WO2003066858A1 - Elements de transfert de ribozymes de transepissage derives de sequences codant des proteines fluorescentes - Google Patents

Elements de transfert de ribozymes de transepissage derives de sequences codant des proteines fluorescentes Download PDF

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WO2003066858A1
WO2003066858A1 PCT/GB2003/000475 GB0300475W WO03066858A1 WO 2003066858 A1 WO2003066858 A1 WO 2003066858A1 GB 0300475 W GB0300475 W GB 0300475W WO 03066858 A1 WO03066858 A1 WO 03066858A1
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mrna
fluorescent protein
cleavage
ribozymes
ribozyme
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Matthew John Andrew Wood
Sarah Louise Everatt
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Isis Innovation Limited
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • 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
    • C12N2310/124—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes based on group I or II introns

Definitions

  • the present invention relates to model systems, and more particularly to model systems for transgenic work in animals.
  • Ribozymes, antisense strategies and, more recently, deoxyribozymes, or DNAzymes have all been employed as powerful tools to interfere with gene expression at the RNA level.
  • Ribozymes are RNA molecules capable of catalysis, while DNAzymes are similar to ribozymes but man-made and comprise deoxyribonucleotides.
  • antisense strategies antisense RNA binds to mRNA in a sequence dependent manner, the cell destroys the resulting double stranded RNA and, therefore, the expression of the gene is reduced. All of these strategies exploit complementary Watson-Crick base pairing in order to target the RNA in a sequence specific manner and, in this way, these technologies can be used to target genes for down-regulation or repair.
  • Hammerhead ribozymes are small catalytic RNA molecules that have the ability to cleave RNA. Hammerhead ribozymes were originally found in plant viroids and virusoids where they form a hammerhead structure to self-cleave during the replication process (Forster et al, (1987) Cold Spring Harbor Symp. Quant. Biol., 52, 249-59). Hammerhead ribozymes have been engineered to cleave in trans, i.e. to cleave other RNA molecules, in a sequence specific manner.
  • the hammerhead ribozyme has a three-stem structure.
  • Stem II is the conserved catalytic core of the ribozyme and is composed of 13 nucleotides, while stems I and III can be designed to bind any nucleotide sequence.
  • the only sequence requirement for cleavage to occur is an NUX motif in the target molecule, where N can be any nucleotide and X can be any nucleotide except for guanine.
  • the ribozyme binds the target via its two binding arms and cleaves the substrate. After dissociation, it is free to react with further substrates. Mg 2 + plays a direct role in the cleavage reaction and must be present for cleavage to occur.
  • Accompanying Figure 3 is a schematic diagram of a hammerhead ribozyme cleaving in trans.
  • Hammerhead ribozymes can be delivered using plasmid or viral based vectors or, more recently, as chemically stabilised molecules, and can, therefore, be delivered in vivo.
  • hammerhead ribozymes have been shown to down- regulate gene expression in vivo, the down-regulation seen is often quite mild and does not persist for a length of time suitable for useful therapy.
  • the group I intron ribozyme from Tetrahymena thermophilia was the first RNA to be shown to exhibit catalytic activity (Cech et al. (1981) Cell, 27(3 Pt 2), 487-96).
  • the group I intron ribozyme is more complex than the self-cleaving hammerhead ribozymes and is significantly larger.
  • Group I intron ribozymes are found in a variety of organisms and act to splice in cis, that is to perform a self splicing reaction. This reaction occurs in two stages whereby 3' and 5' exons flanking the ribozyme sequence are ligated, and a mature RNA transcript is generated.
  • the group I intron has been used in the laboratory to splice in trans, that is to perform a splicing reaction on an RNA molecule distinct from itself.
  • the accompanying Figure 4 is a schematic representation of a group I intron ribozyme-mediated modification of a trans-RNA target.
  • the group I intron ribozyme from Tetrahymena thermophilia is 421nt long with a universally conserved central catalytic core of approximately 200nt surrounded by several less conserved peripheral elements.
  • the group I intron ribozyme has been shown to fold into a small globular structure. The splicing reaction is dependent upon an endogenous guanosine molecule and magnesium ions (c.f. Doherty et al. (2001) Ann. Rev. Biophys. Biomol. Struct., 30, 457-75).
  • the term “repair” is taken to mean altering an mRNA to result in the expression of a different polypeptide from that encoded by the original. "Silencing” and its associated terms are taken to indicate that an mRNA has been down-regulated or prevented from expressing its polypeptide.
  • DNAzymes are catalytic DNA molecules that cleave RNA in a sequence specific manner. DNAzymes are not naturally occurring molecules, and are usually generated by in vitro selection procedures to produce active molecules which generally exceed the catalytic efficiency of ribozymes.
  • the DNAzyme binds the target via the two target binding arms and cleaves between an unpaired purine (A, G) and a paired pyrimidine (C, U) residue.
  • A, G unpaired purine
  • C, U paired pyrimidine
  • the DNAzyme can be individually designed to cleave virtually any target RNA.
  • Effective DNAzymes with binding arms ranging from 7 to 15 nucleotides have been reported (Kurreck et al. (2001) J Biol. Chem., 20, 20). Magnesium ions play a direct role in the cleavage reaction and must be present for cleavage to occur.
  • DNAzymes are more stable than ribozymes, and stability can be further increased by chemical modification of bases. DNAzymes have a much higher catalytic activity than ribozymes. Delivery of chemically synthesised DNAzymes is generally by a transfection-based system, which has tended to limit the use of these molecules therapeutically, but other methods for their delivery are under investigation, and a DNA expression vector has been reported. As the time from design to synthesis of usable DNAzymes is usually only a matter of days, these molecules are useful for in vitro and cell culture work, unlike hammerhead ribozymes, which must be designed, cloned and expressed.
  • Green Fluorescent Protein has been widely exploited by scientists as an expression system (Tsien (1998) Ann. Rev. Biochem., 67, 509-44) as it is easy to quantify and requires no additional steps to visualise. Mutagenesis of the original sequence from the jellyfish Aequorea victoria has resulted in a more stable and brighter protein EGFP (enhanced green fluorescent protein). Further mutagenesis of the EGFP gene has produced the colour variants, EBFP, ECFP and EYFP (Blue, Cyan and Yellow fluorescent proteins respectively - c.f Cormack et al, (1996) Gene, 173(1), 33-8).
  • a catalytic polynucleic acid molecule characterised in that the molecule is capable of catalysing the cleavage of a selected mRNA and replacing the 3' cleavage product with a coding sequence encoding all or part of a fluorescent protein, the resulting mRNA being translatable to express a fluorescent protein.
  • the mRNA encodes a first fluorescent protein and the catalytic molecule encodes a second fluorescent protein, whereby replacement of the 3' cleavage product with a coding sequence encoding part of a second fluorescent protein results in mRNA translatable to express a fluorescent protein different from that encoded by the uncleaved mRNA.
  • a catalytic polynucleic acid molecule characterised in that the molecule is capable of catalysing the cleavage of an mRNA encoding a fluorescent protein and replacing the 3' cleavage product with a coding sequence encoding part of a second fluorescent protein, the resulting mRNA being translatable to express a fluorescent protein different from that encoded by the uncleaved mRNA.
  • the polynucleic acid molecule will generally be a ribozyme or a DNAzyme, and references herein to ribozymes or DNAzymes include references to all such molecules, unless otherwise apparent.
  • Typical ribozymes include hammerhead ribozymes and group I intron ribozymes. More particularly, the catalytic molecule should be able to introduce a coding sequence into an mRNA.
  • Preferred ribozymes of the present invention are capable of tra «_?-splicing target mRNA molecules, and this is generally effected by the ribozyme of the invention comprising a 3' intron encoding the C terminus of a fluorescent protein.
  • This fluorescent protein will be different from the fluorescent protein that is the target of the ribozyme, and will, effectively, be exchanged with the 3' cleavage product of the target mRNA to generate a different fluorescent protein.
  • the fluorescent protein encoded by the intron may be all or part of the protein, but must generally include at least the C terminus thereof, as the intron will generally replace the 3' end of the mRNA. It is not essential that the replaced portion be the same length as that replacing it, provided that the resulting mRNA encodes a protein that is detectable by fluorescence analysis, such as by instrument or by the naked eye.
  • the protein encoded by the target mRNA is also a fluorescent protein, and that both proteins belong to the eGFP family.
  • the resulting protein preferably belongs to this family, also, and the family is discussed more, below. However, especially where one protein differs from another by more than a couple of bases in the mRNA, it is also possible that the resulting protein is a fluorescing hybrid.
  • ribozymes of the present invention may be highly specific for a selected mRNA, or may be selective for two or more fluorescent protein mRNA's, for example.
  • reference to o the target mRNA herein as encoding a fluorescent protein includes the possibility that the target encodes another protein. It is generally preferred that the target encodes a fluorescent protein, but it may be desired to evaluate whether a target mRNA can be successfully repaired by a fluorescent protein, prior to using a therapeutic repair, for example.
  • Ribozymes of the present invention may be used to induce colour changes in selected cells, and it is possible that target cells will express more than one mRNA as fluorescent proteins, in which case, it may be desirable to provide ribozymes specific for one particular fluorescent protein, such as the yellow fluorescent protein (EYFP).
  • EYFP yellow fluorescent protein
  • target cells only express one fluorescent protein, then there will generally be no requirement to provide ribozymes specific for one particular mRNA.
  • the ribozyme be able to target all fluorescent protein mRNA's, as those ribozymes which are specific for selected fluorescent protein mRNA's must target that site on the mRNA which is specific for that mRNA and, if this is on the 5' side of the cleavage site, then the sequence will remain in the final mRNA. This is not important, provided that the resulting mRNA encodes a different coloured fluorescent protein from the original.
  • the intron will generally provide the latter portion of the mRNA that differs from one fluorescent protein to the next.
  • the difference between this and EGFP is only two bases and, provided that the intron comprised in the ribozyme serves to replace these bases in the original mRNA, then the resulting fluorescent protein will be changed from blue to green, or vice versa.
  • the fluorescent proteins are especially useful in the present invention as, although the differences in emission and excitation of these proteins are quite marked, as shown in the accompanying Figure 1, the differences between the sequences of the different coloured proteins, at the genetic level, are very small, and usually only amount to a few bases, as demonstrated in Figure 2.
  • dsRed Another coloured protein has been isolated from Discosoma sp. called dsRed, but has little sequence similarity to the other coloured proteins, also as shown in Figure 2.
  • SEQ ID NO. 1 is the nucleotide sequence of EGFP mRNA, while the protein encoded by this sequence is SEQ ID NO. 2.
  • SEQ ID NOS. 3 and 4 are the nucleotide and protein sequences of EBFP
  • SEQ JJD NOS. 5 and 6 are the nucleotide o and protein sequences of ECFP
  • SEQ ID NOS. 7 and 8 are the nucleotide and protein sequences of EYFP.
  • SEQ ID NO. 9 is the nucleotide sequence of dsRED.
  • SEQ ID NO. 10 is the mRNA sequence and SEQ ID NO. 11 is the ribozyme sequence of Figure 3.
  • SEQ ID NOS. 12 and 13 are the mRNA sequence and DNAzyme sequence, respectively, of Figure 5.
  • SEQ ID NOS. 14, 15 and 16 are the nucleotide sequences of Figures 7, 8 and 9, respectively.
  • SEQ JD NO. 17 is the GCFPRZ1 9 base pair internal guide sequence (IGS).
  • SEQ ID NO. 18 is the 9 base pair internal guide sequence (IGS) of GCFPRZ2.
  • SEQ ID NOS. 19 to 21 are as defined in Example 2.
  • the present invention provides means by which the delivery of ribozymes to a target tissue may be established.
  • a target tissue expresses a selected fluorescent protein
  • a ribozyme of the invention is then delivered to the tissue and the success of delivery established by ascertaining the presence of a modified fluorescent protein.
  • the EGFP mouse provides an excellent model for testing different forms of ribozyme delivery. There is no restriction on the form of delivery tested. The only consideration is to whether the tissue begins to express the fluorescent protein encoded by the test ribozyme, and which can only be expressed if delivery of ribozyme to the tissue was successful.
  • any suitable method may be employed to enable tissue to express a fluorescent protein, or other protein, via appropriate mRNA.
  • Suitable transformation methods are well known in the art, and may involve cloning into cell lines using electroporation or by using appropriate vectors, including plasmids and other vehicles, such as viruses.
  • the resulting cells if eggs, for example, may be grown into whole organisms, or may be replicated into cell lines, for example, if they are another form of tissue.
  • suitable techniques for obtaining cell lines or tissues expressing the desired mRNA are well known in the art. Further, there is no restriction as to the specificity sought for the delivery system under test.
  • ribozyme On a larger scale, it may be desired to deliver ribozyme to the entire body, in which case an animal constitutively expressing the target mRNA in all tissues is desirable for testing. Such an animal may also be used for anything more specific, such as targeting one organ but not another, in which case the absence of the transformed mRNA can also be tested by absence of the fluorescent protein that would necessarily result from such a transformant. Such specificity can descend all the way down to organelles, as discussed below.
  • the present invention allows the determination of success or failure of selected delivery systems for ribozymes and DNAzymes, by positive identification of the results. It is readily feasible to test any number of parameters, including different promoters and delivery methods, such as tissue specific promoters, strong promoters, weak promoters, systemic delivery, retrograde transport and the like.
  • the active presence of a ribozyme of the invention can be measured by the presence of the second fluorescent protein.
  • a tissue may express EGFP and the ribozyme carries the intron for changing EGFP into EBFP. If the ribozyme is successfully delivered to the tissue, then it will be possible to measure the presence of expressed EBFP.
  • ribozymes of the invention Whilst it is possible that successful delivery of ribozymes of the invention will result in substantially 100% conversion of a first mRNA into a second mRNA, it is also likely that not all of the mRNA present in the cell will be converted, so that a mixture of fluorescent proteins will be apparent in the affected tissue. However, by using appropriate fluorescent measuring means, this presents no problems to the person skilled in the art. Such results also provide valuable information regarding the system under test, as it may also provide information as to the level of gene silencing that might be expected by using the delivery system in question, or even the IGS being used, if the target mRNA is also the end target for repair. In such case, the target mRNA may also temporarily carry a fluorescent marker, which can be transformed by a ribozyme of the invention.
  • the present invention envisages the use of ribozymes to change tissue colour, but it is generally preferred that the method of the present invention be employed to assay the effectiveness of means for delivering ribozymes to tissues in vivo.
  • the art is replete with delivery systems that work in theory but which, often for unknown reasons, do not work in practice.
  • the advantage of the present invention is that, by determining the success of delivery of ribozymes of the invention, such delivery systems may then be used to deliver any other ribozymes that it may be desired to deliver to the site in question.
  • the present invention also envisages the use of ribozymes of the invention as markers for the success of the delivery of other ribozymes.
  • a tissue may express a protein that does not normally fluoresce but that, when subjected to ribozymes of the present invention, expresses fluorescent proteins that may be detected.
  • delivery of the ribozymes of the invention can be discontinued and re-continued at any time that it is desired to confirm the success of delivery of ribozyme to the tissue.
  • a suitable target protein to establish successful delivery of a ribozyme is albumin, for example.
  • albumin for example.
  • the present invention further provides a system or method for determining the likely success of delivering a polynucleic acid molecule to a target tissue such that the polynucleic acid molecule is expressed in said tissue, wherein the target tissue expresses a selected mRNA, said system comprising delivering a catalytic polynucleic acid molecule to said tissue in a manner in which it is desired to deliver said polynucleic acid molecule, and wherein the catalytic polynucleic acid molecule is capable of catalysing the cleavage of the selected mRNA into 5' and 3' cleavage products, and replacing the 3' cleavage product with a coding sequence encoding all or part of a fluorescent protein, the resulting mRNA being translatable to express a fluorescent protein, success being determined by assaying the presence of fluorescent protein encoded by said resulting mRNA.
  • a ribozyme can successfully be delivered to a cell or tissue and be expressed therein, then it is extremely likely that another polynucleotide sequence, and especially another ribozyme, such as a repair or silencing ribozyme, can also be successfully delivered and expressed. This allows the skilled person to establish the desirability of using a given delivery method quickly and easily.
  • ribozymes and DNAzymes can readily be investigated using the fluorescent protein, or FP, system described herein both generally, using the system to improve delivery vectors, testing out the kinetics of modified hammerhead species, and specifically, using the system to perfect delivery to a particular cell type, tissue or organ before using the therapeutic ribozyme of interest.
  • the system of the present invention such as using a tr ⁇ 7W-splicing group I intron ribozyme to splice EBFP sequence onto EGFP mRNA, causing an easily quantifiable colour change from green to blue, for example, enables researchers to improve the efficiency, specificity and delivery of ribozymes and deoxyribozymes both generally and specifically.
  • the systems of the present invention permit the optimisation of delivery of these molecules using non-viral methods, for example, and will greatly facilitate the work being undertaken in this area, especially given that the delivery and persistence of expression of DNAzymes are currently considered to be the only major obstacles to their therapeutic use.
  • Stably transfected cell lines expressing EGFP, EYFP, EBFP and ECFP form a preferred embodiment of the present invention, as do host animals expressing these proteins constitutively. It is generally preferred that any line or animal express only one fluorescent protein. It will also be appreciated that the invention extends to such animals and cell lines expressing other fluorescent variants of these proteins. While EGFP, EYFP, EBFP and ECFP are the preferred proteins of the present invention, the invention also extends to other fluorescent members of this family that may result from any future genetic alterations to any one of the family.
  • the family is generally characterised by a substantial homology of the first 195 bases from the 5' end of the mRNA, and the invention extends to any fluorescent protein encoded by an mRNA having the first 195 bases of its base sequence of identical to bases 1 to 195 of SEQ ID NO. 1, or differing therefrom by no more than 5%, and preferably no more than 2%, particularly preferably 1% or less.
  • ribozymes and DNAzymes on these stably transfected cell lines can be analysed in a quantifiable manner using the FACS machine (Fluorescence Activated Cell Sorter), for example. Using such a machine, the actual number of fluorescent cells and the intensity of the fluorescence is readily determined (Cormack et al, (1996) Gene, 173(1), 33-8).
  • FACS machine Fluorescence Activated Cell Sorter
  • RT-PCR analysis of the FP RNA extracted from cells and tissues and, similarly, Western blot analysis of the repaired FP protein may be used, for example, to provide confirmation of results obtained by such processes.
  • Real-time PCR using the TaqManTM machine may be employed to provide absolute quantification and analysis of the action of the catalytic nucleic acid molecules of the present invention.
  • Group I intron ribozymes may be expressed at the desired location by suitable mammalian expression vectors, such as PCDNA 3 , into which they have been cloned, together with suitable control and expression sequences, such as the SV40 or adenovirus promoters, together with an initiation sequence, for example.
  • suitable mammalian expression vectors such as PCDNA 3
  • suitable control and expression sequences such as the SV40 or adenovirus promoters, together with an initiation sequence, for example.
  • suitable expression vectors include inactivated or attenuated retroviruses, or retroviruses having no significant pathological effect on the recipient, but which are capable of infecting cells in the recipient, adenovirus, adeno- associated virus (AAV) and lentivirus, which may be used to express hammerhead and group I intron ribozymes of the invention, for example.
  • adenovirus adeno-associated virus
  • AAV adeno- associated virus
  • lentivirus which may be used to express hammerhead and group I intron ribozymes of the invention, for example.
  • These molecules can be cloned into the expression vectors, such as adenovirus, under the control of a mammalian promoter specific for the synthesis of large amounts of small RNA, such as the U2 snRNA promoter (Ares et al. (1985) Mol Cell Biol., 5(7), 1560-70).
  • DNAzymes may be stabilised by the use of internal phosphothioate bonds. Such molecules are less susceptible to degradation, and can be delivered to the target site with less likelihood of degradation, and will continue to be effective for longer.
  • a suitable DNAzyme is the 10-23 DNAzyme (Santoro et al. (1997) Proc Nat. Acad. Sci. U S A,. 94(9), 4262-6).
  • the 10-23 DNAzyme consists of a catalytic domain of 15 nucleotides with flanking target-binding arms, as illustrated in Figure 3 with respect to hammerhead ribozymes.
  • Living Colours TM subcellular localisation vectors for example, which localise fluorescence to specific organelles or structures within living cells in order to visualise biological processes as they occur.
  • Using such vectors and expression systems enables investigation into the ability of delivery systems to co-localise the ribozymes and DNAzymes with these localised proteins. Delivery of catalytic nucleic acids to specific compartments of the cell in order to enhance the therapeutic activity can then be optimised (Sullenger (1995) Appl Biochem Biotechnol., 54(1-3), 57-61).
  • the present invention enables the detailed construction of such catalytic molecules to be investigated and analysed in detail.
  • a panel of DNAzymes and ribozymes with mismatches on one of the binding arms, on both of the binding arms and mismatches in different positions along the binding arms can be created.
  • Such a panel can then be used to provide a guide to required specificity of sequence for ribozymes to be as selective as required.
  • ribozymes targeted to different sites on the target RNA can be more efficacious, and that this can equally apply to ribozymes of the invention. Such a use can also be exploited to help where multiple therapeutic ribozymes are desired or required, to help determine the efficacy of delivery.
  • Ribozymes which can benefit from the system of the present invention may be any that may be used for therapy, or otherwise, in the human or animal body.
  • such ribozymes may be used to either repair or silence gene expression products, and are applicable, for example, to genetic diseases, such as Huntingdon's, or sickle cell anaemia, and cancer, such as leukaemia, as well as to infectious diseases, such as HIV, for example.
  • trans-splicing ribozymes are applicable where any other ribozyme might be used, so that any disease is a potential target.
  • the system of the present invention enables these molecules to target any organ, tissue or cell type within an FP mouse, for example, and allows optimisation of delivery of the group I intron ribozyme. Even very small amounts of splicing can be visualised using antibodies that are readily available against EGFP and EBFP, for example.
  • Vectors are available that allow an EGFP tag to be inserted at either the N- terminus or C-terminus of a protein of choice.
  • Making such an EGFP/target molecule fusion protein (and hence EGFP/target molecule fusion RNA) and targeting this RNA using a catalytic nucleic acid of the present invention can be used to efficiently co- localise ribozyme/DNAzyme and target, as the resulting hybrid would be readily detectable.
  • the present invention provides a system or method for enabling the selection of the best IGS, or combination of IGS' s for use in repairing a given RNA.
  • the system comprises a panel of ribozymes capable of splicing a full length FP into an RNA, the panel having a ribozyme having all possible variations, or a majority of all possible variations, of the IGS.
  • a 6bp IGS this would be 4 6 , or 4096.
  • Target RNA, transfection reagent and cells can then all be added to the wells and allowed to incubate overnight.
  • FACS analysis can be used to determine which wells contain EGFP; these wells must therefore contain a ribozyme capable of splicing EGFP onto the target RNA in a sequence specific manner.
  • a second 96 well plate containing only those 45 ribozymes present in that well of the initial plate that contained fluorescence, could then be plated out individually (and in duplicate), and treated in the same way as the first, by addition of target RNA, transfection reagent and cells. The plate can be incubated overnight. Any well containing the individual ribozyme capable of splicing EGFP onto the target RNA will contain fluorescence when analysed subsequently analysed using FACS or simple visual observation under the microscope, for example. An optimal IGS can thus be readily detected.
  • the fluorescent proteins referred to herein need not, unless otherwise specifically required, belong to the eGFP family. For example, this includes both dsRED and the original GFP.
  • the catalytic molecules of the invention are also useful in many other situations. For example, they find particular use in confocal microscopy, which can be used to track the movements of proteins within a cell, for example, whilst fluorescent protein markers have been used for this process previously, this has been done at the genetic level.
  • the advantage of the present invention is that it can be used at the transcriptional level, thereby bypassing problems associated with genetic modification.
  • the catalytic molecules of the invention are also useful in visualising the activity of two or more proteins within a cell. Tagging a protein with a dedicated ribozyme or, at least, a ribozyme not capable of tagging a second protein of interest, and then tagging the second protein with a second ribozyme not capable of tagging the first, and wherein the two ribozymes provide for the encoding of different fluorescent proteins, enables the activities and interactions of the two, or more, proteins to be tracked, in vivo, by a confocal microscope, for example, without affecting anything at the genetic level.
  • the invention extends to hosts, such as animals, tissues and cell lines, transformed with genes encoding one or more of the ribozymes of the invention.
  • hosts will not express the fluorescent protein encoded by the ribozyme, in the absence of a target mRNA, and can be used to establish methods of obtaining target mRNA expression, or co-expression in cells expressing the ribozymes of the invention, such as by breeding, fusion, or transformation.
  • Hosts transformed with such genes may have them under suitable control, so that they may be controllably expressed.
  • Such controls may take the form of inducible promoters, such as the tetracycline on/off promoter, or tissue specific promoters, for example.
  • the nucleic acids of this invention have catalytic activity and in a preferred aspect, the target RNA encodes a fluorescent protein, and the catalytic nucleic acid cleaves the RNA to alter the fluorescence.
  • the target RNA is cleaved and the RNA fragments cannot then be used to encode the fluorescent protein.
  • preferred nucleic acids of this invention include hammerhead ribozymes and DNAzymes.
  • the sequence complementary to the target RNA binds through Watson-Crick base pairing and the catalytic core cleaves the target RNA molecule, thus down-regulating the protein, in this case reducing the amount of fluorescent protein (e.g. eGFP) seen in the cells/animal in an easily quantifiable manner.
  • fluorescent protein e.g. eGFP
  • Figure 1 shows the excitation and emission spectra for the proteins EGFP, EBFP, ECFP, EYFP and dsRED using the Living Colours TM detection system
  • Figure 2 is a sequence alignment of the proteins of Figure 1
  • Figure 3 shows the hammerhead ribozyme-mediated trans cleavage of an mRNA molecule
  • Figure 4 is a schematic representation of group I intron ribozyme mediated modification of a trans-RNA target
  • Figure 5 shows the trans cleavage of an mRNA molecule by a 10-23 DNAzyme
  • Figure 6 shows the areas of EGFP mRNA (bases 292-364 ) calculated to display high or low secondary structure using the M-Fold server;
  • Figures 7, 8 and 9 show the EGFP RNA sequence and the position of Hammerhead ribozymes, the position of Group I intron ribozyme IGS, and the position of DNAzymes within this sequence, respectively;
  • Figure 10 shows the results of a cleavage assay of target RNA against increasing concentration of hammerhead ribozyme
  • Figure 11 shows the optimum magnesium concentration for DNAzyme cleavage of
  • Figure 12 shows a time course cleavage assay of DNAzymes
  • FIGS. 13a and 13b show the effects of the addition of varying quantities of
  • RNA is single stranded and forms complex secondary structures that may sometimes render a particular region of the RNA inaccessible.
  • Computer programs such as Michael Zuker's M-Fold server (Zuker (1989) Methods Enzymol., 180, 262-88) can predict, with a degree of accuracy, regions within the RNA sequence that have relatively low secondary structure and, therefore, would theoretically be accessible to therapeutic ribozymes and DNAzymes. Accordingly, this method was employed for EGFP, and the resulting graph is shown in Figure 6, which shows the actual M-Fold output for bases 292-364 ofEGFP.
  • ribozymes are specific for any of the coloured proteins (except dsRED), as the ribozymes were all designed to target regions where the sequences of the coloured proteins are identical. Inactive versions of these ribozymes were also designed, identical to the active ribozyme, other than a C ⁇ G substitution at base 23 of the conserved catalytic core.
  • Figure 7 illustrates the EGFP RNA sequence showing the position of the Hammerhead ribozymes GFPHH1, GFPHH2 and GFPHH3.
  • FIG. 8 illustrates the EGFP RNA sequence, showing the position of the Group I intron ribozyme internal guide sequences (IGS).
  • IGS Group I intron ribozyme internal guide sequences
  • GFP1DZ was designed to the same sequence as the hammerhead ribozyme GFPHH2, although the cleavage site differed.
  • Another DNAzyme was designed to specifically cleave EGFP RNA but not EYFP RNA:
  • Another DNAzyme was designed to specifically cleave EBFP RNA but not EGFP RNA.
  • This DNAzyme has no catalytic sequence or similarity to the EGFP sequence.
  • Cleavage assays were performed in vitro using full length EGFP RNA (in vitro transcribed using Ambion MAXIscriptTM kit) and either hammerhead ribozyme RNA (in vitro transcribed using Ambion MEGASHORTscriptTM kit) or DNAzymes (commercially synthesised by SigmaGenosys).
  • cleavage assays were performed in the presence of magnesium ions (0-50mM) and 50mM Tris-HCl pH 8.0 in a total volume of lO ⁇ l for 2 hours at 37°C.
  • Accompanying Figure 11 determines the optimum magnesium concentration for the DNAzymes, where the values are final magnesium concentration of the reaction in mM, and shows that the optimum magnesium concentration for DNAzyme cleavage of EGFP target RNA is, in all cases, 5mM which is the physiological concentration found in cells and in vivo.
  • Accompanying Figure 12 shows the time course cleavage assay of DNAzymes, where the values shown are the reaction time in minutes. The asterisks denote the time at which the cleavage products first become visible, and it can be seen that there is some variability as cleavage products were not seen using GFP Y-DZ until 60 minutes had elapsed. This does not seem to be a factor in the usefulness of these molecules as can be seen in Figures 13a and 13b, where GFP Y-DZ can be seen to be particularly effective in cell culture.
  • 293 HEK (Human Embryonic Kidney) cells were transiently transfected with l ⁇ g EGFP plasmid under control of either the CMV (Cytomegalovirus) promoter or the SV-40 (Simian Virus 40) promoter.
  • Two group I intron ribozymes were designed, both targeting the EGFP gene and both mediating the splicing of ECFP RNA sequence onto EGFP RNA, thereby effecting a "repair" of green to cyan.
  • GCFPRZ1 has a 9 base pair internal guide sequence (IGS), SEQ ID NO. 17, which is complementary to bases 87 - 96 of the EGFP gene (the "A" residue of the ATG start codon being taken as base 1).
  • the exon of this ribozyme comprises 698 base pairs of the ECFP sequence.
  • GCFPRZ2 has a 9 base pair internal guide sequence (IGS), SEQ ID NO. 18, which is complementary to bases 102 -111 of the EGFP gene (the "A" residue of the ATG start codon being taken as base 1).
  • the exon of this ribozyme comprises 683 base pairs of the ECFP sequence.
  • EGFP RNA Full length EGFP RNA was synthesised using Ambion Megascript SP6 in vitro transcription kit while the ribozymes were synthesised using Ambion Maxiscript T7 in-vitro transcription kit. The RNA was precipitated and quantified.
  • the GCFPRZ1 or GCFPRZ2 ribozyme, as appropriate, (lmM) was preheated in reaction buffer (50mM HEPES (pH 7.0), 150mM NaCl and 5mM MgCl 2 ) at 95°C for 2 minutes.
  • EGFP target RNA (lOOnM) was preheated at 95°C for 2 minutes, in the presence (or absence) of lOO ⁇ M GTP, and then added to the ribozymes and incubated at 37°C for 3 hours.
  • RNAse-free water 5 ⁇ l of 5M sodium acetate and 150 ⁇ l of ice cold ethanol, and incubated at -20°C overnight.
  • the RNA was centrifuged, washed, and resuspended in 30 ⁇ l of RNAse- free water.
  • the RNA from the splicing reactions was subjected to reverse transcription using either random hexamer primers (which transcribes all RNA species present) or GFPrev primer (which transcribes all EGFP RNA's present as well as those of the colour derivatives).
  • the cDNA was resuspended in 20 ⁇ l clean water, l ⁇ l of the cDNA was taken to PCR using EGFP forward primer (which amplifies any of the colour derivatives) and a specific ECFP reverse primer which is designed to be specific for the ECFP sequence.
  • ECFP reverse primer is as follows:
  • the melting temperature (Tm) of this primer is 64.3°C, therefore the annealing temperature for the PCR reaction should be 60°C (Tm -5°C) , however to increase specificity of the primer and to completely ensure absence of binding to the eGFP cDNA, the annealing temperature used was 65°C.
  • the lanes contained the following ingredients:
  • RZ2 trans-splices ECFP sequence onto EGFP RNA - as a PCR product will only be seen if ECFP sequence is present.
  • ECFP sequence can only be present if the splice reaction occurred.
  • RZ2 appeared to work in the absence of GTP. This unexpected result can be explained by the carryover of unincorporated GTP from both of the in vitro transcription reactions, as these would not be removed during the precipitation step. As the amount of GTP required to catalyse the reaction is very small, any carry over could easily cause the reaction to proceed.
  • primer dimers indicates that the PCR reaction should be performed at even higher annealing temperatures.
  • RZ1 In order to ascertain whether RZ1 could be used successfully, a second in vitro transcription of RZ1 RNA was performed. This time, the plasmid template for the transcription reaction was cut with a restriction enzyme further downstream of the stop codon of the ECFP exon, in order to enable the RT reaction to be performed using a reverse primer specific for the trans-spliced products only. This RT-PCR should be more sensitive and allow lower levels of repair to be detected. The PCR reaction was performed this time at 68 °C to further increase specificity.
  • the lanes on the gel contained the following ingredients:
  • M molecular weight marker
  • Lanes 2, 5 and 7 contained PCR products.
  • RZ2 mediated trans-splicing at levels that could be detected via RT-PCR using random hexamers and GFPrev primer
  • RZ1 mediated trans-splicing at levels that could be detected via RT- PCR using a more sensitive splice product specific reverse primer. No primer dimers were observed.
  • a further PCR reaction was performed as a positive control, this time using EGFP forward AND reverse primers.
  • the lanes of the gel contained the following ingredients:
  • M molecular weight marker
  • Lanes 1 to 7 showed the presence of cDNA in all reactions, proving that absence of a PCR product in the "target only" RT-PCR reactions was indeed due to the specificity of the reverse PCR primer for the CFP sequence, not because there was no cDNA present in the reaction. No band should have been, nor was, present in lane 8 (Target only Reverse transcribed with splice specific primer) as the full length EGFP RNA present in this reaction does not have complimentarity to the splice product specific primer and therefore should not undergo transcription into cDNA

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Abstract

L'invention porte sur une molécule d'acide polynucléique catalytique capable de catalyser le clivage d'un ARNm sélectionné en produits de clivage 5' et 3' et de remplacer le produit de clivage 3' par une séquence de codage codant tout ou partie d'une protéine fluorescente, l'ARNm obtenu pouvant être transduit pour exprimer une protéine fluorescente, ce qui permet de réaliser avec succès des procédés d'administration de ribozymes sur des sites exprimant l'ARNm cible.
PCT/GB2003/000475 2002-02-04 2003-02-04 Elements de transfert de ribozymes de transepissage derives de sequences codant des proteines fluorescentes WO2003066858A1 (fr)

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WO2008137814A1 (fr) * 2007-05-04 2008-11-13 Abbott Laboratories Procédés pour évaluer l'administration de molécules d'arn inhibitrices
WO2009054558A1 (fr) * 2007-10-25 2009-04-30 National Cancer Center Procédé pour diagnostiquer une maladie par imagerie moléculaire à l'aide d'un adénovirus contenant un ribozyme de transépissage

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WO2000071701A1 (fr) * 1999-05-24 2000-11-30 New England Biolabs, Inc. Procede de generation de genes non transferables separes capables d'exprimer un produit proteique actif

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008137814A1 (fr) * 2007-05-04 2008-11-13 Abbott Laboratories Procédés pour évaluer l'administration de molécules d'arn inhibitrices
WO2009054558A1 (fr) * 2007-10-25 2009-04-30 National Cancer Center Procédé pour diagnostiquer une maladie par imagerie moléculaire à l'aide d'un adénovirus contenant un ribozyme de transépissage
KR100907106B1 (ko) 2007-10-25 2009-07-09 국립암센터 라이보자임을 함유한 아데노바이러스를 이용한 질병의분자영상진단법
US8541237B2 (en) 2007-10-25 2013-09-24 National Cancer Center Method for diagnosing disease using adenovirus harboring trans-splicing ribozyme by molecular imaging

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