Patent Application: US-201414785229-A

Abstract:
the present invention relates to constructs , vectors , relative host cells and pharmaceutical compositions which allow an effective gene therapy , in particular of genes larger than 5 kb .

Description:
the plasmids used for aav vector production were derived from either the pzac2 . 1 ( 52 ) or paav2 . 1 ( 53 ) plasmids that contain the inverted terminal repeats ( itrs ) of aav serotype 2 ( table 1 ). n . b . cmv : cytomegalovirus promoter ; cba : chicken beta - actin ; rho : human rhodopsin promoter ; rhok : human rhodopsin kinase promoter ; vmd2 : vitelliform macular dystrophy 2 promoter ; egfp : enhanced green fluorescent protein ; abca4 : human atp - binding cassette , sub - family a , member 4 ; myo7a : human myosin viia ; sv40 : simian virus 40 poly - adenilation signal ; bgh : bovine growth hormone poly - adenilation signal ; 3xflag : 3xflag tag ; ha : hemagglutinin tag ; ap : alkaline phosphatase recombinogenic region ; ak : f1 phage recombinogenic region ; ts : trans - splicing ; itr5 : 2 : plasmid with the left itr from aav serotype 5 and the right itr from aav serotype 2 ; itr2 : 5 : plasmid with the left itr from aav serotype 2 and the right itr from aav serotype 5 . when not specified the left and right itr are from aav serotype 2 . normal size and oversize aav vector plasmids contained full length expression cassettes including the promoter , the full - length transgene cds and the poly - adenilation signal ( pa ) ( table 1 ). the two separate aav vector plasmids ( 5 ′ and 3 ′) required to generate dual aav vectors contained either the promoter followed by the n - terminal portion of the transgene cds ( 5 ′ plasmid ) or the c - terminal portion of the transgene cds followed by the pa signal ( 3 ′ plasmid , table 1 ). normal size egfp plasmids were generated by cloning the egfp cds of paav2 . 1 - cmv - egfp plasmid ( 720 bp ) ( 53 ) in pzac2 . 1 ( 52 ); oversize egfp was generated from paav2 . 1 - cmv - egfp ( 53 ) by inserting a dna stuffer sequence of 3632 bp from human abca4 ( nm — 000350 . 2 , bp 1960 - 5591 ) upstream of the cmv promoter and a second dna stuffer sequence of 3621 bp , composed of : murine abca4 ( nm 007378 . 1 , 1066 - 1 and 7124 - 6046 bp ; 2145 total bp ) and human harmonin ( nm153676 . 3 131 - 1606 bp ; 1476 total bp ), downstream of the pa signal ( this construct was used in the experiments of fig1 a , d , fig4 and fig1 ). to generate dual aav vector plasmids , the egfp cds ( 720 bp ) was split into two constructs : one containing the n - terminal cds ( pmid : 9759496 , bp 1 - 393 ) and the other containing the c - terminal cds ( pmid : 9759496 , bp 394 - 720 ). the oversize abca4 plasmids contained the full - length human abca4 cds ( genenm — 000350 . 2 , bp 105 - 6926 ), while the oversize myo7a plasmids contained the full - length human myo7a cds from isoform 1 ( nm — 000260 . 3 , bp 273 - 6920 ). to generate plasmids for dual aav ov vectors the abca4 and myo7a cds were split into two constructs , one containing n - terminal cds ( abca4 : nm — 000350 . 2 , bp 105 - 3588 ; myo7a : nm — 000350 . 2 , bp 273 - 3782 ) and the other containing c - terminal cds ( abca4 : nm — 000350 . 2 , bp 2819 - 6926 ; myo7a : nm — 000350 . 2 , bp 2913 - 6920 ). therefore , the region of homology shared by overlapping vector plasmids was 770 bp for abca4 and 870 bp for myo7a . to generate plasmids for dual aav ov vectors the human cep290 cds was split into two constructs , one containing n - terminal cds ( cep290 : nm — 025114 , bp 345 - 4076 ) and the other containing c - terminal cds ( cep290 : nm — 025114 , bp 3575 - 7784 ). therefore , the region of homology shared by overlapping vector plasmids was 502 bp . to generate trans - splicing and hybrid vector plasmids the abca4 and myo7a cds were split at a natural exon - exon junction . abca4 was split between exons 19 - 20 ( 5 ′ half : nm — 000350 . 2 , 105 - 3022 bp ; 3 ′ half : nm — 000350 . 2 , bp 3023 - 6926 ) and myo7a was split between exons 24 - 25 ( 5 ′ half : nm — 000350 . 2 , bp 273 - 3380 ; 3 ′ half : nm — 000350 . 2 , bp 3381 - 6926 ). the abca4 and myo7a proteins were both tagged at their c - terminus : abca4 with either the 3 × flag ( gactacaaagaccatgacggtgattataaagatcatgacatcgactacaaggatgacgatgacaag ) or hemagglutinin ( ha ) tag ( tatccgtatgatgtgccggattatgcg ); myo7a with the ha tag only . to generate trans - splicing and hybrid vector plasmids the cep290 cds was split at a natural exon - exon junction : between exons 29 - 30 ( 5 ′ half : nm — 025114 , 345 - 3805 ; 3 ′ half : nm — 025114 , 3806 - 7784 ). the cep290 protein was tagged at its c - terminus with the hemagglutinin ( ha ) tag . the splice donor ( sd ) and splice acceptor ( sa ) signals contained in trans - splicing and hybrid dual aav vector plasmids are as follows : the recombinogenic sequence contained in hybrid ap vector plasmids ( present in both first and second plasmids ) were derived from alkaline phosphate ( ap ) genes ( nm — 001632 , bp 823 - 1100 ), as previously described ( 39 ). the recombinogenic sequence contained in hybrid ak vector plasmids ( present in both first and second plasmids ) were derived from the phage f1 genome ( gene bank accession number : j02448 . 1 ; bp 5850 - 5926 ). the ak sequence is : the ubiquitous cmv promoter is that contained in pzac2 . 1 ( 52 ) or paav2 . 1 - cmv - egfp ( 53 ); the ubiquitous cba promoter was derived from paav2 . 1 - cba - egfp ( 11 ), the pr - specific human rho and rhok promoters were derived from paav2 . 1 - rho - egfp and paav2 . 1rhok - egfp , respectively ( 10 ); the rpe - specific vmd2 promoter ( ng — 009033 . 1 , 4870 - 5470 bp ) corresponds to the previously described ecori - xcmi promoter fragment ( 41 ) and was amplified by human genomic dna . to generate dual aav hybrid ak vectors with heterologous itrs from aav serotype 2 and 5 we exchanged the left itr2 of the 5 ′- half plasmid and the right itr2 of the 3 ′- half plasmid with the itr5 ( as depicted in fig1 a ). the plasmids for the production of aav2 vectors with heterologous itrs are the following : pzac5 : 2 - cmv - 5 ′ abca4 - sd - ak , pzac2 : 5 - ak - sd - 3 ′ abca4 - 3 × flag , paav5 : 2 - cba - 5 ′ myo7a - sd - ak and paav2 : 5 - ak - sd - 3 ′ myo7a - ha ( table 1 ). for the purposes of this invention , a coding sequence of abca4 , myo7a and cep290 which are preferably respectively selected from the sequences herein enclosed , or sequences encoding the same amino acid sequence due to the degeneracy of the genetic code , is functionally linked to a promoter sequence able to regulate the expression thereof in a mammalian retinal cell , particularly in photoreceptor cells . suitable promoters that can be used according to the invention include the cytomegalovirus promoter , rhodopsin promoter , rhodopsin kinase promoter , interphotoreceptor retinoid binding protein promoter , vitelliform macular dystrophy 2 promoter , fragments and variants thereof retaining a transcription promoter activity . viral delivery systems include but are not limited to adenoviral vectors , adeno - associated viral ( aav ) vectors , pseudotyped aav vectors , herpes viral vectors , retroviral vectors , lentiviral vectors , baculoviral vectors . pseudotyped aav vectors are those which contain the genome of one aav serotype in the capsid of a second aav serotype ; for example an aav2 / 8 vector contains the aav8 capsid and the aav 2 genome ( auricchio et al . ( 2001 ) hum . mol . genet . 10 ( 26 ): 3075 - 81 ). such vectors are also known as chimeric vectors . other examples of delivery systems include ex vivo delivery systems , which include but are not limited to dna transfection methods such as electroporation , dna biolistics , lipid - mediated transfection , compacted dna - mediated transfection . the construction of an aav vector can be carried out following procedures and using techniques which are known to a person skilled in the art . the theory and practice for adeno - associated viral vector construction and use in therapy are illustrated in several scientific and patent publications ( the following bibliography is herein incorporated by reference : flotte t r . adeno - associated virus - based gene therapy for inherited disorders . pediatr res . 2005 december ; 58 ( 6 ): 1143 - 7 ; goncalves m a . adeno - associated virus : from defective virus to effective vector , virol j . 2005 may 6 ; 2 : 43 ; surace e m , auricchio a . adeno - associated viral vectors for retinal gene transfer . prog retin eye res . 2003 november ; 22 ( 6 ): 705 - 19 ; mandel r j , manfredsson f p , foust k d , rising a , reimsnider s , nash k , burger c . recombinant adeno - associated viral vectors as therapeutic agents to treat neurological disorders . mol ther . 2006 march ; 13 ( 3 ): 463 - 83 ). suitable administration forms of a pharmaceutical composition containing aav vectors include , but are not limited to , injectable solutions or suspensions , eye lotions and ophthalmic ointment . in a preferred embodiment , the aav vector is administered by subretinal injection , e . g . by injection in the subretinal space , in the anterior chamber or in the retrobulbar space . preferably the viral vectors are delivered via subretinal approach ( as described in bennicelli j , et al mol ther . 2008 jan . 22 ; reversal of blindness in animal models of leber congenital amaurosis using optimized aav2 - mediated gene transfer ). the doses of virus for use in therapy shall be determined on a case by case basis , depending on the administration route , the severity of the disease , the general conditions of the patients , and other clinical parameters . in general , suitable dosages will vary from 10 8 to 10 13 vg ( vector genomes )/ eye . aav vectors were produced by the tigem aav vector core by triple transfection of hek293 cells followed by two rounds of cscl2 purification ( 54 ). for each viral preparation , physical titers [ genome copies ( gc )/ ml ] were determined by averaging the titer achieved by dot - blot analysis ( 55 ) and by pcr quantification using taqman ( 54 ) ( applied biosystems , carlsbad , calif .). hek293 cells were maintained in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ) containing 10 % fetal bovine serum and 2 mm l - glutamine ( gibco , invitrogen s . r . l ., milan , italy ). cells were plated in six - well plates at a density of 2 × 10 6 cells / well and transfected 16 hours later with 1 . 3 μg of pdeltaf6 helper plasmid which contains the ad helper genes ( 56 ) using the calcium phosphate method . after 5 hours , cells were washed once with dmem and incubated with aav2 / 2 vectors ( m . o . i : 10 5 gc / cell of each vector ; 1 : 1 co - infection with dual aav vectors resulted in of 2 × 10 5 total gc / cell ) in a final volume of 700 μl serum - free dmem . two hours later 2 ml of complete dmem was added to the cells . cells were harvested 72 hours following infection for western blot analysis . this study was carried out in accordance with the nih guide for the care and use of laboratory animals , the association for research in vision and ophthalmology statement for the use of animals in ophthalmic and vision research , and the italian ministry of health regulation for animal procedures . mice were housed at the institute of genetics and biophysics animal house ( naples , italy ) and maintained under a 12 - hour light / dark cycle ( 10 - 50 lux exposure during the light phase ). c57bl / 6 and balb / c mice were purchased from harlan italy srl ( udine , italy ). albino abca4 −/− mice were generated through successive crosses and backcrosses with balb / c mice ( homozygous for rpe65 leu450 ) ( 57 ) and maintained inbred . breeding was performed crossing homozygous mice . pigmented sh14626sb / 4626sb ( referred to as sh1 −/−) mice were imported from the wellcome trust sanger institute ( cambridge , uk , a kind gift of dr . karen steel ) and back - crossed twice with cba / ca mice purchased from harlan italy srl ( udine , italy ) to obtain heterozygous sh1 +/ 4626sb ( referred to as sh1 +/−) mice to expand the colony . the mice were maintained intercrossed ; breeding was performed crossing heterozygous females with heterozygous males . the pigmented sh1 mice used in this study were either usher 1b affected ( sh1 −/−) or unaffected ( sh1 +/− and sh1 +/+). the genotype for the myo7a 4626sb allele was performed by pcr analysis of genomic dna ( extracted from the mouse tail tip ) followed by dna sequencing . the primers used for the pcr amplification are as follows : fw1 ( gtggagcttgacatctacttgacc ) and rev3 ( agctgaccctcatgactctgc ), which generate a product of 712 bp that was sequenced with the fw1 primer . the large white female pigs used in this study were registered as purebred in the lwherd book of the italian national pig breeders &# 39 ; association ( azienda agricola pasotti , imola , italy ). mice ( 4 - 5 weeks - old ) were anesthetized with an intraperitoneal injection of 2 ml / 100 g body weight of avertin [ 1 . 25 % w / v of 2 , 2 , 2 - tribromoethanol and 2 . 5 % v / v of 2 - methyl - 2 - butanol ( sigma - aldrich , milan , italy )] ( 58 ), then aav2 / 8 vectors were delivered subretinally via a trans - scleral transchoroidal approach as described by liang et al ( 59 ). all eyes were treated with 1 μl of vector solution . the aav2 / 8 doses ( gc / eye ) delivered vary across the different mouse experiments as it is described in the “ results ” section . aav2 / 1 - cmv - human tyrosinase ( 60 ) ( dose : 2 × 10 8 gc / eye ) or aav2 / 5 - cmv - egfp ( encoding normal size egfp , dose : 4 × 10 8 gc / eye ) was added to the aav2 / 8 vector solution that was subretinally delivered to albino ( abca4 −/− and balb / c ) ( fig6 b , 7 - 8 ) or pigmented sh1 mice ( fig1 - 11 ), respectively . this allowed us to mark the rpe within the transduced part of the eyecup , which was subsequently dissected and analyzed . ( fig6 b , 7 - 8 , 10 - 11 ). subretinal delivery of aav vectors to the pig retina was performed as previously described ( 11 ). all eyes were treated with 100 μl of aav2 / 8 vector solution . the aav2 / 8 dose was 1 × 10 10 ( fig3 b ) or 1 × 10 11 gc of each vector / eye ( fig5 b and 16 ) and co - injection of dual aav vectors resulted in a total dose of 2 × 10 10 gc / eye or 2 × 10 11 gc / eye , respectively . samples ( hek293 cells , retinas or eyecups ) for western blot analysis were lysed in ripa buffer ( 50 mm tris - hcl ph 8 . 0 , 150 mm nacl , 1 % np40 , 0 . 5 % na - deoxycholate , 1 mm edta ph 8 . 0 , 0 . 1 % sds ) to extract egfp and myo7a proteins , or in sie buffer ( 250 mm sucrose , 3 mm imidazole ph 7 . 4 , 1 % ethanol , and 1 % np - 40 ) to extract abca4 protein . pig samples ( the treated areas of the retina as well as whole rpe sheets ) were lysed in ripa buffer to extract myo7a from rpe sheets , and in sie buffer to extract myo7a and abca4 from retinas . lysis buffers were supplemented with protease inhibitors ( complete protease inhibitor cocktail tablets , roche , milan , italy ) and 1 mm phenylmethylsulfonyl . after lysis egfp and myo7a samples were denatured at 99 ° c . for 5 minutes in 1 × laemli sample buffer ; abca4 samples were denatured at 37 ° c . for 15 minutes in 1 × laemli sample buffer supplemented with 4m urea . lysates were separated by 7 % ( abca4 and myo7a samples ) or 12 % ( egfp samples ) sds - polyacrylamide gel electrophoresis . the antibodies used for immuno - blotting are as follows : anti egfp ( sc - 8334 , santa cruz , dallas , tex ., usa , 1 : 500 ); anti - 3 × flag ( a8592 , sigma - aldrich , 1 : 1000 ); anti - myo7a ( polyclonal , primm srl , milan , italy , 1 : 500 ) generated using a peptide corresponding to aminoacids 941 - 1070 of the human myo7a protein ; anti - ha antibody ( prb - 101p - 200 , ha . 11 , covance , princeton , n . j ., usa , 1 : 2000 ); anti - β tubulin ( t5201 , sigma aldrich , 1 : 10000 ); anti - filamin a ( catalog # 4762 , cell signaling technology , danvers , mass ., usa , 1 : 1000 ); anti - dysferlin ( dysferlin , clone ham1 / 7b6 , monx10795 , tebu - bio , le perray - en - yveline , france , 1 : 500 ). the quantification of egfp , abca4 and myo7a bands detected by western blot was performed using imagej software ( free download is available at http :// rsbweb . nih . gov / ij /). abca4 and myo7a expression was normalized to filamin a or dysferlin for the in vitro and in vivo experiments , respectively . egfp expression was normalized to β - tubulin or μg of proteins for in vitro and in vivo experiments , respectively . different proteins were used for normalization based on the similarity of their molecular weight to those of the different transgene products . the fundus live - imaging was performed by dilating the eye of c57bl / 6 with a drop of tropicamide 1 % ( visufarma , rome , italy ) and subsequent eye stimulation with a 300 w flash . fundus photographs were taken using a topcon trc - 50ix retinal camera connected to a charge - coupled - device nikon d1h digital camera ( topcon medical system , oakland , n . j ., usa ). to evaluate egfp expression in histological sections , eyes from c57bl / 6 mice or large white pigs ( 11 ) were enucleated one month after aav2 / 8 injection . mouse eyes were fixed in 4 % paraformaldehyde over - night and infiltrated with 30 % sucrose over - night ; the cornea and the lens were then dissected and the eyecups were embedded in optimal cutting temperature compound ( o . c . t . matrix , kaltek , padua , italy ). pig eyes were fixed in 4 % paraformaldehyde for 48 hours , infiltrated with 10 % sucrose for 4 hours , 20 % sucrose for 4 hours and finally 30 % sucrose overnight . then , the cornea , the lens , and the vitreous body were dissected and the egfp - positive portions of the eyecups were embedded in optimal cutting temperature compound ( o . c . t . matrix , kaltek ). serial cryosections ( 10 μm thick ) were cut along the horizontal meridian and progressively distributed on slides . retinal histology pictures were captured using a zeiss axiocam ( carl zeiss , oberkochen , germany ). to analyze melanosome localization in the rpe of pigmented sh1 mice , eyes were enucleated 2 months following the aav injection , fixed in 2 % glutaraldehyde - 2 % paraformaldehyde in 0 . 1m phosphate buffer over - night , rinsed in 0 . 1m phosphate buffer , and dissected under a florescence microscope . the egfp - positive portions of the eyecups were embedded in araldite 502 / embed 812 ( catalog # 13940 , araldite 502 / embed 812 kit , electron microscopy sciences , hatfield , pa ., usa ). semi - thin ( 0 . 5 - μm ) sections were transversally cut on a leica ultratome rm2235 ( leica microsystems , bannockburn , ill ., usa ), mounted on slides , and stained with epoxy tissue stain ( catalog # 14950 , electron microscopy sciences ). melanosomes were counted by a masked operator analyzing 10 different fields / eye under a light microscope at 100 × magnification . retinal pictures were captured using a zeiss axiocam ( carl zeiss ). for electron microscopy analyses eyes were harvested from abca4 −/− or sh1 mice at 3 and 2 months after aav injection , respectively . eyes were fixed in 0 . 2 % glutaraldehyde - 2 % paraformaldehyde in 0 . 1m phem buffer ph 6 . 9 ( 240 mm pipes , 100 mm hepes , 8 mm mgcl 2 , 40 mm egta ) for 2 hours and then rinsed in 0 . 1 m phem buffer . eyes were then dissected under light or fluorescence microscope to select the tyrosinase - or egfp - positive portions of the eyecups of albino ( abca4 −/− and balb / c ) and pigmented sh1 mice , respectively . the transduced portion of the eyecups were subsequently embedded in 12 % gelatin , infused with 2 . 3m sucrose and frozen in liquid nitrogen . cryosections ( 50 nm ) were cut using a leica ultramicrotome em fc7 ( leica microsystems ) and extreme care was taken to align pr connecting cilia longitudinally . measurements of rpe thickness and counts of lipofuscin granules in abca4 −/− eyes were performed by a masked operator ( roman polishchuk ) using the item software ( olympus sys , hamburg , germany ). briefly , rpe thickness was measured in at least 30 different areas along the specimen length using the “ arbitrary line ” tool of item software . the “ touch count ” module of the item software was utilized to count the number of lipofuscin granules in the 25 μm 2 areas distributed randomly across the rpe layer . the granule density was expressed as number of granules per 25 μm 2 . the immuno - gold analysis aimed at testing the expression of abca4 - ha in abca4 −/− samples after aav vector delivery was performed by incubating cryosections successively with monoclonal anti - ha antibody ( mms - 101p - 50 , covance , 1 : 50 ), rabbit anti - mouse igg , and 10 - nm gold particle - conjugated protein a . to quantify rhodopsin localization to the connecting cilium of sh1 pr , cryosections of sh1 mice were successively incubated with anti - rhodopsin antibody ( 1d4 , ab5417 , abcam , cambridge , uk , 1 : 100 ), rabbit anti - mouse igg , and 10 - nm gold particle - conjugated protein a . the quantification of gold density of rhodospin in the connecting cilia was performed by a masked operator using item software ( olympus sys ). briefly , the “ touch count ” module of the item software was used to count the number of gold particles per cilium that were normalized to the cilium perimeter ( nm ) that was measured using the “ closed polygon tool ”. gold density was expressed as gold particles / nm . immunogold labelled cryosections were analyzed under fei tecnai - 12 ( fei , eindhoven , the netherlands ) electron microscope equipped with a veletta ccd camera for digital image acquisition . to assess the recovery from light desensitization eyes were stimulated with 3 light flashes of 1 cd s / m2 and then desensitized by exposure to constant light ( 300 cd / m2 ) for 3 minutes . then , eyes were stimulated over time using the pre - desensitization flash ( 1 cd s / m2 ) at 0 , 5 , 15 , 30 , 45 and 60 minutes post - desensitization . the recovery of rod activity was evaluated by performing the ratio between the b - wave generated post - desensitization ( at the different time points ) and that generated pre - desensitization . the recovery from light desensitization was evaluated in 2 - month - old abca4 −/− mice at 6 weeks post treatment ( fig1 ). data are presented as mean ± standard error of the mean ( s . e . m .). statistical p values & lt ; 0 . 05 were considered significant . one - way anova with post - hoc multiple comparison procedure was used to compare data depicted in : fig2 ( p anova : a . 0 . 0002 ; b . 0 . 0015 ; c . 2 × 10 − 7 ); fig8 b ( p anova : 0 . 076 ); fig1 b ( p anova : 0 . 5 ). as lipofuscin granules ( fig7 b ) and melanosomes ( fig1 b ) were counted , counts were analyzed by deviance from a negative binomial generalized linear models ( 61 ) ( fig7 b : p value analysis of deviance 0 . 03794 ; fig1 b : p value analysis of deviance & lt ;& lt ; 2 × 10 − 10 ). the statistically significant differences between groups determined with the post - hoc multiple comparison procedure are marked by asterisks in the figures . the inventors generated oversize ( oz ), dual aav trans - splicing ( ts ), and hybrid vectors that included either the reporter egfp , the therapeutic abca4 - 3 × flag or the myo7a - ha coding sequences . the inventors also generated dual aav trans - splicing ( ts ), and hybrid vectors that included the therapeutic cep290 tagged at its c - terminus with ha tag . the recombinogenic sequences included in the dual aav hybrid vectors were based on either a previously reported region of the alkaline phosphatase transgene ( ap , dual aav hybrid ap ) ( 39 ) or a 77 bp sequence from the the f1 phage genome ( ak , dual aav hybrid ak ) that the inventors found to be recombinogenic in previous experiments ( colella and auricchio , unpublished data ). the inventors also generated dual aav overlapping ( ov ) vectors for abca4 , myo7a and cep290 . the inventors did not generate dual aav ov vectors for egfp because the efficiency of this approach relies on transgene - specific overlaps for reconstitution ( 38 ) and therefore cannot be extrapolated from one gene to another . instead , for egfp the inventors generated single aav vectors of normal size ( ns ) to compare levels of transgene expression from the various strategies . the constructs generated for production of all aav vectors used in this study are listed in table 1 and a schematic representation of the various approaches is depicted in fig1 . the inventors used aav2 / 2 vectors for the in vitro experiments , with the ubiquitous cytomegalovirus ( cmv ) or chicken beta - actin ( cba ) promoters , which efficiently transduce hek293 cells ( 40 ). in addition , since the use of heterologous itrs from aav serotypes 2 and 5 can increase the productive reassembly of dual aav vectors ( 51 ), the inventors also generated dual aav ak vectors with heterologous itrs ( fig1 a ) encoding abca4 and myo7a . aav vectors with heterologous itrs were packaged in aav capsids from serotype 2 and tested in vitro . in the experiments performed in vivo in the retina , the inventors used aav2 / 8 vectors , which efficiently transduce rpe and pr ( 10 - 12 ) but poorly infect hek293 cells , and either the ubiquitous cba and cmv promoters ( 11 ), or the rpe - specific vitelliform macular dystrophy 2 ( vmd2 ) ( 41 ) or the pr - specific rhodopsin ( rho ) and rhodopsin kinase ( rhok ) promoters ( 10 ) ( table 1 ). the inventors initially compared the efficiency of the various oz , dual aav ov , ts and hybrid ap and ak strategies for aav - mediated large gene transduction in vitro by infecting hek293 cells with the aav2 / 2 vectors [ multiplicity of infection , m . o . i . : 10 5 genome copies ( gc )/ cell of each vector ] with ubiquitous promoters ( cmv for egfp , abca4 - 3 × flag , and cep290 - ha , and cba for myo7a - ha ). cell lysates were analyzed by western blot with anti - egfp ( fig2 a ), - 3 × flag ( to detect abca4 - 3 × flag , fig2 b ), - myo7a ( fig2 c ) and - ha ( to detect cep290 - ha ) ( fig1 a ) antibodies . representative western blots are shown in fig2 a - c and 12 a . all strategies resulted in the expression of proteins of the expected size . as predicted , no bands of the expected size were observed when only one of the dual aav vectors was used for infection ( fig2 a - c and 12 a ). quantification of transgene expression ( fig2 d - f ) showed that the dual aav hybrid ap approach resulted in the lowest levels of transgene expression , while the dual aav ov , ts and hybrid ak approaches were more efficient than the aav oz approach . dual aav ts and hybrid ak approaches confirmed their ability to efficiently express large genes also in the case of cep290 ( fig1 b ). in addition , the use of dual aav ak vectors with heterologous itrs resulted in expression of full - length abca4 and myo7a proteins in vitro ( fig1 ). dual aav ts and hybrid ak but not ov vectors transduce mouse and pig photoreceptors . the inventors then evaluated each of the aav - based systems for large gene transduction in the mouse retina . to test the dual aav ov , which was transgene - specific , the inventors used the therapeutic abca4 and myo7a genes ( fig3 ). the inventors used egfp to evaluate the aav oz and the dual aav ts , hybrid ap and ak approaches ( fig4 ). western blot analysis on retinal lysates , one month after subretinal delivery in c57bl / 6 mice of the dual aav ov vectors ( dose of each vector / eye : 1 . 3 × 10 9 gc ), encoding abca4 - 3 × flag from the ubiquitous cmv promoter , revealed robust protein expression ( fig3 a ). to determine which cell type in the retina expressed abca4 , the inventors used dual aav ov vectors that contained either the pr - specific rho and rhok , or the rpe - specific vmd2 ( dose of each vector / eye : 1 × 10 9 gc ) promoters . the inventors detected abca4 protein expression in retinas injected with the vmd2 but not in those containing the rho and rhok promoters ( fig3 a ). these results were also confirmed in the large white pig retina . the pig retina is an excellent model to evaluate vector efficiency because of its size , which is similar to the human retina , and because it is enriched with cones that are concentrated in a streak - like region whose cone density is comparable to that of the primate macula ( 11 ). the inventors injected large white pig subretinally with dual aav ov vectors encoding abca4 - 3 × flag ( dose of each vector / eye : 1 × 10 10 gc ), and observed abca4 protein expression with the cmv but not the rho promoter ( fig3 b ). similarly , subretinal administration of dual aav ov vectors encoding myo7a - ha resulted in weak myo7a protein expression in the mouse retina with the ubiquitous cba ( dose of each vector / eye : 2 . 5 × 10 9 gc ) and no detectable expression with the rho ( dose of each vector / eye : 3 . 2 × 10 9 gc ) promoter ( fig3 c ). overall , these data suggested that the dual aav ov approach was more efficient for large gene transfer to rpe than to pr , which are a major target of gene therapy for irds , such as stgd and ush1b . to find an aav - based strategy that efficiently transduces large genes in pr , the inventors evaluated the retinal transduction properties of the aav oz and dual aav ts , hybrid ap , and ak approaches . the inventors initially used egfp , which allowed us to easily localize transgene expression in the various retinal cell types including pr as well as to properly compare the levels of aav - based large transgene transduction to those of a single aav ns vector . c57bl / 6 mice were subretinally injected with aav ns , oz and dual aav ts , and hybrid ap and ak vectors ( dose of each vector / eye : 1 . 7 × 10 9 gc ), all encoding egfp under the transcriptional control of the cmv promoter . one month later , fundus photographs showed that the highest levels of fluorescence were obtained with the aav ns , and dual aav ts and hybrid ak approaches ( fig1 ). fluorescence microscope analysis of retinal cryosections showed that detectable levels of rpe or pr transduction could be observed in : 77 % ( 10 / 13 ) retinas injected with aav ns and oz vectors ; 92 % ( 12 / 13 ) retinas injected with dual aav ts , hybrid ap and ak vectors . fig4 shows the best transduced retinas from each of these groups . the most robust levels of pr transduction were obtained with the aav ns and dual aav ts and hybrid ak approaches . the inventors then assessed pr - specific transduction levels in c57bl / 6 mice following subretinal administration of dual aav ts and hybrid ak vectors , which appears the most promising for large gene reconstitution in pr , as well as aav ns vectors for comparison ( dose of each vector / eye : 2 . 4 × 10 9 gc ). all vectors encoded egfp under the transcriptional control of the pr - specific rho promoter . one month after vector administration retinas were cryosectioned and analyzed under a fluorescence microscope ( fig5 a ). all approaches resulted in high levels of pr transduction , which seemed more consistent with the single aav ns vector . the inventors found pr transduction in : 100 % ( 6 / 6 ) of the retinas injected with aav ns ; 60 % ( 9 / 15 ) of the retinas injected with dual aav ts ; 71 % ( 10 / 14 ) of the retinas injected with dual aav hybrid ak . fig5 a shows the best transduced retinas from each of these groups . thus , the inventors conclude that dual aav ts and hybrid ak strategies allow efficient mouse pr transduction although at levels which are lower than those obtained with a ns aav . the inventors then confirmed that subretinal administration of dual aav ts and hybrid ak vectors ( dose of each vector / eye : 1 × 10 11 gc ; egfp - positive retinas out of total injected : 2 / 2 dual aav ts ; 2 / 2 dual aav hybrid ak ) transduced pr of white large pigs ( fig5 b ). in addition , subretinal delivery to the pig retina of dual aav ts and hybrid ak vectors ( dose of each vector / eye : 1 × 10 11 ) resulted in efficient expression of both full - length abca4 - 3 × flag specifically in prs ( fig1 a ) and full - length myo7a - ha in rpe and prs ( fig1 b ) interestingly , dual aav hybrid ak vectors resulted in more consistent expression of the large abca4 and myo7a proteins in prs , compared with dual aav ts vectors ( fig1 ). dual aav vectors improve the retinal phenotype of stgd and ush1b mouse models . to understand whether the levels of pr transduction obtained with the dual aav ts and hybrid ak approaches may be therapeutically relevant , the inventors investigated them in the retina of two mouse models of irds , stgd and ush1b caused by mutations in the large abca4 and myo7a genes , respectively . although the abca4 −/− mouse model does not undergo severe pr degeneration ( 42 ), the absence of the abca4 - encoded all - trans retinal transporter in pr outer segments ( 43 - 44 ) causes an accumulation of lipofuscin in pr as well as in rpe , as result of pr phagocytosis by rpe ( 45 ). as a consequence , both the number of lipofuscin granules in the rpe and the thickness of rpe cells are greater in abca4 −/− mice than in control mice ( 45 ). moreover the abca4 −/− mouse model is characterized by delayed dark adaptation ( 57 , 62 ). since abca4 is expressed specifically in pr , the inventors generated dual aav ts and hybrid ak vectors encoding abca4 - 3 × flag under the transcriptional control of the rho promoter . these vectors were subretinally injected in wild - type c57bl / 6 mice ( dose of each vector / eye : 3 - 5 × 10 9 gc ) and one month later retinas were lysed and analyzed by western blot with anti - 3 × flag antibodies . both approaches resulted in robust yet variable levels of abca4 - 3 × flag expression . abca4 - 3 × flag expression levels were more consistent in retina treated with the dual aav hybrid ak vectors ( fig6 a ). these results were confirmed in large white pigs ( data not shown ). in addition , one month - old albino abca4 −/− mice were injected subretinally with the dual aav hybrid ak rho - abca4 - ha vectors ( dose of each vector / eye : 1 - 3 × 10 9 gc ). three months later , eyes were harvested and immuno - electron microscopy analysis with anti - hemagglutinin ( ha ) antibodies of retinal sections confirmed that immunogold particles were correctly localized in pr outer segments only in animals that were injected with the combination of 5 ′ and 3 ′ dual aav hybrid ak vectors ( fig6 b ). to assess the functionality of the abca4 protein expressed by the dual vectors , the inventors also performed transmission electron microscopy to assess the presence and number of rpe lipofuscin granules ( fig7 ) and rpe thickness ( fig8 ). both were greater in the retina of abca4 −/− mice injected with control vectors than in the retina of wild - type , age - matched balb / c controls , and were reduced or normalized in the eyes injected with the therapeutic dual aav ts or hybrid ak vectors ( fig7 b and 8b ). in addition , the ability of abca4 −/− photoreceptors to recover from light desensitization was significantly improved in the retinas treated with the therapeutic vectors when compared to control retinas ( fig1 ). the inventors then tested pr transduction levels and efficacy of dual aav - mediated myo7a gene transfer in the retina of sh1 mice , the most commonly used model of ush1b ( 23 - 24 , 46 - 48 ). in sh1 mice , a deficiency in the motor myo7a causes the mis - localization of rpe melanosomes ( 47 ), which do not enter into the rpe microvilli , and the accumulation of rhodopsin at the pr connecting cilium ( 48 ). since myo7a is expressed in both rpe and pr ( 22 - 23 ), the inventors then used dual aav ts and hybrid ak vectors expressing myo7a - ha under the transcriptional control of the ubiquitous cba promoter . one month - old wild - type c57bl / 6 mice were injected with the dual aav vectors ( dose of each vector / eye : 1 . 7 × 10 9 gc ) and eyecup lysates were evaluated one month later using western blot analysis with anti - ha antibodies . results showed similarly robust and consistent levels of myo7a expression in retinas treated with both approaches ( fig9 ). taking advantage of our anti - myo7a antibody able to recognize both murine and human myo7a , we compared the levels of myo7a achieved following delivery of dual aav vectors to the sh1 −/− eye to those expressed endogenously in the sh1 +/+ eye ( fig1 ). we used both the cba ( fig1 , left panel , dose of each vector / eye : 1 - 6 × 10 9 gc ) and the rho promoters ( fig1 , right panel , dose of each vector / eye : 2 × 10 9 gc ) to distinguish myo7a expression achieved in both pr and rpe from that in pr alone : the former is about 20 % ( fig1 , left panel ) and the latter up to about 50 % of endogenous myo7a ( fig1 , right panel ). our analysis additionally shows that the levels of myo7a expression achieved in pr by dual aav hybrid ak are higher than those obtained with the dual aav ts vectors despite the number of transduced retinas is similar ( ts - myo7a : 3 retinas positive out of 8 injected ; ak - myo7a : 4 retinas positive out of 8 treated ; fig1 , right panel ). to test the ability of myo7a expressed from dual aav vectors to rescue the defects of the sh1 −/− retina , the inventors then subretinally injected the cba sets of dual aav ts and hybrid ak vectors ( dose of each vector / eye : 2 . 5 × 10 9 gc ) in one month - old sh1 mice . the inventors assessed rpe melanosome ( fig1 ) and rhodopsin localization ( fig1 ) by analysis of semi - thin retinal section and by immuno - electron microscopy , respectively . unlike unaffected sh1 +/−, the sh1 −/− melanosomes do not enter the rpe microvilli after delivery of control vectors ( each single 5 ′ half of the dual - aav strategies , fig1 ). the number of rpe melanosomes correctly localized apically was significantly improved after the delivery of either dual aav ts or hybrid ak vectors encoding myo7a ( fig1 b ). remarkably , the inventors also found that the myo7a expression mediated by dual aav ts and hybrid ak vectors reduced the accumulation of rhodopsin at the connecting cilium of sh1 −/− pr ( fig1 ). while aav - mediated gene therapy is effective in animal models and in patients with inherited blinding conditions ( 5 - 9 , 49 ), its application to diseases affecting the retina and requiring a transfer of genes larger than 5 kb ( referred to as large genes ) is inhibited by aav limited cargo capacity . to overcome this , the inventors compared the efficiency of various aav - based strategies for large gene transduction including : aav oz and dual aav ov , ts and hybrid approaches in vitro and in mouse and pig retina . in previous experiments , inventors selected a 77 bp sequence from the f1 phage genome that the inventors identified for its recombinogenic properties and used in the dual hybrid approach ( ak , dual aav hybrid ak ). the inventors &# 39 ; in vitro and in vivo results show that the dual aav hybrid ak surprisingly outperforms the dual aav hybrid ap and that all dual aav strategies the inventors tested ( with the exception of the dual aav hybrid ap ) outperform aav oz vectors in terms of transduction levels . this may be explained by the homogenous size of the dual aav genome population when compared to oz genomes , which may favor the generation of transcriptionally active large transgene expression cassettes . the dual aav ov approach seems particularly interesting when compared to the ts or hybrid ak approaches as dual aav ov vectors only contain sequences belonging to the therapeutic transgene expression cassette . however , when the inventors administered dual aav ov vectors to the subretinal space of adult mice and pigs , the inventors were only able to detect expression of the large abca4 protein when the ubiquitous or the rpe - specific promoters , but not the pr - specific promoters , were used . this may suggest that the homologous recombination required for dual aav ov reconstitution is more efficient in rpe than pr . this is consistent with the low levels of homologous recombination reported in post - mitotic neurons ( 50 ) and may partially explain the lack of dual aav ov - mediated myo7a transduction recently reported by other groups ( 30 ). the inventors conclude that subretinal administration of dual aav ov vectors should not be used for large gene transfer to pr , although the inventors cannot exclude that sequences that are more recombinogenic than those included in the inventors &# 39 ; dual aav ov abca4 and myo7a vectors may allow efficient homologous recombination in pr . dual aav ts and hybrid ak approaches efficiently transduce mouse and pig pr , differently from what the inventors observed with dual aav ov . this is consistent with the knowledge that the mechanism of large gene reconstitution mediated by dual aav ts and hybrid ak approaches may be via itr - mediated head - to - tail rejoining ( 32 , 35 , 51 ) rather than homologous recombination . the levels of mouse pr transduction the inventors achieved with dual aav ts and hybrid ak is lower and less consistent than with single ns vectors . however , dual aav may be effective for treating inherited blinding conditions that require relatively low levels of transgene expression , i . e . diseases inherited as autosomal recessive . indeed , the inventors show that subretinal delivery of dual aav ts and hybrid ak improves and even normalizes the retinal defects of two animal models of inherited retinal diseases , stgd and ush1b , which are due to mutations in large genes and are attractive targets of gene therapy . the genome size of dual aav vectors is homogenous , which means identity and safety issues related to their use should be less considerable than those related to aav oz vectors , which have heterogeneous genome sizes . in contrast , the inventors detected neither erg or retinal histological abnormalities in the mice that the inventors followed up to 1 - 2 months after dual aav vector delivery ( data not shown ). in conclusion , the inventors identified a new recombinogenic sequence ( ak ) that strikingly improves the performance of the aav dual hybrid vector system . in fact they found that dual aav vectors are efficient both in vitro and in the retina in vivo . while dual aav ov vectors efficiently transduce rpe , they do not transduce pr , whereas dual aav ts and hybrid ak approaches drive efficient large gene reconstitution in both cell types . administration of dual aav ts and hybrid ak approaches improved the retinal phenotype of mouse models of stgd and ush1b , providing evidence of the efficacy of these strategies for gene therapy for these and other blinding conditions , which require large gene transfer to retinal pr as well as rpe . these findings will greatly broaden the application of aav vectors for gene therapies not only to eyes , but also to muscle as well as to other organs and tissues . diseases other than ird caused by defective genes larger than 5 kb include non - 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