WO2005056750A2 - Inversion-duplication d'acides nucleiques et bibliotheques preparees de cette maniere - Google Patents

Inversion-duplication d'acides nucleiques et bibliotheques preparees de cette maniere Download PDF

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WO2005056750A2
WO2005056750A2 PCT/IL2004/001114 IL2004001114W WO2005056750A2 WO 2005056750 A2 WO2005056750 A2 WO 2005056750A2 IL 2004001114 W IL2004001114 W IL 2004001114W WO 2005056750 A2 WO2005056750 A2 WO 2005056750A2
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nucleic acid
stranded
vector
double
ligating
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PCT/IL2004/001114
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Elena Feinstein
Igor Mett
Michael Shtutman
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Quark Biotech, Inc.
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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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • 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
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • 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
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    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/12—Applications; Uses in screening processes in functional genomics, i.e. for the determination of gene function
    • C—CHEMISTRY; METALLURGY
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    • C12N2330/00—Production
    • C12N2330/30—Production chemically synthesised

Definitions

  • the present invention relates to the fields of molecular biology and genetic engineering. More specifically, the invention relates to the field of nucleic acid technology and RNA interference. BACKGROUND OF THE INVENTION
  • Novel high throughput techniques in molecular biology like genome sequencing and microarray expression analysis, provide predominantly structural and correlative expression data, respectively. As such, they do not directly support identification of targets for development of drugs. This dictated a need for creation of high throughput methods specifically aimed at the solution of the problem of target identification in a reliable, efficient, robust, fast and relatively inexpensive manner.
  • GSE genetic suppressor elements
  • a GSE library is delivered into target cells, which are then selected on the basis of development of the desired phenotype by virtue of the GSE expression.
  • Co-assigned US Patent application No. 10/620193 describes a technology platform that utilizes microarrays for identification of functional library clones by evaluating the differences in their redundancy in the cells before and after the phenotypic selection. This method of detection not only ensures the usage of more mild selection procedures but also enables identification of library elements that are either enriched or depleted in the course of phenotypic selection thus supporting both positive and negative selections.
  • GSE libraries are comprised of randomly fragmented cDNA molecules subcloned into expression vectors in random orientation. Such library design is compatible with the above detection platform only in the case of positive selection when library clones enriched during phenotypic selection are sought. However, when negative selection is pursued, GSE library design becomes incompatible with the above methodology since partially overlapping or differently oriented inactive library clones may mask the disappearance of active GSEs.
  • PCT/IL 2004/000515 discloses a novel method of library preparation, also termed OFCEL, specifically compatible with the above-described microarray-based approach for detection of functional clones and, thus, biologically important genes.
  • RNAi RNA interference is a phenomenon describing double-stranded (ds) RNA-dependent gene specific posttranscriptional silencing. Originally, attempts to study this phenomenon and to manipulate mammalian cells experimentally were frustrated by an active, non-specific antiviral defense mechanism which was activated in response to long dsRNA molecules; see Gil et al. 2000,Apoptosis, 5:107-1 14. Later it was discovered that synthetic duplexes of 21 nucleotide RNAs could mediate gene specific RNAi in mammalian cells, without the stimulation of the generic antiviral defense mechanisms. Elbashir et al. Nature 2001, 411 :494- 498; Caplen et al.
  • RNA interference refers to the process of sequence-specific post transcriptional gene silencing.
  • a short primer on RNAi RNA- directed RNA polymerase acts as a key catalyst.
  • RNAi has emerged as one of the most efficient methods for inactivation of genes (Nature Reviews, 2002, v.3, p.737-47; Nature, 2002, v.418,p.244-51). As a method, it is based on the ability of dsRNA species to enter a specific protein complex, where it is then targeted to the complementary cellular RNA and specifically degrades it.
  • dsRNAs are digested into short (17-29 bp) dsRNA fragments (also referred to as short inhibitory RNAs - "siRNAs”) by type III RNAses (DICER, Drosha, etc) (Nature, 2001 , v.409, p.363-6 ; Nature, 2003, .425, p.415-9). These fragments and complementary mRNA are recognized by specific RISC protein complex. The whole process is culminated by endonuclease cleavage of target mRNA (Nature Reviews, 2002, v.3, p.737-47; Curr Opin Mol Ther. 2003 Jun;5(3):217-24).
  • DIER type III RNAses
  • RNA for gene inactivation Some of the advantages of the use of double-stranded RNA for gene inactivation are: • High specificity (Proc Natl Acad Sci U S A. 2003 May 27;100(l l):6347-52; Proc Natl Acad Sci U S A. 2003 May 27;100(11):6289-91). • High efficiency of gene inactivation (even compared to the antisense RNA with the same sequence (Mol. Cell, 2000, v.6, 1077-87; Cell, 2003, v.l 15, 199-208) • Robustness (high frequency of occurrence of biologically active dsRNA fragments vs low frequency of occurrence of biologically active antisense RNA fragments derived from the same cDNA) (Mol. Cell, 2000, v.6, 1077-87)
  • RNAi RNAi-binding protein
  • vector systems have been developed for expressing long and short (17-29 bp) double strand RNA in different organisms from plant to mammals (Nature, 2001,v.411, 494-8; Genes & Dev, 2002, v.16, 948-58).
  • Expression of double-stranded RNA molecules is achieved by introduction of gene-specific inverted repeats under the control of the appropriate promoter (either polIII for short 19-29 bp fragments or polll for long, -500 bp, fragments), which upon expression generates stem-and-loop (hairpin) RNA molecules.
  • RNAi libraries pre- chosen short inhibitory RNAs
  • Preparation of such libraries is expensive and time- consuming since several different siRNAs must be synthesized and tested for their gene inhibitory activity.
  • Another limitation of this approach is that the libraries will fit only known genes and known splice variants.
  • preparation of a pre-chosen RNAi library, which is tissue- or condition-specific, is also problematic.
  • RNA libraries are cell/selection-specific and have the potential to substitute currently employed gene-inhibiting libraries.
  • ⁇ Possibility of significant reduction of library complexity due to high efficiency and robustness of gene inhibitory effects.
  • ⁇ Possibility of more efficient use of microarray-based identification of phenotypically selected library elements supported by: o independence of the dsRNA activity form direction of transcription o possibility to use non-overlapping fragments (reduction of complexity without compromise in efficiency)
  • RNAi expressing libraries include: o Natural cellular non-specific response to double-stranded RNA; o Establishment of a technique enabling efficient generation of double-stranded RNAs in a library-wise manner (one per cell).
  • the subject of the present invention is a process enabling generation of inverted repeat structure mediating the expression of double stranded hairpin RNA species from a cDNA population of any complexity.
  • the inventors of the instant application have discovered processes for preparing plasmid or viral vectors, which encode dsRNA molecules to be employed in RNAi. These vectors and the processes for preparing them are useful in inhibiting the biological activity of polypeptide- encoding genes, and may be used for the preparation of expression libraries, which can serve as a tool in drug target identification and drug screening.
  • the present invention provides processes for preparing plasmid or viral vectors, which encode long dsRNA molecules.
  • the present invention provides kits for performing these methods.
  • the present invention provides vectors, including expression vectors prepared by these processes and libraries comprising a plurality of such vectors.
  • a preferred embodiment of the present invention concerns methodology and processes for duplication-inversion of any double-stranded DNA fragment, such as a cDNA fragment, existing in a mixture of any complexity.
  • the resulting double-stranded cDNA fragment contains the original cDNA fragment, a spacer sequence and the original cDNA fragment positioned in the reverse orientation.
  • RNA expressed from such a structure forms a hairpin (stem-and-loop) structure.
  • Such long double-stranded RNA is processed by intracellular complexes to produce siRNAs thus potentially conferring effective dsRNA-mediated gene silencing (RNAi).
  • exemplary processes of the present invention are: 1 ) Ligation of single-stranded stem-and-loop adaptors to both ends of a DNA fragment. 2) These adaptors may contain pBR and fl origins of replication (one of the adaptors) and a drug resistance marker (another adaptor). 3) Following ligation, the resultant circular single-stranded plasmid contains a given DNA fragment duplicated and positioned both in forward and in reverse (inverted) orientation. 4) Following second strand synthesis and propagation by means of bacteria, double- stranded plasmid DNA is digested to produce a library of inverted repeats separated by a sequence spacer that contains a drug resistance marker to facilitate subsequent re- cloning. 5) This library may be further re-cloned under the control of a eukaryotic (e.g. mammalian) promoter into an appropriate expression vector.
  • a eukaryotic e.g. mammalian
  • Step 1 Preparation of c DNA fragments containing asymmetrical non-palindromic protruding ends.
  • any individual double-stranded-cDNA fragment or any mixture of double-stranded-cDNA fragments (a library), having either "sticky” (protruding) termini (ends) (produced by digestion with restriction enzymes) or “blunt” (non protruding) termini (produced by digestion with restriction enzymes or with DNase I) are ligated to an excess of assymmetrical non-palindromic adaptors, designed to be compatible for ligation with given cDNA fragments, thus providing them with suitable protruding termini.
  • the use of asymmetrical, non-palindromic protruding ends is important for blocking the possibility of self-ligation of double-stranded-cDNA fragments and adaptors (see below).
  • Step 2 Ligation of single-stranded DNA stem-and-loop adaptors to double-stranded DNA molecules for generation of circular single-stranded DNA molecules suitable for further replication in appropriate bacteria strains.
  • One of the adaptors e.g. the left adaptor (L- adaptor) contains an origin of replication such as a pUC origin of replication (Ori), whereas the other adaptor (the right adaptor, R-adaptor) contains a resistance gene such as the ⁇ - lactamase gene and promoter (or the reverse).
  • Each of these sequences is flanked at both sides by a self-complementary region ranging from 10-500 bp, optionally being approximately 50bp, to provide double-stranded stems and compatible "sticky" (overlapping / protruding) ends for double-stranded DNA ligation.
  • the stem regions of the adaptors also contain recognition sites for one or more restriction enzymes to be used in subsequent steps of the procedure.
  • the resulting circular DNA molecule appears to contain both single stranded (pUC origin and ⁇ -lactamase) and double stranded (cDNA fragment itself and self annealed sequences of the adaptors) regions.
  • Step 3 Second strand synthesis.
  • the second strand synthesis is primed with an oligonucleotide complementary to the single stranded region of the DNA molecule obtained during the previous step.
  • the synthesis is performed using commercially available DNA polymerase enzymes with strand displacement capacity.
  • the resulting DNA strand is covalently closed in a circular form using DNA ligase.
  • the resultant double-stranded DNA molecules actually have all the features of plasmid vector DNA (origin of replication and drug resistance gene) and also contain two inverted copies of the initial cDNA fragment separated by the plasmid sequences.
  • Step 4 Recloning of inverted repeats into expression vectors.
  • An appropriate bacteria optionally E.coli
  • E.coli E.coli
  • Restriction endonuclease recognition sites optionally Not I or any other restriction endonuclease site
  • flank the inverted DNA structure are suitable for subsequent re-cloning of the inverted repeats cassette into eukaryotic expression vectors.
  • the present invention provides for a process for production of a vector encoding a double-stranded RNA molecule comprising: a) producing a nucleic acid comprising asymmetrical non-palindromic protruding ends; b) ligating a stem-and-loop single-stranded DNA adaptor to each end of the nucleic acid , to produce a circular single stranded nucleic acid molecule; and c) synthesizing a second strand and ligating its ends, to produce a double stranded nucleic acid plasmid vector.
  • the process may further comprise: d) amplifying the vector by introducing the vector into a bacterium and propagating the bacterium.
  • the process may further comprise: d) amplifying the vector by introducing the vector into a bacterium and propagating the bacterium; and e) re-cloning a portion of the vector into a eukaryotic expression vector
  • the portion of the vector to be re-cloned comprises the inverted repeats of the original nucleic acid, and thus comprises a DNA fragment derived from the vector created in step c) which contains the original nucleic acid of step a), an inverted copy of said nucleic acid of step a), and optionally one of the adaptors of step b) which preferably contains a drug resistance marker gene.
  • the portion of the vector re-cloned in step e) may comprise an inverted duplicated original nucleic acid of step a) whereby the duplicate copies of the original nucleic acid of step a) are separated by a bacterial drug resistance gene.
  • the eukaryotic expression vector employed in step e) may optionally contain a polymerase II promoter.
  • the nucleic acids of step a) may be produced by digestion with a restriction enzyme producing protruding termini, which may be asymmetric, and subsequent ligation of non-palindromic adapters which may be asymmetrical; random digestion and/or physical shearing and subsequent adaptor ligation are also a possibility.
  • One of the adaptors of step b) contains an origin of replication (ori), which may be a bacterial (such as pUC) or phage ori, or both; the other adaptor may contain a drug resistance gene and or a reporter gene (and appropriate promoter(s)) (such as, inter alia, the ⁇ -lactamase gene and promotor or any other drug or antibiotic (such as, inter alia, cycloserin, fosfomycin, glycopeptides, beta-lactams, isoniazid, metronidazole, aminoglycosides, tetracyclines, ampycillin, kanamycin, zeocin, chloramphenicol, macrolides, fusidic acid, lincosamides, mupirocin, linezolid, Rifampicin, Nitrofurans, Sulphonamides, Trimethoprim, etc) resistance gene, preferably suitable for the use with bacteria.
  • ori origin of replication
  • each of the adaptors may be flanked at both sides by a self-complementary region of 10-500 bp, and may comprise recognition sites for several restriction enzymes.
  • the length of the self-complementary region is preferably about 20-250 bp, more preferably about 20-100 bp, even more preferably about 30-80 bp, most preferably about 50 bp.
  • the basic feature of the adaptors which may be used with the processes of the present invention is that one of them contains a bacterial origin of replication (e.g. OriP) and optionally also a filamentous phage origin of replication (e.g, fl) and the other one -a gene conferring drug resistance.
  • a bacterial origin of replication e.g. OriP
  • a filamentous phage origin of replication e.g, fl
  • the adaptors convert it into a single-stranded replication form of phagemid that may then be converted into a double-stranded form containing the original cDNA fragment in the form of inverted repeats.
  • Any adaptor design that provides a bacterial and optionally a phage origin of replication in one adaptor and a selectable marker (e.g. gene conferring drug resistance) in another adaptor may be used with the processes of the present invention; all other sequences may be variable.
  • the structure of the adaptors used with some of the processes of the present invention enable them to be ligated to the required nucleic acids and thus, enable the re-cloning of stem-and-loop structured duplicated-inverted forms of said nucleic acid into a eukaryotic expression vector.
  • One particular non-limiting aspect of the above processes may comprise: a) producing a nucleic acid comprising asymmetrical non-palindromic protruding ends; b) ligating a stem-and-loop single-stranded DNA adaptor to each end of the nucleic acid , to produce a circular single stranded nucleic acid molecule, wherein one of the adaptors contains an FI origin of replication; c) synthesizing a second strand and ligating its ends, to produce a double stranded nucleic acid plasmid vector; d) amplifying the plasmid vector by introducing it into a bacterium and propagating the bacterium; e) re-cloning a portion of the vector into a eukaryotic expression vector; and f) amplifying the single-stranded replication form 1 (RF1) of the plasmid vector of step c) by propagation in a suitable bacterial strain in the presence of a helper phage.
  • the adaptors of step b) may be prepared by their excision, using restriction enzymes, from a self-annealed single-stranded plasmid of step f).
  • origin of replication (borne on one adaptor) and marker gene and promoter (borne on the other adaptor) combination comprises the pUC origin of replication and the beta-lactamase gene and promoter; further, the adaptor which contains the pUC origin of replication may in addition contain an fl origin of replication (which may be derived from the filamentous phage Ml 3).
  • the processes of the present invention may be used to generate a library of vectors which encode double-stranded RNA molecules.
  • the process is the same as described above, and may be performed simultaneously on a population of nucleic acids.
  • the present invention also provides for a process for production of a plurality of vectors encoding double-stranded RNA molecules comprising: a) producing a population of nucleic acids comprising asymmetrical non-palindromic protruding ends; b) ligating a stem and loop single-stranded DNA adaptor to each end of each nucleic acid contained in the population of step a), to produce a circular single stranded nucleic acid molecule; and c) synthesizing a second strand and ligating its ends, to produce double stranded nucleic acid vectors.
  • the process may further comprise: d) amplifying each vector by introducing the vactor into a bacterium and propagating the bacterium.
  • the process may further comprise: e) re-cloning a portion of each vector into a eukaryotic expression vector.
  • All of the processes described herein may also be applied to a population of nucleic acids and result in a population of vectors which encode a plurality of double-stranded RNA molecules which can mediate RNA interference, and/or a population of expression vectors which can express said RNA molecules.
  • an additional important aspect of the present invention concerns the vectors which are produced by the processes disclosed herein.
  • the present invention provides for a vector produced according to any of said processes, which may be an expression vector.
  • An additional embodiment of this aspect provides an expression vector which encodes an RNA molecule that can mediate RNA interference, said RNA molecule preferably being a stem-and-loop double-stranded RNA molecule.
  • an expression vector produced by the processes disclosed herein comprising an inverted duplicated nucleic acid whereby the inverted duplicate copies of said nucleic acid are separated by an origin of replication and a drug resistance gene is also provided; in some embodiments, the origin of replication and the drug resistance gene are derived from bacteria.
  • Any vector prepared according to the processes of the present invention is also considered to be a part of the present invention, as is any library comprising a plurality of vectors prepared by any one of the production methods disclosed herein.
  • the adaptors used in conjunction with the processes of the present invention may be prepared in several ways. Firstly, they may be prepared by the following process, which is also considered part of the present invention: a) producing a nucleic acid comprising non-palindromic protruding ends; b) ligating a stem-and-loop single-stranded DNA adaptor to each end of the nucleic acid , to produce a circular single stranded nucleic acid molecule, wherein one of the adaptors contains an FI origin of replication and each of the adaptors is flanked at both sides by a self complimentary region which contains one or more restriction enzyme recognition sites; c) amplifying the circular single-stranded nucleic acid molecule in an appropriate bacterial strain infected with a helper phage; and d) excising the adaptor region of the single-stranded nucleic acid molecule using restriction enzymes which recognize sites within the self-complimentary region of step b).
  • an additional embodiment of the present invention provides a method of generation of stem-and-loop adaptors by restriction enzyme excision following amplification of the replication form 1 (RF1) of a circular single stranded nucleic acid molecule produced by the processes disclosed herein.
  • RF1 replication form 1
  • the adaptors may be produced using single-stranded PCR techniques known to one of skill in the art, or any PCR strand separation technique known in the art. Adaptors may also be chemically synthesized by any of the methods which are well-known in the art for synthesis nucleic acids. For example, a commercially available machine (available, inter alia, from Applied Biosystems) can be used.
  • the expression vectors used in conjunction with the processes of the present invention may be selected from: a) A retroviral expression vector comprising a Polymeraselll expression cassette for transcription of adenoviral VAI-VAII sequences within its 3' LTR and a kanamycine drug resistance gene. b) An episomal expression vector comprising a Polymeraselll expression cassette for transcription of adenoviral VAI-VAII sequences within its 3' LTR and a kanamycine drug resistance gene and absent polyadenylation sequences.
  • kits for performing the processes of the present invention comprising sets of stem and loop adaptors, one adaptor comprising one or more origin of replication (such as bacterial pUCor phage fl), and the other adaptor comprising a drug resistance gene and/or a reporter gene (and appropriate promoter(s)).
  • the kit may further comprise one or more kineted primers (for second strand synthesis).
  • the kit may further comprise sets of asymmetrical non-palindromic adaptors. Additionally, the kit may further comprise one or more restriction enzymes, which leave a protruding termini corresponding to the protruding termini present in the stem and loop adaptors and/ or the asymmetrical non- palindromic adaptors.
  • the kit may additionally comprise appropriate ligase and polymerase enzymes.
  • the kit may additionally comprise a eukaryotic expression vector (for re-cloning of the resultant cDNA molecule or library of inverted repeats).
  • ligase enzymes include T4 DNA ligase, T4 RNA ligase, Taq DNA ligase, E.Coli DNA ligase, Pfu DNA ligase and Tth DNA ligase, wter alia.
  • polymerase enzymes include Taq DNA polymerase, Vent DNA polymerase, Deep vent DNA polymerase, Pfu DNA polymerase, Bst DNA polymerase and Tth DNA polymerase, inter alia. Protocols for performing ligation and amplification reactions are well known in the art and can be modified as desired by the skilled artisan.
  • nucleic acid as used herein encompasses "polynucleotide” and “oligonucleotide”, and refers to any molecule composed of DNA nucleotides, RNA nucleotides or a combination of both types, i.e. that comprises two or more of the bases guanidine, cytosine, thymidine, adenine, uracil or inosine, inter alia.
  • a nucleic acid may include natural nucleotides, chemically modified nucleotides and synthetic nucleotides, or chemical analogs thereof.
  • a polynucleotide generally has from about 75 to 10,000 nucleotides, optionally from about 100 to 3,500 nucleotides.
  • An oligonucleotide also termed “oligo” for short, refers generally to a chain of nucleotides extending from 2-75 nucleotides.
  • expression vector refers to vectors that have the ability to incorporate and express heterologous nucleic acid fragments in a foreign cell. Many prokaryotic and eukaryotic expression vectors are known and/or commercially available. Selection of appropriate expression vectors is within the knowledge of those having skill in the art.
  • the expression vector used for eukaryotic expression of stem-and-loop dsRNA preferably contains features preventing non-specific cellular response to double-stranded RNA, such as those described in Genes Dev. 2003 Jun 1; 17(11): 1340-5 or J Biol Chem. 1996 Jan 19;271(3): 1756-63 or both.
  • the expression vector used for eukaryotic expression of stem-and-loop double-stranded RNA is an adenoviral vector or other episomal vector.
  • library in the context of the present invention, is meant a set of at least 5 vectors comprising nucleic acids that differ from each other. Libraries can include thousands, tens of thousands, hundreds of thousands, millions and even tens of millions of different elements.
  • RNA interference RNA interference
  • Figure 1 details an exemplary embodiment of the present invention: a general process of inversion-duplication of cDNA fragments via annealing of single-stranded stem-and-loop adaptors to double-stranded DNA fragments resulting in the generation of an expression library of inverted repeats.
  • Figure 2 shows PCR fragments used in the assembly of pRNAiL.
  • Figure 3 details an exemplary assembly process of pRNAiL.
  • Figure 4 shows the results of a verification experiment conducted by digestion of pRNAiL with diagnostic restriction endonucleases.
  • Figure 5 details an exemplary process of generation of single-stranded stem-and-loop adaptors from ssRNAiL.
  • Figure 6 shows the verification of generation of single-stranded stem-and-loop adaptors from ssRNAiL.
  • Figure 7 details the process of an experiment generating an exemplary model library of inverted repeats.
  • Figure 8 shows the results of an experiment analysing an exemplary pRNAi-pl20 clone by digestion with diagnostic restriction endonucleases.
  • Figure 9 details an exemplary process by which a library of inverted repeats is transferred into mammalian expression vectors.
  • Figure 10 shows the features of the retroviral expression vector pR-VAI-kan, designed for expression of RNAi libraries.
  • Figure 1 1 shows the features of the episomal expression vector pE-VAI-kan, designed for expression of RNAi libraries.
  • pBluescript plasmid lacking the multiple cloning sites MCS
  • pBSdM pBluscript KS (+) (Stratagene) plasmid was linearized using Eel 13611 and Acc65 I endonucleases that flank the MCS. a. Acc65I protruding end was "filled in” (i.e., completed to from a non-protruding end) using Klenow fragment of DNA polymerase I. b. The resulting linearized pBS DNA was purified on an agarose gel, self ligated and transformed into XLl-blue E.coli strain. pBSdM (pBS lacking the polylinker) DNA was purified using a standard protocol.
  • a 764bp GFP cDNA fragment was amplified by PCR using pEGFP (Clontech) as a template with the following primers: sense 5'- CGCTGGAAGATGGAACCCGTCTCCATGGTGAGCAAGGGCGAGGAG and antisense 5' - ATTGCTTTTACAGATGCCGTCTCCGGACTTGTACAGCTCGTCCAT. Both oligos contain BsmB I recognition sites.
  • the resultant cDNA fragment was amplified with two different pairs of primers.
  • the resulting pRNAiL plasmid contains four BsmB I sites that flank "Direct” and "Inverted” GFP fragments.
  • Example 2 Preparation of single-stranded partially self-complementary stem-and-loop adaptors The procedure below describes generation of stem-and-loop single-stranded adaptors using pRNAiL.
  • This plasmid is obtained by simultaneous ligation of four separately synthesized DNA fragments containing: 1. fl origin of replication of filamentous phage Ml 3, and pUC origin of replication; 2. ⁇ -lactamase gene under its own promoter; 3. Any DNA fragment (GFP in our case) positioned in direct orientation; 4. The same DNA positioned in an opposite orientation (inverted repeat).
  • flanking sequences contain two restriction sites, BsmB I and Not I, necessary for further construction steps as well as Bpi I restriction site to produce "sticky" ends designed to ensure only one possible way of the pRNAiL plasmid assembly.
  • the outline of the procedure is as follows: pRNAiL is transformed into E.coli XL-1 blue competent cells. Single stranded plasmid DNA is obtained following infection of the bacterial culture with a helper phage and purified using standard protocol. The purified single-stranded-pRNAiL is denatured and self-annealed to form an internal double stranded region as a result of annealing of direct and inverted DNA fragments.
  • This double stranded region is digested with an appropriate endonuclease (BsmB I) to generate two stem-and-loop single stranded adaptors, one containing a plasmid origin of replication (L-adaptor) and the other containing the ⁇ -lactamase gene (R-adaptor).
  • the stem regions provide sticky ends for further ligation steps ( Figure 5).
  • Phage particles were precipitated by overnight incubation with 20% PEG (MW 8,000), 2.5M NaCl (0.25 volume of the supernatant) at 4° C. g.
  • the phage pellet was collected by centrifugation for 15 min at 20.000g at 4° C. h.
  • the resultant single stranded plasmid forms a partially double stranded structure, as shown in Figure 4.
  • L-adaptor pUC ori
  • R- adaptor ⁇ -lactamase
  • the DNA was further digested with Alul (37o C, 1 hour).
  • Alu I cuts 8 times within the GFP cDNA, generating dsDNA fragments with sizes ranging from 20 to 216 bp.
  • the reaction mixture was incubated for 20 min at 80o C, to inactivate the enzymes.
  • a mixture of L and R single stranded looped adaptors was purified from short double- stranded-cDNA fragments using Microcon column (MontageTM PCR, Millipore corporation, cat# UFC7PCR50) according to the manufacturer's recommendations. Elution volume was 20 ul.
  • Single-stranded stem-and-loop adaptors of a similar design may be obtained by any alternative procedure enabling generation of the appropriate amounts of single-stranded DNA, e.g. by a single-stranded PCR based on the access of one of the primers or PCR with one of the primers being biotinylated followed by strand separation on an avidin column or by direct chemical synthesis of the desired molecules.
  • the structure of the stem-and-loop adaptors may vary and is subject to further optimization; said optimized adaptors are also considered to be a part of the present invention.
  • Introduction of splice donor and splice acceptor sites into the loop remaining in the cDNA portion to be transferred into a eukaryotic expression vector are also contemplated. This design will enable shortening of the loop (if necessary) in the course of expression in mammalian cells.
  • the adaptor mixture obtained as described above was used in conjunction with an exemplary insert - a 500 bp fragment of pl20-ctn cDNA.
  • cDNA is used in the exemplary embodiments herein, it is understood that the processes of the present invention can also be carried out on other nucleic acids fragmented as desired with endonucleases, e.g. on genomic DNA, cDNA synthesized on total cellular mRNA, on any individual or mixed cDNA fragments previously subcloned in a different vector or PCR amplified, or on synthetic polynucleotides / oligonucleotides.
  • endonucleases e.g. on genomic DNA, cDNA synthesized on total cellular mRNA, on any individual or mixed cDNA fragments previously subcloned in a different vector or PCR amplified, or on synthetic polynucleotides / oligonucleotides.
  • Exemplary 0.5 kb dsDNA insert was obtained from pl20 catenin cDNA by PCR amplification using the following primers: sense 5' CAGCGTCTCCATGGACGACTCAGAGGTGGAG and antisense 5'- CATCGTCTCCGGACACGGCCCAAGGTCTGGATAT.
  • the primers contained Esp 31 recognition sites and, following restriction with Esp 31, produced sticky ends compatible with the sticky ends of L- or R-adaptors.
  • reaction mixture was incubated for 5 min at 4°C, followed by 5 min at 20°C and by 10 min at 37°C. The reaction was terminated by 10 min incubation at 75°C. e.
  • reaction mixture (20 ⁇ l) was supplemented with: 7 ⁇ l H 2 O 1 ⁇ l BSA (1 mg/ml) 1 ⁇ l dNTPs (2.5 mM) 1 ⁇ l (8u) Bst DNA polymerase (NEB) f.
  • the reaction mixture was incubated at 65° C for 2 hrs, and the reaction was terminated by 10 min incubation at 80° C. g.
  • reaction mixture was supplemented with: 4 ⁇ l H 2 O 4 ⁇ l lOx ligation buffer of NEB 2 ⁇ l (2u) T4 DNA ligase (Amersham), and incubated for 2 hrs at room temperature.
  • Example 4 Preparation of mammalian expression vectors for expression of double-stranded inhibitory RNA (the library of inverted repeats inserted under the control of Pol II promoter)
  • the DNA fragment containing inverted repeats separated by a functional ⁇ -lactamase gene (or0 any complex mixture of such DNA fragments) is next excised from the double-stranded- pRNAiL using a restriction endonuclease (Not I) and may be inserted into mammalian expression vectors. After being introduced into mammalian cells, these vectors are capable of dsRNA expression (stem-and-loop) ( Figure 9). Two types of compatible mammalian expression vectors, retroviral ( Figure 10) and episomal ( Figure 11), were designed. These5 vectors are characterized by the following main features: 1.
  • Example 5 Vectors encoding long double-stranded RNAs as drugs and delivery systems for these drugs ny of the vectors generated by the processes of the present invention may serve as pharmaceutical compositions. These vectors would then typically be admixed with a pharmaceutically acceptable carrier, and formulated so as to maximize solubility and efficacy. Delivery systems aimed specifically at the enhanced and improved delivery of siRNA into mammalian cells have been developed, see, for example, Shen et al (FEBS letters 539: 111-114 (2003)), Xia et al., Nature Biotechnology 20: 1006-1010 (2002), Reich et al., Molecular Vision 9: 210-216 (2003), Sorensen et al. (J.Mol.Biol.
  • siRNA has recently been successfully used for inhibition in primates; for further details see Tolentino et al., Retina 24(1) February 2004 I 132-138.Respiratory formulations for siRNA are described in U.S. patent application No. 2004/0063654 of Davis et el.
  • compositions of the present invention are administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the disease to be treated, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners.
  • the pharmaceutically "effective amount” for purposes herein is thus determined by such considerations as are known in the art.
  • the amount must be effective to achieve improvement including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art.
  • compositions of the present invention can be administered by any of the conventional routes of administration.
  • a compound - such as a vector encoding a long double stranded RNA - can be administered as the compound or as pharmaceutically acceptable salt and can be administered alone or as an active ingredient in combination with pharmaceutically acceptable carriers, solvents, diluents, excipients, adjuvants and vehicles.
  • the compounds can be administered orally, subcutaneously or parenterally including intravenous, intraarterial, intramuscular, intraperitoneally, and intranasal administration as well as intrathecal and infusion techniques. Implants of the compounds are also useful.
  • Liquid forms may be prepared for injection, the term including subcutaneous, transdermal, intravenous, intramuscular, intrathecal, and other parental routes of administration.
  • the liquid compositions include aqueous solutions, with and without organic cosolvents, aqueous or oil suspensions, emulsions with edible oils, as well as similar pharmaceutical vehicles.
  • the compositions for use in the novel treatments of the present invention may be formed as aerosols, for intranasal and like administration.
  • the patient being treated is a warm-blooded animal and, in particular, mammals including man.
  • the pharmaceutically acceptable carriers, solvents, diluents, excipients, adjuvants and vehicles as well as implant carriers generally refer to inert, non-toxic solid or liquid fillers, diluents or encapsulating material not reacting with the active ingredients of the invention and they include liposomes and microspheres.
  • delivery systems useful in the present invention include U. S. Patent Nos. 5,225,182; 5,169,383; 5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224; 4,439,196; and 4,475,196. Many other such implants, delivery systems, and modules are well known to those skilled in the art. In one specific embodiment of this invention topical and transdermal formulations are particularly preferred.
  • the active dose of compound for humans is in the range of from lng/kg to about 20- 100 mg/kg body weight per day, preferably about 0.01 mg to about 2-10 mg/kg body weight per day, in a regimen of one dose per day or twice or three or more times per day for a period of 1 -2 weeks or longer.
  • the present invention also provides for a process of preparing a pharmaceutical composition which comprises:
  • the compound used in the preparation of a pharmaceutical composition is admixed with a carrier in a pharmaceutically effective amount.
  • nucleotides can be introduced to improve the therapeutic properties of the nucleotides. Improved properties include increased nuclease resistance and/or increased ability to permeate cell membranes.
  • the nucleotides can be selected from naturally occurring or synthetically modified bases. Naturally occurring bases include adenine, guanine, cytosine, thymine and uracil.
  • Modified bases of the oligonucleotides include inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl-, 2-propyl- and other alkyl- adenines, 5-halo uracil, 5- halo cytosine, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiuracil, 8-halo adenine, 8- aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8- substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thioalkyl guanines, 8-hydroxyl guanine and other substituted guanines, other aza and deaza adenines, other aza and deaza guanines, 5-trifluoromethyl uracil and 5-trifluor
  • nucleotide analogs can be prepared wherein the structures of the nucleotides are fundamentally altered and are better suited as therapeutic or experimental reagents.
  • An example of a nucleotide analog is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in DNA (or RNA) is replaced with a polyamide backbone similar to that found in peptides.
  • PNA analogs have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vitro. Further, PNAs have been shown to bind more strongly to a complementary DNA sequence than to a DNA molecule. This observation is attributed to the lack of charge repulsion between the PNA strand and the DNA strand.
  • Other modifications that can be made to oligonucleotides include polymer backbones, cyclic backbones, or acyclic backbones.
  • the modification is a modification of the phosphate moiety, whereby the modified phosphate moiety is selected from the group comprising phosphothioate

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Abstract

La présente invention se rapporte à des processus d'inversion-duplication d'acides nucléiques, qui peuvent être mis en oeuvre pour la préparation de vecteurs codant des molécules d'ARN bicaténaires. Ces vecteurs et les processus permettant leur préparation sont utiles pour inhiber l'activité biologique de gènes codant des polypeptides et ils peuvent être utilisés en tant qu'outil pour l'identification d'une cible médicamenteuse.
PCT/IL2004/001114 2003-12-11 2004-12-07 Inversion-duplication d'acides nucleiques et bibliotheques preparees de cette maniere WO2005056750A2 (fr)

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US8785211B2 (en) 2005-11-15 2014-07-22 Isis Innovation Limited Methods using pores
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JP2009082130A (ja) * 2007-10-02 2009-04-23 Genetai Inc 促進された遺伝子発現能力を伴う新規発現ベクターおよびそれを使用するための方法
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