Patent Application: US-201314654325-A

Abstract:
the present invention is based upon the generation of a nucleic acid construct which may be used to generate transgenic non - human animals . the construct exploits a filaggrin based promoter region which directs the expression of a reporter sequence operatively linked thereto . the present invention provides a nucleic acid , said nucleic acid encoding a filaggrin promoter element and a nucleic acid sequence operatively linked thereto .

Description:
the present invention will now be described in detail with reference to the following figures which show : fig1 : schematic of how the flg - 10k promoter construct was made and used to generate transgenic mice ( not to scale ). fig2 : live animal imaging ( under anaesthetic ) shows very strong luciferase expression in footpad and much lower levels elsewhere in the epidermis . fig3 : regional expression of luciferase in the flg - luc2p mouse . fig4 : immunofluorescence localisation of luciferase within mouse footpad epidermis . ( a ) negative control stained only for dna to reveal nuclei ( blue ; dapi stain ). ( b ) endogenous filaggrin staining ( red ). ( c ) wild - type mouse epidermis has no luciferase expression . ( d ) in the flg - luc mouse , luciferase is localised to the granular layers . fig5 : the importance of using left - to - right ratio analysis in using flg - luc2p mice to assess delivery of drugs or gene silencing agents to the footpad epidermis . ( a ) ivis200 imaging of the same mouse on 5 consecutive days shows that while total gene expression varies from day - to - day expression remains consistent between the left and right paws . ( b ) graphical analysis of left / right ratio over 5 days ( n = 5 mice ). fig6 : testing the usefulness of flg - luc2p mice to assess epidermal delivery of drugs or gene silencing agents . ( a ) the left and right footpads of control mice were treated with vehicle only ( phosphate buffered saline , pbs ). ( b ) left paw was injected with either accell sirna or native sirna targeting the luc2p mrna and the right paw was treated with the corresponding native sirna or accell sirna non - targeting control sirna ( nsc4 , an inverted bacterial laczsequence ). a 10 , 146 by human filaggrin promoter fragment was cloned from a genomic bacterial artificial chromosome ( bac ) clone encompassing the entire human locus using recombineering . this clone covers the region from ˜ 10 kb upstream of the transcription start site , all of the 15 by exon 1 ( partial 5 ′ utr ), and ends at an mlu i restriction site just 18 by inside intron 1 . a 483 by fragment containing the last 459 by of intron 1 and the start of exon 2 ( covering the remainder of the 5 ′ utr was generated by pcr and sequence - verified . this fragment had artificial restriction sites added to allow cloning and also the atg of exon 2 was mutated out so that translation will start from the first kozak sequence and atg placed downstream . the two fragments were ligated together via their mlu i sites ( within intron 1 ) to make a construct that consists of & gt ; 10 kb upstream promoter sequence and a cut - down intron 1 . the whole fragment is flanked by an xho i site upstream and a hind iii site downstream and consists of 10 , 623 by of dna . this construct was designated “ flg - 10k ” for convenience . the xho i - hind iii fragment was cloned into pgl4 . 21 so that the flg - 10k promoter drives luc2p expression . luc2p encodes the mammalian codon - optimized , protein destabilized firefly luciferase gene . cloned by recombineering , with addition of xho i and mul i sites in recombination primers xho i ctcgagtcagtgtctggcagtgaatgagctacaaattgttttcatattgc ttacctgaaggccagtgcttgtttagctgctgaagaaaaatagaaacctt atggcatttagaacatagtttattctttaagtgcagaagtgtgtgactta acccttgactggcatggtcttagctcctgtttacaatttggtatcttact gccacaaagagtctgttctatcagtcttacattctctattttcacatcaa tgctggccagttgtgtctaaacactgcaaaagggagggtatataacaaga tatgtctgacttcctgagccatcatggctgggaactcagtttttaagatt tatctagggtccatttggccaagattggggtggggagggtctgttcagtc agttgagggctttaagatttatttttagtttacaggaggaaccatgttca gactaccaaatagtttccaatgctgagcagggctagggccacagacacac actcctgtttccatagaaattcctcaggcaggcctcatgtacttagccag gaccctgctcttgcccccaaaggaatttatccttttaaaagtataatatt cctcttatctgactgttctactgcacagtggggtaaccacagttaacaat atcatatactatatttcaaaatagctattaatagaagagagaattttgaa tattctcaccataaagaaatgacaaatgtttgaagtgattgatatgctaa ttaccctgatttgatcattacataatgtaaacatgtatagaaacatcact taaatataaacatgcacaattactagatgtcagttaaaaacaaaataaaa cttagaaaagtataatgtgatgatactggttcagggatctgatctttaat ccgaaggaaaagaggagacatgaaagaactgaggggagggcttgttattc ctcttcctcccctttctctattaccacccttgcattaacatgcccctggt tatcttgctatgttttttatatttattatgaatgcagaacacagcaaaaa gtaaatagcaattatatcagttctaattctgcttcaagtaatagaacaac tcaaagcttattaaattaaagggagaatgtattggttcacgtaattgaaa agtcttgaaatggggctagcaggctgtactcactccaaggctcaaagtat atcatcaagattgggtgagtctctcaatctctcagctctgcttttccctc 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tacaccatggattttgtgatcttggctctaattagggggccaatatggtg gacatggtgttatttgaatttaaccatttttcatatctcatatgaaaagt tcctagaattaaaaatttcatgaagaaaacaaaaaatagctgaggatttc tagatgcatactgtgaaaagaaatgcaaatattaatagtaaccatgttct atgaattagtttaagcatatacatgaaatgctgttagagtacacgttgca agagtaactaactgatggcctgattagaaaaatgatagaatgaggtaaag aaagaggtagattctgggaaatatggattaaaatgatgaaacaaaaaatg agaaaaaaactaccttaccttctcaagtattgccatagaagagcagaagt catcctgagcaaagctaaaggcaaaaaacctaggctccagactgatacac tggcttgataatccacactgtgagatgtcaccaaaagttgtctgatccag ctgtggggacttggctccaagttgtctggatatcctaaaataatataaat cccttgaaacaagtgctctaagaaatggaggaagccatactttagggaat gctgataaaatgagtggaaatgttggtggtgtgagtccacctctacctca acaaggtcaccactgatgtgagaatccatgaggttaacaaaaacgagcat cattagtctcataaattaacacttgaaggactttggagccttgggtctca gtgattcttctggaaatatggtagtttagggtctttaataatttttttaa cttttgttatgagcagaaattgtaaataacatgtaaatacaactccagag tggttctgatgttcaccttctgaactcatctagttctcaaactactagat tatgcaacccttgtgctcctacagctaaaccacatatactattctgagcc ctcccctaactacacctgggtaagaggagtgtggtgtagtctggtgttgt gctataccaatagtcatggagatatcaatcaaacatctgcctaatctaca aggtgtaggtggtgaattatgaagaatgagtaaccaggagacttacgatg agagaataaagtctaagttgataaaaagttagtgctgagtagatagaggg gtcttcaggaattggaagcttggtgagggtccccatcatccagaataagt actggtcttcagcaggaagaatttaagaatagaagttacctaaagctcat gaattagtatcagttcaattatgctggtaatctgattttgatctttctca gttatgataaaagtctctgcctggtcatgatataaaccttctttgttctt tagtcttccctacttacaagattaaagtcctggcctaaaatatgagccct aaggtcctgctacctagtgacatgatattgttgataccaactctccatta cccactgcctactgaaatctcctattcctgccttctccatcattgagttc tgtccacctgtcagagtctacttcattgggttgccctccctaacctgccc aacaccaactgcttgccaggccgctaacctattccctcttgttctgcacc ttctctcatagtccacttccaatgcatcaccaagatctgcctctaaaatg tatcttctcttcaatcccactgccaatgccttagtttgagattttactat ctcttccttcttttatataaattgcctgtatccagctcaatccgccatcc atacttccagtagttacatttactgaatgaaaatctggtcctgtcacttt tccacttaaaaacccttcagttgccacccaatataaataatatatacatc acttagaggcttccaaggcttccaagccctttccatatgacctttcaggc ctgcctctccctgatctttcccatacaatccaataagtgcttttattgtt ctttaatgttctttaatgtcctttaatgttcagctcaaatattacctccc cttgacctctctaaaagaaaacagcttgcttcctcaagtaaagctattta ctaatacctaaggtattcctaaaggtatttacaaatacctttattataac acatatcatactgtttatatgtatgctttcttcattagattataaactct tggaggacaaggagcatgccttcttcatttacagttctgtgacaagcatg gtgactggcaagtagtcactggcaagaaatgtttcttgaatgaataaatg atccctaaatactgtgacctatctcttagtctgaatttccctcagttacc tgcagaaattttccctctggaatatattcttgtttatctatctgtctacc tgtctgtctgtctgtctattctatctatctataatctccctatatacaaa ggaaacaggtgagaaaggagagtagaagcttatttcaagttccagtccct cctgatctacattctcctgtaattattagcctatgttaccatgtctgaac agaaaatattggtgatgcccttgatcatgaatagctatcatgtctctgtt ctggcttgctcctggatttttttttttccagtttcatgatgctgcatcac tctgtcaccagtcctcactgttcgtttctatggaacatgaaatggtgaat ggtccatcctttactgctgatactagtcattgctgaaacagccaccctaa agatctcacagtctctctgtttataaacatttaagagtcaagtcatgaag gctttctcccttaactacatgggatgatcagttgttttatctgttatttt ttctgttattacttttgtccttaattgttagagaaaactttcatataaca cagttactacataaatgccccctcccttcacatcttagaatgcctctggc catctctgtagttgtacttgagaggtttaataaagtaccataagtttggg aaaattagctttattgatataagcacttcctggagaatatttcttcacta tacaaaaagctcatatttgatcattgttttctctatatctgtctatctct ctcacatacattctgagtgcctgtgtgtgtgtgtgtgtccccagtgactt ctgcatctgtagtagctggaataacatgtgtgtcacatgtacccccacca cacacacacacacacacacacctctgccacgggcttcgttcaattctatt ctcaatttccaaccttgtcagccatcttgatttcctcttctccaaacctc aaagaaactgttaatgagtacccgagaatgaaaatgttgggcatatggaa aaactgaaagacaatccatattgccataaaatggcctgcttttatctgga aaagccttattatcactcacactcattccttctacatcctttacctcctt ctctttgtctttctgtctgtctctcattctgtctctatcacacacacaca cacaacacacacagagagagagagagggagagagagagagagagagagag aaagagagactggctataaagaaagcagcatctgagggcatcaatggtaa ttcgaacattttgtttgcttgggatcaagattcctttccatcaccattgt cctggcaaatataagctgcaagtggaagtgttttagccagatactgctcc accactctatggctaactaagcaggaatatcagcttcaactatgccatgg aattcaagagaatattcaacctggaaaaattctaaccccaaacagcacct cccaaaagatgactacagctcctgtaggaaatcatttaaccacaaattcc aactccccttcactcctacagcctcagtcacacatctcaaagggctgatc cttgaattgtgacaacctgacccacatcagcagcccagaggccagatggc aacacaatgcctttccagcctactgggtaggaaaagggagggacaagcaa ttgaatgattataattgaaatcctgtaatttattatttgtcaataactcc tgccttgggggagttcccttcactccttagcaaatgctggcagccgcaat atgtgaccacagacacctaaaacaccactgaaagcatttcattatgtggc aacaatgatatggagaataagatctctctggagataagaagacatgccac atctcagagttcactattcaacagcaaagaatttgaatagggggaaaaat tccttagacttagggggaaaaattcctgagattctgagggtaaaaagcta gcatgcaagtgggatcagccagactggcaggaagtggggcatgaaaaccc aagaactatcctcctgtttgcagtataatgttacccctgcagttattaaa ctgcacagatcaaataactatttgaactgagctgagctgagtcaagcaaa gaaatgtacttcaatcatagaatagtagctgtcaggttgaaaagggcatc tatcagacatctacccaatgttcctctacctaactcccatcctaattatc cctgtctcatgttcatctccctgtaattgatgcaacctgatgcaacataa agaggatttggagtgaaaagccctgagttggaggcctgggccttgacttt ttaatttactagccataccacattggtccttctagtcttcagctacaatg tgagcagattaaactagttgatgccaagattccatccagttcaaaattct ctaggcaataatagggaactcactatctccaaaaataattatttgcatgt ttggacaattctattaaaaagtcgtcacttacataaagccaaaacatggt ttcttatagtttctacatatttatctgagttctacctgccttgagattat atagtccaagtataatccctcttctatataacagccactcatatagccaa agacagatattgcatctcccatccatctatccatccatccatccatccat ccatcccactgagttctgtttgccactcatctagacaatcattctatcta ctttactcagaacacataccaatttgtccatatccttactgaaaaatggt catcagaactgaacaaagagctacagaaatagtttgataaatgctgagcc aagtacctcaccacctattttgttccagctaatgtgtttattttaatgca gcctaatatcacattatctttttggctatggcatcactctgccactgcca caatattgctttttttactcatgtcttctaccctatccttgtaaagtgta atttggaactggagtcagaattttactttttcaataatattcatttatca gatttgagccattggtgcaacatgataaaatcttcttgaatcctcagtct atcctaaaaatatttgccatctatccaaattttcacctaagttattgata aaatacaatgagcacaacatggtcaggaaaaaagtcctagagcagaccac taaacaataccctccatattgacataattctgcaagatttggggtctttt ggccccaaagaaggtcatatccaagattgcctcattcttgacactctctc aatgctaccttcttcactcactcatagtccatggtctagtctttctgctg atcctgcaatgatttccacagttaagttcacccagacataggtactttga gagaaatcactgtttaaaacattaaaaatataaataaggttaacaaaaaa aggataaaagttcacacccagaacaatatgccatggtggaaataatgtgg gagttggagtctgaataacctcagattaaatcctagttctgtaacttact tgcttggtgaccttgggtgccttatcagtatcagttccctcatctctaaa atagaggaataataatgacctcaaagggttgtcctgatgattaaaattaa tacacgcaaaatcttagcacattcctagcacataatgggtatacaatata gattactatcattattacatcatggtagagacaaaaatgcaataactact ggcataaaaaaattagacccaagaagaaaaaaggagaaaatgaatatctg tgttctacagatgttaaccaatactccacaaaagaaagaatcaatggtct aataaggttaagaacgaggacattaaacacagttttaaaaactcgggcat atttttatctctttgactctgaacaagattggcacacccttgcatccctt atttgatcatgtcccaggcattgctgtagggcccaaggacacaaaggatg actcagacccagtcaatgctagggtgagaagaaacacaggattcaaggca ccagggggcagggggcacacatataaataagtaattaatataaaatatca ccagtgctattgagaggtaaatacagagtatgtgagggaacactgataaa tctggagatgtcaggtaaggctttatgcaggaggtggtaattttgaagaa tcttaaaagataaagagaagttaaggatgagggtctctcaagtcaaggta gtggaggatagcacgcaaagattttgaacagtagtggaccaatatgggct cgtccaagaaatattgctcattctagagtgacttttccatgagatacagg taataagagggacaattgaaaagatacagattcatgcctattgtaaaggg gcttgtacgtcaggtaaaaaagtctgaaattctgcaggcaagaagaaacc agcacagcgaatgacataagatggtaagccagcttaatagtagagagaga ggctagttacgacacttttttttctttaccaatacccaattgaaagatgg tcaggtccagaactaaaattatagcactggaggtagagaataggcctaca taacaaaatctaagcctgagtataattgatacgatgtgacatgagcatga ggggttaaggaaaaggagaaatgaaaatgactttgtctagcttgagacac ccagtgtgtgggggcatcattaccaatatatgggcttctggaggatagta cattttggtaggaggcacaatgtaagttcagttttcaacactatggattg agataaccatgggacatccatatggagatgcaatctgctcaacataacag tgtgtatatcataagacagaccagggataggagtcccctgtatatttatg gcaataaaagaaaattcatggtttaagaaggaagagtatgtggaacatgt ctctgatgccacgatgtaataaccttgaaaacaaagtaaaattacactaa tgagtcttgcctaattaaactcatgctcctagtgatcaccacttctagtt caattgttcacattcttgctctgctttgaaaaattaaaattaaatttgcc tatcctctactgaccataatttctagaagacggcattcatctcatggcaa gttcttcagtacccaaagatggaatacatagattaaaaaagaacatatat gtagatgcttgtgatgttttcctatcataaattgaatttcaagttcttat aaacgtattaatatgtcctactcttctagagacaaggatcaggaagtgta tttatcaatagatatttaccaagcacctgtcaagccaaagtggggttaca gaaaagtaggtatgggccctgcacacaaacaacctgtattagccaaaggg acccttccataaaatttccaatatgtaaacccaaatttggaacttgctga aacaagtacagatgagtacgtgaggaagctgggaagtaaacacaggttgc tggagaaatagaggtggagatatgggtggatctaggtttggttaggaatg aatcagaccatcccacagagggtggctcctccctgcatggggcctgctat aaaagggccattatctcagccttcagtacccagcaggctccttcaggcta cattctatttgctcttttggtgaacaaggtaagaaggaatacgcgt mlu i the flg - 10k - luc2p construct was sent to taconicartemis gmbh under contract to generate a single - copy transgenic mouse via mouse embryonic stem cell gene targeting into the murine rosa26 icous . rosa26 is a site where high - efficiency gene targeting can be achieved and is a locus where at least heterozygous transgene insertion exerts no harmful phenotypic effects . the resultant mice were transported to dundee . indirect immunofluorescence was used to show co - expression of luciferase with endogenous mouse filaggrin . all mice were treated with a single intradermal injection on day 0 and were imaged daily for 6 days . flg - luc2p heterzygous animals were subjected to live animal imaging using the caliper / xenogen ivis200 system , which allows imaging of the luc2p gene bioluminescence signal , following intraperitoneal injection of luciferin ( the luc2p substrate ), thus revealing , in real - time , quantifiable in vivo luciferase gene activity . surprisingly , luc2p reporter gene expression in these mice was largely detected in the footpad epidermis , whereas one would have predicted that the flg promoter would drive expression all over the epidermis . upon covering the footpads and performing longer exposures , luc2p expression can be detected elsewhere in the epidermis but at a much lower level than footpad . the results of live animal imaging ( under anaesthetic ) are shown in fig2 . very strong luciferase expression is detected in the footpad and much lower levels elsewhere in the epidermis . fig2 ( a ) provides a colour bar showing the gene expression range quantified by the ivis200 ( red tones = maximal expression ; blue tones = minimal expression ). footpad expression following a 1 second exposure in the ivis200 is shown in fig2 ( b ). note that expression is also very strong in the forepaws ( covered up here ). when all paws are covered , a 60 second exposure reveals expression in the tail epidermis , as well as shaved back , ears and snout ( see fig2 ( c )). again , when paws are covered , a 60 second exposure reveals expression in the epidermis of tail , shaved stomach , perioral and perianal regions ( see fig2 d ). thus , reporter gene expression is widespread in the epidermis but is vastly greater in the footpads . regional gene expression was also examined by quantitative reverse transcriptase pcr ( qrt - pcr ; applied biosystems taqman gene expression assay ) and western blot for luciferase protein . this confirmed that luc2p expression is very much greater in the footpad epidermis than elsewhere in the mouse epidermis on both the mrna transcript and protein levels ( see fig3 , below ). by qrt - pcr it was shown that luc2p mrna expression is much higher in the footpad compared to other epidermal regions . similarly , fig3 b shows that luciferase protein was detected by western blot in footpad tissue but fell below the limits of detection in other epidermal regions . these results are consistent with what was observed by live animal imaging of the bioluminescence signal derived from luc2p expression ( see fig2 ). to determine if the flg - 10k promoter is able to direct expression to the correct epidermal compartment — the granular layer — where filaggrin is expressed , immunofluorescence staining was performed in mouse footpad epidermis using antibodies against murine filaggrin and luciferase . this revealed that indeed , the flg - 10k promoter directs expression to the correct epidermal cell layers ( see fig4 ). fig4 ( b ) shows that the endogenous filaggrin is localised to the outermost living layers of the epidermis ( granular layers ). in wild - type mouse epidermis fig4 c shows that there is no luciferase expression . in the flg - luc mouse , luciferase is localised to the granular layers , mirroring the expression of endogenous filaggrin ( see fig4 d and compare to fig4 b ). overall , these data show that the flg - 10k promoter , when targeted into the murine rosa26 locus , drives reporter gene expression in the granular layer of the epidermis and that this expression is largely limited to the footpad — the region of the mouse skin that is most similar histologically to human skin . this footpad - localised expression was not predicted and may be a consequence of ( a ) the precise flg - 10k sequence used including its shortened intron 1 and lack of intron 2 ; ( b ) targeting into rosa26 , outside of the epidermal differentiation complex gene cluster where flg is normally located ; and / or ( c ) the fact that the human filaggrin promoter was used here to direct expression in the mouse . the footpad - restricted high - level reporter gene expression in the flg - luc2p mice is particularly useful for analysis of filaggrin gene expression in real - time . the well circumscribed expression in the footpad , with negligible expression elsewhere in the epidermis , makes this mouse model exceptionally well suited to split body studies where one paw receives injected or topical treatment and the other side receives control treatment . for this to be a useful system for testing cutaneous delivery of therapeutics , the left - to - right expression levels must be consistent from day to day . this was investigated by imaging the same set of flg - luc2p mice over time and comparing quantification of bioluminescence signals from the left and right footpads ( fig5 ). this showed that , although there are clear differences in overall gene expression from day to day ( fig5 a ), the left - to - right ratio is very consistent ( fig5 b ). therefore , these mice are highly suitable for split body trials designed to assess delivery of drugs or gene silencing agents to the epidermis . to test the ability of flg - luc mice to act as a model for epidermal delivery of drugs and gene silencing agents , sirna - targeting luc2p was intradermally injected into the left footpad of flg - luc2p mice . control sirna was injected into the right footpad . two types of sirna chemistries were used : native rna or dharmacon &# 39 ; s accell sirna chemistry ( nuclease resistant with a self - delivery moiety to aid cell penetration ). mice were given a single injection of sirna on day 0 and were imaged daily over 6 days . the results are summarised in fig6 . this analysis showed that ( a ) these mice are very useful for assessing epidermal delivery of molecules aimed at silencing gene expression ; ( b ) relative efficacy and longevity of gene silencing agents ( here accell was superior to native rna chemistry ) and similarly , ( c ) small molecules or other agents aimed at up - or down - regulation of filaggrin gene expression can also be assayed in vivo . pbs treatment only had no effect on left - right ratio over the entire time course ; see also green trace fig6 b . in the test mice the left paw was injected with either accell sirna or native sirna targeting the luc2p mrna and the right paw was treated with the corresponding native sirna or accell sirna non - targeting control sirna ( nsc4 , an inverted bacterial lacz sequence ). in both cases , the luc2p - specific sirna knocked down luciferase gene expression . in the case of accell , gene expression was reduced by 80 % and remained at this level for 4 days , after which the left - right ratio returned slowly to 1 . in the case of native sirna , the level of knockdown was only slightly lower but it returned to baseline more quickly . we have developed a transgenic mouse expressing a luciferase reporter gene under control of a modified human filaggrin gene promoter . this is a very valuable animal model that will allow in vivo analysis and validation of chemical compounds that can up - regulate the activity of the human gene . surprisingly , we found that animals modified to harbour the nucleic acid constructs described herein , generated a luciferase reporter gene that is expressed primarily in the footpad epidermis rather than all over the epidermis . thus , these animals allow live imaging of the luciferase reporter in small , circumscribed areas and left - right comparison in vehicle / control experiments such as assessment of topically applied filaggrin upregulation compounds . moreover , the mouse footpad epidermis is most comparable to human skin in terms of tissue architecture . in addition to the study of filaggrin gene regulation , these animals can also be used for the in vivo assessment of cutaneous delivery of gene silencing technologies , such as small interfering rna ( sirna ) or antisense molecules , applied by injection , systemically or topical formulation . flg - luc mice express the luciferase reporter gene , luc2p , primarily in the footpad , the site of mouse epidermis most similar in structure to human epidermis , with low - level negligible expression elsewhere in the epidermis . the flg - 10k promoter directs expression to the granular layer of the epidermis , where filaggrin is normally expressed in mouse or human epidermis . using left - right ratio analysis , these mice are a useful model system for assessing epidermal delivery , and relative efficacy and longevity of gene silencing agents ( e . g . sirna ). similarly , these mice can be used to assess cutaneous delivery of small molecules or other agents aimed at modulating filaggrin gene expression , in real - time , in living animals .