Patent Application: US-7744498-A

Abstract:
the invention is in the field of recombinant genetic materials , especially for use in gene therapy . vectors used for transferring additional genetic information to cells in the field of gene therapy are often based on viruses . a group of viruses which has been proposed to use for transfection is the group of parvoviruses , in particular the use of adeno associated virus has been proposed . the invention provides improved methods and means for gene therapy and for preparing products to be used in gene therapy using parvovirus based materials . the invention particularly provides regulated expression of genes under control of the combination of a repressor moiety and an activator moiety , particularly for expression of products which are toxic to the host cell in which they are expressed . in this way it is possible to achieve stable transfection for expression of parvovirus toxic proteins so that amongst others a packaging cell line for producing recombinant parvovirus , in particular adeno associated virus is provided as well as virus produced therewith .

Description:
the present invention is , in one aspect , based on the discovery of sequences that enable the efficient production of stable cell lines , expressing in a regulated fashion , high levels of rep78 and rep68 . as noted above , it has been shown to be possible to obtain stable clones expressing the rep78 protein in a regulated fashion . however , the frequency with which such clones are found and , more importantly , the level of regulated rep78 expression , is very low . this has severely limited the application and the development of packaging cell lines for raav . the present invention , for the first time , describes the generation of efficient packaging cell lines for the production of recombinant aav . the classical inducible eukaryotic promoters , like , for instance , the mouse methallothionine promoter or the mouse mammary - tumor virus long terminal repeat promoter , respond to heavy metals , heat shock or hormones 31 - 38 . furthermore , the human immunodeficiency virus ( hiv ) long terminal repeat promoter can be induced by the hiv transactivator protein tat or by the adenovirus e1a protein . an inducible expression system , based on hiv promoter induction by tat , has been described in patent application ep 0 455 424 and expression of aav rep proteins , driven by the hiv promoter , was suggested in patent application wo 95 / 13365 . however , all these inducible promoters are , in principle , not ideally suited to inducibly express ( toxic ) genes , since the promoters are not completely silent in the uninduced state 32 and / or because the inducing principle induces pleiotropic effects in the target cells 39 . a new generation of regulated promoters has adapted parts from bacterial transcription units for use in eukaryotic cells 40 - 47 . these artificial transcription units are adapted from the lac repressor - operator - inductor system or the tn10 - specific tetracycline resistance operon from e . coli . three different systems have been described : i ) the prevention of transcription initiation by well placed repressor - operator complexes on the promoter 40 - 42 , 46 ; or ii ) in blocking of rna - polymerase ii during elongation by a repressor - operator complex 43 , 46 ; or iii ) the activation of a minimal tata - box , replenished with operator sequences which can be recognized by an artificial transactivator ( ta ), wherein the ta consists of the operator binding component derived from the lac - repressor or the tetracycline repressor and the transcription activating domain from vp16 , a herpes simplex virus encoded transcription activator 44 , 45 , 47 . binding of the ta to the operator sequences can be effectively inhibited with specific compounds , i . e . iptg for ta , based on the lac - repressor operator binding domain , and tetracycline or tetracycline - related compounds for ta , based on the tetracycline - repressor operator binding domain . specifically , the latter system , where a minimal promoter is combined with operator sequences from the tet - repressor , is suitable for the regulated expression of foreign genes . since , in eukaryotic cells , no functional transactivating proteins bind to the tet - operator sequences , these promoters are practically inactive in the presence of low concentrations of tetracycline or related compounds such as doxycycline 47 , 48 . similarly , suitable expression patterns can be obtained with the system utilizing a ta comprising the operator binding component of the lac - repressor . however , the utility of lac - repressor / operator based systems in mammalian cells is limited , because the inducer compound β - d - thiogalactopyranoside ( iptg ), despite its rapid uptake and intracellular stability , acts rather slowly and inefficient . consequently , such systems give only moderate expression induction ( 47 ). stable cell lines exist that constitutively express the tetracycline - repressor vp16 fusion gene ( ta ). therefore , in one embodiment of the invention , the aav rep gene is placed downstream from the tet operon and this construct is introduced into ta - expressing cells . tetracycline , or related compounds , are present in the medium during cell expansion . aav rep expression can be induced by removal of said compounds from the culture . even though tetracycline and many tetracycline derivatives are nontoxic to eukaryotic cells at the low concentration required to abolish gene expression , their continuous presence is suboptimal in a variety of experimental and industrial setups ; for example , in the breeding of transgenic animals , in gene therapy or in large volume bioreactors , where , for full activation of gene expression , the tetracycline or derivative needs to be washed out rigorously . for these purposes , an inducing effector substance would be preferred over an inhibiting compound and , as such , a modified ta was developed ( gossen et al ., science 268 ( 1995 ): 1766 - 1769 ). several mutations in the tet - repressor resulted in reversal of the binding characteristics of the repressor protein ( binding in the presence of tetracycline and no - binding in its absence ). this new protein is referred to as a reverse tet repressor ( rtetr ). a reverse tetracycline - controlled activator ( rtta ), in which rtetr was fused to vp16 , was developed . cell lines , expressing the rtta , were generated and can be used to express tet - operon controlled transcription units upon induction with tetracycline or tetracycline derivatives ( gossen et al ., science 268 ( 1995 ): 1766 - 1769 ). therefore , in another embodiment of the present invention , the aav rep gene , cloned downstream from the tet operon , is introduced into rtta - expressing cells and aav rep expression can be induced by the addition of tetracycline or related compounds to the medium . the invention is illustrated by the following non - limiting examples , wherein the following materials and methods were employed . the entire disclosures of each of the literature references cited hereinafter are incorporated by reference herein . ligation of the aav - genome lacking the flanking itr to a regulated promoter . we have used a hela cell line htta expressing the ta constitutively 47 to test for regulated rep expression from tet ( o )- rep gene constructs . to this end , we cloned a 4 . 3 kb xbai fragment derived from paav / ad 11 ( a kind gift from dr . r . j . samulski ) containing the entire rep and cap coding domain in the xbai site immediately downstream of the tet ( o ) ( fig2 ). in this construct , designated tet ( o )+ p5 - rep , the tet ( o ) is situated upstream of a - 68 bp p5 - promoter ( measured from the first base of the tata - box ). to completely substitute the p5 - promoter for the tet ( o ), we had to perform a pcr reaction , due to the absence of suitable restriction sites . the upstream primer was 5 &# 39 ;- attaatctagactagtcgcgcagccgccatgccgggg - 3 &# 39 ; ( seq . id no : 1 ) and the downstream primer was 5 &# 39 ;- tgtggaagtagctctctccc - 3 &# 39 ; ( seq . id no : 2 ). the pcr reaction was performed on paav / ad with pfu ™ ( stratagene ) using the buffer and the reaction conditions recommended by the manufacturer . the final construct , tet ( o ) δp5 - rep , was generated by digesting the 299 bp pcr product with sfii and xbai and ligating the resulting 248 bp fragment in a three part ligation with a 3 . 95 kb sfii - xbai fragment from paav / ad into the xbai site immediately downstream of the tet ( o ) construct . the amplified part of the construct was checked by sequencing and found to be as expected . in order to test the expression capabilities of the recombinant aav packaging constructs , it was necessary to determine the activities of the various promoters . for this purpose , the tet ( o ) δp5 - rep and the tet ( o )+ p5 - rep were transfected into htta - cells in the presence or absence of ( 1 μg / ml ) doxycycline and in the absence or presence of adenovirus ts149 ( moi = 20 ). protein was extracted after two days and western blotted . the filters were incubated with the rep78 , rep68 specific monoclonal antibody 7b7 ( a kind gift of dr . r . j . samulski ) ( fig3 ). both the tet ( o ) δp5 - rep and the tet ( o )+ p5 - rep constructs express rep78 and rep68 in a doxycycline regulated fashion in htta cells . both the tet ( o ) δps - rep promoter and the tet ( o )+ p5 - rep promoter are upregulated upon adenovirus infection . rep78 and rep68 expression in the uninduced state ( i . e . with doxycycline and without adenovirus ) is markedly reduced with the tet ( o )+ p5 - rep , as compared to rep78 and rep68 expression from the tet ( o ) δp5 - rep construct under the same conditions . these results prove i ) that it is possible to obtain regulated expression rep78 / rep68 using the artifical tet - operon . ii ) that adenovirus infection up - regulates expression from the rep - gene in both constructs . this is possibly due to a direct effect on the tet ( o ) and the minimal tata - box , or due to an adenovirus responsive element ( an enhancer ) in the aav - genome . another possible mechanism is an adenovirus induced enhanced translation of rep78 / rep68 specific mrna in adenovirus infected cells . iii ) in the tet ( o )+ p5 - rep construct the major late transcription factor ( mtlf ) site and the two yy1 - sites in the p5 promoter are likely to mediate at least part of the observed down - regulation of rep78 and rep68 expression from tet ( o ) + p5 - rep in the uninduced state and the up - regulation of rep78 and rep68 expression from tet ( o ) + p5 - rep in adenovirus infected htta cells . to determine whether the rep - gene expression was functional , the constructs were used to produce recombinant aav . for this purpose the constructs were co - transfected with a plasmid pig - cft , carrying a recombinant aav vector 49 into adenovirus ts149 ( moi = 20 ) infected htta cells . after incubation at 39 ° c . and 10 % co 2 for three days , the cells and the culture supernatant were collected and subjected to three freeze thaw cycles , as described in 49 . cell debri was removed by centrifugation ( 1200 rpm , rt ) and residual adenovirus ts149 was heat inactivated ( 1 hour , 56 ° c .). recombinant aav was titrated in a replication center assay ( rca ) by serial dilution of the recombinant aav stock on adenovirus ts149 and wtaav infected 293 cells 20 , as described in 49 . this assay takes advantage of the fact that recombinant aav - dna is replicated to very large amounts in wtaav and adenovirus infected 293 cells . after one day , the vector dna accumulation in the nucleus of transduced cells is large enough to allow the detection of individual infected cells following hybridization with a vector specific probe . for this purpose , the cells were collected in pbs - edta ( 5 mm ) after one day and single cell suspensions were transferred to a hybond n + nylon membrane . after processing of the filter and hybridization with a neo - probe ( 1002 bp smai fragment ), individual spots point out cells transduced by the recombinant aav - vector . using the rca , the tet ( o ) δp5 - rep packaging construct produced about 20 spots which , adjusted for the dilution , corresponded to a titer of 6 × 10 4 infectious particles ( ip ) per ml , whereas the tet ( o )+ p5 - rep packaging construct produced about 34 spots corresponding to a titer of 1 . 0 × 1 . 0 5 ip per ml ( fig4 ). thus , the aav - protein coding domain is functional in both constructs . it has previously been shown that the adeno - associated virus p5 promoter contains a motif centered at - 60 and + 1 , relative to its initiation site that mediates transactivation by the 13s e1a protein . a cellular factor , yy1 , that binds to the motif was identified , and its cdna was cloned . yy1 is a 414 - amino - acid zinc finger protein that represses transcription when bound upstream of heterologous basal promoters ; e1a - proteins relieve the repression and activate transcription through yy1 50 . to determine whether stable cell lines expressing high levels of rep78 and rep68 in a regulated fashion could be generated , we performed co - transfections in htta cells of tet ( o ) δp5 - rep or the tet ( o )+ p5 - rep with plasmid px343 containing a hygromycin b resistance gene under transcriptional control of the sv40 promoter in a ratio of 10 : 1 respectively transfections were done in the absence or presence of 2 μg / ml tetracycline . two days after transfection , the medium was replaced by a medium containing 400 μg / ml hygromycine b with or without 2 μg / ml tetracycline . this selection medium was refreshed every 3 to 4 days . after two weeks , the colonies in the dishes were trypsinised , seeded in medium without tetracyline and infected with adenovirus ts149 ( moi = 20 ). after two days , the cells were collected and total protein was western blotted . rep - protein expression was analyzed using the rep78 / rep68 specific monoclonal antibody 7b7 . in untreated cells ( lane htta ) and in pbluescript transfected htta cells ( pblue ), no rep - specific bands are detected . in the positive control lane , htta infected with wtaav - 2 , rep78 and rep68 are easily detected . rep68 runs as a double band in sds - page ( m . e . unpublished results ). purified rep78 and rep68 protein ( kindly provided by dr . nick muzyczka ) in the lanes marked as rep78 ; rep68 served as size markers for the respective proteins . when paav / ad 11 was transfected or when the tet ( o ) δp5 - rep construct was transfected , no rep78 or rep68 could be detected in the adenovirus infected pools ( fig5 ). thus , constructs carrying only the p5 - promotor ( paav / ad ) as functional element driving expression of rep7b and rep68 are much less efficient in generating stable cell lines expressing rep78 / rep68 in a regulatable fashion than combinations of a regulatable activator with the p5 - promotor ( tet ( o )+ p5 - rep ). this complies with the notion that even low levels of rep78 and rep68 inhibit cell growth . the tet ( o )+ p5 - rep construct , in contrast , does express significant amounts of rep78 and rep68 in a regulated fashion ( fig5 ). pools generated in the presence of tetracycline , when the tet ( o ) is inactive , express rep78 and rep68 upon removal of the tetracycline from the medium . however , when pools were generated in the absence of tetracycline , when the tet ( o ) is active , no expression of rep78 and rep68 can be detected . these results indicate the presence of sequences in the p5 promoter that down - regulate expression of rep78 and rep68 , probably coinciding with the adenovirus major late transcription factor ( mtlf ) and the two yy1 - sites 50 in the aav p5 - promoter . decreased basal level of chloroamphenical acetyltransferase ( cat ) expression of a promoter regulated by both a regulatable transactivator and a regulatable transrepressor . constructs . the constructs popi3cat and pcmvlaci were obtained from stratagene ( la jolla , california , usa ). pcmvlaci contains the e . coli lac repressor under transcriptional control of the cmv promoter . in ptet ( o ) opcat the constitutive rous sarcoma virus ( rsv ) promoter from popi3cat was exchanged for the tet - operon promoter by ligating the 5 . 7 kb snabi - bglii fragment from popi3cat to a 0 . 66 kb . pvuii - bamhi fragment containing the tet - operon + minimal tata - box from the cmv promoter . the latter fragment was obtained from a pbluescript sk + ( stratagene , la jolla , calif ., usa ) containing the clai - bamhi fragment from puhd10 - 3 ( obtained from prof . bujard ) in the clai - bamhi sites of pbluescript sk + . the construct pcmvlacz was used as a transfection efficiency control . it carries the e . coli β - galactosidase gene as a reporter gene under the transcriptional control of a cmv promoter . a reporter gene ( cat ) was used to analyze the activity of a promoter with a regulatable transactivator ( tet - vp16 ) and a regulatable repressor ( laci ). the tet - vp16 system has been described in example 1 of this application . the e . coli lac - repressor / operator / inducer system has been shown to function in mammalian cells ( dueschle , u . hibskind , r . a . and bujard , h . ( 1990 ). science 248 , 480 - 483 ). repression with this system can be obtained via various methods of transcription interference . we used a system , marketed by stratagene ( la jolla , calif ., usa ), in which the transcribing rna - polymerase ii is blocked during elongation by a lac repressor / operator complex . the lac repressor / operator complex can be released from the transcription unit , thus allowing elongation by the addition of isopropyl β - d - thiogalactopyranoside ( iptg ) to the culture medium . in ptet ( o ) opcat , expression of cat can be regulated by adding iptg and / or tetracycline or its analogs such as doxycycline to the medium . our presumption was that a promoter containing both a regulatable transrepressor and transactivating function will show less expression in the off - state . in the tet ( o ) opcat plasmid , this situation is created when the tet - vp16 fusion protein is prevented from binding and the lac - repressor is bound to the transcription unit . to test this presumption , htta cells were transiently transfected with the following constructs : pcmvlaci , ptet ( o ) opcat , pbluescript sk +, and pcmvlacz . transfections and subsequent culture were performed in dmem + 10 % fcs and gentamycin ( 50 μg / ml ). transfections were performed using the calcium phosphate transfection system from life technologies ( breda , the netherlands ) according to the manufacturers description . htta cells ( 3 - 6 10 5 cells per dish ) were seeded into 8 60 mm dishes ( greiner , alphen aan de rijn , the netherlands ) one day prior to transfection . the next day , the medium was replaced by medium and , when indicated , doxycycline ( 1 μg / ml , sigma ) was added . three hours later , 4 dishes received the calcium - phosphate precipitate containing 3 μg pcmvlaci , 1 μg pcmvlacz , 0 . 3 μg ptet ( o ) cat and 2 . 7 ug pbluescript sk + . the other four dishes served as negative controls and were not transfected . fourty hours after transfection , iptg ( 3 mm ) was added to the relevant dishes and approximately 8 hours later the cells were washed with pbs and harvested in 1 ml of fresh pbs by scraping with a rubber policeman . the suspension was transferred to an eppendorf tube and the cells were spun 5 min . at 6000 rpm . the cell pellet was re - suspended in 100 μl 250 mm tris - hci ( ph 7 . 8 ). the cells were lysed by three freeze - thaw cycles and the debri was removed by centrifugation ( 14 krpm , 5 min ., rt ). β - galactosidase activity in the extract was measured as described in sambrook et al ( sambrook j , fritsch ef , maniatis t : molecular cloning : a laboratory manual . 1989 ). samples were diluted in 250 mm tris - hcl ( ph 7 . 8 ) so that they contained the same β - galactosidase activity . from the dilutions , 80 μl was used to determine the cat activity . the cat - assays were performed as described in ( sambrook j , fritsch ef , maniatis t : molecular cloning : a laboratory manual . 1989 ). the thin layer chromatography plates were exposed to photostimulable storage phosphor plates and scanned with a phosphorimager ( molecular dynamics , sunnyvale , calif .) and chloroamphenicol conversion was quantitated . two independent experiments were performed . the results of the experiments are depicted in fig6 . the upper part of fig6 shows the autoradiogram of the cat - assay . lanes 1 to 4 of exp . 1 and 9 to 12 of exp . 2 shows the cat - assay from the transfection and lanes 5 to 8 , exp . 1 and 13 to 16 , exp . 2 show the result from the untransfected control cultures . below the autoradiogram is depicted whether iptg or doxycycline ( dox .) was present (+) or absent (-) during the experiment . the middle part of fig6 show the percentage conversion of chloroamphenical into acytylatedforms from each lane . the lower part of fig6 shows the induction of cat - activity in the samples relative to the cat - activity in the most repressed state (- iptg , + dox .) within the same experiment . in the presence of doxycycline and in the absence of iptg , when the promoter is in its most repressed state , cat - expression can be detected , albeit at a very low level ( fig6 a , lane 2 and lane 10 ). in the absence of doxycycline or the presence of iptg , when only one of the two regulatable elements is in the off state , cat - activity is increased by respectively 19 and 2 . 3 - fold . thus , the combination of a regulatable activator and repressor system within a transcription unit allows more efficient silencing of transcription than when only one regulatable element is present . 2 . berns , k . i . in virology ( eds . chanock , r . m ., hirsch , m . s ., melnick , j . l ,., monath , t . p . & amp ; 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