Patent Application: US-201414774586-A

Abstract:
the present invention relates to at least one agent capable of increasing the level of one or more mirna in a cell or cells of a subject , said mirna comprising the sequence uucccuu , for use in the treatment and / or prevention of a retinal dystrophy , in particular characterized by photoreceptor degeneration , relative pharmaceutical compositions , nucleic acids , vectors and host cells .

Description:
recombinant aav vectors containing the murine precursor sequence of mir - 204 and mir - 211 under the cytomegalovirus ( cmv ) promoter were constructed by a two - step cloning protocol . initially , the cassettes containing the precursor of mir - 204 and mir - 211 were amplified from mouse genomic dna using the following two sets of oligonucleotides : 5 ′- ataagaatgcggccgcctgttcaggacttggctaag - 3 ′ ( seq id no : 17 ) and 5 ′- cgcggatccaacatggggttgttaatctg - 3 ′ ( seq id no : 18 ) for mir - 204 ; 5 ′- ataagaatgcggccgctctgaccatgcaatcacag - 3 ′ ( seq id no : 19 ) and 5 ′- cgcggatccaatggatcagggtggcatc - 3 ′ ( seq id no : 20 ) for mir - 211 . the obtained amplimers were subcloned in the topo ® ta cloning ® vector ( invitrogen ) and released following digestion with not i and bamh i . the fragment was then cloned into the not i - bamh i sites of the paav2 . 1 - cmv - egfp plasmid ( 25 ) and used for the production of aav2 / 8 vectors . the vector in which expression of the precursor mir - 204 is under the control of a photoreceptor - specific promoter ( paav . rho . premir - 204 ) was generated by exchanging the cmv promoter of paav . cmv . premir204 with the rhodopsin ( rho ) promoter sequence . briefly , the sequence corresponding to the human rhodopsin promoter was released from paav2 . 1 - rho - egfp plasmid ( 25 ) by restriction with nhe i and not i and was cloned in the paav . cmv . premir204 backbone , previously digested with the same enzymes . to generate the vectors expressing haipl1 ( paav2 . 1 - cmv - haipl1 ), the coding sequence of the haipl1 gene was amplified from human retina cdna ( biochain , hayward , calif .) using the primers haipl1 - noti - forward ( 5 ′- atatgcggccgccatggatgccgctctgctcct - 3 ′) seq id no : 21 and haipl1 - hindiii - reverse ( 5 ′- acgcgtaagcttttatcagtgctgcagcgagtgcc - 3 ′) seq id no : 22 and cloned into the paav2 . 1 - cmv - egfp following digestion with not i and hind iii . recombinant aav2 / 8 viruses were produced by the tigem aav injection core according to protocols described elsewhere ( 26 ). for each viral preparation , physical titers [ genome copies per milliliter ( gc / ml )] were determined by pcr quantification using taqman ( 27 ). all studies on mice were conducted in strict accordance with the institutional guidelines for animal research and with the association for research in vision and ophthalmology ( arvo ) statement for the use of animal in ophthalmic and vision research . all surgery was performed under anesthesia , and all efforts were made to minimize suffering . postnatal mice were anesthetized by hypothermia for 2 min at 4 ° c . and injected subretinally in the dorsal retinal areas with 1 μl of aav vectors corresponding to 1 × 10 9 genome copies ( gc ). the same individual performed all the surgical procedures to minimize variability in the injection technique . animals were sacrificed by cervical dislocation . adult mice ( were anesthetized with an intraperitoneal injection of avertin ( 1 . 25 % w / v of 2 , 2 , 2 - tribromoethanol and 2 . 5 % v / v of 2 - methyl - 2 - butanol ; sigma - aldrich , st . louis , mo .) at 2 ml / 100 g of body weight , and viral vectors were delivered via a trans - scleral transchoroidal approach , as previously described ( 28 ). mice were injected in one eye with 1 ul of a mix comprised of 9 : 1 v / v aav . cmv . premir204 / 211 and aav . cmv . egf . the contralateral eye was injected with 1 ul of aav . cmv . egfp and served as control . following injection ( 17 - 30 days later ), the extent of transduction was assessed by ophthalmoscopy and eyes were harvested . frozen retinal sections on pen - membrane slices were microdissected using an lmd 6500 microscope . slices were fixed for 2 minutes in precold 75 % ethanol ( etoh ) in depc water , then washed twice in depc water for 30 seconds and stained in meyer &# 39 ; s hematoxylin 7 μm for 1 minute . after the nuclei hematoxylin staining , the slices were washed twice in depc water for 30 seconds and dehydrated in etoh 70 %, etoh 80 %, etoh 90 %, twice etoh 100 % ( 30 seconds each ) and dried on air for 15 minutes . the laser parameters used for the microdissection were : power 60 , aperture 7 , speed 7 , specimen balance 46 and offset 25 . mirna expression analysis in mice administered with the aav . cmv . egfp and aav . cmv . premir204 / 211 constructs was performed on samples from whole retinas and optic cups , respectively . total rna was extracted using the mirneasy kit ( qiagen , inc ., hilden , germany ) according to the manufacturer &# 39 ; s instructions and quantified using the nanodrop 1000 ( thermo fischer scientific , waltham , mass .). rna quality was assessed by gel electrophoresis . quantitative ( q ) reverse transcriptase ( rt -) pcr - based detection of mature mir - 204 , mir - 124a and sno234 was performed using the taqman ® microrna assays ( applied biosystems , foster city , calif .). all reactions were performed in triplicate . the qrt - pcr results , recorded as threshold cycle numbers ( ct ), were normalized to the sno234 reference small rna and the relative fold - change of expression was calculated using the 2 − ddct method . for electrophysiological recordings , mice were dark - adapted for 3 hours , accommodated in a stereotaxic apparatus under dim red light , their pupils dilated with a drop of 1 % tropicamide ( alcon laboratories , inc ., fort worth , tex .) and the body temperature maintained at 37 . 5 ° c . ergs were evoked by 10 - ms flashes of different light intensities ranging from 10 4 to 20 cd · s / m 2 generated through a ganzfeld stimulator ( cso , florence , italy ). to minimize the noise , three different responses evoked by light were averaged for each luminance step ( the time interval between light stimuli was 4 - 5 min ). the electrophysiological signals were recorded with gold - plated electrodes inserted under the lower eyelids in contact with the cornea . electrodes in each eye were referred to a needle electrode inserted subcutaneously at the level of the corresponding frontal region . the different electrodes were connected to a two - channel amplifier . amplitudes of a - and b - waves were plotted as a function of increasing light intensities . after completion of responses obtained in dark - adapted conditions ( scotopic ) the recording session continued with the aim to dissect the cone pathway mediating the light response ( photopic ). to this end , the erg in response to light of 20 cd · s / m 2 was recorded in the presence of a continuous background light ( background light set at 50 cd / m 2 ). for each group , the mean b - wave amplitude was plotted as a function of luminance ( transfer curve ) under scotopic and photopic conditions . mice were sacrificed , and their eyeballs were then harvested and fixed overnight by immersion in 4 % paraformaldehyde ( pfa ). before harvesting the eyeballs , the temporal aspect of the sclerae was marked by cautery in order to orient the eyes with respect to the injection site at the moment of the inclusion . the eyes were infiltrated with 30 % sucrose for cryopreservation and embedded in tissue freezing medium ( o . c . t . matrix , kaltek , padua , italy ) in pairs ( i . e . left and right eye ) to facilitate comparative analysis . for each eye , 150 to 200 serial sections ( 10 μm - thick ) were cut along the horizontal plane and the sections were progressively distributed on 10 slides so that each slide contained 10 to 15 sections , each representative of the whole eye at different levels . the sections were stained with 4 ′, 6 ′- diamidino - 2 - phenylindole ( vectashield , vector lab inc ., peterborough , uk ) and egfp was monitored with a zeiss axiocam ( carl zeiss , oberkochen , germany ) at different magnifications . frozen retinal sections were washed once with pbs and then fixed for 10 min in 4 % pfa . sections were then permeabilized either for 15 min in pbs containing 1 % np - 40 ( for anti - rhodopsin , anti - cone arrestin , anti - glutamine synthetase ) or in citrate buffer ( for the anti - m - and s - opsin ). blocking solution containing 10 % normal goat serum ( sigma - aldrich , st . louis , mo .) was applied for 1 hour . primary antibodies were diluted in pbs and incubated overnight at 4 ° c . the secondary antibody ( alexa fluor ® 594 , anti - rabbit or anti - mouse , 1 : 1000 ; molecular probes , invitrogen , carlsbad , calif .) was incubated for 45 min . the primary antibodies used were anti - hcar ( 29 ), opn1mw ( ab5405 ; millipore ), opn1sw ( ab5407 ; millipore ), rhodopsin ( abcam ) and anti - glutamine synthetase ( mab302 ; millipore ). vectashield ( vector lab inc ., peterborough , uk ) was used to visualize nuclei . sections were photographed using zeiss ( lsm 710 ) confocal microscopy . apoptotic nuclei in frozen retinal sections were detected by the tdt - mediated dutp terminal nick - end labeling kit according to the manufacturer &# 39 ; s instructions ( in situ cell death detection kit , tmr red ; roche ). here , the authors propose to use two mirnas , namely mir - 204 and mir - 211 , to protect the retina from neuronal degeneration . in this study , the authors set to determine the effect that the delivery of mir - 204 / 211 to retinal cells has on the progression of neurodegeneration . towards this goal , the authors decided to deliver the precursor forms of mir - 204 ( premir - 204 ) and of mir - 211 ( premir - 211 ) in the retina ( namely in the subretinal space ) of either wild - type mice or mouse models of retinal degeneration . in particular , the authors used the following injection scheme : each mouse analyzed was injected in one eye with an adeno - associated viral ( aav ) construct containing the precursor sequence of either mir - 204 or of mir - 211 ( see methods for details ). the contralateral eye of each animal was injected , using the same strategy , with an aav construct containing a reporter gene cassette ( enhanced green fluorescent protein , egfp ) only and served as an experimental control . for these experiments , the authors decided to use the aav serotype 2 / 8 , which was previously shown to effectively transduce the mammalian retina , predominantly the retinal pigment epithelium and the photoreceptors ( 19 ). in order to drive the expression of either pre - mir - 204 / 211 or egfp in the injected retina , the authors initially used the constitutive cytomegalovirus ( cmv ) promoter . before carrying out the experiments in mouse models of photoreceptor degeneration , the authors first sought to determine whether the aav - mediated delivery of the precursor sequences of mir - 204 and mir - 211 was followed by proper mirna processing and formation of mir - 204 and mir - 211 mature forms in the transduced photoreceptor cells . towards this goal , the authors injected three wild - type mice with the aav . cmv . premir204 viral construct in one eye and with the control aav . cmv . egfp construct in the contralateral eye . the authors then performed laser capture microdissection ( lcm ) of all the injected eyes in order to specifically collect the outer nuclear layer , which contains photoreceptor cells ( fig1 a ). the authors extracted total rna from the collected samples and measured the expression levels of the mature form of mir - 204 by quantitative ( q ) reverse transcriptase ( rt ) pcr using the taqman microrna assay kit . the authors found that the administration of the aav . cmv . premir204 vector conferred an 1 . 5 to 2 - fold increase of mature mir - 204 compared to the endogenous levels ( fig1 b ). these results demonstrate that administration of the aav . cmv . premir204 vector induced an increase in the levels of properly processed mir - 204 . in order to exclude that the delivery and processing of the premir - 204 transgenes could interfere with the mirna processing machinery in the retina , and therefore affect the physiological endogenous levels of other mirnas , the authors also measured in injected eyes the expression of an unrelated mirna , mir - 124 , which is abundantly expressed in the retina ( 16 ). the authors found that mir - 124 expression levels were not significantly modified in the photoreceptors of injected eyes ( fig1 b ). therefore , the authors conclude that the aav - mediated delivery of premir - 204 leads to appropriate processing and increased expression levels of the mature forms of this mirnas in photoreceptors and does not alter the proper processing and expression of other mirnas expressed in the retina . similar results were obtained with aav - mediated delivery of premir - 211 . aav - mediated delivery of mir - 204 / 211 leads to amelioration of retinal morphology and function in mouse models of photoreceptor degeneration to assess the beneficial effect of mir - 204 / 211 in irds , the authors used the following two mouse models : 1 ) a model for an autosomal recessive form of ird caused by a homozygous null mutation in the aryl hydrocarbon interacting protein like 1 ( aipl1 ) gene ( aipl1 knockout mice ) ( 20 ). mutations in the aipl1 gene are responsible for a severe form of lca in humans . in aipl1 −/− mice the retina develops normally until postnatal day ( p ) 12 . after this stage , rod and cone photoreceptors start to quickly degenerate leading to disorganization , fragmentation and notable size reduction of the photoreceptor outer segments and of the thickness of the onl . the loss both of rod and cone function , due to the impaired phototransduction and the undergoing degeneration , is reflected by a complete absence of electroretinogram ( erg ) in aipl1 −/− mice ( 20 , 21 ). 2 ) a model for an autosomal dominant form of ird ( p347s rhodopsin transgenic mouse ). this transgenic mouse line carries a copy of the human rhodopsin gene harboring a proline - to - serine substitution in position 347 of the protein . this mutation is responsible for a form of rp in human patients . the retinal phenotype in this mouse model is less severe compared to aipl1 −/− mice and an erg response , although severely impaired , can be obtained up to 2 - 3 months of postnatal life ( 22 ). the authors found that delivery of the premir - 204 transgene leads to a notable preservation of retinal structure in the injected eyes of the aipl1 −/− and rhodopsin p347s models , as compared to the egfp - injected contralateral eyes . the authors injected aipl1 −/− mice at postnatal day ( p ) 4 using the above - described scheme and sacrificed the mice at p21 . at this stage , only a single row of photoreceptor nuclei is present in egfp - injected eyes ( fig2 a ). in contrast , the authors observed a significant increase in the number of preserved rows as well as in the density of photoreceptor nuclei in the contralateral eyes of the above animals , which were injected with the aav . cmv . premir204 construct ( fig2 b - c ). these results clearly demonstrate a slower progression of retinal degeneration in mir - 204 injected eyes . moreover , the authors observed an increased staining for both rod ( rhodopsin ) and cone photoreceptor ( cone arrestin and m - opsin ) markers in the onl of eyes injected with the aav . cmv . premir204 vector compared to the contralateral eyes injected with the control vector , as assessed by immunofluorescence analysis ( fig3 and 4 ). finally , the cone structure and outer segments were better preserved in the eyes injected with mir - 204 vector , compared to the contralateral ones , as shown by immunolabelling with cone arrestin ( fig3 c and 3d ). similar results were obtained with aav - mediated delivery of premir - 211 . in order to assess whether the protective effect that mir - 204 / 211 conferred to the retina can be exploited in combination with gene replacement approaches , the authors carried out the injection procedure described above ( i . e . subretinal injection at p4 ) in aipl1 −/− mice combining the aav . cmv . premir204 vector with an aav vector driving expression of the humanaipl1 cdna under the control of cmv . this strategy would enable us to evaluate whether the combination of retinal neuroprotection ( conferred by the delivery of mir - 204 / 211 ) and replacement of the aipl1 gene ( humanaipl1 ) deficiency could have a synergistic therapeutic effect . as shown in fig5 , retinal histological analysis at p30 demonstrated that combination of both mir - 204 and humanaipl1 preserved retinal thickness more efficiently compared to the contralateral eye injected with the humanaipl1 alone . these preliminary data suggest that the combination of both mir - 204 injection and humanaipl1 gene delivery can yield additive therapeutic effects , i . e . slowing down of the progression of retinal degeneration ( mir - 204 effect ) and enhancing potential restoration of aipl1 activity ( humanaipl1 gene supplementation ). the authors injected p347s transgenic mice at p4 following the same procedure described above for the aipl1 −/− mouse and sacrificed the animals at two different time points , i . e ., p30 and p60 . first , the authors detected at p30 a notable improvement of the erg response in the mir - 204 - injected eyes of p347s mice , compared to the contralateral egfp - injected eyes ( fig6 ). the authors found that this improvement was persistent also at p60 , i . e ., two months after the injection ( fig7 ). at both stages ( i . e . p30 and p60 ), the enhanced erg response was prevalent under photopic conditions that reflect cone activity , indicating that cone photoreceptors represented the main target of the beneficial effect provided by the mir - 204 injection . in agreement with the above finding , immunofluorescence analysis revealed a significant preservation in the expression of cone photoreceptor markers , such as opn1mw ( m - opsin ) and opn1sw ( s - opsin ) proteins , in mir - 204 injected eyes as opposed to egfp - injected eyes ( fig8 ). in further support of the above results , the authors also found that the injection of the premir - 204 aav constructs determined a statistically significant decrease in the number of apoptotic cells in the photoreceptor layer compared to egfp - injected eyes , as revealed by tunel staining ( fig9 ). moreover , the authors determined that mir - 204 injection led to a dramatic reduction of retinal gliosis , which represents a physiological response to photoreceptor damage , as assessed by immunofluorescence staining with the anti - glutamine synthetase ( anti - gs6 ) antibody ( fig1 ). the authors expanded these observations by increasing the number of murine retinas analyzed and further confirmed the results ( fig1 ). taken together , these findings strongly suggest that the subretinal delivery of the mir - 204 / 211 vectors slows down the retinal degeneration in p347s and preserves retinal function , particularly in both scotopic and photopic conditions . all the above - described injection experiments were carried out using an aav construct containing the cmv constitutive promoter , which drives transgene expression in all transduced cells . to verify whether the restricted expression of mir - 204 / 211 in photoreceptors was sufficient to ensure the beneficial effect of these two mirnas in ird models , the authors generated an aav construct in which expression of the mir - 204 precursor is under the control of the rhodopsin promoter that drives transgene expression specifically in photoreceptors ( allocca et al , j of virology , 2007 and mussolino et al , gene therapy 2011 ). the authors then injected a group of p347s transgenic mice with this construct ( aav . rho . premir204 ) at p4 following the same strategy used for the aav . cmv . premir204 construct . the authors obtained a notable improvement of the erg response in the rho . mir204 injected eyes compared to the contralateral egfp - injected controls at p30 ( fig1 ). the authors expanded these observations by increasing the number of murine retinas analyzed and further confirmed the results ( fig1 ). similar to what shown for use of the cmv promoter ( fig1 ), also the injection of the rho . mir204 construct did not lead to any significant alteration of the amplitude of ‘ b ’ waves in the retinas of mir - 204 / 211 injected eyes compared to contralateral egfp - injected controls eyes of wild type mice ( fig1 ). the above data clearly demonstrate that the restricted expression of mir - 204 transgenes in photoreceptors is sufficient to ensure the protective role of these two mirnas in ird conditions in vivo . it is important to point out that all the previously described experiments have also been performed with either the premir - 204 or the premir - 211 sequences whose injection produced very similar results in all the models analyzed ( see example in fig1 and 13 ). therefore , the authors conclude that they both exert the same beneficial effect in ird conditions in vivo . all of the above data demonstrate that the injection of mir - 204 / 211 in the retina ( and particularly in photoreceptors ) of in vivo models of irds exerts a protective effect on photoreceptors , particularly on cones , by enhancing their survival . this is particularly surprising when considering the fact that mir - 204 / 211 are not detectable in photoreceptor cells ( fig1 ). the strong effect of mir - 204 / 211 on photoreceptors is further strengthened by the evidence that in the retina of a mouse knockout for mir - 211 , that the authors are currently characterizing , a significant deficit of photoreceptor function was observed ( fig1 ). to assess safety of mir - 204 / 211 delivery on retinal physiology , the authors carried out subretinal aav - mediated delivery of mir - 204 in a large cohort of adult c56bl / 6 mice using the same scheme described above . as shown in fig1 , electroretinographic ( erg ) analysis demonstrated that the ‘ b ’ waves were unaltered in mir - 204 injected eyes compared to contralateral egfp - injected controls eyes one month after the injection . moreover , the authors carried out a similar analysis in wild - type mice injected at p4 and the authors performed erg analysis at p30 . also in this case , the authors could not detect any significant alteration of the amplitude of ‘ b ’ waves in the retinas of mir - 204 injected eyes compared to contralateral egfp - injected controls eyes ( fig1 ). similar results were obtained with aav - mediated delivery of premir - 211 . these data support the safety of mir - 204 / 211 delivery to healthy retinas . the authors propose that the intraretinal administration of mir - 204 / 211 , particularly in photoreceptor cells , exerts a beneficial effect in photoreceptor degeneration , and particularly in irds , including retinitis pigmentosa ( both isolated and syndromic forms ), leber congenital amaurosis , cone - rod dystrophies and cone dystrophies . notably , this demonstrates a therapeutic effect obtained by the administration of individual mirnas in photoreceptors in vivo . in the present invention , the authors demonstrate that mir - 204 / 211 have a protective effect in the process of photoreceptor degeneration and death , which are the primary conditions that underlie inherited retinal dystrophies . in such diseases , abnormal rpe differentiation and proliferation do not play key pathogenic roles . 2 . n . meola , v . a . gennarino , s . banfi , pathogenetics 2 , 7 ( 2009 ). 3 . r . garzon , g . marcucci , c . m . croce , nat rev drug discov 9 , 775 ( october , 2010 ). 4 . j . kota et al ., cell 137 , 1005 ( jun . 12 , 2009 ). 5 . a . care et al ., nat . med . 13 , 613 ( may , 2007 ). 6 . t . thum et al ., nature 456 , 980 ( dec . 18 , 2008 ). 7 . d . cacchiarelli et al ., embo rep 12 , 136 ( feb . 1 , 2011 ). 8 . j . elmen et al ., nature 452 , 896 ( apr . 17 , 2008 ). 9 . r . e . lanford et al ., science 327 , 198 ( jan . 8 , 2010 ). 10 . a . g . seto , int . j . biochem . cell biol . 42 , 1298 ( august , 2010 ). 11 . e . l . berson , invest ophtalmol vis sci 34 , 1659 ( 1993 ). 12 . f . p . cremers , et al ., hum . mol . genet . 11 , 1169 ( may 15 , 2002 ). 13 . c . p . hamel , orphanet j rare dis 2 , 7 ( 2007 ). 14 . m . michaelides , et al . surv . ophthalmol . 51 , 232 ( may - 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