleotide.
  • small, interfering RNA refers to a short (typically less than 30 nucleotides long) double stranded RNA molecule. Typically, the siRNA modulates the expression of a gene to which the siRNA is targeted.
  • substantially pure refers to a preparation comprising at least 50-60% by weight of a given material (e.g., nucleic acid, oligonucleotide, protein, etc.) . More preferably, the preparation comprises at least 75% by weight, and most preferably 90-95% by weight of the given compound. Purity is measured by methods appropriate for the given compound (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like) .
  • gene refers to a nucleic acid comprising an open reading frame encoding a polypeptide, including both exon and (optionally) intron sequences.
  • the nucleic acid may also optionally include non-coding sequences such as promoter or enhancer sequences .
  • the term "intron” refers to a DNA sequence present in a given gene that is not translated into protein and is generally found between exons .
  • the phrase "operably linked, " as used herein, may refer to a nucleic acid sequence placed into a functional relationship with another nucleic acid sequence. Examples of nucleic acid sequences that may be operably linked include, without limitation, promoters, transcription terminators, enhancers or activators and heterologous genes which when transcribed and, if appropriate to, translated will produce a functional product such as a protein, ribozyme or RNA molecule.
  • operably linked may also refer to a nucleic acid sequence placed in functional relationship with a ribozyme such that the catalytic cleavage activity of the ribozyme leads to the release of the operably linked nucleic acid sequence.
  • siRNA small interfering RNA
  • the siRNA molecules of the invention are typically between 12-30 nucleotides in length. More preferably, siRNA molecules are about 20, 21, 22, and 23 nucleotides in length.
  • the siRNA molecules may comprise a sequence identical or at least 90% identical to any portion of the target gene whose expression is to be modulated including coding and non-coding sequences.
  • One method for identifying an siRNA target site within the target gene comprises scanning for AA dinucleotide sequences downstream of the AUG start codon and identifying the AA dinucleotide and the adjacent 3' 19 nucleotides as an siRNA target (see Ambion® Guidelines; Austin, TX; www.rockefeller.edu/labheads/tuschl/sirna; see also, in general, Ausubel et al . , supra) .
  • the identified sequence may be searched against a genome database such as GenBank® (maintained by The National Center for Biotechnology Information; NCBI) by a program such as BLAST® (Basic Local Alignment Search Tool) to ensure the sequence does not have significant homology to other genes .
  • An shRNA of the invention typically comprises the sense and antisense strands of an siRNA connected by a sequence of nucleotides sufficiently long enough to allow for the formation of a hairpin structure caused by the hybridization of the sense and antisense strands of the siRNA.
  • the connecting sequence of nucleotides is between about 5 and 15 nucleotides in length.
  • RNA constructs comprising a ribozyme and an shRNA may be interchanged for. the ribozyme-siRNA constructs described herein above and below.
  • Small oligonucleotides such as those described herein are highly susceptible to degradation by assorted nucleases. Moreover, such molecules may be unable to enter cells because of insufficient membrane permeability.
  • oligonucleotides that are modified in various ways to increase stability and membrane permeability.
  • Modified oligoribonucleotides such as 2 ' protected, may be utilized in the present invention (Dharmacon Research; Lafayette, CO) .
  • ribozyme- siRNAs may be produced by expression of DNA sequences cloned into plasmid or retroviral vectors. Using standard methodology known to those skilled in the art, it is possible to maintain the ribozyme-siRNA in any convenient cloning vector (see Ausubel et al . , eds . Current Protocols in Molecular Biology, John Wiley and Sons, Inc.
  • RNA polymerase II promoters may be employed inasmuch as the activity of the fused ribozyme will release the siRNA construct.
  • RNA polymerase II promoters include, without limitation, the cytomegalovirus (CMV) and simian virus 40 (SV40) promoters.
  • CMV cytomegalovirus
  • SV40 simian virus 40
  • Antibiotic resistance markers may also be included in these vectors to enable selection of transformed cells. These may include, for example, genes that confer hygromycin, neomycin or ampicillin resistance.
  • Ribozyme-siRNA constructs and ribozyme-siRNA- encoding vectors may be administered to cells or cell lines by any method such as, without limitation, transfection, electroporation, lipofection, and transduction (see, for example, Ausubel et al . , supra) .
  • Ribozyme-siRNA-encoding vectors and ribozyme-siRNA constructs as described herein are generally administered to a patient as a pharmaceutical preparation.
  • patient refers to human or animal subjects.
  • the pharmaceutical preparation comprising the ribozyme-siRNA construct or vectors encoding the ribozyme-siRNA constructs of the invention are conveniently formulated for administration with an acceptable medium such as water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) , dimethyl sulfoxide (DMSO) , oils, detergents, suspending agents or suitable mixtures thereof.
  • concentration of the ribozyme-siRNA in the chosen medium may depend on the hydrophobic or hydrophilic nature of the medium, as well as the length and other properties of the ribozyme-siRNA molecule. Solubility limits may be easily determined by one skilled in the art.
  • biologically acceptable medium includes any and all solvents, dispersion media and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation, as exemplified in the preceding paragraph.
  • the use of such media for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the ribozyme-siRNA molecules to be administered, its use in the pharmaceutical preparation is contemplated. Selection of a suitable pharmaceutical preparation depends upon the method of administration chosen. For example, ribozyme-siRNA molecules and vectors encoding the same may be administered by direct injection into the desired tissue.
  • a pharmaceutical preparation comprises the ribozyme-siRNA molecule dispersed in a medium that is compatible with the selected tissue.
  • Ribozyme-siRNA molecules and vectors encoding the same may be administered parenterally by intravenous injection into the blood stream, by subcutaneous, intramuscular, or intraperitoneal injection, or any other method known in the art.
  • Pharmaceutical preparations for parenteral injection are commonly known in the art. If parenteral injection is selected as a method for administering the ribozyme-siRNA molecules, steps must be taken to ensure that sufficient amounts of the molecules reach their target cells to exert a biological effect.
  • the lipophilicity of the ribozyme-siRNA molecules, or the pharmaceutical preparation in which they are delivered may have to be increased so that the molecules can arrive at their target locations.
  • the ribozyme- siRNA molecules may have to be delivered in a cell- targeted carrier so that sufficient numbers of molecules will reach the target cells.
  • Methods for increasing the lipophilicity of a molecule are known in the art, and include the addition of lipophilic groups to the ribozyme-siRNA molecules.
  • ribozyme-siRNA molecules and vectors encoding the same may be administered to cells ex vivo and, optionally, the cells may be returned to the patient.
  • Ribozyme-siRNA molecules of the present invention may be encapsulated in a lipophilic, targeted carrier, such as a liposo e.
  • a lipophilic, targeted carrier such as a liposo e.
  • One technique is to use as a carrier for the oligonucleotide a liposomal preparation containing the cationic lipid N-[l-(2,3- dioleyloxy)propyl] -N,N,N-trimethyl ammonium chloride (D OT MA; lipofectin) .
  • a pharmaceutical preparation of the invention may be formulated in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to a physically discrete unit of the pharmaceutical preparation appropriate for the patient undergoing treatment .
  • Each dosage should contain a quantity of active ingredient calculated to produce the desired effect in association with the selected pharmaceutical carrier.
  • Dosage units may be proportionately increased or decreased based on the weight of the patient .
  • Appropriate concentrations for alleviation of a particular pathological condition may be determined by dosage concentration curve calculations, as known in the art.
  • the appropriate dosage unit for the administration of ribozyme-siRNA molecules may be determined by evaluating the toxicity of the ribozyme-siRNA molecules in animal models.
  • concentrations of ribozyme-siRNA pharmaceutical preparations may be administered to mice and the minimal and maximal dosages may be determined based on comparing obtaining desired results as opposed to side effects as a result of the treatment.
  • the pharmaceutical preparation comprising the ribozyme-siRNA molecules may be administered at appropriate intervals, for example, twice a day until the pathological symptoms are reduced or alleviated, after which the dosage may be reduced to a maintenance level .
  • the appropriate interval in a particular case would normally depend on the condition of the patient. While the above discussion refers to the delivery of ribozyme-siRNA molecules, it will be apparent to those skilled in the art that the methods described would also be suitable for the delivery of the vector constructs encoding ribozyme-siRNA molecules. If an allosteric ribozyme is employed in the ribozyme-siRNA construct, the effector may be administered to the patient to modulate the production of the siRNA as desired.
  • the effector may be delivered to the patient systemically or locally and may be delivered by any suitable method known in the art, such as certain of the methods provided hereinabove.
  • the effector may be administered as a mature compound or, where applicable, as a vector containing a sequence encoding for the effector wherein the expression of the encoding sequence may also be regulated.
  • kits Comprising Ribozyme-siRNA Molecules
  • the present invention also encompasses kits for use in effecting the controlled production of an siRNA of interest.
  • kits comprise a recombinant vector containing a nucleic acid sequence encoding a ribozyme operably linked to a promoter suitable for expression in a desired host cell or in vi tro transcription and a multiple cloning site suitable for cloning a nucleic acid encoding a strand of the siRNA of interest so that the cleavage activity of the ribozyme produces the strand of the siRNA.
  • the promoter is preferably a strong promoter and may be constitutive or regulated.
  • RNA polymerase II promoters include, but are not limited to, RNA polymerase II promoters, the T7 RNA polymerase promoter, and the RNA polymerase III promoters U6 and Hi.
  • the ribozyme is preferably an allosteric ribozyme, such as the theophylline regulated HDV ribozyme described hereinbelow.
  • the kit may contain more than one recombinant vector wherein the vectors comprise different ribozymes and/or different multiple cloning sites.
  • the recombinant vectors may be provided in any suitable buffer.
  • kits may further comprise a composition comprising an effector capable of modulating the activity of the allosteric ribozyme, buffers suitable for the cleavage of the ribozyme-siRNA molecule, frozen stocks of host cells, and instruction material.
  • the kit may also further comprise at least one reagent suitable for the in vi tro transcription of the recombinant vector.
  • kits may also further comprise at least one reagent suitable for introducing the recombinant vector or transcribed R ⁇ A product into test samples, cells, or subjects.
  • an "instructional material" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the composition of the invention for performing a method of the invention.
  • the instructional material of the kit of the invention can, for example, be affixed to a container which contains a kit of the invention to be shipped together with a container which contains the kit.
  • the instructional material can be shipped separately from the container with the intention that the instructional material and kit be used cooperatively by the recipient.
  • EXAMPLE 1 Ribozyme-Mediated siR ⁇ A Production Materials and Methods DNA templates for in vitro transcription of ribozyme-siRNA constructs were prepared from synthetic oligonucleotides by PCR amplification using templates and primers that correspond to the sequences shown in Fig. 1.
  • the primer for each coding strand additionally contained the promoter sequence 5 ' -TAATACGACTCACTATA-3 ' (SEQ ID NO: 22) for T7 RNA polymerase (T7 RNAP) .
  • the siRNA is generated by the self-cleavage activity of two HDV ribozymes. Each ribozyme contains either the sense (S) or antisense (A) strand of the siRNA as its 5 ' -cleavage product.
  • HDV ribozymes S w and A wt possess wild-type self-cleavage activity.
  • the modified HDV ribozyme sequence corresponds to that of the genomic HDV ribozyme containing an abbreviated P4 stem-loop sequence (boxed) and lacking nucleotides 41-43 ( ⁇ CAA) which are not requisite for self-cleavage activity (Fig. 1; Wadkins, T.S. and M.D. Been (1997) Nucleic Acids Res. 25:4085-4092).
  • Ribozymes S mut and A mut contain a C75U point mutation (encircled) that obviates self-cleavage activity (Fig. 1; Nakano, S.I., et al . (2000) Science.
  • Ribozymes S theo and A theo contain a theophylline-binding domain and communication module sequence (cm + theo6) in place of P4 (boxed) and exhibit theophylline-dependent self-cleavage activity (Fig. 1; Soukup, G.A. and R.R. Breaker (2000) Curr. Opin. Struct. Biol. 10:318-325).
  • the siRNA sequences appended to each ribozyme construct are designed to target EGFP mRNA at sights beginning 109 and 237 nt downstream of the initiation codon (Fig. 1; siRNA-1 and siRNA-2, respectively) .
  • DNA templates for in vi tro transcription of short hairpin RNAs were prepared by annealing complementary synthetic oligonucleotides that contain the T7 RNAP promoter sequence and correspond to the shRNA sequences shown in Fig. 4A.
  • DNA template for dsRNA that represents nucleotides 12-328 of the open reading frame for EGFP was prepared by PCR amplification using pdlEGFP-Nl (BD Biosciences; Franklin Lakes, NJ) with primers 5 ' -TAATACGACTCACTATAGGGCGAGGAGCTGTTCAC-3 ' (SEQ ID NO: 23) and 5 ' -TAATACGACTCACTATAGGGTCTTGTAGTTGCCGTC-3 ' (SEQ ID NO: 24), where each primer contains the T7 RNAP promoter sequence.
  • Radiolabeled RNAs were transcribed in vi tro from DNA templates using T7 RNAP and [c.- 32 P]-UTP (Amersham; Piscataway, NJ) .
  • transcription reactions (25 ⁇ l) contained DNA template(s), 50 mM Tris-HCl (pH 7.5 @23°C), 15 mM MgCl 2 , 5 mM DTT, 2 mM spermidine, 1 mM each NTP, 6.25 p ol [ ⁇ - 32 P] -UTP ( ⁇ 2.5 ⁇ Ci), and -500 U T7 RNAP. Following incubation at 37°C for 2 hours, reactions were equally divided and products were promptly separated by denaturing (8 M urea) or native 10% polyacrylamide gel electrophoresis (PAGE) .
  • RNAs were prepared by transcription in appropriately scaled reactions (50 ⁇ l) that contained 25 pmol [o.- 32 P]-UTP (-_ ⁇ 10 ⁇ Cl) .
  • RNAs were purified by PAGE, eluted, precipitated with ethanol, and dissolved in water prior to subsequent manipulation.
  • Precursor ribozyme-siRNA transcripts were isolated by denaturing 10% PAGE.
  • the dsRNA was generated by annealing transcripts isolated by denaturing 6% PAGE.
  • shRNAs were isolated by native 10% PAGE.
  • Allosteric ribozyme-siRNA constructs were assayed by incubating purified ribozyme (s) in the absence or presence of 200 ⁇ M theophylline in solution containing 50 mM Tris-HCl (pH 7.5 at 23°C) , 25 mM KC1, and 5 mM MgCl 2 . Following incubation at 37°C for 2 hours, reactions were equally divided and products were promptly separated by denaturing and native 10% PAGE. Recombinant Dicer reactions were performed according to manufacturer specifications (Gene Therapy Systems; San Diego, CA) . RNAs were incubated in the absence or presence of 2 U Dicer in 20 ⁇ L reactions for approximately 15 hours at 37°C. Samples were equally divided and products were promptly separated by denaturing and native 10% PAGE.
  • the wild-type sense (S wt ) ribozyme- siRNA-1 construct (SEQ ID NO : 1) is comprised of a modified HDV ribozyme sequence (from the G nucleotide 3' of the arrowhead through the 3' end of the sequence; SEQ ID NO: 3) and the sense strand of siRNA-1 (from the 5' end through the U nucleotide 5 ' of the arrowhead; SEQ ID NO: 4) .
  • the wild-type antisense (A wt ) ribozyme-siRNA-1 construct (SEQ ID NO: 2) comprises the same modified HDV ribozyme sequence (SEQ ID NO: 3) and the antisense strand of siRNA-1 (from the 5' end through the U nucleotide 5' of the arrowhead; SEQ ID NO : 5) .
  • the mutant sense (S mut ) ribozyme-siRNA-1 construct (SEQ ID NO: 6) and the mutant antisense (A mut ) ribozyme-siRNA-1 construct (SEQ ID NO: 7) contain a C to U point mutation at position 75 of the wild-type ribozyme (see Fig.
  • siRNA i.e. duplex
  • Fig. 2 bands X, Y, and Z
  • spontaneous formation of the siRNA is evident only by co-transcription of S and A wt (Fig. 2B; lane 5) .
  • Fig. 1 the wild-type sense (S t ) ribozyme-siRNA-2 construct (SEQ ID NO: 8) comprises the modified HDV ribozyme sequence (SEQ ID NO: 3) and the sense strand of siRNA-2 (SEQ ID NO: 10) .
  • the wild-type antisense (A t ) ribozyme-siRNA-2 construct comprises the modified HDV ribozyme sequence (SEQ ID NO: 3) and the antisense strand of siRNA-2 (SEQ ID NO: 11) .
  • the mutant sense (S mut ) ribozyme-siRNA-2 construct (SEQ ID NO: 12) and the mutant antisense (A mut ) ribozyme-siRNA-2 construct (SEQ ID NO: 13) contain the C to U point position at position 75 which obviates their self-cleavage activity.
  • the ribozyme-siRNA-2 constructs were analyzed by denaturing and native PAGE of total radiolabeled transcription products (Figs. 2C and 2D). Denaturing PAGE reveals that S t and A wt are observed to produce siRNA strands by self-cleavage (Fig. 2C; lanes 1, 3, and 5-7). Furthermore, native PAGE demonstrates that the siRNA strands spontaneously associate to form the siRNA when co-transcribed (Fig. 2D; lane 5) . Transcription of S wt alone is observed to produce a species whose mobility is similar to the siRNA by native PAGE (Fig. 2D; lane 1) .
  • HDV ribozyme-mediated production of siRNA-2 is similar to, if not slightly more efficient, than siRNA-1 production, thereby demonstrating ribozyme function that is independent of siRNA sequence.
  • the ability of allosteric HDV ribozymes to regulate the production of siRNAs in a ligand-dependent manner was examined by reacting or co-reacting purified ribozymes in the absence or presence of the effector theophylline.
  • the sense theophylline ribozyme-siRNA-1 construct (S theo ; SEQ ID NO: 14) comprises the sense strand of siRNA-1 (SEQ ID NO: 4) and an theophylline responsive HDV ribozyme (SEQ ID NO: 25) wherein the P4 stem-loop sequence (SEQ ID NO: 15) of the "wild-type" modified HDV ribozyme (SEQ ID NO: 3) is replaced by a sequence comprising a theophylline- binding domain and communication module (SEQ ID NO: 16) .
  • the antisense theophylline ribozyme-siRNA-1 construct (A the0 ; SEQ ID NO: 17) comprises the same theophylline responsive HDV ribozyme and the antisense strand of siRNA-1.
  • S theo and A the ° constructs were also made comprising the sense and antisense strands of siRNA-2, respectively (SEQ ID NOs : 18 and 19, respectively).
  • Analysis of ribozyme cleavage products by denaturing PAGE demonstrates that S theo and A theo produce the corresponding siRNA-1 strands in a theophylline-dependent manner either alone (Fig. 3A; lanes 1-4) or in combination (Fig. 3A; lanes 5 and 6) .
  • Fig. 3B shows that siRNA-1 is formed in a theophylline- dependent manner only when s the0 and A theo are reacted in combination (Fig. 3C; lanes 5 and 6) .
  • Theophylline- dependent HDV ribozymes producing siRNA-2 strands exhibit similar ligand-dependent self-cleavage activity and siRNA production by both denaturing and native PAGE (Figs. 3B and 3C, lanes 7-12).
  • Figs. 3B and 3C shows that ligand-dependent self-cleavage activity and siRNA formation appear to be slightly more efficient for ribozymes producing siRNA-2 relative to those producing siRNA-1.
  • the utility of allosteric ribozymes to regulate siRNA production and control R ⁇ Ai in a ligand-dependent manner may depend, in part, upon the ability of the precursor ribozyme-siRNAs and their resulting complexes to avert premature siR ⁇ A release and evade processing by the double-strand-specific ribonuclease Dicer.
  • precursor ribozyme-siR ⁇ A complexes are substrates for recombinant Dicer in vi tro, the products of extensive Dicer reactions were examined by both denaturing and native PAGE.
  • a 317-bp (nucleotides 12-328 of the open reading frame encoding EGFP) segment of double-stranded R ⁇ A and short hairpin R ⁇ As (shR ⁇ As) that correspond to siR ⁇ A-1 and siRNA-2 were utilized (shRNA-1 (SEQ ID NO: 20) and shRNA-2 (SEQ ID NO: 21), respectively; Fig. 4A) .
  • shRNA-1 SEQ ID NO: 20
  • shRNA-2 SEQ ID NO: 21
  • Fig. 4A denaturing and native PAGE demonstrates that double-stranded RNA is degraded by Dicer to form siRNAs comprised of ⁇ 21-nt fragments (Figs. 4B and 4C, lanes 1 and 2) .
  • recombinant Dicer is observed to process both shRNA-1 and shRNA-2 in vi tro to form siRNAs comprised of ⁇ 21-nt fragments (Figs. 4B and 4C, lanes 3-6).
  • inactive precursor ribozyme-siRNA complexes comprised of A mut and S mut are poor substrates for Dicer (Figs. 4B and 4C, lanes 7-10) although the ribozymes appear to associate as evidenced by the appearance of higher molecular weight species on the native gel (Fig.

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Abstract

L'invention concerne des compositions et des procédés de production régulée de petits ARN interférents (ARNsi) et des procédés d'utilisation de ceux-ci.
PCT/US2004/017034 2003-05-29 2004-05-28 Production de petits arn interferents (arnsi) regulee par les ribozymes et procedes d'utilisation de ceux-ci WO2005001039A2 (fr)

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US8367815B2 (en) 2007-08-28 2013-02-05 California Institute Of Technology Modular polynucleotides for ligand-controlled regulatory systems
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US9029524B2 (en) 2007-12-10 2015-05-12 California Institute Of Technology Signal activated RNA interference
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JP2010522572A (ja) * 2008-03-27 2010-07-08 インダストリー−アカデミック コーオペレーション ファウンデーション、ダンコック ユニバーシティー テオフィリンによって標的特異的ｒｎａ置換活性が調節されるアロステリックトランス−スプライシンググループｉリボザイム
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US9145555B2 (en) 2009-04-02 2015-09-29 California Institute Of Technology Integrated—ligand-responsive microRNAs
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EP4019636A1 (fr) * 2011-12-21 2022-06-29 Apse Llc Procédés d'utilisation de vlp avec des capsides résistants aux hydrolases
US20160177299A1 (en) * 2013-06-19 2016-06-23 Apse Llc Compositions and methods using capsids resistant to hydrolases
US10428329B2 (en) * 2013-06-19 2019-10-01 Apse, Inc. Compositions and methods using capsids resistant to hydrolases
US9822361B2 (en) * 2013-06-19 2017-11-21 Apse, Inc. Compositions and methods using capsids resistant to hydrolases
WO2018237372A1 (fr) * 2017-06-23 2018-12-27 Cornell University Molécules d'arn, procédés de production d'arn circulaire, et procédés de traitement
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