WO2006030237A1 - Transcription specifique du tissu par l'arn polymerase iii - Google Patents

Transcription specifique du tissu par l'arn polymerase iii Download PDF

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WO2006030237A1
WO2006030237A1 PCT/GB2005/003599 GB2005003599W WO2006030237A1 WO 2006030237 A1 WO2006030237 A1 WO 2006030237A1 GB 2005003599 W GB2005003599 W GB 2005003599W WO 2006030237 A1 WO2006030237 A1 WO 2006030237A1
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pol
nucleic acid
rna
acid molecule
pol iii
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Joanna Beatrice Wilson
Claire Evangeline Repellin
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The University Court Of The University Of Glasgow
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic

Definitions

  • the invention relates to eukaryotic gene transcription, and in particular to the tissue-specific transcription of genes by RNA polymerase III.
  • RNA polymerase I RNA polymerase I
  • polymerase II RNA polymerase II
  • poly III RNA polymerase III
  • Pol I synthesises rRNAs, which are the precursors of the main ribosomal RNAs.
  • Pol II transcribes all protein-encoding genes leading to a messenger RNA (mRNA) .
  • mRNA messenger RNA
  • Pol III synthesizes 5s rRNA, tRNAs, 7 SL RNA, U6 SnRNA and a few other small stable RNAs.
  • the regulatory sequences of pol II genes often include an enhancer, which is usually located upstream of the coding sequence or alternatively in introns or even 3' of the coding sequence itself.
  • An enhancer is composed of DNA sequence motifs which bind transcription factors to facilitate the transcription of pol II genes. These transcription factors may be present in all cells or only in some specific cell types. Thus enhancers can give tissue specificity to pol II transcription.
  • the immunoglobulin heavy chain (IgH) enhancer E ⁇ directs the expression of heterologous genes to the B cell compartment.
  • RNA pol II gene Another classical feature of a pol II gene is the presence of a TATAA box, which is located approximately 30bp upstream of the start site of the transcript and enables initiation at the correct site.
  • Pol II genes may or may not have introns, and all have a 3' AATAAA polyadenylation (pA) signal which triggers the addition of 80 to 250 adenylate residues at the end of the mRNA. Termination signals can also be found at the end of some pol II genes.
  • pA polyadenylation
  • Termination signals can also be found at the end of some pol II genes.
  • the most striking and unusual feature of the promoters used by RNA pol III is that the majority require sequence elements downstream of the RNA start site within the transcribed region. These internal control regions are generally discontinuous structures composed of essential DNA sequence blocks separated by non-essential regions (8) .
  • Three basic types of promoter are used by RNA pol III, designated types I, II and III.
  • the type I promoter requires three internal elements for efficient transcription: an A block, an intermediate element (IE) and a C block. This promoter arrangement is unique to 5s RNA.
  • RNA pol III The most common promoter arrangement used by RNA pol III is found in the tRNA genes and is the type II promoter. It consists of two highly conserved sequence blocks, A and B, within the transcribed region. The A block of type I and II promoters are homologous. The location of block B is variable.
  • RNA pol III templates lack any requirement for intragenic promoter elements. These are referred to as type III promoters and require a TATA-like box, a proximal sequence element (PSE) and a distal sequence element (DSE) for efficient expression. All the pol III genes are terminated by a TTTT termination signal (with no pA tail) .
  • the expression of a pol III transcript is usually constitutive and not tissue specific.
  • the pol III RNAs are short and non-coding. Therefore this makes pol III promoters ideal for vectors where expression of short non-coding RNA is required.
  • the Hl pol III promoter is used in vectors for the generation of double stranded RNA (dsRNA) hairpin-loop structures, such as the pSilencer vector from Ambion.
  • dsRNA double stranded RNA
  • Meissner et al . (7) have described an attempt to modulate pol III expression from a type III promoter. They mutated the TATA-like box of a human U6 gene promoter so that it was incapable of binding the endogenous transcription factor TBP but acquired the ability to bind a mutant form of TBP. However, this approach is limited by the need to supply the mutant TBP into the expression system.
  • RNA interference RNA interference
  • EBERl and EBER2 are non-polyadenylated, non-translated RNAs of Epstein-Barr virus (EBV) .
  • the EBER genes are transcribed by pol III (9) but do not fall neatly into any of the 3 pol III promoter types. For instance, they have A and B blocks which are characteristic of type II promoters and are essential for transcription (See figure 1) .
  • efficient transcription of the EBER genes also requires 3 upstream elements: a TATA-like box (TGTA, -28 to -23, relative to the start site for transcription for EBERl) an ATF binding site (-55 to -40 for EBERl) and an SpI binding site (-77 to -56 for EBERl) .
  • the ATF and SpI sites are usually associated with sequences transcribed by RNA polymerase II (5)
  • Successive deletions of the 3 upstream elements showed a significant reduction in EBERl transcription (10), thus suggesting that both pol II and pol III elements are necessary for optimal EBER transcription.
  • tissue-specific RNA pol III transcription may be achieved by combining RNA pol III regulatory elements with transcriptional regulatory elements normally associated with pol II genes.
  • the present invention provides a nucleic acid molecule comprising an expression cassette, the expression cassette comprising a target sequence to be transcribed by RNA pol III operably linked to RNA pol III transcriptional regulatory elements, said expression cassette further comprising a pol II transcriptional regulatory element which provides selective transcription of genes by RNA pol II in a target cell or tissue, whereby said target sequence is selectively transcribed by RNA pol III in said target cell or tissue.
  • the nucleic acid molecule (or "construct") of the invention is typically a DNA molecule. It comprises a target sequence to be transcribed into RNA by RNA pol III.
  • the target sequence will typically be operably linked at least to one or more pol III promoter elements and a pol III terminator element in order that the target sequence can be transcribed by pol III.
  • the nucleic acid molecule further comprises a pol II transcriptional regulatory element.
  • the pol II element is capable of conferring tissue- specific expression on genes transcribed by RNA pol II.
  • the present inventors have found that, by combining such a regulatory element with a pol III promoter, tissue specific pol III transcription may be achieved.
  • the pol II transcriptional regulatory element is a pol II enhancer element.
  • it may bind to a transcription factor capable of stimulating transcription of the target sequence by pol III in the target cell or tissue.
  • the transcription factor is endogenous to the target cell or tissue.
  • the enhancer may provide B cell specific expression.
  • the enhancer may be an immunoglobulin E ⁇ enhancer.
  • the E ⁇ enhancer used in the constructs described in the Examples was first described in reference 17 and has the sequence:
  • RNA pol III transcriptional regulatory elements of the construct may consists entirely of intragenic or downstream elements, located downstream of the transcriptional initiation site.
  • the construct may comprise an A block.
  • the construct further comprises either a B block or C block.
  • Constructs comprising an A and C box preferably also comprise an Intermediate Element (IE) located between the two.
  • IE Intermediate Element
  • the pol III transcriptional regulatory elements may further comprise one or more upstream elements, which may include a TATA-like box.
  • the pol III regulatory elements may consist entirely of upstream elements.
  • the construct may comprise at least a TATA-like box.
  • the construct will also comprise either a Proximal Sequence Element (PSE) , a Distal Sequence Element (DSE), or both.
  • PSE Proximal Sequence Element
  • DSE Distal Sequence Element
  • the pol III regulatory elements may comprise some or all elements of an EBER promoter. Preferably these include the A and B blocks (located downstream of the initiation site) , and preferably also the TATA-like box (located upstream of the initiation site) . The SpI and/or the ATF binding sites may also be included as required.
  • EBER promoter elements examples include:
  • the target sequence may encode a RNA molecule capable of inhibiting the expression of a target gene in the same cell.
  • the encoded RNA molecule may be an antisense RNA molecule, RNAi molecule or ribozyme.
  • the encoded RNA molecule is capable of inhibiting EBER expression in an Epstein Barr Virus (EBV)- infected cell, and is expressed specifically in B cells.
  • EBV Epstein Barr Virus
  • Such constructs may be used for the treatment of conditions associated with EBV infection, and in particular with EBV- associated tumours.
  • EBV-associated tumours include Burkitt's lymphoma (BL), which is common in children in equatorial Africa, and nasopharyngeal carcinoma (NPC) , which is common in adult men in Southern China and in ethnically related populations.
  • BL Burkitt's lymphoma
  • NPC nasopharyngeal carcinoma
  • EBV-associated cancers of the Western world include Hodgkin' s disease (HD) and certain AIDS related B-cell lymphomas.
  • HD Hodgkin' s disease
  • AIDS related B-cell lymphomas In these EBV-infected tumours, the virus expresses different genes. In all tumour cells described, two small non-coding, RNA pol III transcribed genes called EBERl and EBER2 are expressed. It is believed that the function of these genes in viral infection is a defence mechanism against the immune system, inhibiting interferon induction, thereby allowing infected cell survival.
  • EBERl binds to and inhibits PKR, a protein kinase capable of inducing interferon synthesis when activated by double stranded RNA.
  • PKR a protein kinase capable of inducing interferon synthesis when activated by double stranded RNA.
  • the invention provides the use of a nucleic acid as described above in the preparation of a medicament for the
  • nucleic acid construct as described herein for use in a method of medical treatment.
  • This aspect of the invention is not limited to the treatment of conditions associated with EBV infection, but extends to any condition which may be treated by expression of a suitable RNA (e.g. an antisense, RNAi, siRNA or ribozyme molecule) in a target cell.
  • a suitable RNA e.g. an antisense, RNAi, siRNA or ribozyme molecule
  • an expression vector comprising a nucleic acid construct as described herein.
  • the vector may be a linear molecule (e.g. one designed for homologous recombination into a host cell chromosome) , a plasmid or a viral vector (e.g. a retroviral or adenoviral vector) .
  • the invention provides a cell comprising a nucleic acid or an expression vector as described herein.
  • non-human transgenic animal comprising a nucleic acid or an expression vector as described herein.
  • the invention further provides a method of modulating transcription of a target sequence operably linked to RNA pol III transcriptional regulatory elements, the method comprising operably linking said target sequence to a pol II transcriptional regulatory element which provides selective transcription of genes by RNA pol II in a target cell or tissue, whereby said target sequence is selectively transcribed by RNA pol III in said target cell or tissue.
  • the method may be performed by introducing a pol II regulatory element into the chromosome of a cell, e.g. by homologous recombination.
  • the target sequence may, for example, be a gene endogenous to the cell which is already transcribed by pol III. Introduction of the pol II regulatory element then modulates expression of that gene, such that it is transcribed in a tissue-specific manner.
  • the method may further comprise the step of generating a non- human transgenic animal from the resulting cell.
  • Figure 1 shows three variant constructs comprising an E ⁇ enhancer linked to the EBERl gene.
  • Constructs A and B comprise all regulatory sequence elements found in wild type EBERl, including the SpI binding site, ATF binding site and TGTA box located upstream of the transcriptional initiation site, and the A and B boxes located downstream.
  • construct A p670
  • construct B p671
  • construct C p672
  • the SpI and ATF sites are deleted and the E ⁇ enhancer is placed adjacent the TGTA box. All three constructs have been shown to express in B-cells in culture.
  • construct B Tissue-specific expression of construct B has been confirmed in vivo in transgenic mice.
  • Transgenic mice carrying constructs A and C have also been successfully generated and confirmed to express the respective constructs.
  • Figure 3 shows a Northern blot of mRNA extracted from mice positive and negative for the p671 transgene. Tissues from 4 month old positive and negative siblings were collected. RNA was extracted from the different positive and negative lymphoid tissues as described in section 4 of experimental procedures. For small tissues, such as MLNs, PLNs, and Peyer' s patches, tissues from 2 positive and 2 negative animals were pooled for the RNA extraction. DNAse I treatment was performed on lO ⁇ g of RNA followed by acid phenol extraction. An RT-PCR was performed on 2 ⁇ g of RNA as described in section 4 of experimental procedures. A control for the PCR reaction was performed using an EBERl plasmid as template.
  • RT-PCR products Half of the RT-PCR products was electrophoresed through an agarose gel followed by Southern blotting. For the PCR control, one tenth of the reaction was used. The Southern blot was hybridised with an EBERl probe. In the negative tissues, faint background signal may be seen. In the transgenic positive tissues, expression is evident in spleen, BM, MLNs, PLNs, P.Patches and thymus, but not in liver or other tissues (data not shown) .
  • Figure 4 illustrates the use of p670 (see Figure 1) to carry a siRNA capable of targeting EBER 1.
  • the transcribed EBER 1 sequence of p670 is replaced with a sense sequence targeting the start and A box of EBER 1, followed by a loop and an antisense sequence.
  • a schematic diagram is shown in Panel A.
  • the sequence itself is shown in Panel B. Transcribed sequence derived from EBERl is underlined. Within the transcribed region, the B box of EBERl is shown in italics.
  • the siRNA sequence is shown in normal capitals; the bold font shows those parts of the siRNA sequence which will hybridise to one another Upstream of the transcribed region, the SpI, ATF and TGTA box of EBERl are shown in italicised capitals.
  • the sequence of the E ⁇ enhancer is shown in Panel C.
  • RNA pol III is located in the nucleoplasm and synthesises the precursors of 5S ribosomal RNA, tRNAs and other small nuclear and cytosolic RNAs.
  • the pol III promoter elements used in the constructs of the invention may be derived from the promoter of any gene transcribed by pol III. These include, but are not limited to, type I, II and III pol III promoters. A naturally-occurring pol III promoter or a portion or fragment thereof may be used. Alternatively, a pol III promoter may be assembled de novo using individual promoter elements.
  • a type I promoter typically comprises an A block, a C block and an Intermediate Element (IE) located between them. All of these elements are located intragenically, i.e. downstream of the transcriptional initiation site.
  • Type I promoters are found, for example, in 5S ribosomal RNA genes from Xenopus laevis and Drosophila. They are thought to be unique to 5S rRNA genes.
  • a typical type II promoter comprises an A block and a B block, also located intragenically, i.e. downstream of the transcriptional initiation site.
  • the A block is homologous to that of the type I promoter; the location of the B block is variable.
  • Type II promoters are the most common pol III promoter type. Examples can be found in most tRNA genes, adenoviral VA genes, and many families of eukaryotic repetitive sequence including AIu repeats.
  • a typical type III promoter comprises a TATA-like box, a Proximal Sequence Element (PSE) and a Distal Sequence Element (DSE) , all of which are located upstream of the transcriptional start site.
  • PSE Proximal Sequence Element
  • DSE Distal Sequence Element
  • U6 snRNA genes The most well-characterised type III promoter is associated with a human U6 snRNA gene.
  • Example include the EBV EBER 1 and 2 promoters. These promoters possess intragenic A and B blocks, as seen in type II promoters, which are essential for transcription. They also possess three elements upstream of the transcriptional start site, namely a TATA-like box (TGTA) , an ATF binding site and an SpI binding site.
  • TGTA TATA-like box
  • a pol III promoter in a construct of the present invention may have numerous configurations. For example, it may consist entirely of elements located downstream of the transcriptional start site. These elements may be intragenic, or may extend downstream of the transcriptional termination site, particularly when the transcript is relatively short. Alternatively, they may consist entirely of upstream elements, or may comprise a combination of both element types.
  • a promoter consisting entirely of downstream regulatory elements normally comprises at least an A block. Typically, it further comprises either a B block or C block. Constructs comprising an A and C box preferably also comprise an Intermediate Element located between the two.
  • a promoter comprising both downstream and upstream elements typically comprises some or all of the elements of an intragenic promoter as described above, in combination with one or more upstream elements, which may include a TATA-like box, a PSE and/or a DSE.
  • a promoter may consist entirely of upstream elements.
  • Such promoters preferably comprise at least a TATA- like box. They may also comprise either or both of a PSE and a DSE. Additionally or alternatively, A, B and/or C blocks may be functional when placed upstream of the transcriptional start site.
  • the precise configuration of the promoter is not critical for the present invention, as long as the target sequence is capable of being selectively transcribed by pol III in the target tissue or cell type when the promoter is supplemented by the pol II regulatory element.
  • selective transcribed is meant that the transcription is specific to that tissue, rather than being constitutive, throughout all (or substantially all) cells or tissues of the organism.
  • the construct will also contain other features necessary for pol III transcription, such as a pol III termination site.
  • a string of 4 or 5 U residues in the transcript is sufficient to serve as a termination signal.
  • Pol III transcripts are not polyadenylated and typically do not encode proteins. Thus they are readily distinguished from pol II transcripts, which are all polyadenylated apart from histone- encoding transcripts. Whether a transcript is produced by pol III or pol I can be determined by testing the sensitivity of transcription to the presence of ⁇ -amanitin. Pol III transcription is inhibited by ⁇ - amanitin (as is pol II transcription) ; however pol I transcription is unaffected by ⁇ -amanitin.
  • Genes transcribed by pol III are generally expressed constitutively, in substantially all cells and tissues.
  • the present invention provides tissue-specific pol III transcription by adding a pol II transcriptional regulatory element to the basal pol III promoter.
  • the constructs of the invention comprise a pol II regulatory element.
  • pol II is also located in the nucleoplasm. However, pol II is responsible for synthesising the precursors of all mRNAs from protein-coding genes, as well as some small nuclear RNAs. Pre-mRNAs generated by pol II are processed by cap formation, polyadenylation and splicing, as is well known to those skilled in the art.
  • Genes transcribed by pol II have a number of regulatory sequences which control initiation of transcription.
  • Many pol II promoters have a TATA box (consensus sequence TATA(A/T)A(A/T) ) located 25 to 30 bases upstream of the initiation site.
  • Some genes instead possess an initator element overlapping the transcriptional start site. These elements are required for formation of the transcription complex and initiation of transcription.
  • Other sequence elements are often required for efficient pol II transcription. These are often located within 100 to 200 bases of the initiation site. Examples include the SPl and CCAAT boxes, as well as the ATF box found in the EBER promoters.
  • pol II promoter Together with the TATA box or initiation element, these may be regarded as the pol II promoter. These generally do not control transcription in a tissue specific way and are generally not suitable for use as pol II regulatory elements in the context of the present invention.
  • pol II genes also possess other regulatory elements, including repressor and enhancer sequences. Certain of these elements confer tissue-specific regulation on the transcription of the genes to which they are linked. Most if not all tissues express structural genes characteristic of themselves, which are either not expressed, or are expressed only at very low levels, in other tissues. Their transcription by pol II is controlled by tissue specific regulatory elements.
  • tissue specific regulatory elements For example, the human immunoglobulin heavy chain enhancer E ⁇ is capable of directing pol II transcription of a gene specifically in the B cell compartment.
  • pol II elements used in the constructs of the invention are capable of conferring tissue-specific expression on a gene transcribed by pol II. Numerous examples of such elements are known.
  • tissue-specific transcription or tissue-specific expression is meant that the transcription occurs at significant levels in one or more particular (“target”) cell or tissue types, but at low levels or not at all in others.
  • the level of transcription in the target cell or tissue type is at least 5 fold, and preferably at least 10, 100 or 1000 fold higher than the level of transcription in one or more other cell or tissue types of the organism. More preferably, the level of transcription is at least 5, 10, 100 or 1000 fold higher than the level of transcription in substantially any other cell or tissue type of the organism.
  • transcription of the target sequence is not detectable (above background levels) in cells or tissues other than the target type.
  • the level of transcription may be determined by Northern blot, RT-PCR, in situ hybridisation or any other appropriate technique.
  • the pol II element may function in a number of different ways. It may repress or inhibit transcription in cells or tissues other than the target cell or tissue type. In such cases the pol III promoter may be capable of driving constitutive pol III transcription of the target sequence in the absence of the pol II element. Such elements may be regarded as repressors.
  • the pol II element may stimulate transcription in the target cell or tissue type.
  • it may bind to one or more transcription factors expressed preferentially (or exclusively) in the target cell or tissue.
  • the pol II element increases the basal level of expression from the pol III promoter.
  • the pol III promoter is constructed such that the pol II element is required for pol III transcription to occur.
  • a pol III promoter is used which is insufficient to drive pol III transcription in the target cell or tissue in the absence of the pol II element, but where the combination of pol III promoter and pol II element is capable of driving pol III transcription.
  • the pol II element may work by a combination of the above mechanisms.
  • it may inhibit transcription in cells and tissues other than the target cell or tissue, while also stimulating transcription in the target cell or tissue.
  • the E ⁇ enhancer works in this way, inhibiting expression in non-lymphoid tissue while stimulating expression in lymphoid tissue.
  • API binding sites are found in many pol II enhancers. Binding of a c-Jun/c-Fos dimer (expressed in certain cells) to the site may result in transcriptional activation of a linked gene. By contrast, binding of JunB (expressed in different cells) in place of c-Jun may lead to repression.
  • Pol II elements which stimulate transcription in the desired tissue/cell type (whether or not they also inhibit transcription in other cells and tissues) may be regarded as pol II enhancers.
  • tissue specific pol II regulatory elements such as enhancers, contain binding sites for multiple factors which interact in complex ways in order to achieve tissue-specific transcription.
  • the pol II regulatory elements for use in the present invention may be composed of a plurality of binding sites capable of binding to a number of different protein factors.
  • the term "pol II regulatory element” should be construed as referring to the minimal nucleic acid sequence required to confer tissue specific expression on a gene transcribed by pol II.
  • the pol II element may be endogenous to the cell. That is to say, at least one copy of the element is found elsewhere in the genomic DNA of the cell (or in the wild type genomic DNA of the species from which the cell is derived) , operably linked to a gene transcribed by pol II to provide tissue-specific expression of that gene.
  • pol II elements which are not endogenous to the cell may also be used.
  • enhancer elements from other species may be used, as these elements often display a high level of evolutionary conservation and so function in other species. Mutants or derivatives of naturally-occurring sequences may also be used as long as they retain their function.
  • Pol II elements derived from viruses e.g. the cytomegalovirus enhancer
  • transcription of viral genes in host cells often shows remarkable tissue specificity.
  • Prokaryotic elements may also be linked to pol II elements in order to confer conditional expression. Where the pol II element binds one or more transcription factors which stimulate transcription of the target sequence, that transcription factor is preferably endogenous to, and naturally expressed in, that cell or tissue (i.e. the transcription factor is expressed there without the need for genetic manipulation) .
  • constructs of the invention may be used in any context in which it is desirable to produce a pol III transcript in a tissue-specific manner.
  • Pol III termination is highly specific, and does not result in a polyadenylated transcript, unlike the majority of pol II transcripts. These properties make pol III transcripts extremely useful, for example, as antagonists of gene expression.
  • Such antagonists typically have one or more stretches of sequence complementary to the DNA encoding the gene to be downregulated and/or its mRNA or pre-mRNA transcript. They include antisense RNA molecules, RNAi molecules and ribozymes. See, e.g. ref. 16 for examples of RNAi and other molecules which have been expressed under the control of pol III promoters.
  • Antisense oligonucleotides hybridise with complementary sequences of RNA generally by Watson-Crick base pairing.
  • the resultant double stranded complex prevents translation of the message into protein product either by steric blocking at the ribosome or activation of RNase H that cleaves the RNA strand of the duplex.
  • Molecules capable of binding to the translation initiation site of the coding DNA e.g. between the -10 and +10 regions of the target gene, may also be used.
  • the complete sequence corresponding to the coding sequence need not be used. For example fragments of sufficient length may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding sequence to optimise the level of antisense inhibition. It may be advantageous to include the initiating methionine ATG codon, and perhaps one or more nucleotides upstream of the initiating codon. A further possibility is to target a conserved sequence of a gene, e.g. a sequence that is characteristic of one or more genes, such as a regulatory sequence.
  • the sequence employed may be 500 nucleotides or less, possibly about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, or about 100 nucleotides. It may be possible to use oligonucleotides of much shorter lengths, 14-23 nucleotides, although longer fragments, and generally even longer than 500 nucleotides are preferable where possible.
  • sequence employed in a down-regulation of gene expression in accordance with the present invention may be a wild-type sequence (e.g. gene) selected from those available, or a mutant, derivative, variant or allele, by way of insertion, addition, deletion or substitution of one or more nucleotides, of such a sequence.
  • the sequence need not include an open reading frame or specify an RNA that would be translatable.
  • RNA interference is a two step process. First, dsRNA is cleaved within the cell to yield short interfering RNAs (siRNAs) of about 21-23nt length with 5' terminal phosphate and 3' short overhangs ( ⁇ 2nt) The siRNAs target the corresponding mRNA sequence specifically for destruction (Zamore P.D. Nature Structural Biology, 8, 9, 746-750, (2001) . See also Fire (1999) Trends
  • Figure 4 illustrates a construct based on p670 ( Figure 1) which uses the EBV EBER 1 promoter, supplemented by a pol III E ⁇ enhancer, to express a siRNA targeting EBER 1. This construct will direct expression to lymphoid cells.
  • Ribozymes are enzymatic RNA molecules capable of catalysing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridisation of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage.
  • the composition of ribozyme molecules must include one or more sequences complementary to the target protein mRNA, and must include the well known catalytic sequence responsible for mRNA cleavage. For this sequence, see US Pat. No. 5,093,246, which is incorporated by reference herein in its entirety.
  • engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyse endonucleolytic cleavage of RNA sequences encoding target proteins.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the molecule of interest for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short sequences of between 15 and 20 ribonucleotides corresponding to the region of the target protein gene containing the cleavage site may be evaluated for predicted structural features, such as secondary structure, that may render the oligonucleotide sequence unsuitable. The suitability of candidate sequences may also be evaluated by testing their accessibility to hybridise with complementary oligonucleotides, using ribonuclease protection assays.
  • the constructs may be used to create transgenic animals.
  • a nucleic acid construct according to the invention may be introduced into the genomic DNA of the animal in order to obtain an animal which displays tissue-specific, pol Ill-driven expression of the target sequence. This raises the possibility of creating animals in which expression of desired genes is inhibited in a tissue-specific manner, by use of target sequences encoding appropriate antisense, RNAi or ribozyme molecules.
  • a suitable pol II regulatory element may be introduced (e.g. by homologous recombination) such that it becomes operably linked to a pol III promoter already present in the genomic DNA of the subject in order to create a construct as described herein.
  • a suitable pol II regulatory element may be introduced (e.g. by homologous recombination) such that it becomes operably linked to a pol III promoter already present in the genomic DNA of the subject in order to create a construct as described herein.
  • constructs are not limited to inhibition of gene expression. It may be desirable to express pol III transcripts for other purposes. For example, pol III transcripts which mimic elements of the HIV genome have been proposed for therapeutic use. These molecules would serve as "decoy" RNAs, competing for binding proteins and so preventing the proteins from binding the viral genome to perform their function in viral replication.
  • This technology may be applied as desired to any appropriate non- human animal subject, whether vertebrate or invertebrate, including trypanosomes, insects (e.g. Drosophila) , worms (e.g. Caenorhabditis elegans) , fish (e.g. zebrafish) , reptiles, amphibians (e.g., frogs and toads, especially Xenopus laevis) and mammals, including rodents (mice, rats, guinea pigs, hamsters) lagomorphs (e.g. rabbits), domestic and agricultural animals (e.g.
  • insects e.g. Drosophila
  • worms e.g. Caenorhabditis elegans
  • fish e.g. zebrafish
  • amphibians e.g., frogs and toads, especially Xenopus laevis
  • mammals including rodents (mice, rats, guinea
  • dogs, cats, horses, sheep, goats, cows, and primates including monkeys (e.g., marmoset, baboon) and apes (e.g., gorilla, chimpanzee, orangutang, gibbon) .
  • monkeys e.g., marmoset, baboon
  • apes e.g., gorilla, chimpanzee, orangutang, gibbon
  • human cells may be manipulated in vitro using the constructs and methods described herein, and that humans may be treated using the therapeutic methods described.
  • constructs of the invention may find use in methods of therapy, and so may be formulated as pharmaceutical compositions.
  • compositions may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes or topical application.
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may include a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • Administration is preferably in a "prophylactically effective amount” or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual.
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Suitable carriers, adjuvants, excipients, etc. can be found in standard pharmaceutical texts, for example Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.
  • the constructs of the invention may be used in methods of gene therapy, and may be introduced into cells of a recipient by any suitable means, such that the target sequence is transcribed by pol III in the desired cells.
  • the construct may be introduced in the form of naked DNA, which is taken up by some cells of animal subjects, including muscle cells of mammals.
  • the construct will generally be carried by a pharmaceutically acceptable carrier alone.
  • the construct may also encapsulated, e.g. by formulation in an inert polymer or a liposome particle.
  • Such methods of gene therapy further include the use of recombinant viral vectors such as adenoviral or retroviral vectors which comprise a construct of the invention.
  • viral vectors may be delivered to the body in the form of packaged viral particles.
  • the viral vectors may themselves be targeted to the appropriate cells.
  • Gene therapy methods are widely documented in the art and may be adapted for use in the expression of the required sequence.
  • PCR reactions were performed on pLexIII plasmid containing EBERl sequence (gift from Dr Mike Clemens) with Pfu polymerase enzyme.
  • the cycling conditions are shown in the table below:
  • the first EBERl construct contains the upstream sequences of EBERl (to -322) and EBERl (called p670, using CRl and CR4 primers) .
  • the second has only the upstream elements required for EBERl expression (SpI, ATF and the TGTA box) and EBERl (called p671, using CR2 and CR4 primers) and the third construct has only the TGTA box of EBERl and EBERl sequence (called p672, using CR3 and CR4 primers) (see figure 1) .
  • the PCR products were digested with £coRI and Xhol and then ligated into pcDNA3.1A, which was linearised with EcoRI and Xhol enzymes (other commercially available vectors could equally be used as the plasmid backbone) .
  • the new plasmids were transformed into DH5 cells. After performing a minipreparation of plasmid DNA from several colonies the plasmids with the correct restriction pattern were sequenced to confirm that the correct product had been obtained. An EndoFree preparation of the plasmid DNA was performed according to the manufacturer's instructions (Qiagen) .
  • Xbal linear fragments were used as transgenes for microinjection of zygotes at 2 and 5 ⁇ g/ml.
  • B6D2.Fl mouse embryos 48 and 24 hours prior to microinjections B6D2.F1 mice were injected with 5 units of pregnant mare's serum gonadotropin (PMS) and chorionic gonadotropin (hCG) respectively. The mice were set up with B6D2.F1 stud males the day before microinjections. Embryos were harvested and placed in M2 medium (Sigma) and at
  • Mouse tail DNA was isolated by digestion with proteinase K and tail solution at 55 0 C shaking overnight and the genomic DNA was purified according to the protocol described by Wilson et al.
  • the DNA was then digested with the appropriate restriction enzyme and electrophoresed through an agarose gel.
  • RNA extraction, Northern blots and reverse-transcriptase (RT)-PCR 4.
  • RT-PCR products were electrophoresed on a 2% agarose gel and generally followed by Southern blotting using an EBERl probe.
  • Quantitative-RT-PCR (QPCR):
  • Quantitative PCR was usually performed following an RT reaction.
  • the SYBRGreen kit from ABgene was used and QPCR was performed using CR8 and CR9 EBERl primers and l/5 th of the RT reaction as template.
  • the cycling conditions were the same as the ones described in 4 except for a hot start of 15 minutes instead of 5 and a plate read analysis of the samples after each cycle.
  • a standard curve was also performed alongside the samples. Each sample for the standard curve had a known quantity of EBERl template. From the standard curve equation the initial quantity of cDNA in each sample could be calculated.
  • the reactions were performed on the DNA engine OpticonTM from MJ Research and the results analysed using Opticon programme.
  • the murine B cell line used for transient transfections of the different EBERl constructs was generated in the lab from an EBV latent gene-positive tumour and is referred to as 39.415.
  • the cells were grown in RPMI medium complemented with 10% FCS, 2% penicillin/streptomycin and 2% 1-glutamine (Gibco) .
  • the cells were incubated at 37°C and 5% CO 2 . They were passaged every 2 to 3 days.
  • transient transfections 10 7 cells were centrifuged and resuspended in 250 ⁇ l of serum free medium (SFM) . 5 ⁇ g of DNA was used for the transfections and added to a final volume of 50 ⁇ l of SFM and placed in a 4mm cuvette (CLP) . The cells were then added and mixed to the DNA in the cuvette, which was placed on ice for 5 minutes. Electroporation was performed using the Gene Pulser® electroporation apparatus (Biorad) . The parameters used for the transfection were 250V and 960 ⁇ F. Following electroporation the cells were placed at 37°C for 10 minutes and then added to 10ml of medium. The cells were either harvested at 24 or 48 hours post transfection.
  • SFM serum free medium
  • CLP 4mm cuvette
  • EBERl constructs Three different EBERl constructs were designed to test if spacing between the elements is critical and if the SPl and ATF sites are essential for efficient expression.
  • the vector backbone used was pcDNA3.
  • IA Invitrogen
  • the immunoglobulin heavy chain (IgH) enhancer E ⁇ enhancer (17) was cloned upstream of the three different EBERl constructs (see figure 1) as described in experimental procedures (section 1) to direct the expression to the B cell compartment. This enhancer has been previously used in the laboratory to direct the expression of pol II transcripts encoding several heterologous genes to the B-cell compartment (1, 11, 13) .
  • the TGTA box of EBERl was maintained in all constructs as it has been shown that this element is important for EBER2 transcription by RNA pol III (6) and therefore may also be critical for EBERl expression.
  • the plasmids containing the different EBERl constructs were sequenced before the preparation of the transgene to check that no mutations had been generated during construction.
  • the transgene was prepared using endotoxin free plasmid DNA preparations so as not to trigger an immune response in tissue culture assays or in vivo, which could obscure any effect due to EBERl expression.
  • each construct was tested in culture.
  • the different constructs were transiently transfected into EBER-negative B cells.
  • RNA extraction the level of expression of EBERl in the three different constructs was determined by reverse transcriptase PCR (RT-PCR) , quantitative- RT-PCR (QPCR) and Northern blots. All three assays showed expression of the different constructs in culture (figure 2) .
  • Pronuclear microinjections were performed using B6D2.F1 mouse embryos and the surviving embryos were implanted in pseudopregnant ICR females. 6 founders for transgene 670, 3 founders for 671 and 10 founders for 672 were generated. Several of the founders have been bred to generate lines which are currently under investigation for expression analysis of the transgene and phenotype. Initially, 2 of the lines carrying the p671 transgene were checked for expression of the transgene. RNA extraction from collected tissues from transgenic positive mice and negative siblings was followed by RT-PCR.
  • the expression in the lines is specific to the lymphoid tissues and the brain (figure 3) and no other tissues tested (liver, muscle, ovaries, uterus, testis, kidneys, small intestine, stomach, heart, lung, trachea, oesophagus, tongue, nasopharyngeal region, dorsal skin, ears, salivary gland) .
  • This demonstrates that the combination of pol II and pol III elements is functional and that the pol II E ⁇ enhancer is compatible with pol III elements.
  • Two of three lines carrying p670 and 6 of 8 lines carrying p672 have also been shown to express their respective transgenes.
  • RNA pol III genes are abundantly and ubiquitously expressed. Therefore use of these promoters with heterologous genes in transgenic animals or in DNA vaccination may well be lethal or undesirable. Several experimental approaches might require a targeted expression of the gene of interest. This is especially true in therapeutic protocols. Therefore we have developed a vector combining pol II and pol III elements to express a pol III gene in a tissue specific manner. In a proof of concept we have used this vector to express EBERl in B cells in culture and in the lymphoid tissues of transgenic mice.
  • a different pol II tissue specific enhancer element could be used, simply by switching the E ⁇ motif with another in order to direct the expression of the gene of interest to a different tissue.
  • a different gene could be used in the vector; for instance, other pol III genes with A and B boxes could readily replace the EBERl sequence.
  • RNAi RNA interference
  • RNAi is a process through which exposure of dsRNA leads to the silencing of the homologous gene, most often post-transcriptionally (4) .
  • RNA silencing was first observed in plants and this phenomenon was termed post- transcriptional gene silencing (PTSG) .
  • PTSG post- transcriptional gene silencing
  • RNAi is a widespread phenomenon which has been intensively used to characterise the functions of genes in Caenorhabditis elegans, Drosophila and trypanosomes. RNAi can be performed in mammalian cells using 21- nucleotide siRNA duplexes which do not induce an interferon response (3) .
  • RNAi transgenes expressing short hairpin RNA could be used as an alternative to generating mouse knock-out (or more accurately, "knock-down") models.
  • tissue specificity would be desirable.
  • RNAi has enormous therapeutic potential and could be used in vivo to silence disease or other deleterious genes.
  • the siRNA sequence of the target gene could be inserted before and after the B box of EBERl using the B box sequence as the loop of the "hairpin".
  • Epstein-Barr virus small RNA (EBER) genes unique transcription units that combine RNA polymerase II and III promoter elements. Cell 57:825-34.
  • a lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes. Cell 33:729-740.

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Abstract

L'invention concerne des matières et des méthodes permettant la transcription spécifique du tissu de séquences d'ADN souhaitées par l'ARN polymérase III. Des éléments régulateurs transcriptionnels qui dirigent la transcription spécifique du tissu par l'ARN polymérase II sont combinés à des éléments régulateurs transcriptionnels de promoteurs qui dirigent la transcription par l'ARN polymérase III, afin d'obtenir une transcription spécifique du tissu par l'ARN polymérase III. L'invention concerne également des constructions comprenant lesdits promoteurs hybrides, particulièrement utiles pour l'expression de molécules d'ARN telles que des ARNsi et des ribozymes.
PCT/GB2005/003599 2004-09-17 2005-09-19 Transcription specifique du tissu par l'arn polymerase iii WO2006030237A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003023015A2 (fr) * 2001-09-13 2003-03-20 California Institute Of Technology Procede d'expression de petites molecules d'arn antivirales a l'interieur d'une cellule

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003023015A2 (fr) * 2001-09-13 2003-03-20 California Institute Of Technology Procede d'expression de petites molecules d'arn antivirales a l'interieur d'une cellule

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
CHESNOKOV I ET AL: "Flanking sequences of an Alu source stimulate transcription in vitro by interacting with sequence-specific transcription factors.", JOURNAL OF MOLECULAR EVOLUTION. JAN 1996, vol. 42, no. 1, January 1996 (1996-01-01), pages 30 - 36, XP009056110, ISSN: 0022-2844 *
GABRIELSEN O S ET AL: "RNA polymerase III (C) and its transcription factors.", TRENDS IN BIOCHEMICAL SCIENCES. NOV 1991, vol. 16, no. 11, November 1991 (1991-11-01), pages 412 - 416, XP002352131, ISSN: 0968-0004 *
IMLER J L ET AL: "Negative regulation contributes to tissue specificity of the immunoglobulin heavy-chain enhancer.", MOLECULAR AND CELLULAR BIOLOGY. JUL 1987, vol. 7, no. 7, July 1987 (1987-07-01), pages 2558 - 2567, XP009056303, ISSN: 0270-7306 *
MARTIGNETTI J A ET AL: "BC1 RNA: transcriptional analysis of a neural cell-specific RNA polymerase III transcript.", MOLECULAR AND CELLULAR BIOLOGY. MAR 1995, vol. 15, no. 3, March 1995 (1995-03-01), pages 1642 - 1650, XP002352127, ISSN: 0270-7306 *
MURPHY S ET AL: "Common mechanisms of promoter recognition by RNA polymerases II and III.", TRENDS IN GENETICS : TIG. APR 1989, vol. 5, no. 4, April 1989 (1989-04-01), pages 122 - 126, XP002352128, ISSN: 0168-9525 *
ROY A M ET AL: "Upstream flanking sequences and transcription of SINEs", JOURNAL OF MOLECULAR BIOLOGY, LONDON, GB, vol. 302, no. 1, 8 September 2000 (2000-09-08), pages 17 - 25, XP004469113, ISSN: 0022-2836 *
WEINBERGER J ET AL: "Localization of a repressive sequence contributing to B-cell specificity in the immunoglobulin heavy-chain enhancer.", MOLECULAR AND CELLULAR BIOLOGY. FEB 1988, vol. 8, no. 2, February 1988 (1988-02-01), pages 988 - 992, XP009056302, ISSN: 0270-7306 *

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