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WO2011046700A1 - Activateur activant cftr - Google Patents

Activateur activant cftr Download PDF

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Publication number
WO2011046700A1
WO2011046700A1 PCT/US2010/048872 US2010048872W WO2011046700A1 WO 2011046700 A1 WO2011046700 A1 WO 2011046700A1 US 2010048872 W US2010048872 W US 2010048872W WO 2011046700 A1 WO2011046700 A1 WO 2011046700A1
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Prior art keywords
cftr
nucleic acid
dhs
gene
promoter
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Ann Harris
Christopher J. Ott
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Childrens Memorial Hospital
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Childrens Memorial Hospital
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4712—Cystic fibrosis

Definitions

  • the present invention relates generally to the field of the cystic fibrosis transmembrane conductance regulator (CFTR), and in particular to the field of the human CFTR gene.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • genes include distal enhancers that may reside large distances from the gene promoters they control. Variations in these enhancer/promoter interactions and the nuclear localization of the genes they regulate are thought to be contributing factors in the diversity of transcriptional profiles between different cell types. Moreover, they are important in adjusting these profiles throughout cellular differentiation and development (Ragoczy et al., 2003,
  • Chromosome Res 1 1 : 513-525 Chromosome Res 1 1 : 513-525. These long-range associations are facilitated by the looping of chromatin whereby regulatory elements come together with requisite nuclear factors to function within 'transcriptional hubs' where transcriptional activity is coordinated (de Laat and Grosveld, 2003, Chromosome Res. 1 1 : 447-459).
  • CF cystic fibrosis
  • transmembrane conductance regulator CFTR gene, which encompasses approximately 189 kb of genomic DNA, shows a complex pattern of expression with both spatial and temporal regulation.
  • the flanking loci, ASZ1 and CTTNBP2 show very different tissue-specific expression in comparison to CFTR.
  • Cystic fibrosis is one of the most common serious inherited diseases amongst Caucasians.
  • CF is an autosomal recessive syndrome this means that around 1 in 2,000 live births are affected.
  • CF affects many organs including sweat glands (cystic fibrosis sufferers have salty sweat), the gut and the pancreas (85% of CF patients are pancreatic insufficient and require enzyme supplements in the diet).
  • sweat glands cystic fibrosis sufferers have salty sweat
  • the gut and the pancreas 85% of CF patients are pancreatic insufficient and require enzyme supplements in the diet.
  • cystic fibrosis patients accumulate mucus in the airways which is only relieved by regular physiotherapy.
  • This mucus serves as a substrate for bacterial infection resulting in lung damage.
  • the gene for cystic fibrosis was identified and sequenced in 1989 and subsequent studies showed that the gene product is a cAMP-activated chloride channel in the membrane of specialized epithelial cells and some other cell types.
  • the CFTR gene encompasses approximately 189 kb at human chromosome 7q31 .2 and mutations within it cause the common genetic disease cystic fibrosis (CF) (Rommens et ai, 1989, Science 245: 1059-1065).
  • CFTR encodes a membrane-associated chloride ion channel that is expressed at the highest levels in chloride-secreting epithelial cells of the small intestine, pancreas, and male genital duct, and at lower levels in the respiratory epithelium and certain other sites.
  • the CFTR promoter has been characterized as a
  • the systems preferably include an isolated nucleic acid element that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:1 , and a CFTR gene promoter that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:2.
  • the systems preferably regulate enhanced expression of the CFTR gene.
  • the nucleic acid element may be recombinant.
  • the CFTR gene promoter may be recombinant.
  • the CFTR gene promoter may be native.
  • the isolated nucleic acid element may be operably linked to the CFTR gene promoter.
  • the systems may further include isolated nucleic acid elements comprising the CFTR gene enhancer sequence of intron 1 .
  • Isolated nucleic acid elements that include functional sequences are disclosed.
  • the functional sequences are contained within the sequence of SEQ ID NO: 1 .
  • the nucleic acid elements regulate expression of the CFTR gene.
  • Vectors containing these nucleic acid elements are also disclosed.
  • Isolated nucleic acid constructs that are up to 50 kbp (kilobase pairs) in length comprising the coding sequence of the CFTR gene together with an isolated nucleic acid element that is at least 95% identical to the nucleic acid sequence of SEQ ID NO:1 , are disclosed. Also disclosed are vectors that comprise these isolated nucleic acid constructs.
  • the methods include formulating the above nucleic acid constructs into delivery vectors suitable for administration to the mammals.
  • Methods for regulating expression of a CFTR gene in mammals are disclosed.
  • the methods include delivering the delivery vectors described above to the mammals, thereby regulating the expression of the delivered CFTR gene in the mammals.
  • FIG. 1 is a schematic diagram of the identification of DNase I hypersensitive sites (DHS) within the CFTR locus in cell types relevant to CF pathology:
  • DHS DNase I hypersensitive sites
  • A Averaged DNase-chip hybridization data from three (Caco2, skin fibroblasts), two (HT29, primary tracheal epithelial cells and NHBE cells), or a single (primary epididymis) experiment, analyzed with ACME statistical software;
  • B CFTR mRNA levels measured by qRT-PCR (quantitative reverse transcription- polymerase chain reaction), where each value is relative to the amount of detected skin fibroblast transcript.
  • Figure 2 depicts graphs illustrating the long-range interactions between the CFTR promoter and specific DHS measured with q3C
  • Figure 3 depicts bar charts illustrating how intronic enhancers act cooperatively to augment the CFTR basal promoter:
  • A Caco2 cells transfected with pGL3B luciferase reporter constructs containing the 787 bp CFTR basal promoter and fragments of the DHS regions found in introns 1 , 10a, b, 11 , or +15.6 cloned into the enhancer site of the vector in either forward or reverse orientations;
  • B Caco2 cells transfected with the single enhancer luciferase reporter constructs (as in A), in addition to constructs containing multiple combinations of two or three DHS regions cloned into the enhancer site.
  • Figure 4 is a graph showing by chromatin immunoprecipitation (ChIP) how the transcription factor HNF1 and the transcriptional
  • coactivator p300 associate with multiple DHS of the CFTR locus in Caco2 cells.
  • Figure 5 is a graph illustrating how EcoRI 3C (EcoRI chromatin conformation capture) detects interactions between the CFTR promoter and elements 3' of gene.
  • Figure 6 is a graph showing enrichment of Rad21 at -20.9, +6.8 and +48.9 kb DHS by ChIP. DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS
  • the present invention relates to compositions and methods for the regulation of the expression of the CFTR gene, which encodes cystic fibrosis transmembrane conductance regulator (CFTR).
  • the CFTR gene is a human CFTR gene.
  • the present invention provides for the identification of one or more DNA sequences that interact with the promoter of a CFTR gene to regulate its expression.
  • the present invention provides for the identification of one or more DNA sequences that interact with the promoter of a CFTR gene to enhance its expression.
  • the present invention discloses nucleic acid elements as regulatory elements of the expression of the CFTR gene.
  • the regulatory elements may be in their forward, 5' to 3' orientation. In other embodiments of the present invention, the regulatory elements may be in a reverse, 3' to 5' orientation.
  • CFTR means cystic fibrosis transmembrane conductance regulator.
  • the CFTR is a human CFTR.
  • CFTR gene refers to the gene that provides the polynucleotide sequence for making a protein called the cystic fibrosis transmembrane conductance regulator (CFTR).
  • CFTR gene refers to the human CFTR gene.
  • the present invention specifically contemplates the human CFTR gene and the human CFTR protein.
  • the present invention provides evidence that the cystic fibrosis transmembrane conductance regulator (CFTR) locus adopts a looped conformation to facilitate expression of the CFTR gene.
  • the complex looped structure of the CFTR locus is present to varying degrees in cells that express the CFTR gene, and is absent from cells in which the CFTR gene is inactive.
  • the promoter region closely interacts with sequences in the middle of the gene about 100 kb from the promoter and with regions 3' to the locus that are about 200 kb away. These interacting regions correspond to prominent DNase I hypersensitive sites within the locus.
  • the present invention provides regulatory elements outside the basal promoter region of the CFTR gene, which contribute to its diverse expression profile.
  • the present invention discloses the present invention
  • nucleic acid sequences that interact with the promoter of a CFTR gene to enhance its expression in a number of human primary cell types relevant to cystic fibrosis (CF) pathology.
  • These nucleic acid sequences are preferably DNA sequences. Because these nucleic acid sequences enhance the expression of the CFTR gene, they may be viewed as CFTR-activating enhancers.
  • the present invention discloses the use of novel technologies to comprehensively map potential regulatory elements of the CFTR locus and to establish their mechanism of action.
  • the present invention contemplates the use of a variety of vectors that can be recombinantly engineered to express the nucleic acid elements that act as regulatory elements of the expression of the CFTR gene.
  • a variety of vectors can be useful for practicing the invention.
  • the term "vector” is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • a plasmid which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to as “recombinant expression vectors" (or simply, "expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), also bacterial or human artificial chromosomes which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • bacterial or human artificial chromosomes which serve equivalent functions.
  • the identified nucleic acid sequences can be used in reporter gene constructs.
  • One utility of such gene constructs is their use to recruit proteins that modify chromatin structure.
  • the identified nucleic acid sequences can be used cooperatively in reporter gene constructs to recruit proteins that modify chromatin structure.
  • the present invention discloses the use of high-resolution tiled microarrays for the detection of multiple intronic and extragenic DNase I hypersensitive sites (DHS), regions of open chromatin that are depleted of nucleosomes and are often associated with gene regulatory elements. It was discovered that several of these DHS regions bind both tissue-specific and general transcription factors and also possess cooperative enhancer activity in vitro. Moreover, in vivo, these enhancers interact directly with the CFTR promoter region. Thus, in some aspects of the invention, recent advances in methodology to evaluate regulatory elements in the human genome in vivo, combined with a biological approach to the expression and function of CFTR, have enabled performing the first in-depth study of the organization of the entire CFTR locus.
  • DHS DNase I hypersensitive sites
  • the properties of key regulatory elements for CFTR are demonstrated, as is demonstrated a novel example of a transcriptionally active human gene adopting distinct conformations in different cell types. It is also disclosed that the active CFTR locus exists in a complex conformation involving the direct interaction of several intronic enhancers with the promoter. These interactions are most evident in epithelial cells of intestinal lineage where CFTR is most highly expressed; however, they are also apparent in epithelial cells derived from the respiratory system, which generally express CFTR at lower levels. The very low levels of CFTR expression observed in airway cell types (such as those shown in Figure 1 B) may be achieved primarily via promoter-mediated mechanisms or by inhibition of promoter-intronic enhancer contacts.
  • intestinal and genital duct cells appear to require the contribution of the intronic enhancers in order to maintain high CFTR expression.
  • These promoter-enhancer interactions do not occur in skin fibroblasts, which lack cis regulatory elements detectable by DHS mapping in the CFTR locus.
  • GM06990 B lymphoblastoid
  • primary H9 undifferentiated embryonic stem cells
  • CD4+ T cells CD4+ T cells
  • K562 erythroleukemia
  • CFTR locus A novel aspect of the CFTR locus is that key interacting elements lie within introns in addition to those distal to the gene.
  • the model for the active CFTR locus described herein predicts that the interaction of multiple enhancers and both tissue-specific and general transcription factors confers the complex regulation of CFTR expression.
  • HNF1 tissue-specific transcription factors interacting with the enhancers in intestinal cells
  • HNF1 tissue-specific transcription factors interacting with the enhancers in intestinal cells
  • the inventors previously showed to be involved in CFTR expression both in vitro and in vivo, through interactions at multiple intronic sites (Mouchel et al., 2003, Biochem. J. 378: 909-908.
  • HNF1 has been implicated in a similar mechanism of transcriptional regulation in the human ADH gene, where it binds a distal enhancer and augments gene expression (Su et al., 2006, J. Biol. Chem. 281 : 19809- 19821 ).
  • one of the general transcription factors involved in the CFTR transcriptional hub is the widely expressed factor p300.
  • This protein has histone acetyl transferase (HAT) activity and is known to be a marker of transcriptionally active elements including promoters and enhancers.
  • HAT histone acetyl transferase
  • p300-enriched sites using tiled microarrays Heintzman et al., 2009, Nature 459:108-112
  • it was discovered that several regions close to the intron 11 enhancer are associated with p300 binding in several cell CFTR- expressing cell types, corroborating the data from Caco2 cells.
  • this region may mark the end of the active domain.
  • other cells such as Caco2, which lack the +6.8 kb DHS and show very low occupancy of CTCF in this region may recruit the +15.6 kb DHS region and the end of the looped domain could then be provided by a more distal CTCF binding site at (+48.9 kb:116950000).
  • the precise definition of elements at the 3' end of the locus that interact with the CFTR promoter cannot be provided by the 3C data presented here, since one Hind III fragment encompasses both the +6.8 kb region and the +15.6kb DHS.
  • the present invention provides as a new compound a regulatory nucleic acid element, which regulates the
  • the nucleic acid element is preferably located within intron 11 of the CFTR gene.
  • the sequence of the regulatory nucleic acid element includes the 1 ,514 nucleotides located at positions 116,821 ,781-116,823,295 on human chromosome 7, described herein as the nucleic acid sequence of SEQ ID NO:1.
  • the sequence of the nucleic acid element includes at least 75% of the 1 ,514 nucleotides of SEQ ID NO:1 , preferably at least 85% of the 1 ,514 nucleotides of SEQ ID NO:1 , and more preferably at least 95% of the 1 ,514 nucleotides of SEQ ID NO:1.
  • one or more parts of the regulatory nucleic acid element are located in intron 11 , whereas other part or parts of the sequence are located in an adjacent exon, i.e. in one or more of sequences flanking intron 11.
  • the adjacent exon is exon 11 of the CFTR gene.
  • a preferred 1 ,514 nucleotides long nucleic acid element may include 1 ,457 nucleotides from intron 11 , and 57 nucleotides from exon 11.
  • the nucleic acid element preferably interacts with the promoter of the CFTR gene, regulating the expression of the CFTR gene. More preferably, the nucleic acid element enhances the expression of the CFTR gene.
  • the invention also includes: Nucleic acid elements containing up to 200 nucleotide residues including at least one defined oligonucleotide and having at least 70% homology with part or all of the sequence shown in SEQ ID NO:1 ; nucleic acid elements that are capable of controlling expression of the human CFTR gene and which have at least 70% homology with part or all of the sequence shown in SEQ ID NO:1 ; nucleic acid elements that are capable of controlling expression of the CFTR gene and which include a functional sequence at least about 10 nucleotides in length, preferably about 50 nucleotides in length, and more preferably about 250 nucleotides in length and contained within one of the sequences set out above; nucleic acid elements that are capable of regulating expression of the CFTR gene and which are derived exclusively or mainly from intron 11 of the CFTR gene, in particular from the 1 ,514 bp intron 11 sequence as shown in SEQ
  • one or more of the above nucleic acid elements is used as a part of a system for the regulation of the expression of the CFTR gene.
  • one or more regulatory nucleic acid elements are used in conjunction with the promoter of the CFTR gene, to regulate the expression of the CFTR gene.
  • the sequence of the CFTR promoter is shown as the 787 nucleotides long sequence of SEQ ID NO:2.
  • the sequence of the CFTR promoter includes at least 75% of the nucleic acid sequence of SEQ ID NO:2, preferably at least 85% of the nucleic acid sequence of SEQ ID NO:2, and more preferably at least 95% of the nucleic acid sequence of SEQ ID NO:2.
  • the present invention contemplates combinations of various embodiments of the nucleic acid element disclosed above and various embodiments of the CFTR promoter.
  • the present invention provides novel DHS that were detected in the CFTR gene, both in cell lines that had been evaluated previously by classical methods and in primary tracheal epithelial cells that were investigated here for the first time.
  • a novel DHS lies in intron 11. This enhancer lies almost exactly at the midpoint of the locus, and its association with the promoter results in the formation of two -100 kb chromosome loops.
  • One skilled in the art can further determine the molecular basis of the enhancer activity in the intron 11 DHS element and additional transcription factors that directly bind to this region.
  • HNF1 binds directly to the tissue-specific enhancer in the intron 1 DHS (Ott et al., 2009, J. Cell. Mol. Med. 13: 680-692) and this factor is also a strong candidate for involvement at the intron 11 DHS element since the ⁇ 1.5 kb region contains a number of predicted binding sites for HNF1 and is enriched by ChIP with an antibody specific for this factor.
  • HNF1 recruits and coordinates the other nuclear factors necessary for CFTR
  • the newly discovered enhancer sequence of intron 11 may be used in conjunction with the enhancer sequence of intron 1 (see e.g. Harris, U.S. Patent No.
  • the invention describes the analysis of elements associated with DHS that show enhancer activity and that interact directly with the CFTR promoter.
  • DHS elements associated with DHS that show enhancer activity and that interact directly with the CFTR promoter.
  • a number of other DHS were identified in each of the cell types that do not encompass such elements. The inventors previously showed that some of these sites had modest enhancer activity (Phylactides et al., 2002, Eur. J. Biochem. 269: 553-559), though others appear to be associated with cell type-specific CFTR expression by alternative mechanisms, as yet unidentified. It is also possible that some of these DHS may contain regulatory elements for other genes, particularly since many of the sites do not show a direct correlation with CFTR expression.
  • embodiments of the invention described herein are mainly focused on regulatory elements detected within the CFTR locus itself, it is possible that other cis and/or trans elements outside this region also coordinate regulation of the CFTR gene.
  • the present invention includes unexpected results.
  • enhancers act cooperatively to augment the CFTR basal promoter.
  • the diversity in DNase-chip data generated from the airway- derived cell types is of interest. Not wanting to be bound by the following theory, it may suggest different regulatory mechanisms in these cells.
  • the NHBE cells which are a mixture of bronchial and tracheal epithelial cells, provide evidence for similar CFTR regulatory mechanisms to those of intestinal epithelial cells.
  • the pHTE cultures of tracheal cells alone exhibit a different set of DHS and moreover, show only slight evidence for intra-locus looping.
  • each of these lung cell types can be investigated further, in order to dissect their CFTR regulatory pathways.
  • the model for CFTR gene expression that is revealed by the data presented herein provides a paradigm for other large genes with multiple regulatory elements lying within both introns and intergenic regions.
  • the observations disclosed herein can enable novel approaches to designing regulated transgenes for tissue-specific gene therapy protocols.
  • endogenous enhancers such as those identified herein may be incorporated into gene therapy vectors in order to provide stable expression, at appropriate levels, in the relevant cell types.
  • identification of the specific transcription factors acting upon genes such as CFTR will begin to reveal the cellular signaling pathways involved in regulating transcript levels, which might also be targeted for therapeutic benefit.
  • the present invention contemplates gene therapy.
  • the CFTR gene is about 189 kb in length.
  • the coding sequence comprises 27 exons and is in total about 6.5 kb in length.
  • most or all of the coding sequence of a gene is delivered into target cells.
  • regulatory regions and/or introns or intron fragments may be delivered to cells in the context of the coding sequence.
  • a regulatory element intraonic enhancer
  • the expression of the CFTR gene is greatly enhanced. It is known that the CFTR gene promoter by itself is not very active.
  • a variety of gene delivery systems may be used for cystic fibrosis gene therapy, including, e.g., adenoviruses, liposomes, and retroviral vectors.
  • the idea of gene therapy is that the DNA in question is introduced into human cells, in an episomal (non-integrated) or integrated form, and will there express the CFTR protein which acts as a chloride ion channel.
  • the DNA administered in the course of gene therapy will in principle be expressed in all human cells in which it becomes incorporated. This is because the viral and other promoters hitherto used do not have any cell specificity. But the desired effect of gene therapy is that the CFTR gene should be expressed in, and only in, those cells where it would be expressed in a normal healthy individual.
  • the invention provides a nucleic acid construct up to 15 kbp in length comprising the coding sequence of the CFTR gene together with a nucleic acid element as described above.
  • the specified maximum length of 15 kbp is not critical, and longer constructs are envisaged which are, however, much shorter than the complete CFTR gene.
  • the length of the construct is such that the construct can conveniently be incorporated in a viral or other vector for administration.
  • the nucleic acid regulator that is provided in this construct is preferably present between exon 11 and exon 12 of the coding sequence of the CFTR gene.
  • elements of this kind sometimes have powerful long-distance effects that are independent of their position or orientation in the gene. So it is envisaged that this element may
  • the regulator is positioned so as to be capable of modulating expression of the CFTR gene.
  • a CFTR gene promoter sequence is also present, operably linked to the CFTR gene and generally in a functional position at the upstream end of the coding sequence of the gene.
  • the invention provides a vector containing a nucleic acid construct as defined; and a method of treating by gene therapy a patient suffering from cystic fibrosis which method comprises administering to the patient this vector.
  • the invention further provides a method of preparing an agent for treating cystic fibrosis by gene therapy, which method comprises bringing a nucleic acid construct as defined into a form suitable for administration.
  • a method of preparing an agent for treating cystic fibrosis by gene therapy comprises bringing a nucleic acid construct as defined into a form suitable for administration.
  • the following steps are envisaged: 1 ) Place the CFTR cDNA in a vector where it is driven by its own promoter rather than by a viral one, despite the relative weakness of the CFTR promoter; 2) Modify the CFTR cDNA by PCR-mutagenesis to insert the splice donor site at the end of exon 11 (within intron 11 ) and the splice acceptor site of exon 12 (also within intron 11 ).
  • the human colon carcinoma cell lines Caco2 and HT29, primary skin fibroblasts (ATCC# GM08333) were grown by standard methods.
  • Primary human fetal male epididymis cells and primary tracheal epithelial cells were cultured as previously described (Harris et al., 1991 , Development 113: 305-310; Davis P et al., 1990, Am. J. Physiol. 258: C71- C76).
  • Normal Human Bronchial Epithelial (NHBE) cells were a mixture of primary human bronchial and tracheal epithelial cells (Lonza, CC-2541 ).
  • NHBE Normal Human Bronchial Epithelial
  • DNase-chip was performed as previously described (Crawford et al., 2006, Nat. Methods 3: 503-509), with modifications (Ott et al., 2009, J. Cell. Mol. Med. 13: 680-692) and experiment analyzed with ACME statistical software (Scacheri et al., 2006, Meth. Enzymol. 411 : 270- 282).
  • DNase-chip data will be publicly available at the Encyclopedia of DNA Elements (ENCODE) Consortium web site, University of California Santa Cruz, Santa Cruz, CA (The ENCODE Project Consortium, 2007, Nature 447: 799-816) and will also be deposited at the Gene Expression Omnibus (GEO) database, National Center for Biotechnology Information (NCBI), U.S. National Library of Medicine, Bethesda, MD.
  • ENCODE Encyclopedia of DNA Elements
  • NCBI National Center for Biotechnology Information
  • CFTR expression was assayed as described previously using Taqman ® primer/probe set spanning CFTR exons 5 and 6 (TAQEX5/6) (Mouchel et al., 2003, Biochem. J. 378: 909-908).
  • Transient promoter/enhancer reporter assays Sequences encompassing the DHS in introns 10a, b, 11 , and at +15.6, were amplified by PCR using Pfu DNA polymerase (Stratagene, La Jolla, CA). Primers are shown in Table 2. Reporter assays were performed by standard methods using a reporter gene construct driven by the 787 bp CFTR minimal promoter (Smith et al., 1996, J. Biol. Chem. 271 : 9947-9954; Phylactides et al., 2002, Eur. J. Biochem. 269: 553-559).
  • ChIP Chromatin immunoprecipitation
  • Primer sequences The primer sequences and locations used for RT-PCR, plasmid cloning, 3C, and ChIP are listed in Table 2.
  • DNase-chip was used to identify DHS in a number of cell types relevant to CFTR expression including primary human tracheal and bronchial epithelial cells, primary human fetal epididymis epithelial cells, and the human colon carcinoma cell lines Caco2 and HT29, all of which express CFTR. Human skin fibroblasts that do not express CFTR were also evaluated, to provide an example of the chromatin structure of the transcriptionally inactive CFTR locus.
  • Figure 1 shows identification of DHS within the CFTR locus in cell types relevant to CF pathology.
  • Figure 1 (A) shows averaged DNase- chip hybridization data from three (Caco2, skin fibroblasts), two (HT29, primary tracheal epithelial cells and NHBE cells), or a single (primary epididymis) experiment was analyzed with ACME statistical software (Scacheri et al, 2006, Meth. Enzymol 411 : 270-282).
  • a major DHS was identified at the CFTR promoter (Pr) in all cells that express the gene; several specific DHS of interest were detected including those in intron 1 (Intl ) and intron 11 (Int11 ).
  • CTTBP2 gene The zero point of the x-axis represents the beginning of the first CFTR exon.
  • the y-axis for each DHS track represents -loglO(P-value) between 0-16 as determined by ACME.
  • DNase-chip experiments were performed on independent cultures of skin fibroblasts and Caco2 cells, two experiments were carried out on primary tracheal and NHBE (bronchial and tracheal) epithelial cells and HT29 cells and one experiment evaluated primary epididymis cells (Figure 1A).
  • the DNase hypersensitivity tracks represent averaged data where multiple experiments were performed. The data demonstrate that each cell type has a specific and unique pattern of DHS along the locus.
  • the promoter region of the gene is hypersensitive in each CFTR- expressing cell type, but is DNase-resistant in the non-expressing skin fibroblasts. Outside the extended promoter region multiple DHS were identified, including those that are ubiquitous, common to several cell types, or cell-type specific.
  • DHS were also detected 3' to the locus at +15.6 kb, +21.5 kb, +36.6 kb and +48.9 kb from the translational stop.
  • strong DHS were detected in intron 11 (1811 + 0.8 kb) and at +15.6 kb, in addition to the +21.5 kb, 36.6 kb and 48.9 kb sites.
  • the intron 11 DHS was also detected in Caco2, HT29, and primary epididymis cells. Both colon carcinoma cell lines, Caco2 and HT29, also showed the +15.6 kb DHS.
  • intron 1 (185 + 10 kb) that the inventors previously showed to contain an intestinal-specific enhancer element in vitro and in vivo was detected in both Caco2 and HT29 cells. Additional cell-line specific DHS are evident in intron 10 (1716 + 13.2 kb) in Caco2 cells and intron 18 (3600 + 1.6 kb) in HT29.
  • the intron 10 site encompasses two closely spaced DHS at 1716 + 13.2 kb and +13.7 kb, that were characterized in the inventors' previous work (Smith et al., 2000, Genomics 64: 90-96; Phylactides et al., 2002, Eur. J. Biochem.
  • DHS located -35 kb and -44 kb with respect to the CFTR translational start site in primary epididymis cells. These upstream DHS are closer to the neighboring gene ASZ1 (ankyrin repeat, SAM and basic leucine zipper) and may be involved in its regulation.
  • the CFTR locus is organized in a complex looped structure in vivo.
  • Quantitative chromosome conformation capture (q3C), as described in Hagege et al., 2007, Nat. Protocols 2: 1722-1733, was used to test the hypothesis that c/s-acting elements located within DHS interact with each other and the CFTR promoter to regulate gene expression (Figure 2).
  • FIG. 2 shows long-range interactions between the CFTR promoter and specific DHS measured with q3C.
  • the organization of the CFTR locus is displayed above the graphs.
  • Each assayed Hind III fragment is represented by a gray bar with the restriction sites and primer locations along the top of each graph.
  • the 'bait' region of the CFTR promoter is schematically shown, which includes a primer and Taqman probe adjacent to the 5' Hind III site; this Hind III fragment spans the identified CFTR transcriptional start sites.
  • the x-axis in each graph represents position relative to the translational start site; the y-axis represents interaction frequency relative to the interaction frequency between two Hind III fragments within the ubiquitously expressed ERCC3 gene.
  • Below each graph the major DHS of each cell type (see Figure 1 ) are represented by bars. Data for each cell type are from a single representative 3C experiment (each experiment performed at least twice), error bars represent SEM of at least 2 PCR reactions for each fragment.
  • the DHS in intron 10 may be directly interacting with the promoter or exhibiting a bystander effect due to the stronger association of the intron 11 DHS with the promoter as these DHS map within about 17 kb of each other.
  • the interactions were moderate in HT29, primary epididymis, and NHBE cells. Several of these interactions were weakly evident in the primary tracheal epithelial cells which display a different pattern of DHS. Although other intronic DHS exist in these cells, none were found to interact with the promoter region.
  • CFTR intron 11 An element within CFTR intron 11 is a strong transcriptional enhancer.
  • the DNase-chip analysis revealed a strong DHS within CFTR intron 11 at 1811 + 0.8 kb in several cell types. Since the inventors previously demonstrated enhancer function associated with several intronic DHS in the CFTR gene (Smith et al., 1996, J. Biol. Chem. 271 : 9947-9954; Phylactides et al., 2002, Eur. J. Biochem. 269: 553-559;
  • Figure 3 shows how regulatory elements, i.e. intronic enhancers in this case, act cooperatively to augment the CFTR basal promoter.
  • Figure 3A Caco2 cells were transfected with pGL3B luciferase reporter constructs containing the 787 bp CFTR basal promoter and fragments of the DHS regions found in introns 1 , 10a, b, 11 , or +15.6 cloned into enhancer site of the vector in either forward or reverse orientations.
  • Figure 3B Caco2 cells were transfected with the single enhancer luciferase reporter constructs (as in Figure 3A), in addition to constructs containing multiple combinations of two or three DHS regions cloned into the enhancer site.
  • the fragment encompassing the DHS in intron 11 enhanced CFTR promoter activity almost 20-fold in the forward and reverse orientation.
  • the intron 1 enhancer (Ott et al., 2009, J. Cell. Mol. Med. 13: 680-692) acts as a modest enhancer with about 5-fold effect on the CFTR promoter, while the +15.6 kb DHS region does not show enhancer activity.
  • HNF1 hepatocyte nuclear factor 1
  • Figure 4 shows how HNF1 and p300 associate with multiple DHS of the CFTR locus in Caco2 cells.
  • Figure 5 is a graph illustrating how EcoRI 3C detects interactions between CFTR promoter and elements 3' of the gene. 3C was carried out as described except using EcoRI instead of Hindi II . Data shown are from CF7 ⁇ R-expressing HT-29 cells.
  • Figure 6 is a graph showing enrichment of Rad21 at -20.9, +6.8 and +48.9 kb DHS by ChlP. Experiments were carried out as described using Caco2 chromatin and rabbit anti-Rad21 antibody.
  • ⁇ denotes distance from last coding base (4574) in the CFTR transcript.
  • TAQEX6a R ATGAGGAGTGCCACTTGCAAA 15
  • FAM denotes FAM labeling of the sequence.
  • TAMRA denotes TAMRA labeling of the sequence.

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Abstract

La présente invention concerne de nouvelles compositions et de nouveaux procédés pour le contrôle de l'expression d'un gène CFTR, en particulier le gène CFTR humain. L'invention concerne un nouvel élément régulateur de gène CFTR capable d'augmenter l'activité du promoteur de gène CFTR, et des constructions d'acide nucléique comprenant l'élément conjointement avec la région codante du gène CFTR. L'élément et les constructions contenant celui-ci sont utiles en thérapie génique pour traiter la mucoviscidose.
PCT/US2010/048872 2009-10-15 2010-09-15 Activateur activant cftr Ceased WO2011046700A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020086836A1 (en) * 1996-03-20 2002-07-04 Ann Harris Cftr gene regulator
US20070161029A1 (en) * 2005-12-05 2007-07-12 Panomics, Inc. High throughput profiling of methylation status of promoter regions of genes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020086836A1 (en) * 1996-03-20 2002-07-04 Ann Harris Cftr gene regulator
US20070161029A1 (en) * 2005-12-05 2007-07-12 Panomics, Inc. High throughput profiling of methylation status of promoter regions of genes

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] 1 March 2004 (2004-03-01), HILLIER ET AL.: "Homo Sapiens BAC Clone CTB-68P20 from 7, Complete Sequence.", retrieved from http://www.ncbi.nlm.nih.gov/nuccore/1809237 Database accession no. AC000111 *
OTT ET AL.: "Intronic Enhancers Coordinate Epithelial-Specific Looping of the Active CFTR Locus.", PNAS., vol. 106, no. 47, 24 November 2009 (2009-11-24), pages 19934 - 19939 *

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