Patent Application
20080026985
I. Field of the Invention
The present invention relates to the control of gene expression and, in particular, it relates to methods of, and means for, suppressing the expression of a particular, selected gene.
II. Related Art
The ability to selectively suppress the expression of a gene is useful in many areas of biology, for example in methods of treatment where the expression of the gene may be undesirable; in preparing models of disease where lack of expression of a particular gene is associated with the disease; in modifying the phenotype in order to produce desirable properties. Thus, the ability to selectively suppress the expression of a gene may allow the “knockout” of human genes in human cells (whether wild type or mutant) and the knockout of eukaryotic genes in studies of development and differentiation.
Present methods of attempting to suppress the expression of a particular gene fall into three main categories, namely antisense technology, ribozyme technology and targeted gene deletion brought about by homologous recombination.
Antisense techniques rely on the introduction of a nucleic acid molecule into a cell which typically is complementary to a mRNA expressed by the selected gene. The antisense molecule typically suppresses translation of the mRNA molecule and prevents the expression of the polypeptide encoded by the gene. Modifications of the antisense technique may prevent the transcription of the selected gene by the antisense molecule binding to the gene's DNA to form a triple helix.
Ribozyme techniques rely on the introduction of a nucleic acid molecule into a cell which expresses a RNA molecule which binds to, and catalyses the selective cleavage of, a target RNA molecule. The target RNA molecule is typically a mRNA molecule, but it may be, for example, a retroviral RNA molecule.
Antisense- and ribozyme-based techniques have proven difficult to implement and they show varying degrees of success in target gene suppression or inactivation. Furthermore, these two techniques require persistent expression or administration of the gene-inactivating agent.
Targeted gene deletion by homologous recombination requires two gene-inactivating events (one for each allele) and is not easily applicable to primary cells, particularly for example primary human mammary cells which can only be maintained in culture for a few passages. Targeted gene deletion has remained difficult to perform in plants. The cre-lox mediated site-specific integration has been the method of choice although the efficiency of specific integrative events is low (Alberts et al (1995) Plant J. 7, 649-659; Vergunst & Hooykass (1998) Plant Mol. Biol. 38, 393-406; Vergunst et al (1998) Nucl. Acids Res. 26, 2729-2734).
These major shortcomings in existing technology have led us to seek an alternative strategy.
Acute promyelocytic leukaemia (APL) is underlined by the involvement of mutant retinoic acid receptor (RAR) proteins, formed by gene fusions brought about by chromosomal translocations. Molecular analysis of one APL subset has identified a fusion between the RARα gene and a Kruppel-like zinc finger gene named promyelocytic leukaemia zinc finger (PLZF). Further investigations have shown that the resulting PLZF-RARα fusion protein functions as a gene repressor by targeting histone deacetylation of retinoic acid regulated genes. Several studies have shown that this repression is mediated by the PLZF portion of the fusion protein, which interacts with a complex of proteins which includes the components N-CoR, SMRT, Sin3 and HDAC and which in turn results in the recruitment of the histone deacetylase (HDAC) complex to target genes (see, for example, Grignani et al (1998) Nature 391, 815-818; Chen et al (1993) EMBO J. 12, 1161-1167; Razin (1998) EMBO J. 17, 4905-4908; David et al (1998) Oncogene 16, 2549-2556; and Lin et al (1998) Nature 391, 811-814). HDAC directed gene inactivation, therefore results from the targeted assembly of components, some of which have been identified (eg N-CoR, SMRT, Sin3 etc) making a gene inactivating complex which mediates its effect through histone deacetylation.
Although this work shows that in certain forms of APL fusion proteins are able to recruit histone deacetylase activity which appears to have the effect of inactivating the expression of certain genes, no-one has suggested that a method can be devised based on recruitment of histone deacetylase or other means of inactivating chromatin in order to selectively suppress expression of a chosen target gene or a set of genes. Surprisingly, we have shown that this can be achieved.
RARα-PLZF and RARα-PML fusion proteins are known from studies of acute promyelocytic leukaemia (APL) and are described in, for example, Grignani et al (1998) Nature 391, 815-818.
Fusions of GAL4 with a portion of PLZF protein, and LexA DNA binding domain fused to various fragment of Sin 3A are described in David et al (1998) Oncogene 16, 2549-2556 which, for the avoidance of doubt, are excluded from the polypeptide of the present invention.
Fusions of the GAL4 DNA binding domain and PLZF-RARα are described in Lin et al (1998) Nature 391, 811-814 which, for the avoidance of doubt, are excluded from the polypeptide of the present invention.
Fusions of the GAL4 DNA binding domain with N-CoR or portions thereof, or with the C terminal domain of the T3Rβ1 receptor molecule (thyroid hormone receptor molecule), and LexA DNA binding domain fused with the C terminal domain of the T3Rα or RARα (retinoic acid receptor) receptor molecules, which, for the avoidance of doubt, are excluded from the polypeptide of the present invention, are described in Hörlein et al (1995) Nature 377, 397-404. Fusions of the GAL4 DNA binding domain with the C terminal domain of vErbA (viral oncogene erbA of the avian erythroblastosis virus (AEV)), T3R and RAR receptor molecules are also mentioned. These polypeptides are also, for the avoidance of doubt, excluded from the polypeptide of the present invention.
There is no suggestion in David et al (1998) Oncogene 16, 2549-2556, Lin et al (1998) Nature 391, 811-814 or Hörlein et al (1995) Nature 377, 397-404 that polypeptides comprising a nucleic acid binding portion and a chromatin inactivation portion can be designed and engineered to bring about the selective suppression of a chosen gene. Rather, David et al (1998) and Lin et al (1998) are both studies of gene repression in acute promyelocytic leukaemia, and Hörlein et al (1995) relates to the identification of N-CoR.
A first aspect of the invention provides a polypeptide comprising a nucleic acid binding portion which binds to a site present in a eukaryotic genome and a chromatin inactivation portion provided that when the nucleic acid binding portion is a DNA binding portion of RARα the chromatin inactivation portion is not a portion of PLZF protein and is not a portion of PML protein; and provided that when the nucleic acid binding portion is a DNA binding portion of the Saccharomyces cerevisiae GAL4 protein the chromatin inactivation portion is not a portion of PLZF protein, the C-terminal domain of vErbA, T3R, T3β1 or RAR, or N-CoR or a portion of N-CoR; and provided that when the nucleic acid binding portion is a DNA binding portion of the Escherichia coli LexA the chromatin inactivation portion is not mSin3, or the C-terminal domain of T3Rα or RARα.
The polypeptides of the invention may be useful in methods and uses provided by further aspects of the invention, discussed in more detail below. In particular, the polypeptides of the invention may be useful in a method of suppressing the expression of a selected gene in a eukaryotic cell the method comprising introducing into the cell (a) a polypeptide comprising a nucleic acid binding portion which binds to a site at or associated with the selected gene which site is present in a eukaryotic genome and a chromatin inactivation portion, or (b) a polynucleotide encoding said polypeptide.
It is preferred if the polypeptides of the invention are hybrid polypeptides which do not occur in nature. For example, it is preferred if the nucleic acid binding portion is derived from one protein and that the chromatin inactivation portion is derived from a different protein and that the molecular configuration does not arise in nature, for example through chromosome translocation events. The proteins from which the nucleic acid binding portion and the chromatin inactivation portion are derived may be from the same species (for example, as is described in more detail below, the nucleic acid binding portion may be a DNA binding portion of a human steroid receptor protein such as oestrogen receptor (ER) and the chromatin inactivation portion may be a portion of human PLZF) or they may be from different species (for example a bacterial DNA binding protein may be fused to a portion of human PLZF).
Thus, in a particular preferred embodiment the polypeptide of the invention is one which is produced by genetic engineering means wherein the nucleic acid binding portion and the chromatin inactivation portion are selected as is described in more detail below.
It is preferred if the nucleic acid binding portion is not the Saccharomyces cerevisiae GAL4 protein or a DNA-binding portion thereof, and it is preferred if the nucleic acid binding portion is not the Escherichia coli LexA protein or a DNA-binding portion thereof.
In relation to the first aspect of the invention the site present in a eukaryotic genome is a site which is at or associated with a selected gene or genes whose expression it is desirable to suppress or inactivate. It is preferred if the site is a site which is naturally present in a eukaryotic genome. However, as is discussed in more detail below, the site may be one which has been engineered into the genome, or it may be a site associated with an inserted viral sequence. The site engineered into the genome to be in the vicinity of the gene whose expression is to be suppressed may be a site from the same species (but present elsewhere in the genome) or it may be a site present in a different species. By “genome” we include not only chromosomal DNA but other DNA present in the eukaryotic cell, such as DNA which has been introduced into the cell, for example plasmid or viral DNA. It is preferred if the nucleic acid binding portion can bind to chromosomal DNA or, as is described in more detail below, to RNA transcribed from chromosomal DNA.
The chromatin inactivation portion may be any polypeptide or part thereof which directly or indirectly leads to chromatin inactivation. By “directly” leading to chromatin inactivation we mean that the polypeptide or part thereof itself acts on the chromatin to inactivate it. By “indirectly” leading to chromatin inactivation we mean that the polypeptide or part thereof does not itself act on the chromatin but rather it is able to recruit or promote a cellular component to do so.
Chromatin inactivation generally results in the suppression or inactivation of gene expression. Chromatin inactivation is typically a localised event such that suppression or inactivation of gene expression is restricted to, typically, one or a few genes. Thus, the chromatin inactivation portion is any suitable polypeptide which, when part of the polypeptide of the invention and when targeted to a selected gene by the nucleic acid binding portion, locally inactivates the chromatin associated with the selected gene so that expression of the gene is inactivated or suppressed. Histone deacetylation is associated with chromatin inactivation and so it is particularly preferred if the chromatin inactivation portion facilitates histone deacetylation. Targeted deacetylation of histones associated with a given gene leads to gene inactivation in an, essentially, irreversible manner. By “suppression” or “inactivation” of gene expression we mean that in the presence of the polypeptide of the invention the expression of the selected, targeted gene is at least five-fold, preferably at least ten-fold, more preferably at least 100-fold, and most preferably at least 1000-fold lower than in the absence of the polypeptide of the invention under equivalent conditions. Gene expression can be measured using any suitable method including using reverse transcriptase-polymerase chain reaction (RT-PCR), RNA hybridisation, RNAse protection assays, nuclear run-off assays and alteration of chromatin as judged by DNAse 1 hypersensitivity.
In animal and plant cells histone deacetylation is brought about by the so-called histone deacetylase complex (HDAC) which contains, in addition to one or more histone deacetylase enzymes, ancillary proteins which are involved in the formation and function of the complex. In addition, there are other protein components which although they may not be part of HDAC they bind to or otherwise interact with HDAC and help facilitate histone deacetylation.
Deacetylation and acetylation of histones is a well-known phenomenon which is reviewed in the following: Chen & Li (1998) Crit. Rev. Eukaryotic Gene Expression 8, 169-190; Workman & Kingston (1998) Ann. Rev. Biochem. 67, 545-579; Perlmann & Vennstrom (1995) Nature 377, 387-; Wolfe (1997) Nature 387, 16-17; Grunstein (1997) Nature 389, 349-352; Pazin & Kadonaga (1997) Cell 89, 325-328; DePinho (1998) Nature 391, 533-536; Bestor (1998) Nature 393, 311-312; and Grunstein (1998) Cell 93, 325-328.
The polypeptide composition of the HDAC complex is currently under investigation. Polypeptides which may form part of, or are associated with, certain HDAC complexes include histone deacetylase 1 (HDAC1) Taunton et al (1996) Nature 272, 408-441); histone deacetylase 2 (HDAC2) (Yang et al (1996) Proc. Natl. Acad. Sci. USA 93, 12845-12850); histone deacetylase 3 (HDAC3) (Dangond et al (1998) Biochem. Biophys. Res. Comm. 242, 648-652); N-CoR (Horlein et al (1995) Nature 377, 397-404); SMRT (Chen & Evans (1995) Nature 377, 454-457); SAP30 (Zhang et al (1998) Molecular Cell 1, 1021-1031). Sin3 (Ayer et al (1995) Cell 80, 767-776; Schreiber-Agus et al (1995) Cell 80, 777-786) SAP18 (Zhang et al (1997) Cell 89, 357-364); and RbAp48 (Qian et al (1993) Nature 364, 648-652). All of these papers are incorporated herein by reference. It is believed that there may be further components of the HDAC complex or which interact with the HDAC complex which are, as yet, undiscovered. It is envisaged that these too will be useful in the practice of the invention.
PLZF has been shown to interact with N-CoR and SMRT, which in turn recruit a HDAC complex. PLZF will also directly interact with HDAC (Lin et al (1998) Nature 391, 811-814; Grignani et al (1998) Nature 391, 815-818; David et al (1998) Oncogene 16, 2549-2556).
Complexes formed which contain any of N-CoR, SMRT, Sin3, SAP18, SAP30 and histone deacetylase are described in Heinzel et al (1997) Nature 387, 43-48; Alland et al (1997) Nature 387, 49-55; Hassig et al (1997) Cell 89, 341-347; Laherty et al (1997) Cell 89, 349-356; Zhang et al (1997) Cell 89, 357-364; Kadosh & Struhl (1997) Cell 89, 365-371; Nagy et al (1997) Cell 89, 373-380; and Laherty et al (1998) Molecular Cell 2, 33-42. All of these papers are incorporated herein by reference.
Thus, it is particularly preferred if the component of a HDAC complex or the polypeptide which binds to or facilitates recruitment of a HDAC complex is any one of PLZF, SMRT, Sin3, SAP18, SAP30 or N-CoR, or HDACs including HDAC1, HDAC2 or HDAC3. It will be appreciated that it may not be necessary for all of the polypeptides to be present so long as a functional portion thereof is present. For example, with respect to histone deacetylase enzymes (for example, HDAC1, HDAC2 or HDAC3) the functional portion may be a portion that retains histone deacetylase activity or it may be a portion which contains a binding site for other components of a HDAC complex or a portion which otherwise recruits the HDAC complex and promotes histone deacetylation. Similarly, with respect to other components of the HDAC complex or polypeptides which bind to the HDAC complex the functional portion may be a portion which contains a binding site for other components of the HDAC complex. To date six mammalian HDAC genes have been described (Grozinger et al (1999) Proc. Natl. Acad. Sci. USA 96, 4868-4873), it is believed that any one or more of these may be useful in the practise of the present invention.
It is preferred that the chromatin inactivation portion is not N-CoR or a portion thereof; or the C-terminal domain of the vErbA, T3R (including T3Rβ1 or T3Rα) or RAR (including RARα) receptor molecule, particularly if the nucleic acid binding portion is the Saccharomyces cerevisiae GAL4 protein or a DNA-binding portion thereof, or the Escherichia coli LexA protein or a DNA-binding portion thereof.
It is believed that binding motifs are present within the components of the HDAC complex or within polypeptides which bind the HDAC complex and these motifs may be sufficient to act as chromatin inactivation portions in the polypeptide of the invention since they may facilitate histone deacetylation by recruiting a HDAC complex.
Furthermore, it will be appreciated that variants of a component of the HDAC complex or variants of a polypeptide which binds to the HDAC complex may be used. Suitable variants include not only functional portions as described above, but also variants in which amino acid residues have been deleted or replaced or inserted provided that the variant is still able to facilitate histone deacetylation. Thus, suitable variants include variants of histone deacetylase in which the amino acid sequence has been modified compared to wild-type but which retain their ability to deacetylate histones. Similarly, suitable variants include variants of, for example, Sin3 or PLZF in which the amino acid sequence has been modified compared to wild-type but which retain their ability to interact with or in the HDAC complex. Similarly, variants of other proteins interacting with components of the HDAC complex and other transcription factors that can bring about gene inactivation through HDAC activity may be used.
All or parts of the Rb, MAD and MeCpG2 proteins may interact with the HDAC complex.
While most work has been done on HDAC complexes and polypeptides involved in recruiting HDAC complexes in mammalian systems, the fundamental nature of the system is such that functionally equivalent polypeptides are expected to be found in other eukaryotic cells, in particular in other animal cells and in plant cells. For example,
It is not necessary that the chromatin inactivation portion is from the same cell type or species as the cell into which the polypeptide (or polynucleotide encoding the polypeptide) is introduced but it is desirable if it is since such a chromatin inactivation portion may be able to inactivate chromatin more effectively in that cell.
It is particularly preferred if the chromatin inactivation portion of the polypeptide is PLZF, a portion of PLZF that can facilitate histone deacetylation, or a polypeptide, or portion of a polypeptide, known to cause gene activation via histone deacetylation. For example, the portion of PLZF in PLZF-RARα which is involved in APL is believed to interact with N-CoR and SMRT.
It is also particularly preferred if the chromatin inactivation portion is a polypeptide with histone deacetylase enzyme activity such as contained in HDAC1, HDAC2 or HDAC3.
The nucleic acid binding portion may be any suitable binding portion which binds to a site present in a eukaryote, such as a plant or animal, genome. It is particularly preferred that the nucleic acid binding portion is able to bind to a site which is at or associated with a selected gene whose expression is to be suppressed by the presence of the chromatin inactivating portion of the polypeptide of the invention. It is preferred that the nucleic acid binding portion binds selectively to the desired site. There may be one or more desired sites to which the nucleic acid binding portion may bind. For example, the polypeptide of the invention may be used to suppress the expression of a group of genes which each have a binding site for a common DNA binding portion (for example, are under the controls of a steroid hormone receptor such as the oestrogen receptor (ER)). For the avoidance of doubt, the site present in the eukaryote may be a naturally occurring site, or it may be a site which has been engineered to be there. The site need not be originally from the same or any other eukaryote. For example, it may be a bacterial repressor binding site which has been engineered to be present in the DNA of the eukaryotic cell, or it may be a mammalian steroid hormone receptor binding site which has been engineered into plant cells. However, it is preferred if the site to which the nucleic acid binding portion binds is naturally present in the eukaryotic cell and is present in its natural position in the genome.
The nucleic acid binding portion may be a DNA binding portion or an RNA binding portion. Proteins which have the ability to bind either DNA or RNA in a sequence selective manner are well known in the art and some are described in more detail below. In the case of the RNA binding portion, the site present in the eukaryotic genome which binds the RNA binding portion is, typically, nascent RNA being transcribed from DNA at the selected site for inactivation. The RNA may be that which is being transcribed by the gene whose expression is to be suppressed, or it may be that which is being transcribed by a gene adjacent to, or at least close to, the gene whose expression is to be suppressed. It is preferred that the RNA binding portion binds to an RNA sequence which is at or close to the 5′ end of the transcript. It will be appreciated that whilst being transcribed, nascent RNA remains at or close to its site of transcription and that if the site of transcription is at or close to the gene whose expression is to be suppressed, using an RNA binding portion in the polypeptide of the invention facilitates the localisation of the chromatin inactivation portion to the desired site.
The DNA binding portion may be all or a DNA-binding portion of a zinc-finger DNA binding protein or it may be all or a DNA-binding portion of a helix-turn-helix DNA binding protein.
Suitably the DNA binding portion may be all or a DNA-binding portion of an animal or plant DNA binding protein, or it may be all or a DNA binding portion of a bacterial or yeast DNA binding protein which has been engineered to bind to one or more sites in the plant or animal genome. Bacterial or yeast DNA binding proteins are less preferred and it is particularly preferred if the DNA binding protein does not contain a DNA binding portion of wild-type Saccharomyces cerevisiae GAL4 or wild-type Escherichia coli LexA.
Any DNA binding protein with the ability to bind DNA through a DNA recognition sequence may be used. This includes DNA binding proteins, and engineered DNA binding proteins, such as engineered zinc finger proteins and helix-turn-helix DNA binding proteins.
Databases listing transcription factors and their binding sites are listed below:
http://www.embl-heidelberg.de/srs5bin/cgi-bin/wgetz?-fun+pagelibinfo+-info+TFFACTOR
http://www.embl-heidelberg.de/srs5bin/cgi-bin/wgetz?-fun+pagelibinfo+-info+TFSITE
http://www.embl-heidelberg.de/srs5bin/cgi-bin/wgetz?-fun+pagelibinfo+-info+TFCELL
http://www.embl-heidelberg.de/srs5bin/cgi-bin/wgetz?-fun+pagelibinof+-info+TFCLASS
http://www.embl-heidelberg.de/srs5bin/cgi-bin/wgetz?-fun+pagelibinfo+-info+TFMATRIX
http://www.embl-heidelberg.de/srs5bin/cgi-bin/wgetz?-fun+pagelibinfo+-info+TFGENE
It is believed that all or part of the listed transcription factors may be useful in the practice of the invention.
Other gene regulatory proteins which may be useful in the practice of the invention include virally encoded DNA binding proteins such as those required for regulating viral and cellular gene expression and/or viral replication. These include but are not limited to the large T antigen of polyoma viruses, the E2 protein of papillomaviruses and the ICP4, ICP0 protein of herpesviruses.
Sequence specific RNA binding proteins, which bind to nascent RNA, may be engineered to bring about gene inactivation by the methods of the invention through HDAC complex formation in the proximity of transcriptionally active chromatin. For example, the transcriptionally active chromatin may be proviral and the RNA binding protein one which binds to transcribed proviral RNA. The tat protein of HIV is an example of an RNA binding protein.
In plants, DNA binding proteins are involved in, amongst other things, floral development, cold regulation/adaptation, and plant responses to ethylene or pathogens. Thus, the polypeptides of the invention, and the methods of the invention described below, may be used to analyse the role of these genes in these developmental and other processes.
A particularly preferred embodiment is wherein the DNA binding portion is all or a DNA binding part of a nuclear receptor DNA binding protein such as a steroid hormone receptor protein.
The nuclear receptor DNA binding protein superfamily includes oestrogen receptor (ER), androgen receptor (AR), progesterone receptor (PR), retinoic acid receptor (RAR) and the like (see Mangelsdorf et al (1995) Cell 83, 835-839 for a review and nomenclature).
It is particularly preferred if the steroid hormone receptor protein is estrogen receptor (ER).
As noted, DNA binding proteins may be engineered so as to bind to a particular, selected target DNA sequence which is at or associated with a selected gene. In one embodiment of the invention the DNA binding protein is one which has been engineered to bind to a site which is present in a mutant gene sequence within the plant or animal cell but is not present in the equivalent wild type sequence. For example, and as is discussed in more detail below, the engineered DNA binding portion may bind selectively to a dominant negative, mutated gene, such as a mutant oncogene and, upon binding, chromatin inactivation occurs and suppresses the expression of the mutant oncogene. Examples of oncogenes mutated in human cancer include RAS (H-ras) and Bcl-10.
Typically, the DNA binding portion and the chromatin inactivation portion are fused such that the fusion polypeptide may be encoded by a nucleic acid molecule. Suitably, the DNA binding portion and the chromatin inactivation portion are joined so that both portions retain their respective activities such that the polypeptide may bind to a site present in a plant or animal genome and, upon binding, the chromatin inactivation portion is still able to inactivate chromatin. The two portions may be joined directly, but they may be joined by a linker peptide. Suitable linker peptides are those that typically adopt a random coil conformation, for example the polypeptide may contain alanine or proline or a mixture of alanine plus proline residues. Preferably, the linker contains between 10 and 100 amino acid residues, more preferably between 10 and 50 and still more preferably between 10 and 20. In any event, whether or not there is a linker between the portions of the polypeptide the polypeptide is able to bind its target DNA and is able to inactivate chromatin thereby selectively suppressing or inactivating gene expression.
A further aspect of the invention provides a polynucleotide encoding a polypeptide of the invention. In particular, the invention provides a polynucleotide wherein the nucleic acid binding portion and the chromatin inactivation portion are fused such that the fusion polypeptide is encoded by a single open reading frame of the polynucleotide. The polynucleotide may be DNA or RNA; DNA is preferred. DNA may or may not contain introns but, in any case, the polynucleotide encodes a polypeptide of the invention.
Polynucleotides which encode suitable nucleic acid binding portions, particularly DNA binding portions are known in the art or can be readily designed from known sequences such as from known sequences contained in scientific publications or contained in nucleotide sequence databases such as the GenBank, EMBL and dbEST databases. Polynucleotides which encode suitable chromatin inactivation portions are known in the art or can readily be designed from known sequences and made. Polynucleotide sequences encoding various suitable chromatin inactivation portions are given above in the references which refer to the polypeptides or are available from GenBank or EMBL or dbEST. A reference for PLZF is Chen et al (1993) EMBO J. 12, 1161-1167.
Polynucleotides which encode suitable linker peptides can readily be designed from linker peptide sequences and made.
Thus, polynucleotides which encode the polypeptides of the invention can readily be constructed using well known genetic engineering techniques.
A variety of methods have been developed to operably link polynucleotides, especially DNA, to other polynucleotides, including vectors, for example via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.
Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. The DNA segment, generated by endonuclease restriction digestion as described earlier, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3′-single-stranded termini with their 3′-5′-exonucleolytic activities, and fill in recessed 3′-ends with their polymerising activities.
The combination of these activities therefore generates blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyse the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.
Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, Conn., USA.
A desirable way to modify the DNA encoding the polypeptide of the invention is to use the polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487-491. This method may be used for introducing the DNA into a suitable vector, for example by engineering in suitable restriction sites, or it may be used to modify the DNA in other useful ways as is known in the art.
In this method the DNA to be enzymatically amplified is flanked by two specific primers which themselves become incorporated into the amplified DNA. The said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.
Methods of joining a polynucleotide to a nucleic acid vector are, of course, applicable to joining any polynucleotides.
The DNA (or in the case of retroviral vectors, RNA) is then expressed in a suitable host to produce a polypeptide of the invention. Thus, the DNA encoding the polypeptide of the invention may be used in accordance with known techniques, appropriately modified in view of the teachings contained herein, to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of the polypeptide of the invention. Such techniques include those disclosed in U.S. Pat. Nos. 4,440,859 issued 3 Apr. 1984 to Rutter et al, 4,530,901 issued 23 Jul. 1985 to Weissman, 4,582,800 issued 15 Apr. 1986 to Crowl, 4,677,063 issued 30 Jun. 1987 to Mark et al, 4,678,751 issued 7 Jul. 1987 to Goeddel, 4,704,362 issued 3 Nov. 1987 to Itakura et al, 4,710,463 issued 1 Dec. 1987 to Murray, 4,757,006 issued 12 Jul. 1988 to Toole, Jr. et al, 4,766,075 issued 23 Aug. 1988 to Goeddel et al and 4,810,648 issued 7 Mar. 1989 to Stalker, all of which are incorporated herein by reference.
The DNA (or in the case of retroviral vectors, RNA) encoding the polypeptide of the invention may be joined to a wide variety of other DNA sequences for introduction into an appropriate host. The companion DNA will depend upon the nature of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration is desired.
Generally, the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. If necessary, the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, although such controls are generally available in the expression vector. The vector is then introduced into the host through standard techniques. Generally, not all of the hosts will be transformed by the vector. Therefore, it will be necessary to select for transformed host cells. One selection technique involves incorporating into the expression vector a DNA sequence, with any necessary control elements, that codes for a selectable trait in the transformed cell, such as antibiotic resistance. Alternatively, the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.
Host cells that have been transformed by the recombinant DNA of the invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression of the polypeptide, which can then be recovered.
Many expression systems are known, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae), filamentous fungi (for example Aspergillus), plant cells, animal cells and insect cells.
The vectors include a prokaryotic replicon, such as the ColE1 ori, for propagation in a prokaryote, even if the vector is to be used for expression in other, non-prokaryotic, cell types. The vectors can also include an appropriate promoter such as a prokaryotic promoter capable of directing the expression (transcription and translation) of the genes in a bacterial host cell, such as E. coli, transformed therewith.
A promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with exemplary bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention. It is preferred that the promoter is one which can be regulated. It is particularly preferred if the promoter is an inducible promoter which can be selectively induced at an appropriate time once the vector has been introduced into the eukaryotic cell. It will be appreciated that upon induction, the polypeptide of the invention may be expressed in the cell and exert its effect. In this situation, induction of expression of the polypeptide of the invention leads to suppression of the targeted gene. Inducible promoters are known in the art for many eukaryotic cells including plant and animal cells. These include heat-shock-, glucocorticoid-, oestradiol-, and metal-inducible promoter systems.
Typical prokaryotic vector plasmids are pUC18, pUC19, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, Calif., USA) and pTrc99A and pKK223-3 available from Pharmacia, Piscataway, N.J., USA.
A typical mammalian cell vector plasmid is pSVL available from Pharmacia, Piscataway, N.J., USA. This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-1 cells.
An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia. This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.
Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (YIps) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (YCps).
Plant transformation vectors are well known in the art. For example, vectors for Agrobacterium-mediated transformation are available from the Centre for the Application of Molecular Biology to International Agriculture, GPO Box 3200, Canberra, ACT 2601, Australia (cambia@cambia.org.au).
The present invention also relates to a host cell transformed with a polynucleotide vector construct of the present invention. The host cell can be either prokaryotic or eukaryotic. Bacterial cells are preferred prokaryotic host cells and typically are a strain of E. coli such as, for example, the E. coli strains DH5 available from Bethesda Research Laboratories Inc., Bethesda, Md., USA, and RR1 available from the American Type Culture Collection (ATCC) of Rockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cells include plant, yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic and kidney cell lines. Yeast host cells include YPH499, YPH500 and YPH501 which are generally available from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA. Preferred mammalian host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkey kidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293 cells which are human embryonic kidney cells. Preferred insect cells are Sf9 cells which can be transfected with baculovirus expression vectors.
Protoplasts for transformation are typically generated as required by methods known in the art. Plant cell lines are not generally available. However, one cell line which is commonly used is the Bright Yellow 2 cell line from tobacco (BY2; Mu et al (1997) Plant Mol. Biol. 34, 357-362).
Transformation of appropriate cell hosts with a DNA construct of the present invention is accomplished by well known methods that typically depend on the type of vector used. With regard to transformation of prokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110 and Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Transformation of yeast cells is described in Sherman et al (1986) Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, N.Y. The method of Beggs (1978) Nature 275, 104-109 is also useful. With regard to vertebrate cells, reagents useful in transfecting such cells, for example calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877, USA. With regard to plant cells and whole plants three plant transformation approaches are typically used (J. Draper and R. Scott in D. Grierson (ed.), “Plant Genetic Engineering”, Blackie, Glasgow and London, 1991, vol. 1, pp 38-81):
i) Agrobacterium-mediated transformation, using the Ti plasmid of A. tumefaciens and the Ri plasmid of A. rhizogenes (P. Armitage, R. Walden and J. Draper in J. Draper, R. Scott, P. Armitage and R. Walden (eds.), “Plant Genetic Transformation and Expression—A Laboratory Manual”, Blackwell Scientific Publications, Oxford, 1988, pp 1-67; R. J. Draper, R. Scott and J. Hamill ibid., pp 69-160);
Agrobacterium-mediated transformation is also described in Hooykaas & Schilperoot (1992) Plant Mol. Biol. 19, 15-38; Zupan & Zambryski (1995) Plant Physiol. 107, 1041-1047; and Baron & Zambryski (1996) Curr. Biol. 6, 1567-1569.
ii) DNA-mediated gene transfer, by polyethylene glycol-stimulated DNA uptake into protoplasts, by electroporation, or by microinjection of protoplasts or plant cells (J. Draper, R. Scott, A. Kumar and G. Dury, ibid., pp 161-198). Direct gene transfer into protoplasts is also described in Neuhaus & Spangenberg (1990) Physiol. Plant 79, 213-217; Gad et al (1990) Physiol. Plant 79, 177-183; and Mathur & Koncz (1998) Method Mol. Biol. 82, 267-276;
iii) transformation using particle bombardment (D. McCabe and P. Christou, Plant Cell Tiss. Org. Cult., 3, 227-236 (1993); P. Christou, Plant J., 3, 275-281 (1992)).
Some species are amenable to direct transformation, avoiding a requirement for tissue or cell culture (Bechtold et al (1993) Life Sciences, C.R. Acad. Sci. Paris 316, 1194-1199).
Agrobacterium-mediated transformation is generally less effective for monocotyledonous plants for which approaches ii) and iii) are therefore preferred. However, Agrobacterium is capable of transferring DNA to some monocotyledenous plants if tissues containing “competent” cells are infected (see Hiei et al (1997) Plant Mol. Biol. 35, 205-218). In all approaches a suitable selection marker, such as kanamycin- or herbicide-resistance, is preferred or alternatively a screenable marker (“reporter”) gene, such as 3-glucuronidase or luciferase (see J. Draper and R. Scott in D. Grierson (ed.), “Plant Genetic Engineering”, Blackie, Glasgow and London, 1991, vol. 1 pp 38-81). Electroporation is also useful for transforming and/or transfecting cells and is well known in the art for transforming yeast cell, bacterial cells, insect cells, vertebrate cells and some plant cells (eg barley cells, see Lazzeri (1995) Methods Mol. Biol. 49, 95-106).
For example, many bacterial species may be transformed by the methods described in Luchansky et al (1988) Mol. Microbiol. 2, 637-646 incorporated herein by reference. The greatest number of transformants is consistently recovered following electroporation of the DNA-cell mixture suspended in 2.5×PEB using 6250V per cm at 25 μFD.
Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente (1990) Methods Enzymol. 194, 182.
Successfully transformed cells, i.e. cells that contain a DNA construct of the present invention, can be identified by well known techniques. For example, cells resulting from the introduction of an expression construct of the present invention can be grown to produce the polypeptide of the invention. Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method such as that described by Southern (1975) J. Mol. Biol. 98, 503 or Berent et al (1985) Biotech. 3, 208. Alternatively, the presence of the protein in the supernatant can be detected using antibodies as described below.
In addition to directly assaying for the presence of recombinant DNA, successful transformation can be confirmed by well known immunological methods when the recombinant DNA is capable of directing the expression of the polypeptide. For example, cells successfully transformed with an expression vector produce polypeptides displaying appropriate antigenicity. Samples of cells suspected of being transformed are harvested and assayed for the protein using suitable antibodies.
Thus, in addition to the transformed host cells themselves, the present invention also contemplates a culture of those cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium.
In relation to plants, it is envisaged that the invention includes single cell derived cell suspension cultures, isolated protoplasts or stable transformed plants. In the latter case it is preferred if the polypeptide of the invention is expressed using an inducible promoter system to avoid potentially lethal effects of gene down-regulation during regeneration of homozygous plants.
Although the polypeptides or polynucleotides of the invention may be introduced into any suitable host cell, it will be appreciated that they are primarily designed to be effective in appropriate animal or plant cells, particularly those that have one or more sites within their DNA to which the polypeptide of the invention may bind.
Thus, the animal of plant cells which contain a polypeptide (or polynucleotide) of the invention whose presence suppresses the expression of a particular gene, or the animals or plants containing these cells, may be considered to have the gene “knocked out” in the sense that it can no longer be expressed. The chromatin inactivation by histone deacetylation may be irreversible.
It will be readily appreciated that introduction of a polypeptide of the invention into an animal or plant cell, or introduction and expression of a polynucleotide encoding a polypeptide of the invention in an animal or plant cell, will allow targeting of the polypeptide to an appropriate binding site within the DNA (and which is bound by the DNA-binding portion of the polypeptide) and allow for the chromatin at or associated with the target binding site to be inactivated so as to lead to suppression or inactivation of gene expression. Typically, the polypeptide of the invention is selected so that it targets a selected gene. Thus, suitably, the targeted gene has a site which is bound by the DNA binding portion of the polypeptide associated with it. The site which is so bound may be within the gene itself, for example within an intron or within an exon of the gene; or it may be in a region 5′ of the transcribed portion of the gene, for example within or adjacent to a promoter or enhancer region; or it may be in a region 3′ of the transcribed portion of the gene.
Genes regulated by oestrogen receptor (ER) include the progesterone receptor (PR) gene and the PS2 (trefoil related protein) gene. Thus, the method of the invention may be used to inactivate the PR gene or the PS2 gene when the DNA binding portion of the compound of the invention is at least the DNA-binding portion of ER. Anti-oestrogen therapy is used in the treatment of breast cancer. The full repertoire of oestrogen regulated genes involved in breast cancer is presently unknown. It is generally considered that anti-oestrogen therapy results in the altered expression of key oestrogen regulated genes involved in breast cancer cell growth and transformation. The methods of the invention described below may provide an alternative, potentially more effective, way of regulating the expression (particularly inhibiting) of oestrogen-responsive genes. It may be that for certain DNA binding portions, in a given plant or animal cell there is only one target site and the expression of only one gene is suppressed by the chromatin inactivation portion. However, there may be more than one target site and introduction of a polypeptide (or polynucleotide) of the invention may lead to suppression of expression of a number of genes.
Thus, a further aspect of the invention provides a method of suppressing the expression of a selected gene in a eukaryotic cell the method comprising introducing into the cell (a) a polypeptide comprising a nucleic acid binding portion which binds to a site at or associated with the selected gene which site is present in a eukaryotic genome and a chromatin inactivation portion, or (b) a polynucleotide encoding said polypeptide.
Suitably, the polypeptide is a polypeptide of the invention as described above. Also suitably, the polynucleotide is a polynucleotide of the invention as described above. It is preferred if the preferred polypeptides or polynucleotides of the invention are used in the method. In particular, it is preferred that the chromatin inactivation portion is PLZF or a portion thereof, for example a portion that can facilitate histone deacetylation. Although it may be a site which has been engineered into the cell, it is preferred if the site at or associated with the selected gene is naturally present in the eukaryotic genome. Preferably, the eukaryotic cell is a plant cell or an animal cell.
The ability to suppress the expression of a selected gene is useful in many areas of biology.
Typically, when the gene whose expression is suppressed is in an animal cell, the animal cell is a cell within an animal and the method of the invention is used to suppress the expression of a selected gene in an animal. For the avoidance of doubt, animal in this context includes human. Examples of particular uses in animal cells include allele-specific inactivation of oncogenic proteins such as mutant Ras and mutant Bcl-10; inhibition of oestrogen receptor regulated gene expression in breast cancer; inhibition of androgen receptor; inhibition of genes of interest for developmental studies; inhibition of genes for developing transgenic models of human diseases; and inhibition of genes involved in tissue modelling, as found in cancer and wound healing.
Also typically, the plant cell is a cell within a plant and the method of the invention is used to suppress the expression of a selected gene in a plant.
In one embodiment, the method of the invention is used to suppress the expression of socially or environmentally unacceptable or undesirable genes in commercially engineered transgenic plants. Such genes may include, for example, antibiotic or herbicide selectable marker genes. In this embodiment, the gene in the transgenic plant is targeted for silencing.
In a further embodiment of the invention novel plant architecture or floral morphology may be achieved by targeting some known homeotic genes involved in these developmental pathways.
Suitably, the method of the invention is used to suppress or inactivate the expression of a gene whose expression it is desirable to suppress or inactivate. Such genes include oncogenes, viral genes including genes present in proviral genomes and so the method in relation to animals may constitute a method of medical treatment. Oncogenes may be overexpressed in certain cancers and it may be desirable to suppress their expression. Some oncogenes are oncogenic by virtue of having an activating mutation. Using the method of the invention the selective suppression of expression of a mutant oncogene may be achieved using a DNA binding portion that selectively binds to the mutant oncogene sequence and wherein the chromatin inactivation portion inactivates the chromatin in which the oncogene resides or with which it is associated so that expression of the mutant oncogene is suppressed. Suppression of oncogene overexpression or of mutant (especially activated) oncogene expression is generally desirable in treating cancers in which the oncogenes play a role. Mutant oncogenes which may be targeted by the method of the invention include Ras and Bcl-10. These may be targeted by engineered DNA binding proteins capable of recognising the mutated genes in a sequence specific manner.
The expression of viral genes in an animal or plant cell is generally undesirable since this expression is often associated with pathogenesis. The nucleic acid of certain viruses may be formed into chromatin and the expression of such viral genes may be controlled by modification of this chromatin. For example, retroviral proviruses (ie DNA copies of retroviral RNAs) are often incorporated into animal and plant genomes where they become part of the chromatin, for example, integrated HIV provirus and integrated human papillomavirus. Gypsy and Copia-like retrotransposons appear to be widely distributed in the plant kingdom. Copia-like retrotransposons, or at least their reverse transcriptase domains, appear broadly distributed in higher plants while the Gypsy-like elements (which share their organisation with the retroviruses but lack retroviral envelope domains) are less abundant (Suoniemi et al (1998) Plant J. 13, 699-705). Integration of viral DNA into the plant genome has been demonstrated for geminiviral DNA into the tobacco nuclear genome (Bejarano et al (1996) Proc. Natl. Acad. Sci. USA 93, 759-764). Potential retroviruses have also recently been described in plants (Wright & Voytus (1998) Genetics 149, 703-715). Using the method of the invention the selective suppression of expression of a viral gene may be achieved. Engineered DNA binding proteins, or RNA binding proteins, such as HIV tat protein, may be used to target a chromatin inactivation portion and lead to proviral genome inactivation by binding to nascent genomic RNA transcripts, achieving histone deacetylation by proximity.
Certain genetic diseases are caused by dominant mutations, such as achondroplasia. Suppression of expression of the mutant allele may be useful in treating these diseases. Using the method of the invention the selective suppression of expression of the mutant allele may be achieved using a DNA binding portion that selectively binds to the mutant allele sequence and wherein the chromatin inactivation portion inactivates the chromatin in which the mutant allele resides or with which it is associated so that expression of the mutant allele is suppressed.
These methods of the invention typically and preferably involve the transfer of a polynucleotide encoding said polypeptide into an animal or plant cell.
Gene transfer systems known in the art may be useful in the practice of the methods of the present invention in which the polynucleotide of the invention is introduced into a cell either within or outwith an animal body. Such an introduction of a polynucleotide may be therapeutically useful and constitutes a form of gene therapy. These include viral and nonviral transfer methods. A number of viruses have been used as gene transfer vectors, including papovaviruses, eg SV40 (Madzak et al (1992) J. Gen. Virol. 73, 1533-1536), adenovirus (Berkner (1992) Curr. Top. Microbiol. Immunol. 158, 39-61; Berkner et al (1988) BioTechniques 6, 616-629; Gorziglia and Kapikian (1992) J. Virol. 66, 4407-4412; Quantin et al (1992) Proc. Natl. Acad. Sci. USA 89, 2581-2584; Rosenfeld et al (1992) Cell 68, 143-155; Wilkinson et al (1992) Nucleic Acids Res. 20, 2233-2239; Stratford-Perricaudet et al (1990) Hum. Gene Ther. 1, 241-256), vaccinia virus (Moss (1992) Curr. Top. Microbiol. Immunol. 158, 25-38), adeno-associated virus (Muzyczka (1992) Curr. Top. Microbiol. Immunol. 158, 97-123; Ohi et al (1990) Gene 89, 279-282), herpes viruses including HSV and EBV (Margolskee (1992) Curr. Top. Microbiol. Immunol. 158, 67-90; Johnson et al (1992) J. Virol. 66, 2952-2965; Fink et al (1992) Hum. Gene Ther. 3, 11-19; Breakfield and Geller (1987) Mol. Neurobiol. 1, 337-371; Freese et al (1990) Biochem. Pharmacol. 40, 2189-2199), and retroviruses of avian (Brandyopadhyay and Temin (1984) Mol. Cell. Biol. 4, 749-754; Petropoulos et al (1992) J. Virol. 66, 3391-3397), murine (Miller (1992) Curr. Top. Microbiol. Immunol. 158, 1-24; Miller et al (1985) Mol. Cell. Biol. 5, 431-437; Sorge et al (1984) Mol. Cell. Biol. 4, 1730-1737; Mann and Baltimore (1985) J. Virol. 54, 401-407; Miller et al (1988) J. Virol. 62, 4337-4345), and human origin (Shimada et al (1991) J. Clin. Invest. 88, 1043-1047; Helseth et al (1990) J. Virol. 64, 2416-2420; Page et al (1990) J. Virol. 64, 5370-5276; Buchschacher and Panganiban (1992) J. Virol. 66, 2731-2739). To date most human gene therapy protocols have been based on disabled murine retroviruses.
Nonviral gene transfer methods known in the art include chemical techniques such as calcium phosphate coprecipitation (Graham and van der Eb (1973) Virology 52, 456-467; Pellicer et al (1980) Science 209, 1414-1422); mechanical techniques, for example microinjection (Anderson et al (1980) Proc. Natl. Acad. Sci. USA 77, 5399-5403; Gordon et al, 1980; Brinster et al (1981) Cell 27, 223-231; Constantini and Lacy (1981) Nature 294, 92-94); membrane fusion-mediated transfer via liposomes (Felgner et al (1987) Proc. Natl. Acad. Sci. USA 84, 7413-7417; Wang and Huang (1989) Biochemistry 28, 9508-9514; Kaneda et al (1989) J. Biol. Chem. 264, 12126-12129; Stewart et al (1992) Hum. Gene Ther. 3, 267-275; Nabel et al, 1990; Lim et al (1992) Circulation 83, 2007-2011); and direct DNA uptake and receptor-mediated DNA transfer (Wolff et al (1990) Science 247, 1465-1468; Wu et al (1991) J. Biol. Chem. 266, 14338-14342; Zenke et al (1990) Proc. Natl. Acad. Sci. USA 87, 3655-3659; Wu et al, 1989b; Wolff et al (1991) BioTechniques 11, 474-485; Wagner et al, 1990; Wagner et al (1991) Proc. Natl. Acad. Sci. USA 88, 4255-4259; Cotten et al (1990) Proc. Natl. Acad. Sci. USA 87, 4033-4037; Curiel et al (1991a) Proc. Natl. Acad. Sci. USA 88, 8850-8854; Curiel et al (1991b) Hum. Gene Ther. 3, 147-154). Viral-mediated gene transfer can be combined with direct in vivo gene transfer using liposome delivery, allowing one to direct the viral vectors to the tumour cells and not into the surrounding nondividing cells. Alternatively, the retroviral vector producer cell line can be injected into tumours (Culver et al (1992) Science 256, 1550-1552). Injection of producer cells would then provide a continuous source of vector particles. This technique has been approved for use in humans with inoperable brain tumours.
Other suitable systems include the retroviral-adenoviral hybrid system described by Feng et al (1997) Nature Biotechnology 15, 866-870, or viral systems with targeting ligands such as suitable single chain Fv fragments.
In an approach which combines biological and physical gene transfer methods, plasmid DNA of any size is combined with a polylysine-conjugated antibody specific to the adenovirus hexon protein, and the resulting complex is bound to an adenovirus vector. The trimolecular complex is then used to infect cells. The adenovirus vector permits efficient binding, internalization, and degradation of the endosome before the coupled DNA is damaged.
Liposome/DNA complexes have been shown to be capable of mediating direct in vivo gene transfer. While in standard liposome preparations the gene transfer process is nonspecific, localized in vivo uptake and expression have been reported in tumour deposits, for example, following direct in situ administration (Nabel (1992) Hum. Gene Ther. 3, 399-410).
Gene transfer techniques which target DNA directly to a target cell or tissue, is preferred. Receptor-mediated gene transfer, for example, is accomplished by the conjugation of DNA (usually in the form of covalently closed supercoiled plasmid) to a protein ligand via polylysine. Ligands are chosen on the basis of the presence of the corresponding ligand receptors on the cell surface of the target cell/tissue type. These ligand-DNA conjugates can be injected directly into the blood if desired and are directed to the target tissue where receptor binding and internalization of the DNA-protein complex occurs. To overcome the problem of intracellular destruction of DNA, coinfection with adenovirus can be included to disrupt endosome function.
It may be advantageous if the polypeptide of the invention is expressed in the target cell using an inducible promoter. Examples of suitable inducible promoters include those that can be induced by heat shock, glucocorticoids, oestradiol and metal ions.
Preferably, the method of suppressing the expression of a selected gene is used to suppress expression of a gene in a human cell; in one particularly preferred embodiment the human cell is within a human body.
However, the method of the invention may involve the modification of animal cells (including human cells) outside of the body of an animal (ie an ex vivo treatment of the cells) and the so modified cells may be reintroduced into the animal body.
From the foregoing, it will be appreciated that the method of the invention may be useful to suppress the activity of a plurality of selected genes. In particular, the method of the invention may be used to suppress the activity of a group of genes whose expression is controlled, at least to a large extent, by a single transcription factor. For example, the method may be used to suppress oestrogen-regulated genes as is described in more detail in the Examples.
A further aspect of the invention provides use of a polypeptide comprising a nucleic acid binding portion which binds to a site at or associated with a selected gene which site is naturally present in a eukaryotic genome and a chromatin inactivation portion in the manufacture of an agent for suppressing the expression of the selected gene in a eukaryotic cell.
A still further aspect of the invention provides use of a polynucleotide encoding a polypeptide comprising a nucleic acid binding portion which binds to a site at or associated with a selected gene which site is naturally present in a eukaryotic genome and a chromatin inactivation portion in the manufacture of an agent for suppressing the expression of the selected gene in a eukaryotic cell.
It will be appreciated that it is particularly preferred if the polypeptide or polynucleotide is used in the preparation of a medicament for suppressing the expression of a selected gene in an animal. For the avoidance of doubt, by “animal” we include human.
A further aspect of the invention provides a method of treating a patient in need of suppression of the expression of a selected gene, the method comprising administering to the patient an effective amount of a polypeptide comprising a nucleic acid binding portion which binds to a site at or associated with the selected gene and a chromatin inactivation portion.
A still further aspect of the invention provides a method of treating a patient in need of suppression of the expression of a selected gene, the method comprising administering to the patient an effective amount of a polynucleotide encoding a polypeptide comprising a nucleic acid binding portion which binds to a site at or associated with the selected gene and a chromatin inactivation portion.
It will be appreciated that suppression of the expression of a selected gene is useful where the expression or overexpression of the selected gene is undesirable and contributes to a disease state in the patient. Examples of undesirable expression of a gene include the expression of certain activated oncogenes in cancer.
Suppression of the expression of the ER upregulated genes is desirable in the treatment of breast cancer. Similarly, suppression of the expression of the androgen receptor (AR)-regulated genes is desirable in the treatment of prostate cancer.
Further aspects of the invention provides use of a polypeptide comprising a nucleic acid binding portion which binds to a site at or associated with a selected gene and a chromatin inactivation portion in the manufacture of a medicament for suppressing the expression of a selected gene in a patient in need of such suppression; and the use of a polynucleotide encoding a polypeptide comprising a nucleic acid binding portion which binds to a site at or associated with a selected gene and a chromatin inactivation portion in the manufacture of a medicament for suppressing the expression of a selected gene in a patient in need of such suppression.
Still further aspects of the invention provides a polypeptide comprising a nucleic acid binding portion which binds to a site at or associated with a selected gene and a chromatin inactivation portion for use in medicine; and a polynucleotide encoding a polypeptide comprising a nucleic acid binding portion which binds to a site at or associated with a selected gene and a chromatin inactivation portion for use in medicine. Thus, the polypeptide or polynucleotide are packaged and presented for use in medicine.
Yet still further aspects of the invention provide a pharmaceutical composition polypeptide comprising a nucleic acid binding portion which binds to a site at or associated with a selected gene and a chromatin inactivation portion and a pharmaceutically acceptable carrier; and a pharmaceutical composition comprising a polynucleotide encoding a polypeptide comprising a nucleic acid binding portion which binds to a site at or associated with a selected gene and a chromatin inactivation portion and a pharmaceutically acceptable carrier.
By “pharmaceutically acceptable” is included that the formulation is sterile and pyrogen free. Suitable pharmaceutical carriers are well known in the art of pharmacy.
In the drawings:
The invention will now be described in more detail with reference to the following Figures and Examples wherein:
lacZ together with 2 μg of the expression plasmid pSG5 (lanes 1-2), or 1μ of pSG5 (lanes 3-4, 9-12), AR (lanes 3-8), PLZF-AR (lanes 5-6, 9-10) or PLZF (lanes 7-8, 11-12), as indicated, in the presence or absence of R1881 (1 nM), prepared in ethanol. R1881 is a potent activator of the androgen receptor, for example see Quarmby et al (1990) Mol. Endocrinol. 4, 1399-4107. Ethanol was added to the control samples (-).
Work was carried out to produce an analogue of the PLZF-RARα gene fusion found in acute promyelocytic leukaemia (Guidez et al (1998) Blood 91, 2634-2642), in which the RARα portion was replaced by an equivalent region of the human estrogen receptor (ERα). To do this, a 1392 bp region of PLZF coding region was amplified by PCR from a full length cDNA clone using a generic oligonucleotide primer to 5′ flanking cloning vector sequence (T7 primer;) and a primer complementary to PLZF sequences encompassed by bases 1441-1446 of the sequence of Chen et al (1993) EMBO J. 12, 1161-1167, with additional bases added to the 3′ end, so as to introduce an in-frame XhoI restriction enzyme site Primer PLZF R; ccgctcgagCTGAATGAGCCAGTAAGTGCATTCTC) (SEQ ID NO:1). Similarly, a 1407 bp region of a human ERα cDNA clone (HEG0; Tora et al 1989 EMBO J. 8, 1981-1986) was amplified by PCR using primers which introduced an in frame XhoI site into 5′ coding region and a BamHI site into the 3′ untranslated region (Primers ER F1; CCGCTCGAGggccaaattcagataatcgac (SEQ ID NO:2) and ER R1; ccgtgtgggaTccagggagctctca) (SEQ ID NO:3)). PLZF and ER PCR products were restriction enzyme digested with EcoRI and XhoI and XhoI and BamHI respectively. The digest products were purified and ligated with pSG5 expression vector DNA (Stratagene) previously digested with the restriction enzymes EcoRI and BamHI. The ligation product was used to transform E. coli bacteria and plasmid DNA prepared from individual clones.
Recombinant pSG5 plasmids containing PLZF-ER gene fusion DNA were initially identified by restriction enzyme digestion and were subsequently confirmed by DNA sequence analysis. The resultant cloned PLZF-ER gene encodes the first 455 amino acids of PLZF, fused in frame with amino acids 151-595 of human ERα sequence. This clone was used in experiments to address expression and subsequent inhibition of ER regulated gene activity by the PLZF-ER fusion protein, as shown in
g of pSG5 (lane 1), human ERα (HEG0, lane 2), HEG19 (lane 3), PLZF-ERα (lane 4) or PLZF (lane 5). (A) Lysates were prepared in HS buffer and gel shifts performed using [32P]-labelled ERE, followed by fractionation on a 5% non-denaturing polyacrylamide gel and visualised by autoradiography. The uncomplexed ERE is seen at the bottom of the gel whilst receptor-complexed ERE migrates more slowly. (B) Lysates prepared for (A) were run on a 10% SDS-polyacrylamide gel, proteins were transferred to nitrocellulose and immunoblotting performed using a monoclonal antibody F3, raised against the C-terminal portion of human ERα. The band at 68 kDal seen in each lane is a cross-reacting protein unrelated to the oestrogen receptor. Molecular mass marker sizes are listed on the right in kDal.
The ability of PLZF-ERα to activate transcription of oestrogen responsive genes was tested in the oestrogen receptor-negative COS-1 cells. COS-1 cells were transiently transfected with an oestrogen responsive reporter gene 17M-ERE-G-CAT. Activation of gene expression results in synthesis of the bacterial chloramphenicol acetyl transferase (CAT) protein whose enzymatic activity can be assayed in vitro using 14C-labelled chloramphenicol and acetyl CoA. Acetylation of 14C-chloramphenicol can be visualised using thin layer chromatography and autoradiography. Quantitation is performed using phosphorimager analysis (Bio-Rad, UK) (see Metzger et al (1995) Mol. Endocrinol. 9, 579-591 for details of procedures). Normalization for variations in transfection efficiency is carried out by co-transfection of the reporter gene with a β-galactosidase expression plasmid (pCH110). As well as the two reporter genes the cells were transfected with the expression vector pSG5 (lanes 1-3), HEG0 (lanes 4-6), HEG19 (lanes 7-9), PLZF-ERα (lanes 10-12) or PLZF (lanes 13-15). Ligands (17β-oestradiol (10 nM, lanes 2, 5, 8, 11, 14) or 4-hydroxytamoxifen (100 nM, lanes 3, 6, 9, 12, 15) were added. The ligands were prepared in ethanol so an equal volume of ethanol was added to the no ligand controls (lanes 1, 4, 7, 10, 13). The results show that the CAT reporter gene activity is increased in the presence of E2 when HEG0 or HEG19 are expressed (lanes 3-6 and 7-9, respectively), HEG19 being a less potent activator than the full-length receptor (HEG0), as expected. PLZF has little if any effect on CAT activity (lanes 13-15). PLZF-ERα also does not activate the CAT reporter gene. Indeed some repression of the background CAT activity seen with pSG5 is observed (lanes 10-12, compare with lanes 1-3). Certainly no activation is seen with PLZF-ERα in the presence of 17β-oestradiol. These results indicate that PLZF-ERα is unable to activate expression of oestrogen responsive genes despite having an ability to bind to oestrogen response elements.
In order to investigate whether PLZF-ERα can inhibit transactivation by endogenous ER, we transiently transfected MCF-7 cells. This is a breast cancer-derived cell line that expresses the oestrogen receptor and requires oestrogen for growth. MCF-7 cells were transiently transfected with an oestrogen responsive reporter gene 17M-ERE-G-CAT and pCH110, together with pSG5 (lanes 1-3), HEG19 (lanes 4-6), PLZF-ERα (lanes 7-9) or PLZF (lanes 10-12) as described for COS-1 cells above. Ligands were added as appropriate (see
From Example 1 it can be concluded that the RARα portion of PLZF-RARα can be replaced by another related DNA binding protein activity.
PLZF-ERα protein retains the ability to bind specifically to the oestrogen receptor DNA binding element. PLZF-ERα displays little, if any ability to activate gene expression.
Replacing the RARα DNA binding activity redirects gene inactivation to the binding specificity of the new DNA binding domain.
PLZF-ERα is able to compete with endogenous ERα in a breast cancer cell lines to over-ride oestrogen-activated gene expression.
PLZF-ERα inhibits oestrogen responsive gene expression in an oestrogen-independent manner.
A second analogue of the PLZF-RARα gene fusion was produced in which the RARα portion was replaced by an equivalent region of the human androgen receptor (AR). A 1146 bp region of a human AR cDNA clone (Tilley et al 1989 Proc. Natl. Acad. Sci. USA 86, 327-331) was amplified by PCR using primers which introduced an in frame XhoI site into the 5′ coding region and a BamHI site immediately following the stop codon (Primers AR F1; ggagctcgagggTTGGAGACTGCCAGGGACC (SEQ ID NO:4) and AR R1; gtgaggatccTCACTGGGTGTGGAAATAGATGG) (SEQ ID NO:5)). The AR PCR product was restriction enzyme digested with XhoI and BamHI and ligated with XhoI/BamHI digested PLZF-ER to replace the ER portion with AR. The ligation product was used to transform E. coli bacteria and plasmid DNA prepared from individual clones. Recombinant pSG5 plasmids containing PLZF-AR gene fusion DNA were initially identified by restriction enzyme digestion and were subsequently confirmed by DNA sequence analysis. The resultant cloned PLZF-AR gene encodes the first 455 amino acids of PLZF, fused in frame with amino acids 537-917 of human AR. Transient transfections in COS-1 cells, followed by immunoblotting of cell extracts with antibodies directed against PLZF or AR were used to confirm expression and expected size.
lacZ together with 2 μg of the expression plasmid pSG5 (lanes 1-2), or 1 μg of pSG5 (lanes 3-4, 9-12), AR (lanes 3-8), PLZF-AR (lanes 5-6, 9-10) or PLZF (lanes 7-8, 11-12), as indicated, in the presence or absence of R1881 (1 nM), prepared in ethanol. R1881 was purchased from DuPont, USA. Ethanol was added to the control samples (−). The reporter gene has been described in Schule et al (1988) Science 242, 1418-1420.
From these experiments it can be concluded that the PLZF-androgen receptor fusion inhibits androgen receptor-mediated transcription activation.
A plasmid vector is produced which encodes the PLZF-AR fusion protein as described in Example 2 under the control of the PSA gene promoter which allows for selective expression in prostate tissue. The plasmid is prepared in a sterile and pyrogen-free form and is formulated into liposomes. The plasmid DNA-containing liposomes are administered into the vicinity of the prostate. Plasmid DNA is taken up by the prostate cancer cells and androgen receptor-mediated transcription is suppressed selectively in prostate cells.
A retroviral vector is produced which encodes the PLZF-ER fusion protein as described in Example 1. The retroviral vector is administered into the site of the breast tumour. Retroviral RNA is taken up by the breast cancer cells and oestrogen receptor-mediated transcription is suppressed selectively in breast cells.
Female and male (non-castrated) balb-c nude mice, 6-8 weeks old, and adult female wistar NMU rats were obtained from Harlan (Oxon, UK) and housed in Containment level 0 in Central Biological Services, Hammersmith Campus, Imperial College London. 60-day release (0.72 mg) 17 β-estradiol pellets, and 30- and 60-day release (42 mg) tetracycline pellets were purchased from Innovation Research of America (Sarasota, USA). Matrigel was obtained from Becton-Dickinson (Oxon, UK).
Establishment of LNCaP Tet Off and LNCaP Tet Off-PLZF-AR Cell Lines
Phoenix cells (obtained from Dr Nolan, Stanford, USA) are derived from 293 human kidney fibroblasts and express the Moloney Murine Leukaemia Virus gag, pol and env proteins, allowing the packaging of plasmids encoding the viral packaging signal into retroviruses that can be used to infect human cells. Phoenix cells were transiently transfected with appropriate retroviral plasmids (pRevTet Off or pRevTRE-PLZF-AR) using the calcium phosphate method (Invitrogen, Renfrew, UK). Thirty-six hours later, the culture media containing packaged retroviruses were collected and added to LNCaP cells. Twenty-four later the medium was changed and infected cells were allowed to recover for a further 24 hours, at which time fresh medium containing 300 μg/ml G418 (selected for clones that had stably integrated pRevTet Off) and 200 μg/ml hygomycin B (selected for clones that had also stably integrated pRevTRE-PLZF-AR) was added, as appropriate. When infecting with pRevTRE retroviruses, doxycycline (1 μg/ml) was present throughout.
Selection was maintained until discrete colonies of resistant cells developed. Colonies were ringed in permanent ink on the underside of the plates to allow easy identification. The plates were washed in PBS and individual colonies were picked using sterile cloning disks, (Labcor Corp, Maryland, USA). For this procedure, the filter paper disks were soaked in trypsin-EDTA solution and applied to individual ringed colonies for 2-3 minutes, using sterile forceps, before the disks were transferred into 24 well plates containing 1 ml/well of culture media. During subsequent culture the cells migrated off the disks onto the plastic surface of the well. Cloned, confluent cultures were trypsinised and re-seeded into T25 flasks and subsequently T75 flasks. Hygromycin B and/or G418 was used in the culture medium throughout the cell cloning process and subsequent routine culture, to maintain stable integration of the transfected DNA. Doxycycline was also added as appropriate.
After selection, 200 μg/ml G418 was used in cultures for maintenance. For cells transduced with pRevTet Off and pRevTRE-PLZF-AR cultures contained in addition hygromycin B at a concentration of 150 μg/ml. Doxycycline at a concentration of 1 μg/ml was also added in these cultures.
Cell Growth Assays
Growth of the MCF-7 Tet Off and MCF-7 Tet Off transfected with PLZF-ER clones 13 and 23 (JP13 and 23) cell lines was determined by evaluation of cell proliferation. Cells were plated onto 6-well culture plates at 50,000 cells/well in 2 mls of complete culture medium. 24 hours later, the culture medium was removed, the cells washed twice with 1×PBS, and 2 mls of phenol-red free DMEM, 5% DSS, P/S/G, 100 μg/ml G418, and in the case of JP13 and JP23, 80 μg/ml hygromycin B added. 72 hours later (day 0) estradiol (10−8M) or equal volume of drug vehicle (100% ethanol), and doxycycline (1 g/ml) were added to cultures as appropriate. Every 48 hours, cells were harvested by suspension using trypsin in 0.02% EDTA solution. Cell counts were performed in triplicate (4 readings per well) using a haemocytometer, with trypan blue exclusion to identify viable cells. Media was changed every 72 hours for remaining wells. This procedure was repeated for 3 independent experiments and the mean values of the counts were determined.
Growth of LNCaP, LNCaP Tet Off and LNCaP Tet Off-PLZF-AR cell lines was determined in a similar way with the following modifications: cells were cultured with 2 mls of phenol-red free RPMI, 5% DSS and P/S/G, supplemented with 200 μg/ml G418 in the case of LNCaP Tet Off and LNCaP Tet Off-PLZF-AR lines, and additionally 150 μg/ml hygromycin B in the case LNCaP Tet Off-PLZF-AR lines. Seventy-two hours later (day 0) R1881 (10−9M) or equal volume of drug vehicle (100% ethanol), and doxycycline (1 μg/ml) were added to cultures as appropriate.
Cell Cycle Analysis
Cells were trypsinised to a single cell population. After pelleting, the cells were washed in cold 1×PBS. The cell pellet was resuspended in 1 ml of 1×PBS and the resulting suspension centrifuged at 1000 rpm for 5 minutes. The PBS was removed and 1 ml of cold 70% ethanol added dropwise while vortexing to ensure that cells were fixed as single cells. Fixed cells were stored at 4° C. until analysis.
Prior to analysis cells were washed twice with 1×PBS and then treated with RNase (100 μg/ml) for 15 minutes. The cells were finally treated with propidium iodide (50 μg/ml) and left for 1 hour in the dark prior to analysis. Analysis of propidium iodide staining were performed using a FACScan instrument (Becton-Dickinson, Oxon, UK). For each sample 1×106 cells were analysed using Win MD1 version 2.8 software (Becton-Dickinson).
Screening for Inducible LNCaP Tet Off Clones
Viral supernatant containing the luciferase gene was prepared from the pRevTRE-Luc vector and used to infect all LNCaP Tet Off clones (previously passaged 4-6 times). LNCaP Tet Off clones were seeded onto 12-well culture plates at a density of 50,000 cells per well 24 hours prior to infection. Infection was also carried out of LNCaP as a negative control, and MCF-7 Tet Off and CHO-AA8-Luc Tet Off as positive controls. The latter is a pre-made double stable Tet Off cell line included in the Clontech RevTet Off System pack that exhibits induction of luciferase upon removal of tetracycline or doxycycline from the culture medium. After 24 hours of incubation, media were changed and doxycycline (1 μg/ml) was added to appropriate cultures. Cells were incubated for a further 72 hours. For performing luciferase assays the cells were washed twice with 1×PBS. Lysis buffer (60 μl; Luclite Plus kit, Packard) was added to each well and incubated on ice for 10 minutes. A volume of 20 μl of each lysate was added to 20 μl of the 2× luclite substrate (Luclite Plus kit) in 96 well plates and lucifearse activities were determined using a luminometer (Becton-Dickinson, Oxon, UK). Luciferase activity was determined for triplicate wells and the mean calculated for all LNCaP Tet Off clones.
Functional Assay for PSA Protein
LNCaP and derived cell lines were plated out in 12-well plates at 100,000 cells/well in 1.5 mls of maintenance culture media, composition described above. 24 hours later, the culture medium was removed, the cells washed twice with 1×PBS, and 1.5 mls of phenol-red free RPMI, 5% DSS and P/S/G, supplemented with the appropriate selective antibiotics. After 72 hours (day 0), R1881 (10−9M) and doxycycline (1 μg/ml) were added as appropriate. On days 2, 4, 6, and 8 media were removed from cultures and sent in sealed tubes to the Department of Medical Oncology at Charing Cross Hospital for radioimmune assay for PSA. R1881 and doxycycline were replenished in cultures every 48 hours.
Nude Mouse Studies
Slow-release estradiol and tetracycline pellets were implanted subcutaneously, under aseptic conditions, in the flank under isofluorane general anaesthesia. The skin incision was closed with a single Michel clip, which was removed after 7-10 days. After approximately 24 hours, MCF-7 Tet Off and JP23 cells were harvested following trypsinization and suspended in 1×PBS. LNCaP and LNCaP Tet off-PLZF-AR cells were suspended in Matrigel after trypsinization. Cells were injected subcutaneously into the flank. Animals were monitored for ill-effects. Tumours were allowed to grow to a maximum of 15 mm in diameter, at which time the animals were sacrificed, and tumours resected and snap frozen in liquid nitrogen. All work was carried out in accordance with the provisions of the Animals (Scientific Procedures) Act, 1986 of the United Kingdom (HMSO, London, UK, 1990) and with appropriate local ethical and Health and Safety approval.
Rat NMU Studies
Rat mammary tumours induced by the carcinogen, nitrosomethylurea (NMU), have the hormone sensitivity characteristic of human breast tumours (Williams et al., 1981). This model has been established and validated in studies to evaluate new anti-cancer drugs, including those with anti-estrogenic effects. After injection of NMU, approximately 70% of animals develop mammary tumours (which are often multiple) within 3 months.
Female wistar rats, bred and injected with NMU at Harlan, UK, were obtained. The animals had been given 5 mg NMU per 100 g body weight on days 0, 14 and 28. The animals were transferred within 2 months of day 0. Adenoviruses encoding PLZF-ERα and PLZF were prepared as described by He et al., 1998, by Professor Nicholas Lemoine's Group, Department of Cancer Medicine, Imperial College London. Adenovirus encoding PLZF-ERα or PLZF (109 pfu in 0.2 mls) was injected intratumorally in multiple angles and planes. Animals were monitored for ill-effects. Tumours were allowed to grow to a maximum of 20 mm in diameter, at which time the animals were sacrificed, and tumours resected and snap frozen in liquid nitrogen. All work was carried out in accordance with the provisions of the Animals (Scientific Procedures) Act, 1986 of the United Kingdom (HMSO, London, UK, 1990) and with appropriate local ethical and Health and Safety approval.
Tumour Measurement
Tumour dimensions were determined twice a week (unless stated otherwise) by measuring two diameters at right angles with Vernier callipers.
Tumour volume was estimated using the following formula:
Tumour volume=(d1)2×(d2)/2
Where d1 is the length and d2 is the width.
The two-tailed Student's t-test was used to determine whether differences in tumour volume in different treatment groups were statistically significant.
MCF-7 cells form tumours when implanted in nude mice. However, tumour formation is absolutely dependent on E2, requiring co-implantaion of slow-release E2 pellets. Further, tumour formation can be inhibited by coadministration of anti-estrogens such as tamoxifen. Having established that the growth of MCF-7 Tet Off cells is blocked by PLZF-ERα, its potential for inhibiting tumor formation in nude mice was next investigated. For this, a single 60-day release estradiol pellet was subcutaneously implanted in to 10 female balb-c nude mice. Twenty-four hours later, 5×106 MCF-7 Tet Off cells (in 0.1 mls of 1×PBS) were injected per animal. Eight out of the 10 animals developed tumours starting 17 days after injection of cells showing that the MCF-7 Tet Off cell line forms tumours in nude mice.
Slow-release estradiol pellets were now subcutaneously implanted into 2 groups of 12 female balb-c nude mice. Twenty-four hours later, 5×106 MCF-7 Tet Off cells (in 0.1 mls of 1×PBS) were inoculated subcutaneously into one group of mice and an equal number of JP23 cells (in 0.1 mls of 1×PBS) injected into the other group. One of the animals in the JP23 injected group died during general anaesthesia. The animals were subsequently monitored for tumour growth twice weekly. Nude mice were sacrificed after 60 days, or when the tumour reached 15 mm. Small tumour deposits were first noted in 4 of the mice injected with MCF-7 Tet Off cells on day 24. Two further mice developed tumours between days 28 and 32. Four of the mice in this group were either found dead or wer culled due to illness, including 2 with tumours (between days 39 and 52). The mean tumour volume increased over time. None of the group of mice injected with JP23 cells developed tumours, but 5 of them were either found dead or had to be culled because of illness (between days 5 and 56).
The above study suggests that PLZF-ERα expression inhibits MCF-7 Tet Off tumour formation in nude mice. It is possible, however, that lack of tumour formation was due to changes in MCF-7 Tet Off cells during viral transduction and establishment of the JP23 cell line, that are independent of PLZF-ERα expression. In order to investigate this possibility a further study was carried out in which 48 female balb/c nude mice were randomly divided into 3 groups. All mice were implanted with a single 60-day release estradiol pellet. Each animal in one of the groups was also implanted with a single 42 mg 60-day release tetracycline pellet. Twenty-four hours later, this and a second group of animals were injected with 5×106 JP23 cells, grown previously throughout in medium containing doxycycline. The remaining group was injected with the same number of MCF-7 Tet Off cells. Nude mice were sacrificed after 60 days, or when the tumour reached 15 mm.
Tumours arose in 12 out of 16 mice injected with MCF-7 Tet Off. Thirteen out of 16 animals in the tetracycline group injected with JP23 formed tumours, whereas only one animal in the group injected with JP23 without tetracycline pellets formed a tumour which on further analysis was shown to no longer express PLZF-ERα. The growth of tumours in the JP23 injected group that had received tetracycline was greater than in the group injected with MCF-7 Tet Off cells. In this study there were no premature animal deaths due to illness.
PLZF-ERα Causes Shrinkage of Rat Mammary Tumours (
So far, the effect of PLZF-ERα on tumour development and progression in nude mice had been studied. Whilst these studies showed a clear anti-tumour effect of PLZF-ERα, an investigation of the ability of PLZF-ERα to induce shrinkage of spontaneous, pre-established tumours would demonstrate its potential as a possible therapeutic agent. Rat mammary tumours induced by the carcinogen, nitrosomethylurea (NMU), have the hormone sensitivity characteristic of human breast tumours (Williams et al., 1981). This model has been established and validated in studies to evaluate new anti-cancer drugs, including those with anti-estrogenic effects. After injection of NMU, approximately 70% of animals develop mammary tumours (which are often multiple) within 3 months.
The effect of direct intratumoral delivery of adenovirus encoding PLZF-ERα in comparison to adenovirus encoding PLZF was studied using the rat NMU model. The recombinant pad-Track-CMV/padEasy system as described by He et al., 1998, was used by Professor Nicholas Lemoine's Group, Department of Cancer Medicine, Imperial College London, to generate adenoviruses encoding PLZF-ERα and PLZF. Sixty-nine female wistar rats, bred and injected with NMU at Harlan UK, were obtained. The animals had been given 5 mg NMU per 100 g body weight on days 0, 14 and 28. The animals were transferred to the Central Biological Services Facility, Hammersmith Campus, Imperial College London, within 2 months of day 0. Forty-three of these animals developed tumours. Rats were randomized when tumour size reached 10 mm to receive either adenovirus encoding PLZF-ERα or PLZF (109 pfu in 0.2 mls). Virus was injected intratumorally in multiple angles and planes on day 0 and day 1. Growth of tumours was then monitored for 4 weeks. Tumours were allowed to grow to a maximum of 20 mm in diameter. Animals culled within the first 2 weeks of the first viral injection were excluded from the analysis. Also, mice that developed tumour ulceration after the injection of virus were taken out of the study.
Ad-PLZF injected tumours, with one exception, all continued to grow with time. Those tumours injected with Ad-PLZF-ERα, however, generally demonstrated a reduced rate of growth in the first two weeks after viral injection. This was followed by a period of tumour stasis or, in some cases, tumour regression. Mean +/−SE tumour volume of the group of animals injected with virus encoding PLZF increased by 9.9 fold after 4 weeks, whereas the mean +/−SE tumour volume of the Ad-PLZF-ERα injected group was only 4.6 fold greater after 4 weeks than at the commencement of the experiment. PLZF-ERα resulted in tumour stasis or regression in 4 out of 14 rats compared with only 1 of 15 animals treated with virus encoding PLZF. Statistical significance was observed between the two treatment groups (p<0.015) from 28 days after injection. The above results demonstrate that direct intratumoral delivery of PLZF-ERα causes progressive tumour shrinkage.
Similar results were obtained for JP23 cultured in the presence of dox, following addition of E2, i.e. compared with cultures treated with ethanol there was an increased proportion of cells in S and G2. In the absence of dox, however, the percentage of E2-treated JP23 cells in the S and G2 phases was similar to that obtained in the absence of E2 for JP23 and MCF-7 Tet Off cells, with a similarly higher proportion in G1 as shown in
LNCaP Tet Off Cells (
The above studies illustrate the activity of PLZF-ERα, a fusion protein designed to inhibit the action of estrogen-regulated genes in an estrogen-responsive breast cancer cell line. Clearly, such studies are facilitated by the use of a tetracycline-responsive gene expression system, as this allows investigation of the cellular response to Gene ICE where constitutive expression is a problem. Using the methods described in section 2.2.13, pRev Tet Off retrovirus were generated and was used to infect LNCaP cells. The resulting cells were selected in media containing G418, and cloned. Thirty-one LNCaP Tet Off clones were initially obtained, of which 13 survived repeated passaging.
Screening for Inducible LNCaP Tet Off Clones with RevTRE-luc Virus (
In order to determine which of the 13 LNCaP G418-resistant clones expressed tTA, the LNCaP Tet Off clones were infected with RevTRE-luc virus to identify G418-resistant clones that have high inducibility of tetracycline-regulated gene expression with low background activity. RevTRE-luc encodes a tetracycline-regulated firefly luciferase gene (Clontech, Oxon, UK) which enables the tetracycline regulation of tTA expressing clones to be readily established. Each clone exhibited induction of luciferase activity in the absence of doxycycline, varying from 3.8 to 10.6 fold. Ten of the clones displayed over 5 fold induction with clone 3 showing the highest-fold induction of luciferase activity Infection was also carried out of LNCaP as a negative control, and the commercially obtained MCF-7 Tet Off (which showed 15.5 fold induction) and CHO-AA8-Luc Tet Off (11.0 fold induction) as positive controls. The latter is a pre-made double stable Tet Off cell line included in the Clontech RevTet Off System pack that exhibits induction of luciferase upon removal of tetracycline or doxycycline from the culture medium.
3.7 Generation of cell lines stably expressing PLZF-AR In order to investigate the ability of PLZF-AR to inhibit androgen responses in the androgen-regulated LNCaP prostate cancer cell line, PLZF-AR cloned into the mammalian expression plasmid pSG5 has previously been generated and used for the studies outlined in section 1.12.2 (Pike et al., 2004). The PLZF-AR coding sequences in pSG5 were PCR amplified using primers encoding BamH1 sites, to allow cloning into the BamH1 site in pRevTRE. Insertion and correct orientation were confirmed by restriction digestion and automated DNA sequencing of the full PLZF-AR coding sequences. Using the methods described in section 2.2.13, retroviruses encoding PLZF-AR and encoding Tet Off were simultaneously used to infect LNCaP. Also pRevTRE-PLZF-AR was used to infect the previously constructed LNCaP Tet Off clone 3 cell line. To ensure that PLZF-AR was not expressed in the cells during the selection period, 1 μg/ml dox was added to the culture medium along with the selective antibiotics, hygromycin B to a concentration of 200 μg/ml and the maintenance concentration (200 μg/ml) of G418. The medium was changed every 2-3 days to maintain effective levels of dox, hygromycin B and G418.
From cultures that were simultaneously infected with retroviruses encoding PLZF-AR and Tet Off, 24 clones that survived the selection, were picked and cell lines derived for further evaluation. The growth of these lines was slow, with the cells tending to aggregate and seed poorly on passaging. Subsequently, only 3 clones survived, with just one clone (DM3) demonstrating an acceptable rate of growth for subsequent growth studies.
Following infection of the LNCaP Tet Off line with pRevTRE-PLZF-AR, 17 clones were picked, with only 5 lines surviving repeated passaging.
Expression of PLZF-AR mRNA in Stably Transduced LNCaP Cell Lines (
To investigate whether PLZF-AR mRNA was expressed in an inducible manner in any of the cell lines from dual infection with tTA and PLZF-AR retroviruses, RNA was prepared from each cell line cultured either in the presence or absence of dox (1 μg/ml) for 72 hours. CDNA was prepared from the RNA samples and PCR carried out using primers designed to amplify a 340 bp product from PLZF-AR, with the 5′ primer being derived from PLZF sequences and the 3′ primer from the AR sequences, ensuring that only PLZF-AR, but not endogenous PLZF or AR mRNA is detected.
Expression of PLZF-AR Protein in a Stably Infected LNCaP Cell Line (
Western blotting analysis was performed to investigate whether the PLZF-AR fusion protein could be detected in LNCaP Tet Off-PLZF-AR clone 3 (DM3).
Subcellular Localisation of PLZF-AR in Stably Transduced LNCaP Cells
Previous studies carried out using immunofluorescence staining of COS-1 cells transiently transfected with PLZF-AR showed that PLZF-AR is cytoplasmic in the absence of ligand and is nuclear in the presence of androgens such as R1881 (Pike et al., 2004). Further, similar studies with JP13 and JP23 cell lines showed that maximal expression of PLZF-ERα is observed 72-96 hours (See
PLZF-AR Inhibits PSA Expression in LNCaP (
Prostate specific antigen (PSA) is an androgen-regulated gene. The protein product, conveniently measured in the serum by radioimmune assay, is used clinically as a marker of disease activity.
LNCaP, LNCaP Tet Off clone 3 and DM3 cells were seeded in RPMI without phenol red containing charcoal stripped serum in the presence or absence of R1881, and the presence or absence of dox. Culture media were removed after days 2, 4, 6 and 8 and assayed for PSA. For all three cell lines there was a negligible rise in PSA levels with time in the absence of ligand. In LNCaP and LNCaP Tet Off cells, PSA levels rose progressively with time in the presence of R1881. Similarly, with DM3 in the presence of androgen and dox there was a steady rise in PSA levels with time. However, in the presence of R1881 but absence of dox, with PLZF-AR expressed, the rise in PSA levels was inhibited, a statistically significant difference (p<0.05) being detectable within 2 days. After 8 days the PSA level 2.6-fold less than observed in cultures treated with both R1881 and doxycycline. In DM3, even in the presence of dox, the maximal level of PSA was less than 50% of that observed for LNCaP and LNCaP Tet Off treated with R1881 after 8 days.
The observation that there was some rise in PSA levels in LNCaP Tet Off-PLZF-AR cultures treated with androgen but no doxycycline may be due to the Tet Off system being slightly leaky in the “off” state or due to the clone not being a pure population of PLZF-AR transduced cells.
PLZF-AR Inhibits the Androgen-Dependent Growth of LNCaP Cells (
The above data suggests that PLZF-AR inhibits PSA protein expression in LNCAP cells. Given that LNCaP cell growth is androgen-dependent, with the effects of androgen being mediated by changes in gene expression brought about upon binding AR, expression of PLZF-AR in LNCaP cells and the consequent inhibition of the expression of androgen-regulated genes would be expected to result in inhibition of LNCaP growth. In order to assess the effect of PLZF-AR on androgen-dependent growth of LNCaP cells, growth assays were carried out in the presence and absence of R1881 and/or dox. For this 50,000 LNCaP, LNCaP Tet Off and DM3 cells were plated in each well of 6-well plates in RPMI containing 10% fetal calf serum (FCS). Twenty-four hours later, the medium was replaced by RPMI lacking phenol red and containing 5% dextran-coated charcoal stripped FCS. Phenol red was omitted as it has been shown to be weakly androgenic, whilst treatment of fetal calf serum with dextran-coated charcoal strips the serum of steroid hormones. Seventy-two hours later, R1881 and dox were added, as appropriate and this stage was taken as day 0, Since R1881 was prepared in ethanol, an equal volume of ethanol was added to the no R1881 controls.
In the absence of R1881, LNCaP and LNCaP Tet Off cells did not show an appreciable increase in cell number, whereas R1881 stimulated their growth, with cell number doubling approximately every two days over the time course of the experiment. There was no appreciable difference in the R1881-induced growth of LNCaP or LNCaP Tet Off cells in the presence or absence of dox. In the presence of dox, the DM3 cell line showed an enhanced rate of proliferation in the presence of androgen, with no appreciable growth in the absence of R1881. However, in the absence of dox, with PLZF-AR expressed (and hence AR-regulated genes silenced), growth was largely inhibited. By day 4 this growth inhibition could clearly be noted. By day 10, there was a 2.5-fold difference in cell numbers in cultures treated with R1881 and doxycycline compared to those treated with R1881 alone (p<0.05). The observation that there was a slight rise in cell numbers in cultures grown in the presence of androgen but absence of dox, is consistent with the results obtained in the functional assay for PSA expression. Also, in keeping with the PSA data, the absolute number of DM3 cells after 10 days in plus androgen and dox conditions was around 50% compared to androgen-treated LNCaP and LNCaP Tet Off cultures.
The Effect of PLZF-AR on Tumour Growth in Nude Mice (
Having established that the expression of PSA and growth of LNCaP cells is blocked by PLZF-AR, its potential for inhibiting tumour formation in nude mice was next investigated. LNCaP cells form tumours when implanted in non-castrated mice (so that there is endogenous circulating androgen) nude mice, though the development of tumours can be disappointing. The use of Matrigel which is a solubilized basement membrane preparation extracted from EHS mouse sarcoma, greatly enhances LNCaP tumorgenicity in nude mice (Lim et al., 1993). Twenty-four non-castrated- male balb-c nude mice were obtained and were randomly divided into two groups. In to each animal of one group, 5×106 LNCaP cells (in 0.2 mls of Matrigel) were injected. The same number of DM3 cells (suspended in Matrigel) were injected into the animals of the second group. In the LNCaP group, 10 animals developed tumours appearing two weeks after cell inoculation, compared with 6 animals in the PLZF-AR transduced cell line.
The tumour dimensions were subsequently monitored weekly for 10 weeks at which time the animals were sacrificed. In the LNCaP injected group, a progressive rise in mean tumour volume was observed as expected. In the LNCaP Tet Off-PLZF-AR injected group there was a significantly inhibited rate of tumour growth compared with the LNCaP group (p<0.05).
The above study suggests that PLZF-AR expression inhibits LNCaP tumour formation in nude mice. It is possible, however, that reduced rate of tumour progression was due to changes in LNCaP cells during viral transduction and establishment of the DM3 cell line, that are independent of PLZF-AR expression.
| Number | Date | Country | Kind |
|---|---|---|---|
| 9915126.8 | Jun 1999 | JP | national |
| PCT/GB00/02497 | Jun 2000 | GB | national |
This application is a continuation-in-part of application Ser. No. 10/019,520, the entirety of which is deemed incorporated herein by reference, filed Jun. 10, 2002, entitled “CONTROL OF GENE EXPRESSION” and which, in turn, is a continuation of co-pending PCT International Application No. PCT/GB00/02497, having an International filing date of Jun. 26, 2000 and having a priority date of Jun. 30, 1999 and entitled “CONTROL OF GENE EXPRESSION”.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10019520 | Jun 2002 | US |
| Child | 11712311 | Feb 2007 | US |
