Patent Application: US-80780201-A

Abstract:
an improved tet - repressible system is described in which the transgene is expressed under the control of a promoter which is activated upon binding of a fusion protein composed of a reverse tet repressor / activation domain to tet operator sequences located immediately upstream of the transgene promoter . the tet operator sequences are substantially free of interferon inducible response elements . the reverse tet repressor is fused to an activation domain which lacks signals for protein clearance , thus extending expression of the fusion protein . suitably , the teto sequences are immediately upstream of a quiet promoter .

Description:
the present invention provides novel nucleic acid sequences for an adeno — associated virus of serotype 1 ( aav - 1 ). also provided are fragments of these aav - 1 sequences . among particularly desirable aav - 1 fragments are the inverted terminal repeat sequences ( itrs ), rep and cap . each of these fragments may be readily utilized , e . g ., as a cassette , in a variety of vector systems and host cells . such fragments may be used alone , in combination with other aav - 1 sequences or fragments , or in combination with elements from other aav or non - aav viral sequences . in one particularly desirable embodiment , a cassette may contain the aav - 1 itrs of the invention flanking a selected transgene . in another desirable embodiment , a cassette may contain the aav - 1 rep and / or cap proteins , e . g ., for use in producing recombinant ( raav ) virus . thus , the aav - 1 sequences and fragments thereof are useful in production of raav , and are also useful as antisense delivery vectors , gene therapy vectors , or vaccine vectors . the invention further provides nucleic acid molecules , gene delivery vectors , and host cells which contain the aav - 1 sequences of the invention . also provided a novel methods of gene delivery using aav vectors . as described herein , the vectors of the invention containing the aav - 1 capsid proteins of the invention are particularly well suited for use in applications in which the neutralizing antibodies diminish the effectiveness of other aav serotype based vectors , as well as other viral vectors . the raav vectors of the invention are particularly advantageous in raav readministration and repeat gene therapy . these and other embodiments and advantages of the invention are described in more detail below . as used throughout this specification and the claims , the term “ comprising ” is inclusive of other components , elements , integers , steps and the like . the aav - 1 nucleic acid sequences of the invention include the dna sequences of seq id no : 1 ( fig1 a - 1 f ), which consists of 4718 nucleotides . the aav - 1 nucleic acid sequences of the invention further encompass the strand which is complementary to seq id no : 1 , as well as the rna and cdna sequences corresponding to seq id no : 1 and its complementary strand . also included in the nucleic acid sequences of the invention are natural variants and engineered modifications of seq id no : 1 and its complementary strand . such modifications include , for example , labels which are known in the art , methylation , and substitution of one or more of the naturally occurring nucleotides with an analog . further included in this invention are nucleic acid sequences which are greater than 85 %, preferably at least about 90 %, more preferably at least about 95 %, and most preferably at least about 98 - 99 % identical or homologous to seq id no : 1 . the term “ percent sequence identity ” or “ identical ” in the context of nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence . the length of sequence identity comparison may be over the full - length sequence , or a fragment at least about nine nucleotides , usually at least about 20 - 24 nucleotides , at least about 28 - 32 nucleotides , and preferably at least about 36 or more nucleotides . there are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity . for instance , polynucleotide sequences can be compared using fasta , a program in gcg version 6 . 1 . fasta provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences ( pearson , 1990 , herein incorporated by reference ). for instance , percent sequence identity between nucleic acid sequences can be determined using fasta with its default parameters ( a word size of 6 and the nopam factor for the scoring matrix ) as provided in gcg version 6 . 1 , herein incorporated by reference . the term “ substantial homology ” or “ substantial similarity ,” when referring to a nucleic acid or fragment thereof , indicates that , when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid ( or its complementary strand ), there is nucleotide sequence identity in at least about 95 - 99 % of the sequence . also included within the invention are fragments of seq id no : 1 , its complementary strand , cdna and rna complementary thereto . suitable fragments are at least 15 nucleotides in length , and encompass functional fragments which are of biological interest . certain of these fragments may be identified by reference to fig1 a - 1c . examples of particularly desirable functional fragments include the aav - 1 inverted terminal repeat ( itr ) sequences of the invention . in contrast to the 145 nt itrs of aav - 2 , aav - 3 , and aav - 4 , the aav - 1 itrs have been found to consist of only 143 nucleotides , yet advantageously are characterized by the t - shaped hairpin structure which is believed to be responsible for the ability of the aav - 2 itrs to direct site - specific integration . in addition , aav - 1 is unique among other aav serotypes , in that the 5 ′ and 3 ′ itrs are identical . the full - length 5 ′ itr sequences of aav - 1 are provided at nucleotides 1 - 143 of seq id no : 1 ( fig1 a ) and the full - length 3 ′ itr sequences of aav - 1 are provided at nt 4576 - 4718 of seq id no : 1 ( fig1 c ). one of skill in the art can readily utilize less than the full - length 5 ′ and / or 3 ′ itr sequences for various purposes and may construct modified itrs using conventional techniques , e . g ., as described for aav - 2 itrs in samulski et al , cell , 33 : 135 - 143 ( 1983 ). another desirable functional fragment of the aav - 1 genome is the p5 promoter of aav - 1 which has sequences unique among aav p5 promoters , while maintaining critical regulatory elements and functions . this promoter is located within nt 236 - 299 of seq id no : 1 ( fig1 a ). other examples of functional fragments of interest include the sequences at the junction of the rep / cap , e . g ., the sequences spanning nt 2306 - 2223 , as well as larger fragments which encompass this junction which may comprise 50 nucleotides on either side of this junction . still other examples of functional fragments include the sequences encoding the rep proteins . rep 78 is located in the region of nt 334 - 2306 of seq id no : 1 ; rep 68 is located in the region of nt 334 - 2272 , and contains an intron spanning nt 1924 - 2220 of seq id no : 1 . rep 52 is located in the region of nt 1007 - 2304 of seq id no : 1 ; rep 40 is located in the region of nt 1007 - 2272 , and contains an intron spanning nt 1924 - 2246 of seq id no : 1 . also of interest are the sequences encoding the capsid proteins , vp 1 [ nt 2223 - 4431 of seq id no : 1 ], vp2 [ nt 2634 - 4432 of seq id no : 1 ] and vp3 [ nt 2829 - 4432 of seq id no : 1 ]. other fragments of interest may include the aav - 1 p19 sequences , aav - 1 p40 sequences , the rep binding site , and the terminal resolute site ( trs ). the invention further provides the proteins and fragments thereof which are encoded by the aav - 1 nucleic acids of the invention . particularly desirable proteins include the rep and cap proteins , which are encoded by the nucleotide sequences identified above . these proteins include rep 78 [ seq id no : 5 ], rep 68 [ seq id no : 7 ], rep 52 [ seq id no : 9 ], rep 40 [ seq id no : 11 ], vp1 [ seq id no : 13 ], vp2 [ seq id no : 15 ], and vp3 [ seq id no : 17 ] and functional fragments thereof while the sequences of the rep and cap proteins have been found to be closely related to those of aav - 6 , there are differences in the amino acid sequences ( see table 1 below ), as well as differences in the recognition of these proteins by the immune system . however , one of skill in the art may readily select other suitable proteins or protein fragments of biological interest . suitably , such fragments are at least 8 amino acids in length . however , fragments of other desired lengths may be readily utilized . such fragments may be produced recombinantly or by other suitable means , e . g ., chemical synthesis . the sequences , proteins , and fragments of the invention may be produced by any suitable means , including recombinant production , chemical synthesis , or other synthetic means . such production methods are within the knowledge of those of skill in the art and are not a limitation of the present invention . in another aspect , the present invention provides vectors which utilize the aav - 1 sequences of the invention , including fragments thereof , for delivery of a heterologous gene or other nucleic acid sequences to a target cell . suitably , these heterologous sequences ( i . e ., a transgene ) encode a protein or gene product which is capable of being expressed in the target cell . such a transgene may be constructed in the form of a “ minigene ”. such a “ minigene ” includes selected heterologous gene sequences and the other regulatory elements necessary to transcribe the gene and express the gene product in a host cell . thus , the gene sequences are operatively linked to regulatory components in a manner which permit their transcription . such components include conventional regulatory elements necessary to drive expression of the transgene in a cell containing the viral vector . the minigene may also contain a selected promoter which is linked to the transgene and located , with other regulatory elements , within the selected viral sequences of the recombinant vector . selection of the promoter is a routine matter and is not a limitation of this invention . useful promoters may be constitutive promoters or regulated ( inducible ) promoters , which will enable control of the timing and amount of the transgene to be expressed . for example , desirable promoters include the cytomegalovirus ( cmv ) immediate early promoter / enhancer [ see , e . g ., boshart et al , cell , 41 : 521 - 530 ( 1985 )], the rous sarcoma virus ltr promoter / enhancer , and the chicken cytoplasmic β - actin promoter [ t . a . kost et al , nucl . acids res ., 11 ( 23 ): 8287 ( 1983 )]. still other desirable promoters are the albumin promoter and an aav p5 promoter . optionally , the selected promoter is used in conjunction with a heterologous enhancer , e . g ., the β - actin promoter may be used in conjunction with the cmv enhancer . yet other suitable or desirable promoters and enhancers may be selected by one of skill in the art . the minigene may also desirably contain nucleic acid sequences heterologous to the viral vector sequences including sequences providing signals required for efficient polyadenylation of the transcript ( poly - a or pa ) and introns with functional splice donor and acceptor sites . a common poly - a sequence which is employed in the exemplary vectors of this invention is that derived from the papovavirus sv - 40 . the poly - a sequence generally is inserted in the minigene downstream of the transgene sequences and upstream of the viral vector sequences . a common intron sequence is also derived from sv - 40 , and is referred to as the sv40 t intron sequence . a minigene of the present invention may also contain such an intron , desirably located between the promoter / enhancer sequence and the transgene . selection of these and other common vector elements are conventional [ see , e . g ., sambrook et al , “ molecular cloning . a laboratory manual ”, 2d edit ., cold spring harbor laboratory , new york ( 1989 ) and references cited therein ] and many such sequences are available from commercial and industrial sources as well as from genebank . the selection of the transgene is not a limitation of the present invention . suitable transgenes may be readily selected from among desirable reporter genes , therapeutic genes , and optionally , genes encoding immunogenic polypeptides . examples of suitable reporter genes include β - galactosidase ( β - gal ), an alkaline phosphatase gene , and green fluorescent protein ( gfp ). examples of therapeutic genes include , cytokines , growth factors , hormones , and differentiation factors , among others . the transgene may be readily selected by one of skill in the art . see , e . g ., wo 98 / 09657 , which identifies other suitable transgenes . suitably , the vectors of the invention contain , at a minimum , cassettes which consist of fragments of the aav - 1 sequences and proteins . in one embodiment , a vector of the invention comprises a selected transgene , which is flanked by a 5 ′ itr and a 3 ′ itr , at least one of which is an aav - 1 itr of the invention . suitably , vectors of the invention may contain a aav - 1 p5 promoter of the invention . in yet another embodiment , a plasmid or vector of the invention contains aav - 1 rep sequences . in still another embodiment , a plasmid or vector of the invention contains at least one of the aav - 1 cap proteins of the invention . most suitably , these aav - 1 - derived vectors are assembled into viral vectors , as described herein . in one aspect , the present invention provides a recombinant aav - 1 viral vector produced using the aav - 1 capsid proteins of the invention . the packaged raav - 1 virions of the invention may contain , in addition to a selected minigene , other aav - 1 sequences , or may contain sequences from other aav serotypes . methods of generating raav virions are well known and the selection of a suitable method is not a limitation on the present invention . see , e . g ., k . fisher et al , j . virol ., 70 : 520 - 532 ( 1993 ) and u . s . pat . no . 5 , 478 , 745 . in one suitable method , a selected host cell is provided with the aav sequence encoding a rep protein , the gene encoding the aav cap protein and with the sequences for packaging and subsequent delivery . desirably , the method utilizes the sequences encoding the aav - 1 rep and / or cap proteins of the invention . in one embodiment , the rep / cap genes and the sequences for delivery are supplied by co - transfection of vectors carrying these genes and sequences . in one currently preferred embodiment , a cis ( vector ) plasmid , a trans plasmid containing the rep and cap genes , and a plasmid containing the adenovirus helper genes are co - transfected into a suitable cell line , e . g ., 293 . alternatively , one or more of these functions may be provided in trans via separate vectors , or may be found in a suitably engineered packaging cell line . an exemplary cis plasmid will contain , in 5 ′ to 3 ′ order , aav 5 ′ itr , the selected transgene , and aav 3 ′ itr . in one desirable embodiment , at least one of the aav itrs is a 143 nt aav - 1 itr . however , other aav serotype itrs may be readily selected . suitably , the full - length itrs are utilized . however , one of skill in the art can readily prepare modified aav itrs using conventional techniques . similarly , methods for construction of such plasmids is well known to those of skill in the art . a trans plasmid for use in the production of the raav - 1 virion particle may be prepared according to known techniques . in one desired embodiment , this plasmid contains the rep and cap proteins of aav - 1 , or functional fragments thereof . alternatively , the rep sequences may be from another selected aav serotype . the cis and trans plasmid may then be co - transfected with a wild - type helper virus ( e . g ., ad2 , ad5 , or a herpesvirus ), or more desirably , a replication — defective adenovirus , into a selected host cell . alternatively , the cis and trans plasmid may be co - transfected into a selected host cell together with a transfected plasmid which provides the necessary helper functions . selection of a suitable host cell is well within the skill of those in the art and include such mammalian cells as 293 cells , hela cells , among others . alternatively , the cis plasmid and , optionally , the trans plasmid , may be transfected into a packaging cell line which provides the remaining helper functions necessary for production of a raav containing the desired aav - 1 sequences of the invention . an example of a suitable packaging cell line , wherein an aav - 2 capsid is desired , is b - 50 , which stably expresses aav - 2 rep and cap genes under the control of a homologous p5 promoter . this cell line is characterized by integration into the cellular chromosome of multiple copies ( at least 5 copies ) of p5 - rep - cap gene in a concatomer form . this b - 50 cell line was deposited with the american type culture collection depositary , 10801 university boulevard , manassas , va . 20110 - 2209 , on sep . 18 , 1997 under accession no . crl - 12401 pursuant to the provisions of the budapest treaty . however , the present invention is not limited as to the selection of the packaging cell line . exemplary transducing vectors based on aav - 1 capsid proteins have been tested both in vivo and in vitro , as described in more detail in example 4 . in these studies , it was demonstrated that recombinant aav vector with an aav - 1 virion can transduce both mouse liver and muscle . these , and other aav - 1 based gene therapy vectors which may be generated by one of skill in the art are beneficial for gene delivery to selected host cells and gene therapy patients since the neutralization antibodies of aav - 1 present in much of the human population exhibit different patterns from other aav serotypes and therefore do not neutralize the aav - 1 virions . one of skill in the art may readily prepare other raav viral vectors containing the aav - 1 capsid proteins provided herein using a variety of techniques known to those of skill in the art . one may similarly prepare still other raav viral vectors containing aav - 1 sequence and aav capsids of another serotype . one of skill in the art will readily understand that the aav - 1 sequences of the invention can be readily adapted for use in these and other viral vector systems for in vivo , ex vivo or in vivo gene delivery . particularly well suited for use in such viral vector systems are the aav - 1 itr sequences , the aav - 1 rep , the aav - 1 cap , and the aav - 1 p5 promoter sequences . for example , in one desirable embodiment , the aav - 1 itr sequences of the invention may be used in an expression cassette which includes aav - 1 5 ′ itr , a non - aav dna sequences of interest ( e . g ., a minigene ), and 3 ′ itr and which lacks functional rep / cap . such a cassette containing an aav - 1 itr may be located on a plasmid for subsequent transfection into a desired host cell , such as the cis plasmid described above . this expression cassette may further be provided with an aav capsid of a selected serotype to permit infection of a cell or stably transfected into a desired host cell for packaging of raav virions . such an expression cassette may be readily adapted for use in other viral systems , including adenovirus systems and lentivirus systems . methods of producing ad / aav vectors are well known to those of skill in the art . one desirable method is described in pct / us95 / 14018 . however , the present invention is not limited to any particular method . another aspect of the present invention is the novel aav - 1 p5 promoter sequences which are located in the region spanning nt 236 - 299 of seq id no : 1 . this promoter is useful in a variety of viral vectors for driving expression of a desired transgene . similarly , one of skill in the art can readily select other fragments of the aav - 1 genome of the invention for use in a variety of vector systems . such vectors systems may include , e . g ., lentiviruses , retroviruses , poxviruses , vaccinia viruses , and adenoviral systems , among others . selection of these vector systems is not a limitation of the present invention . in yet another aspect , the present invention provides host cells which may be transiently transfected with aav - 1 nucleic acid sequences of the invention to permit expression of a desired transgene or production of a raav particle . for example , a selected host cell may be transfected with the aav - 1 p5 promoter sequences and / or the aav - 1 5 ′ itr sequences using conventional techniques . providing aav helper functions to the transfected cell lines of the invention results in packaging of the raav as infectious raav particles . such cell lines may be produced in accordance with known techniques [ see , e . g , u . s . pat . no . 5 , 658 , 785 ], making use of the aav - 1 sequences of the invention . alternatively , host cells of the invention may be stably transfected with a raav expression cassette of the invention , and with copies of aav - 1 rep and cap genes . suitable parental cell lines include mammalian cell lines and it may be desirable to select host cells from among non - simian mammalian cells . examples of suitable parental cell lines include , without limitation , hela [ atcc ® depositary , accession no . ccl 2 ], a549 [ atcc ® depositary , accession no . ccl 185 ], kb [ ccl 17 ], detroit [ e . g ., detroit 510 , ccl 72 ] and wi - 38 [ ccl 75 ] cells . these cell lines are all available from the american type culture collection depositary , 10801 university boulevard , manassas , va . 20110 - 220910801 university boulevard , manassas , va . 20110 - 2209 usa . other suitable parent cell lines may be obtained from other sources and may be sued to construct stable cell lines containing the p5 and / or aav rep and cap sequences of the invention . recombinant vectors generated as described above are useful for delivery of the dna of interest to cells . in another aspect , the present invention provides a method for delivery of a transgene to a host which involves transfecting or infecting a selected host cell with a recombinant viral vector generated with the aav - 1 sequences ( or functional fragments thereof ) of the invention . methods for delivery are well known to those of skill in the art and are not a limitation of the present invention . in one desirable embodiment , the invention provides a method for aav — mediated delivery of a transgene to a host . this method involves transfecting or infecting a selected host cell with a recombinant viral vector containing a selected transgene under the control of sequences which direct expression thereof and aav - 1 capsid proteins . optionally , a sample from the host may be first assayed for the presence of antibodies to a selected aav serotype . a variety of assay formats for detecting neutralizing antibodies are well known to those of skill in the art . the selection of such an assay is not a limitation of the present invention . see , e . g ., fisher et al , nature med ., 3 ( 3 ): 306 - 312 ( march 1997 ) and w . c . manning et al , human gene therapy , 9 : 477 - 485 ( mar . 1 , 1998 ). the results of this assay may be used to determine which aav vector containing capsid proteins of a particular serotype are preferred for delivery , e . g ., by the absence of neutralizing antibodies specific for that capsid serotype . in one aspect of this method , the delivery of vector with aav - 1 capsid proteins may precede or follow delivery of a gene via a vector with a different serotype aav capsid protein . thus , gene delivery via raav vectors may be used for repeat gene delivery to a selected host cell . desirably , subsequently administered raav vectors carry the same transgene as the first raav vector , but the subsequently administered vectors contain capsid proteins of serotypes which differ from the first vector . for example , if a first vector has aav - 2 capsid proteins , subsequently administered vectors may have capsid proteins selected from among the other serotypes , including aav - 1 , aav - 3a , aav - 3b , aav - 4 and aav - 6 . thus , a raav - 1 - derived recombinant viral vector of the invention provides an efficient gene transfer vehicle which can deliver a selected transgene to a selected host cell in vivo or ex vivo even where the organism has neutralizing antibodies to one or more aav serotypes . these compositions are particularly well suited to gene delivery for therapeutic purposes . however , the compositions of the invention may also be useful in immunization . further , the compositions of the invention may also be used for production of a desired gene product in vitro . the above - described recombinant vectors may be delivered to host cells according to published methods . an aav viral vector bearing the selected transgene may be administered to a patient , preferably suspended in a biologically compatible solution or pharmaceutically acceptable delivery vehicle . a suitable vehicle includes sterile saline . other aqueous and non - aqueous isotonic sterile injection solutions and aqueous and non - aqueous sterile suspensions known to be pharmaceutically acceptable carriers and well known to those of skill in the art may be employed for this purpose . the viral vectors are administered in sufficient amounts to transfect the cells and to provide sufficient levels of gene transfer and expression to provide a therapeutic benefit without undue adverse effects , or with medically acceptable physiological effects , which can be determined by those skilled in the medical arts . conventional and pharmaceutically acceptable routes of administration include , but are not limited to , direct delivery to the liver , oral , intranasal , intravenous , intramuscular . subcutaneous , intradermal , and other parental routes of administration . routes of administration may be combined , if desired . dosages of the viral vector will depend primarily on factors such as the condition being treated , the age , weight and health of the patient , and may thus vary among patients . for example , a therapeutically effective human dosage of the viral vector is generally in the range of from about 1 ml to about 100 ml of solution containing concentrations of from about 1 × 10 9 to 1 × 10 16 genomes virus vector . a preferred human dosage may be about 1 × 10 13 to 1 × 10 16 aav genomes . the dosage will be adjusted to balance the therapeutic benefit against any side effects and such dosages may vary depending upon the therapeutic application for which the recombinant vector is employed . the levels of expression of the transgene can be monitored to determine the frequency of dosage resulting in viral vectors , preferably aav vectors containing the minigene . optionally , dosage regimens similar to those described for therapeutic purposes may be utilized for immunization using the compositions of the invention . for in vitro production , a desired protein may be obtained from a desired culture following transfection of host cells with a raav containing the gene encoding the desired protein and culturing the cell culture under conditions which permits expression . the expressed protein may then be purified and isolated , as desired . suitable techniques for transfection , cell culturing , purification , and isolation are known to those of skill in the art . the following examples illustrate several aspects and embodiments of the invention . the replicated form dna of aav - 1 was extracted from 293 cells that were infected by aav - 1 and wild type adenovirus type 5 . aav - free 293 cells and 84 - 31 cells were provided by the human application laboratory of the university of pennsylvania . these cells were cultured in dulbecco &# 39 ; s modified eagle medium with 10 % fetal bovine serum ( hyclone ), penicillin ( 100 u / ml ) and streptomycin at 37 ° c . in a moisturized environment supplied with 5 % c 02 . the 84 - 31 cell line constitutively expresses adenovirus genes e1a , e1b , e4 / orf6 , and has been described previously [ k . j . fisher , j . virol ., 70 : 520 - 532 ( 1996 )]. aav - 1 ( atcc vr - 645 ) seed stock was purchased from american type culture collection ( atcc , manassas , va .). aav viruses were propagated in 293 cells with wild type ad5 as a helper virus . the recombinant aav viruses were generated by transfection using an adenovirus free method . briefly , the cis plasmid ( with aav itr ), trans plasmid ( with aav rep gene and cap gene ) and helper plasmid ( pfδ13 , with essential regions from the adenovirus genome ) were simultaneously co - transfected into 293 cells in a ratio of 1 : 1 : 2 by calcium phosphate precipitation . the pfδ13 helper plasmid has an 8 kb deletion in the adenovirus e2b region and has deletions in most of the late genes . this helper plasmid was generated by deleting the rsrii fragment from pfg140 ( microbix , canada ). typically , 50 μg of dna ( cis : trans : pfδ13 at ratios of 1 : 1 : 2 , respectively ) was transfected onto a 15 cm tissue culture dish . the cells were harvested 96 hours post - transfection , sonicated and treated with 0 . 5 % sodium deoxycholate ( 37 ° c . for 10 min ). cell lysates were then subjected to two rounds of a cscl gradient . peak fractions containing aav vector were collected , pooled , and dialyzed against pbs before injecting into animals . to make raav virus with aav - 1 virion , the pav1h or p5e18 ( 2 / 1 ) was used as the trans plasmid to provide rep and cap function . for the generation of raav based on aav - 2 , p5e18 was used as the trans plasmid since it greatly improved the raav yield . this plasmid , p5e18 ( 2 / 2 ), expresses aav - 2 rep and cap and contains a ps promoter relocated to a position 3 ′ to the cap gene , thereby minimizing expression of rep78 and rep68 . the strategy was initially described by li et al , j . virol ., 71 : 5236 - 5243 ( 1997 ). p5e18 ( 2 / 2 ) was constructed in the following way . the previously described pmmtv - trans vector ( i . e ., the mouse mammary tumor virus promoter substituted for the p5 promoter in an aav - 2 - based vector ) was digested with smai and clai , filled in with the klenow enzyme , and then recircularized with dna ligase . the resulting construct was digested with xbai , filled in , and ligated to the blunt - ended bamhi - xbai fragment from pcr - p5 , constructed in the following way . the p5 promoter of aav was amplified by pcr and the amplified fragment was subsequently cloned into pcr2 . 1 ( invitrogen ) to yield pcr - p5 . the helper plasmid pav1h was constructed by cloning the bfai fragment of paav - 2 into pbluescript ii - sk (+) at the bcorv and smai sites . the 3 . 0 - kb xbai - kpni fragment from p5e18 ( 2 / 2 ), the 2 . 3 - kb xbai - kpni fragment from pav1h , and the 1 . 7 - kb kpni fragment from p5e18 ( 2 / 2 ) were incorporated into a separate plasmid p5e18 ( 2 / 1 ), which contains aav - 2 rep , aav - 1 cap , and the aav - 2 p5 promoter located 3 ′ to the cap gene . plasmid p5e18 ( 2 / 1 ) produced 10 - to 20 - fold higher quantities of the vector than pav1h ( i . e ., 10 12 genomes / 50 15 - cm 2 plates ). hirt dna extraction was performed as described in the art with minor modification [ r . j . samulski et al ., cell , 33 : 135 - 143 ( 1983 )]. more particularly , hirst solution without sds was used instead of using original hirt solution containing sds . the amount of sds present in the original hirst solution was added after the cells had been fully suspended . to construct aav - 1 infectious clone , the hirt dna from aav - 1 infected 293 cells was repaired with klenow enzyme ( new england biolabs ) to ensure the ends were blunt . the treated aav - 1 hirt dna was then digested with bamhi and cloned into three vectors , respectively . the internal bamhi was cloned into pbluescript ii - sk + cut with bamhi to get pav1 - bm . the left and right fragments were cloned into pbluescript ii - sk + cut with bamhi + ecorv to obtain pav1 - bl and pav1 - br , respectively . the aav sequence in these three plasmids were subsequently assembled into the same vector to get aav - 1 infectious clone paav - 1 . the helper plasmid for recombinant aav - 1 virus generation was constructed by cloning the bfa 1 fragment of paav - 1 into pbluescript ii - sk + at the ecorv site . analysis of the hirt dna revealed three bands , a dimer at 9 . 4 kb , a monomer at 4 . 7 kb and single - stranded dna at 1 . 7 kb , which correlated to different replication forms of aav - 1 . the monomer band was excised from the gel and then digested with bamhi . this resulted in three fragments of 1 . 1 kb , 0 . 8 kb and 2 . 8 kb . this pattern is in accordance with the description by bantel - schaal and zur hausen , virol ., 134 ( 1 ): 52 - 63 ( 1984 ). the 1 . 1 kb and 2 . 8 kb bamhi fragments were cloned into pbluescript - ks (+) at bamhi and ecorv site . the internal 0 . 8 kb fragment was cloned into bamhi site of pbluescript - ks (+). these three fragments were then subcloned into the same construct to obtain a plasmid ( paav - 1 ) that contained the full sequence of aav - 1 . the paav - 1 was then tested for its ability to rescue from the plasmid backbone and package infectious virus . the paav - 1 was then transfected to 293 cells and supplied with adenovirus type as helper at moi 10 . the virus supernatant was used to reinfect 293 cells . for southern blot analysis , hirt dna was digested with dpni to remove bacteria - borne plasmid and probed with internal bamhi fragment of aav - 1 . the membrane was then washed at high stringency conditions , which included : twice 30 minutes with 2 × ssc , 0 . 1 % sds at 65 ° c . and twice 30 minutes with 0 . 1 × ssc , 0 . 1 % sds at 65 ° c . the membrane was then analyzed by both phosphor image and x - ray autoradiography . the results confirmed that paav - 1 is indeed an infectious clone of aav serotype 1 . the entire aav - 1 genome was then determined by automatic sequencing and was found to be 4718 nucleotides in length ( fig1 a - 1 f ). for sequencing , an abi 373 automatic sequencer as used to determine the sequences for all plasmids and pcr fragments related to this study using the fs dye chemistry . all sequences were confirmed by sequencing both plus and minus strands . these sequences were also confirmed by sequencing two independent clones of pav - bm , pav - bl and pav - br . since the replicated form of aav - 1 dna served as the template for sequence determination , these sequences were also confirmed by sequencing a series of pcr products using original aav - 1 seed stock as a template . the length of aav - 1 was found to be within the range of the other serotypes : aav - 3 ( 4726 nucleotides ), aav - 4 ( 4774 nucleotides ), aav - 2 ( 4681 nucleotides ), and aav - 6 ( 4683 nucleotides ). the aav - 1 genome exhibited similarities to other serotypes of adeno - associated viruses . overall , it shares more than 80 % identity with other known aav viruses as determined by the computer program megalign using default settings [ dnastar , madison , wis .]. the key features in aav - 2 can also be found in aav - 1 . first , aav - 1 has the same type of inverted terminal repeat which is capable of forming t - shaped hairpin structures , despite the differences at the nucleotide level ( fig2 and 3 ). the sequences of right itrs and left itrs of aav - 1 are identical . the aav tr sequence is subdivided into a , a ′, b , b ′, c , c ′, d and d ′ [ bern , cited above ]. these aav itr sequences are also virtually the same as those found in aav - 6 right itr , there being one nucleotide difference in each of a and a ′ sequence , and the last nucleotide of the d sequence . second , the aav - 2 rep binding motif [ gctcgctcgctcgctg ( seq id no : 20 )] is well conserved . such motif can also be found in the human chromosome 19 aav - 2 pre - integration region . finally , non - structural and structural coding regions , and regulatory elements similar to those of other aav serotypes also exist in aav - 1 genome . although the overall features of aav terminal repeats are very much conserved , the total length of the aav terminal repeat exhibits divergence . the terminal repeat of aav - 1 consists of 143 nucleotides while those of aav - 2 , aav - 3 , and aav - 4 are about 145 or 146 nucleotides . the loop region of aav - 1 itr most closely resembles that of aav - 4 in that it also uses tct instead of the ttt found in aav - 2 and aav - 3 . the possibility of sequencing error was eliminated using restriction enzyme digestion , since these three nucleotides are part of the saci site ( gagctc ; nt 69 - 74 of seq id no : 1 ). the p5 promoter region of aav - 1 shows more variations in nucleotide sequences with other aav serotypes . however , it still maintains the critical regulatory elements . the two copies of yy1 [ see , fig1 a - 1 f ] sites seemed to be preserved in all known aav serotypes , which have been shown to be involved in regulating aav gene expression . in aav - 4 , there are 56 additional nucleotides inserted between yy1 and e - box / usf site , while in aav - 1 , there are 26 additional nucleotides inserted before the e - box / usf site . the p19 promoter , p40 promoter and polya can also be identified from the aav - 1 genome by analogy to known aav serotypes , which are also highly conserved . thus , the analysis of aav terminal repeats of various serotypes showed that the a and a ′ sequence is very much conserved . one of the reasons may be the rep binding motif ( gctc ) 3 gctg [ seq id no : 20 ]. these sequences appear to be essential for aav dna replication and site - specific integration . the same sequence has also been shown to be preserved in a monkey genome [ samulski , personal communication ]. the first 8 nucleotides of the d sequence are also identical in all known aav serotypes . this is in accordance with the observation of the srivastava group that only the first 10 nucleotides are essential for aav packaging [ x . s . wang et al , j . virol ., 71 : 3077 - 3082 ( 1997 ); x . s . wang et al , j . virol ., 71 : 1140 - 1146 ( 1997 )]. the function of the rest of the d sequences still remain unclear . they may be somehow related to their tissue specificities . the variation of nucleotide in b and c sequence may also suggest that the secondary structure of the itrs is more critical for its biological function , which has been demonstrated in many previous publications . the nucleotide sequences of aav - 1 , obtained as described above , were compared with known aav sequences , including aav - 2 , aav - 4 and aav - 6 using dna star megalign . this comparison revealed a stretch of 71 identical nucleotides shared by aav - 1 , aav - 2 and aav - 6 . see , fig1 a - 1f . this comparison further suggested that aav - 6 is a hybrid formed by homologous recombination of aav - 1 and aav - 2 . see , fig3 a and 3b . these nucleotides divide the aav - 6 genome into two regions . the 5 ′ half of aav - 6 of 522 nucleotides is identical to that of aav - 2 except in 2 positions . the 3 ′ half of aav - 6 including the majority of the rep gene , complete cap gene and 3 ′ itr is 98 % identical to aav - 1 . biologically , such recombination may enable aav - 1 to acquire the ability to transmit through the human population . it is also interesting to note that the itrs of aav - 6 comprise one aav - 1 itr and one aav - 2 itr . the replication model of defective parvovirus can maintain this special arrangement . studies on aav integration have shown that a majority of aav integrants carries deletions in at least one of the terminal repeats . these deletions have been shown to be able to be repaired through gene conversion using the other intact terminal repeat as a template . therefore , it would be very difficult to maintain aav - 6 as a homogenous population when an integrated copy of aav - 6 is rescued from host cells with helper virus infection . the aav - 6 with two identical aav - 2 itrs or two identical aav - 1 itrs should be the dominant variants . the aav - 6 with two aav - 1 itrs has been observed by russell &# 39 ; s group [ rutledge , cited above ( 1998 )]. so far there is no report on aav - 6 with two aav - 2 itrs . acquirement of aav - 2 p5 promoter by aav - 6 may have explained that aav - 6 have been isolated from human origin while aav - 1 with the same virion has not . the regulation of p5 promoter between different species of aav may be different in vivo . this observation suggests the capsid proteins of aav were not the only determinants for tissue specificity . although it is clear that aav - 6 is a hybrid of aav - 1 and aav - 2 , aav - 6 has already exhibited divergence from either aav - 1 or aav - 2 . there are two nucleotide differences between aav - 6 and aav - 2 in their first 450 nucleotides . there are about 1 % differences between aav - 6 and aav - 1 in nucleotide levels from nucleotides 522 to the 3 ′ end . there also exists a quite divergent region ( nucleotide 4486 - 4593 ) between aav - 6 and aav - 1 ( fig1 a - 1 f ). this region does not encode any known proteins for aavs . these differences in nucleotide sequences may suggest that aav - 6 and aav - 1 have gone through some evolution since the recombination took place . another possible explanation is that there exists another variant of aav - 1 which has yet to be identified . so far , there is no evidence to rule out either possibility . it is still unknown if other hybrids ( aav - 2 to aav - 4 , etc .) existed in nature . the coding region of aav - 1 was deduced by comparison with other known aav serotypes . table 1 illustrates the coding region differences between aav - 1 and aav - 6 . the amino acid residues are deduced according to aav - 2 . with reference to the amino acid position of aav - 1 , table 1 lists the amino acids of aav - 1 which have been changed to the corresponding ones of aav - 6 . the amino acids of aav - 1 are shown to the left of the arrow . reference may be made to seq id no : 5 of the amino acid sequence of aav - 1 rep 78 and to seq id no : 13 for the amino acid sequence of aav - 1 vp1 . it was surprising to see that the sequence of the aav - 1 coding region is almost identical to that of aav - 6 from position 452 to the end of coding region ( 99 %). the first 508 nucleotides of aav - 6 have been shown to be identical to those of aav - 2 [ rutledge , cited above ( 1998 )]. since the components of aav - 6 genome seemed to be aav - 2 left itr — aav - 2 p5 promoter — aav - 1 coding region — aav - 1 right itr , it was concluded that aav - 6 is a naturally occurred hybrid between aav - 1 and aav - 2 . recombinant gene transfer vectors based on aav - 1 viruses were constructed by the methods described in example 1 . to produce a hybrid recombinant virus with aav - 1 virion and aav - 2 itr , the aav - 1 trans plasmid ( pav1h ) and the aav - 2 cis - lacz plasmid ( with aav - 2 itr ) were used . the aav - 2 itr was used in this vector in view of its known ability to direct site - specific integration . also constructed for use in this experiment was an aav - 1 vector carrying the green fluorescent protein ( gfp ) marker gene under the control of the immediate early promoter of cmv using pav1h as the trans plasmid . 84 - 31 cells , which are subclones of 293 cells ( which express adenovirus e1a , e1b ) which stably express e4 / orf5 , were infected with raav - 1 gfp or raav - lacz . high levels of expression of gfp and lacz was detected in the cultured 84 - 31 cells . this suggested that raav - 1 based vector was very similar to aav - 2 based vectors in ability to infect and expression levels . the performance of aav - 1 based vectors was also tested in vivo . the raav - 1 cmv - α1at virus was constructed as follows . the ecori fragment of pat85 ( atcc ) containing human α1 - antitrypsin ( α1at ) cdna fragment was blunted and cloned into pcr ( promega ) at a smai site to obtain pcr - α1at . the cmv promoter was cloned into pcr - α1at at the xbai site . the a1b - α1at expression cassette was removed by xhoi and clai and cloned into pav1h at the xbai site . this vector plasmid was used to generate aav - 1 - cmv - α1at virus used in the experiment described below . for screening human antibodies against aav , purified aav virus is lysed with ripa buffer ( 10 mm tris ph 8 . 2 , 1 % triton x - 100 , 1 % sds , 0 . 15 m nacl ) and separated in 10 % sds - page gel . the heat inactivated human serum was used at a 1 to 1000 dilution in this assay . the raav - 1 cmv - α1at viruses were injected into rag - 1 mice through tail vein injection at different dosages . the concentration of human α1 - antitrypsin in mouse serum was measured using elisa . the coating antibody is rabbit anti - human human α1 - antitrypsin ( sigma ). the goat - antihuman α1 - antitrypsin ( sigma ) was used as the primary detection antibodies . the sensitivity of this assay is around 0 . 3 ng / ml to 30 ng / ml . the expression of human α - antitrypsin in mouse blood can be detected in a very encouraging level . this result is shown in table 2 . raav - 1 cmv - lacz viruses were also injected into the muscle of c57bl6 mice and similar results were obtained . collectively , these results suggested that aav - 1 based vector would be appropriate for both liver and muscle gene delivery . simple and quantitative assays for neutralizing antibodies ( nab ) to aav - 1 and aav - 2 were developed with recombinant vectors . a total of 33 rhesus monkeys and 77 normal human subjects were screened . wild - caught juvenile rhesus monkeys were purchased from covance ( alice , tex .) and labs of virginia ( yemassee , s . c .) and kept in full quarantine . the monkeys weighed approximately 3 to 4 kg . the nonhuman primates used in the institute for human gene therapy research program are purposefully bred in the united states from specific - pathogen - free closed colonies . all vendors are u . s . department of agriculture class a dealers . the rhesus macaques are therefore not infected with important simian pathogens , including the tuberculosis agent , major simian lentiviruses ( simian immunodeficiency virus and simian retroviruses ), and cercopithecine herpesvirus . the animals are also free of internal and external parasites . the excellent health status of these premium animals minimized the potential for extraneous variables . for this study , serum was obtained from monkeys prior to initiation of any protocol . nab titers were analyzed by assessing the ability of serum antibody to inhibit the transduction of reporter virus expressing green fluorescent protein ( gfp ) ( aav1 - gfp or aav2 - gfp ) into 84 - 31 cells . various dilutions of antibodies preincubated with reporter virus for 1 hour at 37 ° c . were added to 90 % confluent cell cultures . cells were incubated for 48 hours and the expression of green fluorescent protein was measured by fluoroimaging ( molecular dynamics ). nab titers were calculated as the highest dilution at which 50 % of the cells stained green . analysis of nab in rhesus monkeys showed that 61 % of animals tested positive for aav - 1 ; a minority ( 24 %) has nab to aav - 2 . over one - third of animals had antibodies to aav - 1 but not aav - 2 ( i . e ., were monospecific for aav - 1 ), whereas no animals were positive for aav - 2 without reacting to aav - 1 . these data support the hypothesis that aav - 1 is endemic in rhesus monkeys . the presence of true aav - 2 infections in this group of nonhuman primates is less clear , since cross - neutralizing activity of an aav - 1 response to aav - 2 can not be ruled out . it is interesting that there is a linear relationship between aav - 2 nab and aav - 1 nab in animals that had both . for these neutralization antibody assays , human serum samples were incubated at 56 ° c . for 30 min to inactivate complement and then diluted in dmem . the virus ( raav or rad with either lacz or gfp ) was then mixed with each serum dilution ( 20 ×, 400 ×, 2000 ×, 4000 ×, etc .) and incubated for 1 hour at 37 ° c . before applied to 90 % confluent cultures of 84 - 31 cells ( for aav ) or hela cells ( for adenovirus ) in 96 - well plates . after 60 minutes of incubation at culture condition , 100 μl additional media containing 20 % fcs was added to make final culture media containing 10 % fcs . the human neutralizing antibodies against these three viruses seemed to be unrelated since the existence of neutralizing antibodies against aav are not indications for antibodies against adenovirus . however , aav requires adenovirus as helper virus , in most of the cases , the neutralizing antibodies against aav correlated with the existence of neutralizing antibodies to adenovirus . among the 77 human serum samples screened , 41 % of the samples can neutralize the infectivity of recombinant adenovirus based on ad5 . 15 / 77 ( 19 %) of serum samples can neutralize the transduction of raav - 1 while 20 / 77 ( 20 %) of the samples inhibit raav - 2 transduction at 1 to 80 dilutions or higher . all serum samples positive in neutralizing antibodies for aav - 1 in are also positive for aav - 2 . however , there are five ( 6 %) raav - 2 positive samples that failed to neutralize raav - 1 . in samples that are positive for neutralizing antibodies , the titer of antibodies also varied in the positive ones . the results from screening human sera for antibodies against aavs supported the conclusion that aav - 1 presents the same epitome as that of aav - 2 to interact with cellular receptors since aav - 1 neutralizing human serums can also decrease the infectivity of aav - 2 . however , the profile of neutralizing antibodies for these aavs is not identical , there are additional specific receptors for each aav serotype . the recombinant aav - 1 vectors of the invention and the recombinant aav - 2 vectors [ containing the gene encoding human α1 - antitrypsin ( α1at ) or murine erythropoietin ( epo ) from a cytomegalovirus - enhanced β - actin promoter ( cb )] were evaluated in a direct comparison to equivalent copies of aav - 2 vectors containing the same vector genes . recombinant viruses with aav - 1 capsids were constructed using the techniques in example 1 . to make raav with aav - 1 virions , pav1h or p5e18 ( 2 / 1 ) was used as the trans plasmid to provide rep and cap functions . for the generation of the raav based on aav - 2 , p5e18 ( 2 / 2 ) was used as the trans plasmid , since it greatly improved the raav yield . [ early experiments indicated similar in vivo performances of aav - 1 vectors produced with pav1h and p5e19 ( 2 / 1 ). all subsequent studies used aav - 1 vectors derived from p5e18 ( 2 / 1 ) because of the increased yield .] equivalent stocks of the aav - 1 and aav - 2 vectors were injected intramuscularly ( 5 × 10 1 genomes ) or liver via the portal circulation ( 1 × 10 11 genomes ) into immunodeficient mice , and the animals ( four groups ) were analyzed on day 30 for expression of transgene . see , fig4 a and 4b . aav - 2 vectors consistently produced 10 - to 50 - fold more serum erythropoietin or α1 - antitrypsin when injected into liver compared to muscle . ( however , the aav - 1 - delivered genes did achieve acceptable expression levels in the liver .) this result was very different from that for aav - 1 vectors , with which muscle expression was equivalent to or greater than liver expression . in fact , aav - 1 outperformed aav - 2 in muscle when equivalent titers based on genomes were administered . c57bl / 6 mice ( 6 - to 8 - week old males , jackson laboratories ) were analyzed for aav mediated gene transfer to liver following intrasplenic injection of vector ( i . e ., targeted to liver ). a total of 10 11 genome equivalents of raav - 1 or raav - 2 vector were injected into the circulation in 100 μl buffered saline . the first vector contained either an aav - 1 capsid or an aav - 2 capsid and expressed α1at under the control of the chicken β - actin ( cb ) promoter . day 28 sera were analyzed for antibodies against aav - 1 or aav - 2 and serum α1at levels were checked . animals were then injected with an aav - 1 or aav - 2 construct expressing erythropoietin ( epo , also under the control of the cb promoter ). one month later sera was analyzed for serum levels of epo . the following groups were analyzed ( fig5 a - 5 d ). in group 1 , vector 1 was aav - 2 expressing α1at and vector 2 was aav - 2 expressing epo . animals generated antibodies against aav - 2 following the first vector administration which prevented the readministration of the aav - 2 based vector . there was no evidence for cross - neutralizing the antibody to aav - 1 . in group 2 , vector 1 was aav - 1 expressing α1at while vector 2 was aav - 1 expressing epo . the first vector administration did result in significant α1at expression at one month associated with antibodies to neutralizing antibodies to aav - 1 . the animals were not successfully readministered with the aav - 1 epo expressing construct . in group 3 , the effectiveness of an aav - 2 vector expressing epo injected into a naive animal was measured . the animals were injected with pbs and injected with aav - 2 epo vector at day 28 and analyzed for epo expression one month later . the neutralizing antibodies were evaluated at day 28 so we did not expect to see anything since they received pbs with the first vector injection . this shows that in naive animals aav - 2 is very efficient at transferring the epo gene as demonstrated by high level of serum epo one month later . group 4 was an experiment similar to group 3 in which the animals originally received pbs for vector 1 and then the aav - 1 expressing epo construct 28 days later . at the time of vector injection , there obviously were no antibodies to either aav - 1 or aav - 2 . the aav - 1 based vector was capable of generating significant expression of epo when measured one month later . group 5 is a cross - over experiment where the initial vector is aav - 2 expressing α1at followed by the aav - 1 construct expressing epo . the animals , as expected , were efficiently infected with the aav - 2 vector expressing α1at as shown by high levels of the protein in blood at 28 days . this was associated with significant neutralizing antibodies to aav - 2 . importantly , the animals were successfully administered aav - 1 following the aav - 2 vector as shown by the presence of epo in serum 28 days following the second vector administration . at the time of this vector administration , there was high level aav - 2 neutralizing antibodies and very low cross - reaction to aav - 1 . the level of epo was slightly diminished possibly due to a small amount of cross - reactivity . group 6 was the opposite cross - over experiment in which the initial vector was aav - 1 based , whereas the second experiment was aav - 2 based . the aav - 1 vector did lead to significant gene expression of α1at , which also resulted in high level aav - 1 neutralizing antibody . the animals were very efficiently administered aav - 2 following the initial aav - 1 vector as evidenced by high level epo . a substantially identical experiment was performed in muscle in which 5 × 10 10 genomes were injected into the tibialis anterior of c57bl / 6 mice as a model for muscle directed gene therapy . the results are illustrated in fig6 a - 6d and are essentially the same as for liver . in summary , this experiment demonstrates the utility of using an aav - 1 vector in patients who have pre - existing antibodies to aav - 2 or who had initially received an aav - 2 vector and need readministration . this example illustrates the construction of recombinant aav vectors which contain aav - 1 itrs of the invention . an aav - 1 cis plasmid is constructed as follows . a 160 bp xho - nrui aav - 1 fragment containing the aav - 1 5 ′ itr is obtained from pav1 - bl . pav1 - bl was generated as described in example 1 . the xho - nrui fragment is then cloned into a second pav1 - bl plasmid at an xbai site to provide the plasmid with two aav - 1 itrs . the desired transgene is then cloned into the modified pav - 1bl at the nrui and bamhi site , which is located between the aav - 1 itr sequences . the resulting aav - 1 cis plasmid contains aav - 1 itrs flanking the transgene and lacks functional aav - 1 rep and cap . recombinant aav is produced by simultaneously transfecting three plasmids into 293 cells . these include the aav - 1 cis plasmid described above ; a trans plasmid which provides aav rep / cap functions and lacks aav itrs ; and a plasmid providing adenovirus helper functions . the rep and / or cap functions may be provided in trans by aav - 1 or another aav serotype , depending on the immunity profile of the intended recipient . alternatively , the rep or cap functions may be provided in cis by aav - 1 or another serotype , again depending on the patient &# 39 ; s immunity profile . in a typical cotransfection , 50 μg of dna ( cis : trans : helper at ratios of 1 : 1 : 2 , respectively ) is transfected onto a 15 cm tissue culture dish . cells are harvested 96 hours post transfection , sonicated and treated with 0 . 5 % sodium deoxycholate ( 37 ° for 10 min ). cell lysates are then subjected to 2 - 3 rounds of ultracentrifugation in a cesium gradient . peak fractions containing raav are collected , pooled and dialyzed against pbs . a typical yield is 1 × 10 13 genomes / 10 9 cells . using this method , one recombinant virus construct is prepared which contains the aav - 1 itrs flanking the transgene , with an aav - 1 capsid . another recombinant virus construct is prepared with contains the aav - 1 itrs flanking the transgene , with an aav - 2 capsid . all publications cited in this specification are incorporated herein by reference . while the invention has been described with reference to a particularly preferred embodiments , it will be appreciated that modifications can be made without departing from the spirit of the invention . such modifications are intended to fall within the scope of the claims . cattttgacc gcgaaatttg aacgagcagc agcc atg ccg ggc ttc tac gag atc 355 gtg atc aag gtg ccg agc gac ctg gac gag cac ctg ccg ggc att tct 403 gac tcg ttt gtg agc tgg gtg gcc gag aag gaa tgg gag ctg ccc ccg 451 gat tct gac atg gat ctg aat ctg att gag cag gca ccc ctg acc gtg 499 asp ser asp met asp leu asn leu ile glu gln ala pro leu thr val gcc gag aag ctg cag cgc gac ttc ctg gtc caa tgg cgc cgc gtg agt 547 ala glu lys leu gln arg asp phe leu val gln trp arg arg val ser aag gcc ccg gag gcc ctc ttc ttt gtt cag ttc gag aag ggc gag tcc 595 tac ttc cac ctc cat att ctg gtg gag acc acg ggg gtc aaa tcc atg 643 tyr phe his leu his ile leu val glu thr thr gly val lys ser met gtg ctg ggc cgc ttc ctg agt cag att agg gac aag ctg gtg cag acc 691 val leu gly arg phe leu ser gln ile arg asp lys leu val gln thr atc tac cgc ggg atc gag ccg acc ctg ccc aac tgg ttc gcg gtg acc 739 ile tyr arg gly ile glu pro thr leu pro asn trp phe ala val thr aag acg cgt aat ggc gcc gga ggg ggg aac aag gtg gtg gac gag tgc 787 tac atc ccc aac tac ctc ctg ccc aag act cag ccc gag ctg cag tgg 835 gcg tgg act aac atg gag gag tat ata agc gcc tgt ttg aac ctg gcc 883 ala trp thr asn met glu glu tyr ile ser ala cys leu asn leu ala gag cgc aaa cgg ctc gtg gcg cag cac ctg acc cac gtc agc cag acc 931 cag gag cag aac aag gag aat ctg aac ccc aat tct gac gcg cct gtc 979 atc cgg tca aaa acc tcc gcg cgc tac atg gag ctg gtc ggg tgg ctg 1027 ile arg ser lys thr ser ala arg tyr met glu leu val gly trp leu gtg gac cgg ggc atc acc tcc gag aag cag tgg atc cag gag gac cag 1075 val asp arg gly ile thr ser glu lys gln trp ile gln glu asp gln gcc tcg tac atc tcc ttc aac gcc gct tcc aac tcg cgg tcc cag atc 1123 aag gcc gct ctg gac aat gcc ggc aag atc atg gcg ctg acc aaa tcc 1171 gcg ccc gac tac ctg gta ggc ccc gct ccg ccc gcg gac att aaa acc 1219 aac cgc atc tac cgc atc ctg gag ctg aac ggc tac gaa cct gcc tac 1267 gcc ggc tcc gtc ttt ctc ggc tgg gcc cag aaa agg ttc ggg aag cgc 1315 aac acc atc tgg ctg ttt ggg ccg gcc acc acg ggc aag acc aac atc 1363 gcg gaa gcc atc gcc cac gcc gtg ccc ttc tac ggc tgc gtc aac tgg 1411 ala glu ala ile ala his ala val pro phe tyr gly cys val asn trp acc aat gag aac ttt ccc ttc aat gat tgc gtc gac aag atg gtg atc 1459 thr asn glu asn phe pro phe asn asp cys val asp lys met val ile tgg tgg gag gag ggc aag atg acg gcc aag gtc gtg gag tcc gcc aag 1507 gcc att ctc ggc ggc agc aag gtg cgc gtg gac caa aag tgc aag tcg 1555 ala ile leu gly gly ser lys val arg val asp gln lys cys lys ser tcc gcc cag atc gac ccc acc ccc gtg atc gtc acc tcc aac acc aac 1603 atg tgc gcc gtg att gac ggg aac agc acc acc ttc gag cac cag cag 1651 met cys ala val ile asp gly asn ser thr thr phe glu his gln gln ccg ttg cag gac cgg atg ttc aaa ttt gaa ctc acc cgc cgt ctg gag 1699 cat gac ttt ggc aag gtg aca aag cag gaa gtc aaa gag ttc ttc cgc 1747 tgg gcg cag gat cac gtg acc gag gtg gcg cat gag ttc tac gtc aga 1795 trp ala gln asp his val thr glu val ala his glu phe tyr val arg aag ggt gga gcc aac aaa aga ccc gcc ccc gat gac gcg gat aaa agc 1843 gag ccc aag cgg gcc tgc ccc tca gtc gcg gat cca tcg acg tca gac 1891 gcg gaa gga gct ccg gtg gac ttt gcc gac agg tac caa aac aaa tgt 1939 ala glu gly ala pro val asp phe ala asp arg tyr gln asn lys cys tct cgt cac gcg ggc atg ctt cag atg ctg ttt ccc tgc aag aca tgc 1987 ser arg his ala gly met leu gln met leu phe pro cys lys thr cys gag aga atg aat cag aat ttc aac att tgc ttc acg cac ggg acg aga 2035 glu arg met asn gln asn phe asn ile cys phe thr his gly thr arg gac tgt tca gag tgc ttc ccc ggc gtg tca gaa tct caa ccg gtc gtc 2083 aga aag agg acg tat cgg aaa ctc tgt gcc att cat cat ctg ctg ggg 2131 cgg gct ccc gag att gct tgc tcg gcc tgc gat ctg gtc aac gtg gac 2179 arg ala pro glu ile ala cys ser ala cys asp leu val asn val asp ctg gat gac tgt gtt tct gag caa taa atgacttaaa ccaggt atg gct gcc 2231 gat ggt tat ctt cca gat tgg ctc gag gac aac ctc tct gag ggc att 2279 cgc gag tgg tgg gac ttg aaa cct gga gcc ccg aag ccc aaa gcc aac 2327 cag caa aag cag gac gac ggc cgg ggt ctg gtg ctt cct ggc tac aag 2375 tac ctc gga ccc ttc aac gga ctc gac aag ggg gag ccc gtc aac gcg 2423 tyr leu gly pro phe asn gly leu asp lys gly glu pro val asn ala gcg gac gca gcg gcc ctc gag cac gac aag gcc tac gac cag cag ctc 2471 aaa gcg ggt gac aat ccg tac ctg cgg tat aac cac gcc gac gcc gag 2519 lys ala gly asp asn pro tyr leu arg tyr asn his ala asp ala glu ttt cag gag cgt ctg caa gaa gat acg tct ttt ggg ggc aac ctc ggg 2567 cga gca gtc ttc cag gcc aag aag cgg gtt ctc gaa cct ctc ggt ctg 2615 gtt gag gaa ggc gct aag acg gct cct gga aag aaa cgt ccg gta gag 2663 cag tcg cca caa gag cca gac tcc tcc tcg ggc atc ggc aag aca ggc 2711 cag cag ccc gct aaa aag aga ctc aat ttt ggt cag act ggc gac tca 2759 gln gln pro ala lys lys arg leu asn phe gly gln thr gly asp ser gag tca gtc ccc gat cca caa cct ctc gga gaa cct cca gca acc ccc 2807 gct gct gtg gga cct act aca atg gct tca ggc ggt ggc gca cca atg 2855 gca gac aat aac gaa ggc gcc gac gga gtg ggt aat gcc tca gga aat 2903 tgg cat tgc gat tcc aca tgg ctg ggc gac aga gtc atc acc acc agc 2951 trp his cys asp ser thr trp leu gly asp arg val ile thr thr ser acc cgc acc tgg gcc ttg ccc acc tac aat aac cac ctc tac aag caa 2999 thr arg thr trp ala leu pro thr tyr asn asn his leu tyr lys gln atc tcc agt gct tca acg ggg gcc agc aac gac aac cac tac ttc ggc 3047 tac agc acc ccc tgg ggg tat ttt gat ttc aac aga ttc cac tgc cac 3095 tyr ser thr pro trp gly tyr phe asp phe asn arg phe his cys his ttt tca cca cgt gac tgg cag cga ctc atc aac aac aat tgg gga ttc 3143 phe ser pro arg asp trp gln arg leu ile asn asn asn trp gly phe cgg ccc aag aga ctc aac ttc aaa ctc ttc aac atc caa gtc aag gag 3191 gtc acg acg aat gat ggc gtc aca acc atc gct aat aac ctt acc agc 3239 acg gtt caa gtc ttc tcg gac tcg gag tac cag ctt ccg tac gtc ctc 3287 ggc tct gcg cac cag ggc tgc ctc cct ccg ttc ccg gcg gac gtg ttc 3335 gly ser ala his gln gly cys leu pro pro phe pro ala asp val phe atg att ccg caa tac ggc tac ctg acg ctc aac aat ggc agc caa 3380 met ile pro gln tyr gly tyr leu thr leu asn asn gly ser gln gcc gtg gga cgt tca tcc ttt tac tgc ctg gaa tat ttc cct tct 3425 ala val gly arg ser ser phe tyr cys leu glu tyr phe pro ser cag atg ctg aga acg ggc aac aac ttt acc ttc agc tac acc ttt 3470 gln met leu arg thr gly asn asn phe thr phe ser tyr thr phe gag gaa gtg cct ttc cac agc agc tac gcg cac agc cag agc ctg 3515 glu glu val pro phe his ser ser tyr ala his ser gln ser leu gac cgg ctg atg aat cct ctc atc gac caa tac ctg tat tac ctg 3560 aac aga act caa aat cag tcc gga agt gcc caa aac aag gac ttg 3605 asn arg thr gln asn gln ser gly ser ala gln asn lys asp leu ctg ttt agc cgt ggg tct cca gct ggc atg tct gtt cag ccc aaa 3650 leu phe ser arg gly ser pro ala gly met ser val gln pro lys aac tgg cta cct gga ccc tgt tat cgg cag cag cgc gtt tct aaa 3695 asn trp leu pro gly pro cys tyr arg gln gln arg val ser lys aca aaa aca gac aac aac aac agc aat ttt acc tgg act ggt gct 3740 tca aaa tat aac ctc aat ggg cgt gaa tcc atc atc aac cct ggc 3785 ser lys tyr asn leu asn gly arg glu ser ile ile asn pro gly act gct atg gcc tca cac aaa gac gac gaa gac aag ttc ttt ccc 3830 thr ala met ala ser his lys asp asp glu asp lys phe phe pro atg agc ggt gtc atg att ttt gga aaa gag agc gcc gga gct tca 3875 aac act gca ttg gac aat gtc atg att aca gac gaa gag gaa att 3920 aaa gcc act aac cct gtg gcc acc gaa aga ttt ggg acc gtg gca 3965 lys ala thr asn pro val ala thr glu arg phe gly thr val ala gtc aat ttc cag agc agc agc aca gac cct gcg acc gga gat gtg 4010 val asn phe gln ser ser ser thr asp pro ala thr gly asp val cat gct atg gga gca tta cct ggc atg gtg tgg caa gat aga gac 4055 his ala met gly ala leu pro gly met val trp gln asp arg asp gtg tac ctg cag ggt ccc att tgg gcc aaa att cct cac aca gat 4100 val tyr leu gln gly pro ile trp ala lys ile pro his thr asp gga cac ttt cac ccg tct cct ctt atg ggc ggc ttt gga ctc aag 4145 aac ccg cct cct cag atc ctc atc aaa aac acg cct gtt cct gcg 4190 aat cct ccg gcg gag ttt tca gct aca aag ttt gct tca ttc atc 4235 acc caa tac tcc aca gga caa gtg agt gtg gaa att gaa tgg gag 4280 ctg cag aaa gaa aac agc aag cgc tgg aat ccc gaa gtg cag tac 4325 leu gln lys glu asn ser lys arg trp asn pro glu val gln tyr aca tcc aat tat gca aaa tct gcc aac gtt gat ttt act gtg gac 4370 aac aat gga ctt tat act gag cct cgc ccc att ggc acc cgt tac 4415 ctt acc cgt ccc ctg taattacgtg ttaatcaata aaccggttga ttcgtttcag 4470 met pro gly phe tyr glu ile val ile lys val pro ser asp leu asp glu his leu pro gly ile ser asp ser phe val ser trp val ala glu glu gln ala pro leu thr val ala glu lys leu gln arg asp phe leu val gln trp arg arg val ser lys ala pro glu ala leu phe phe val gln phe glu lys gly glu ser tyr phe his leu his ile leu val glu thr thr gly val lys ser met val leu gly arg phe leu ser gln ile arg asp lys leu val gln thr ile tyr arg gly ile glu pro thr leu thr gln pro glu leu gln trp ala trp thr asn met glu glu tyr ile ser ala cys leu asn leu ala glu arg lys arg leu val ala gln his pro asn ser asp ala pro val ile arg ser lys thr ser ala arg tyr met glu leu val gly trp leu val asp arg gly ile thr ser glu lys ile met ala leu thr lys ser ala pro asp tyr leu val gly pro ala pro pro ala asp ile lys thr asn arg ile tyr arg ile leu glu leu asn gly tyr glu pro ala tyr ala gly ser val phe leu gly trp ala gln lys arg phe gly lys arg asn thr ile trp leu phe gly pro ala phe tyr gly cys val asn trp thr asn glu asn phe pro phe asn asp cys val asp lys met val ile trp trp glu glu gly lys met thr ala thr thr phe glu his gln gln pro leu gln asp arg met phe lys phe glu leu thr arg arg leu glu his asp phe gly lys val thr lys gln glu val lys glu phe phe arg trp ala gln asp his val thr glu val ala his glu phe tyr val arg lys gly gly ala asn lys arg pro ala asp arg tyr gln asn lys cys ser arg his ala gly met leu gln met leu phe pro cys lys thr cys glu arg met asn gln asn phe asn ile cys phe thr his gly thr arg asp cys ser glu cys phe pro gly val ser glu ser gln pro val val arg lys arg thr tyr arg lys leu cys met ala ala asp gly tyr leu pro asp trp leu glu asp asn leu ser gln gln leu lys ala gly asp asn pro tyr leu arg tyr asn his ala asn leu gly arg ala val phe gln ala lys lys arg val leu glu pro ser gly asn trp his cys asp ser thr trp leu gly asp arg val ile tyr lys gln ile ser ser ala ser thr gly ala ser asn asp asn his his cys his phe ser pro arg asp trp gln arg leu ile asn asn asn tyr val leu gly ser ala his gln gly cys leu pro pro phe pro ala asp val phe met ile pro gln tyr gly tyr leu thr leu asn asn gly ser gln ala val gly arg ser ser phe tyr cys leu glu tyr phe pro glu glu val pro phe his ser ser tyr ala his ser gln ser leu asp arg gly ser pro ala gly met ser val gln pro lys asn trp leu pro gly pro cys tyr arg gln gln arg val ser lys thr lys thr asp asn gly arg glu ser ile ile asn pro gly thr ala met ala ser his lys lys glu ser ala gly ala ser asn thr ala leu asp asn val met ile thr gly asp val his ala met gly ala leu pro gly met val trp gln asp arg asp val tyr leu gln gly pro ile trp ala lys ile pro his lys glu asn ser lys arg trp asn pro glu val gln tyr thr ser asn tyr ala lys ser ala asn val asp phe thr val asp asn asn gly leu atg ccg ggc ttc tac gag atc gtg atc aag gtg ccg agc gac ctg gac 48 met pro gly phe tyr glu ile val ile lys val pro ser asp leu asp gag cac ctg ccg ggc att tct gac tcg ttt gtg agc tgg gtg gcc gag 96 glu his leu pro gly ile ser asp ser phe val ser trp val ala glu aag gaa tgg gag ctg ccc ccg gat tct gac atg gat ctg aat ctg att 144 gag cag gca ccc ctg acc gtg gcc gag aag ctg cag cgc gac ttc ctg 192 glu gln ala pro leu thr val ala glu lys leu gln arg asp phe leu gtc caa tgg cgc cgc gtg agt aag gcc ccg gag gcc ctc ttc ttt gtt 240 val gln trp arg arg val ser lys ala pro glu ala leu phe phe val cag ttc gag aag ggc gag tcc tac ttc cac ctc cat att ctg gtg gag 288 gln phe glu lys gly glu ser tyr phe his leu his ile leu val glu acc acg ggg gtc aaa tcc atg gtg ctg ggc cgc ttc ctg agt cag att 336 thr thr gly val lys ser met val leu gly arg phe leu ser gln ile agg gac aag ctg gtg cag acc atc tac cgc ggg atc gag ccg acc ctg 384 arg asp lys leu val gln thr ile tyr arg gly ile glu pro thr leu ccc aac tgg ttc gcg gtg acc aag acg cgt aat ggc gcc gga ggg ggg 432 aac aag gtg gtg gac gag tgc tac atc ccc aac tac ctc ctg ccc aag 480 act cag ccc gag ctg cag tgg gcg tgg act aac atg gag gag tat ata 528 thr gln pro glu leu gln trp ala trp thr asn met glu glu tyr ile agc gcc tgt ttg aac ctg gcc gag cgc aaa cgg ctc gtg gcg cag cac 576 ser ala cys leu asn leu ala glu arg lys arg leu val ala gln his ctg acc cac gtc agc cag acc cag gag cag aac aag gag aat ctg aac 624 ccc aat tct gac gcg cct gtc atc cgg tca aaa acc tcc gcg cgc tac 672 pro asn ser asp ala pro val ile arg ser lys thr ser ala arg tyr atg gag ctg gtc ggg tgg ctg gtg gac cgg ggc atc acc tcc gag aag 720 met glu leu val gly trp leu val asp arg gly ile thr ser glu lys cag tgg atc cag gag gac cag gcc tcg tac atc tcc ttc aac gcc gct 768 tcc aac tcg cgg tcc cag atc aag gcc gct ctg gac aat gcc ggc aag 816 atc atg gcg ctg acc aaa tcc gcg ccc gac tac ctg gta ggc ccc gct 864 ile met ala leu thr lys ser ala pro asp tyr leu val gly pro ala ccg ccc gcg gac att aaa acc aac cgc atc tac cgc atc ctg gag ctg 912 pro pro ala asp ile lys thr asn arg ile tyr arg ile leu glu leu aac ggc tac gaa cct gcc tac gcc ggc tcc gtc ttt ctc ggc tgg gcc 960 asn gly tyr glu pro ala tyr ala gly ser val phe leu gly trp ala cag aaa agg ttc ggg aag cgc aac acc atc tgg ctg ttt ggg ccg gcc 1008 gln lys arg phe gly lys arg asn thr ile trp leu phe gly pro ala acc acg ggc aag acc aac atc gcg gaa gcc atc gcc cac gcc gtg ccc 1056 ttc tac ggc tgc gtc aac tgg acc aat gag aac ttt ccc ttc aat gat 1104 phe tyr gly cys val asn trp thr asn glu asn phe pro phe asn asp tgc gtc gac aag atg gtg atc tgg tgg gag gag ggc aag atg acg gcc 1152 cys val asp lys met val ile trp trp glu glu gly lys met thr ala aag gtc gtg gag tcc gcc aag gcc att ctc ggc ggc agc aag gtg cgc 1200 gtg gac caa aag tgc aag tcg tcc gcc cag atc gac ccc acc ccc gtg 1248 atc gtc acc tcc aac acc aac atg tgc gcc gtg att gac ggg aac agc 1296 acc acc ttc gag cac cag cag ccg ttg cag gac cgg atg ttc aaa ttt 1344 thr thr phe glu his gln gln pro leu gln asp arg met phe lys phe gaa ctc acc cgc cgt ctg gag cat gac ttt ggc aag gtg aca aag cag 1392 glu leu thr arg arg leu glu his asp phe gly lys val thr lys gln gaa gtc aaa gag ttc ttc cgc tgg gcg cag gat cac gtg acc gag gtg 1440 glu val lys glu phe phe arg trp ala gln asp his val thr glu val gcg cat gag ttc tac gtc aga aag ggt gga gcc aac aaa aga ccc gcc 1488 ala his glu phe tyr val arg lys gly gly ala asn lys arg pro ala ccc gat gac gcg gat aaa agc gag ccc aag cgg gcc tgc ccc tca gtc 1536 gcg gat cca tcg acg tca gac gcg gaa gga gct ccg gtg gac ttt gcc 1584 gac agg tac caa aac aaa tgt tct cgt cac gcg ggc atg ctt cag atg 1632 asp arg tyr gln asn lys cys ser arg his ala gly met leu gln met ctg ttt ccc tgc aag aca tgc gag aga atg aat cag aat ttc aac att 1680 leu phe pro cys lys thr cys glu arg met asn gln asn phe asn ile tgc ttc acg cac ggg acg aga gac tgt tca gag tgc ttc ccc ggc gtg 1728 cys phe thr his gly thr arg asp cys ser glu cys phe pro gly val tca gaa tct caa ccg gtc gtc aga aag agg acg tat cgg aaa ctc tgt 1776 ser glu ser gln pro val val arg lys arg thr tyr arg lys leu cys gcc att cat cat ctg ctg ggg cgg gct ccc gag att gct tgc tcg gcc 1824 tgc gat ctg gtc aac gtg gac ctg gat gac tgt gtt tct gag caa taa 1872 met pro gly phe tyr glu ile val ile lys val pro ser asp leu asp glu his leu pro gly ile ser asp ser phe val ser trp val ala glu glu gln ala pro leu thr val ala glu lys leu gln arg asp phe leu val gln trp arg arg val ser lys ala pro glu ala leu phe phe val gln phe glu lys gly glu ser tyr phe his leu his ile leu val glu thr thr gly val lys ser met val leu gly arg phe leu ser gln ile arg asp lys leu val gln thr ile tyr arg gly ile glu pro thr leu thr gln pro glu leu gln trp ala trp thr asn met glu glu tyr ile ser ala cys leu asn leu ala glu arg lys arg leu val ala gln his pro asn ser asp ala pro val ile arg ser lys thr ser ala arg tyr met glu leu val gly trp leu val asp arg gly ile thr ser glu lys ile met ala leu thr lys ser ala pro asp tyr leu val gly pro ala pro pro ala asp ile lys thr asn arg ile tyr arg ile leu glu leu asn gly tyr glu pro ala tyr ala gly ser val phe leu gly trp ala gln lys arg phe gly lys arg asn thr ile trp leu phe gly pro ala phe tyr gly cys val asn trp thr asn glu asn phe pro phe asn asp cys val asp lys met val ile trp trp glu glu gly lys met thr ala thr thr phe glu his gln gln pro leu gln asp arg met phe lys phe glu leu thr arg arg leu glu his asp phe gly lys val thr lys gln glu val lys glu phe phe arg trp ala gln asp his val thr glu val ala his glu phe tyr val arg lys gly gly ala asn lys arg pro ala asp arg tyr gln asn lys cys ser arg his ala gly met leu gln met leu phe pro cys lys thr cys glu arg met asn gln asn phe asn ile cys phe thr his gly thr arg asp cys ser glu cys phe pro gly val ser glu ser gln pro val val arg lys arg thr tyr arg lys leu cys atg ccg ggc ttc tac gag atc gtg atc aag gtg ccg agc gac ctg gac 48 met pro gly phe tyr glu ile val ile lys val pro ser asp leu asp gag cac ctg ccg ggc att tct gac tcg ttt gtg agc tgg gtg gcc gag 96 glu his leu pro gly ile ser asp ser phe val ser trp val ala glu aag gaa tgg gag ctg ccc ccg gat tct gac atg gat ctg aat ctg att 144 gag cag gca ccc ctg acc gtg gcc gag aag ctg cag cgc gac ttc ctg 192 glu gln ala pro leu thr val ala glu lys leu gln arg asp phe leu gtc caa tgg cgc cgc gtg agt aag gcc ccg gag gcc ctc ttc ttt gtt 240 val gln trp arg arg val ser lys ala pro glu ala leu phe phe val cag ttc gag aag ggc gag tcc tac ttc cac ctc cat att ctg gtg gag 288 gln phe glu lys gly glu ser tyr phe his leu his ile leu val glu acc acg ggg gtc aaa tcc atg gtg ctg ggc cgc ttc ctg agt cag att 336 thr thr gly val lys ser met val leu gly arg phe leu ser gln ile agg gac aag ctg gtg cag acc atc tac cgc ggg atc gag ccg acc ctg 384 arg asp lys leu val gln thr ile tyr arg gly ile glu pro thr leu ccc aac tgg ttc gcg gtg acc aag acg cgt aat ggc gcc gga ggg ggg 432 aac aag gtg gtg gac gag tgc tac atc ccc aac tac ctc ctg ccc aag 480 act cag ccc gag ctg cag tgg gcg tgg act aac atg gag gag tat ata 528 thr gln pro glu leu gln trp ala trp thr asn met glu glu tyr ile agc gcc tgt ttg aac ctg gcc gag cgc aaa cgg ctc gtg gcg cag cac 576 ser ala cys leu asn leu ala glu arg lys arg leu val ala gln his ctg acc cac gtc agc cag acc cag gag cag aac aag gag aat ctg aac 624 ccc aat tct gac gcg cct gtc atc cgg tca aaa acc tcc gcg cgc tac 672 pro asn ser asp ala pro val ile arg ser lys thr ser ala arg tyr atg gag ctg gtc ggg tgg ctg gtg gac cgg ggc atc acc tcc gag aag 720 met glu leu val gly trp leu val asp arg gly ile thr ser glu lys cag tgg atc cag gag gac cag gcc tcg tac atc tcc ttc aac gcc gct 768 tcc aac tcg cgg tcc cag atc aag gcc gct ctg gac aat gcc ggc aag 816 atc atg gcg ctg acc aaa tcc gcg ccc gac tac ctg gta ggc ccc gct 864 ile met ala leu thr lys ser ala pro asp tyr leu val gly pro ala ccg ccc gcg gac att aaa acc aac cgc atc tac cgc atc ctg gag ctg 912 pro pro ala asp ile lys thr asn arg ile tyr arg ile leu glu leu aac ggc tac gaa cct gcc tac gcc ggc tcc gtc ttt ctc ggc tgg gcc 960 asn gly tyr glu pro ala tyr ala gly ser val phe leu gly trp ala cag aaa agg ttc ggg aag cgc aac acc atc tgg ctg ttt ggg ccg gcc 1008 gln lys arg phe gly lys arg asn thr ile trp leu phe gly pro ala acc acg ggc aag acc aac atc gcg gaa gcc atc gcc cac gcc gtg ccc 1056 ttc tac ggc tgc gtc aac tgg acc aat gag aac ttt ccc ttc aat gat 1104 phe tyr gly cys val asn trp thr asn glu asn phe pro phe asn asp tgc gtc gac aag atg gtg atc tgg tgg gag gag ggc aag atg acg gcc 1152 cys val asp lys met val ile trp trp glu glu gly lys met thr ala aag gtc gtg gag tcc gcc aag gcc att ctc ggc ggc agc aag gtg cgc 1200 gtg gac caa aag tgc aag tcg tcc gcc cag atc gac ccc acc ccc gtg 1248 atc gtc acc tcc aac acc aac atg tgc gcc gtg att gac ggg aac agc 1296 acc acc ttc gag cac cag cag ccg ttg cag gac cgg atg ttc aaa ttt 1344 thr thr phe glu his gln gln pro leu gln asp arg met phe lys phe gaa ctc acc cgc cgt ctg gag cat gac ttt ggc aag gtg aca aag cag 1392 glu leu thr arg arg leu glu his asp phe gly lys val thr lys gln gaa gtc aaa gag ttc ttc cgc tgg gcg cag gat cac gtg acc gag gtg 1440 glu val lys glu phe phe arg trp ala gln asp his val thr glu val gcg cat gag ttc tac gtc aga aag ggt gga gcc aac aaa aga ccc gcc 1488 ala his glu phe tyr val arg lys gly gly ala asn lys arg pro ala ccc gat gac gcg gat aaa agc gag ccc aag cgg gcc tgc ccc tca gtc 1536 gcg gat cca tcg acg tca gac gcg gaa gga gct ccg gtg gac ttt gcc 1584 gac agg tat ggc tgc cga tgg tta tct tcc aga ttg gct cga gga caa 1632 met pro gly phe tyr glu ile val ile lys val pro ser asp leu asp glu his leu pro gly ile ser asp ser phe val ser trp val ala glu glu gln ala pro leu thr val ala glu lys leu gln arg asp phe leu val gln trp arg arg val ser lys ala pro glu ala leu phe phe val gln phe glu lys gly glu ser tyr phe his leu his ile leu val glu thr thr gly val lys ser met val leu gly arg phe leu ser gln ile arg asp lys leu val gln thr ile tyr arg gly ile glu pro thr leu thr gln pro glu leu gln trp ala trp thr asn met glu glu tyr ile ser ala cys leu asn leu ala glu arg lys arg leu val ala gln his pro asn ser asp ala pro val ile arg ser lys thr ser ala arg tyr met glu leu val gly trp leu val asp arg gly ile thr ser glu lys ile met ala leu thr lys ser ala pro asp tyr leu val gly pro ala pro pro ala asp ile lys thr asn arg ile tyr arg ile leu glu leu asn gly tyr glu pro ala tyr ala gly ser val phe leu gly trp ala gln lys arg phe gly lys arg asn thr ile trp leu phe gly pro ala phe tyr gly cys val asn trp thr asn glu asn phe pro phe asn asp cys val asp lys met val ile trp trp glu glu gly lys met thr ala thr thr phe glu his gln gln pro leu gln asp arg met phe lys phe glu leu thr arg arg leu glu his asp phe gly lys val thr lys gln glu val lys glu phe phe arg trp ala gln asp his val thr glu val ala his glu phe tyr val arg lys gly gly ala asn lys arg pro ala atg gag ctg gtc ggg tgg ctg gtg gac cgg ggc atc acc tcc gag aag 48 met glu leu val gly trp leu val asp arg gly ile thr ser glu lys cag tgg atc cag gag gac cag gcc tcg tac atc tcc ttc aac gcc gct 96 tcc aac tcg cgg tcc cag atc aag gcc gct ctg gac aat gcc ggc aag 144 atc atg gcg ctg acc aaa tcc gcg ccc gac tac ctg gta ggc ccc gct 192 ile met ala leu thr lys ser ala pro asp tyr leu val gly pro ala ccg ccc gcg gac att aaa acc aac cgc atc tac cgc atc ctg gag ctg 240 pro pro ala asp ile lys thr asn arg ile tyr arg ile leu glu leu aac ggc tac gaa cct gcc tac gcc ggc tcc gtc ttt ctc ggc tgg gcc 288 asn gly tyr glu pro ala tyr ala gly ser val phe leu gly trp ala cag aaa agg ttc ggg aag cgc aac acc atc tgg ctg ttt ggg ccg gcc 336 gln lys arg phe gly lys arg asn thr ile trp leu phe gly pro ala acc acg ggc aag acc aac atc gcg gaa gcc atc gcc cac gcc gtg ccc 384 ttc tac ggc tgc gtc aac tgg acc aat gag aac ttt ccc ttc aat gat 432 phe tyr gly cys val asn trp thr asn glu asn phe pro phe asn asp tgc gtc gac aag atg gtg atc tgg tgg gag gag ggc aag atg acg gcc 480 cys val asp lys met val ile trp trp glu glu gly lys met thr ala aag gtc gtg gag tcc gcc aag gcc att ctc ggc ggc agc aag gtg cgc 528 gtg gac caa aag tgc aag tcg tcc gcc cag atc gac ccc acc ccc gtg 576 atc gtc acc tcc aac acc aac atg tgc gcc gtg att gac ggg aac agc 624 acc acc ttc gag cac cag cag ccg ttg cag gac cgg atg ttc aaa ttt 672 thr thr phe glu his gln gln pro leu gln asp arg met phe lys phe gaa ctc acc cgc cgt ctg gag cat gac ttt ggc aag gtg aca aag cag 720 glu leu thr arg arg leu glu his asp phe gly lys val thr lys gln gaa gtc aaa gag ttc ttc cgc tgg gcg cag gat cac gtg acc gag gtg 768 glu val lys glu phe phe arg trp ala gln asp his val thr glu val gcg cat gag ttc tac gtc aga aag ggt gga gcc aac aaa aga ccc gcc 816 ala his glu phe tyr val arg lys gly gly ala asn lys arg pro ala ccc gat gac gcg gat aaa agc gag ccc aag cgg gcc tgc ccc tca gtc 864 gcg gat cca tcg acg tca gac gcg gaa gga gct ccg gtg gac ttt gcc 912 gac agg tac caa aac aaa tgt tct cgt cac gcg ggc atg ctt cag atg 960 asp arg tyr gln asn lys cys ser arg his ala gly met leu gln met ctg ttt ccc tgc aag aca tgc gag aga atg aat cag aat ttc aac att 1008 leu phe pro cys lys thr cys glu arg met asn gln asn phe asn ile tgc ttc acg cac ggg acg aga gac tgt tca gag tgc ttc ccc ggc gtg 1056 cys phe thr his gly thr arg asp cys ser glu cys phe pro gly val tca gaa tct caa ccg gtc gtc aga aag agg acg tat cgg aaa ctc tgt 1104 ser glu ser gln pro val val arg lys arg thr tyr arg lys leu cys gcc att cat cat ctg ctg ggg cgg gct ccc gag att gct tgc tcg gcc 1152 tgc gat ctg gtc aac gtg gac ctg gat gac tgt gtt tct gag caa taa 1200 met glu leu val gly trp leu val asp arg gly ile thr ser glu lys ile met ala leu thr lys ser ala pro asp tyr leu val gly pro ala pro pro ala asp ile lys thr asn arg ile tyr arg ile leu glu leu asn gly tyr glu pro ala tyr ala gly ser val phe leu gly trp ala gln lys arg phe gly lys arg asn thr ile trp leu phe gly pro ala phe tyr gly cys val asn trp thr asn glu asn phe pro phe asn asp cys val asp lys met val ile trp trp glu glu gly lys met thr ala thr thr phe glu his gln gln pro leu gln asp arg met phe lys phe glu leu thr arg arg leu glu his asp phe gly lys val thr lys gln glu val lys glu phe phe arg trp ala gln asp his val thr glu val ala his glu phe tyr val arg lys gly gly ala asn lys arg pro ala asp arg tyr gln asn lys cys ser arg his ala gly met leu gln met leu phe pro cys lys thr cys glu arg met asn gln asn phe asn ile cys phe thr his gly thr arg asp cys ser glu cys phe pro gly val ser glu ser gln pro val val arg lys arg thr tyr arg lys leu cys atg gag ctg gtc ggg tgg ctg gtg gac cgg ggc atc acc tcc gag aag 48 met glu leu val gly trp leu val asp arg gly ile thr ser glu lys cag tgg atc cag gag gac cag gcc tcg tac atc tcc ttc aac gcc gct 96 tcc aac tcg cgg tcc cag atc aag gcc gct ctg gac aat gcc ggc aag 144 atc atg gcg ctg acc aaa tcc gcg ccc gac tac ctg gta ggc ccc gct 192 ile met ala leu thr lys ser ala pro asp tyr leu val gly pro ala ccg ccc gcg gac att aaa acc aac cgc atc tac cgc atc ctg gag ctg 240 pro pro ala asp ile lys thr asn arg ile tyr arg ile leu glu leu aac ggc tac gaa cct gcc tac gcc ggc tcc gtc ttt ctc ggc tgg gcc 288 asn gly tyr glu pro ala tyr ala gly ser val phe leu gly trp ala cag aaa agg ttc ggg aag cgc aac acc atc tgg ctg ttt ggg ccg gcc 336 gln lys arg phe gly lys arg asn thr ile trp leu phe gly pro ala acc acg ggc aag acc aac atc gcg gaa gcc atc gcc cac gcc gtg ccc 384 ttc tac ggc tgc gtc aac tgg acc aat gag aac ttt ccc ttc aat gat 432 phe tyr gly cys val asn trp thr asn glu asn phe pro phe asn asp tgc gtc gac aag atg gtg atc tgg tgg gag gag ggc aag atg acg gcc 480 cys val asp lys met val ile trp trp glu glu gly lys met thr ala aag gtc gtg gag tcc gcc aag gcc att ctc ggc ggc agc aag gtg cgc 528 gtg gac caa aag tgc aag tcg tcc gcc cag atc gac ccc acc ccc gtg 576 atc gtc acc tcc aac acc aac atg tgc gcc gtg att gac ggg aac agc 624 acc acc ttc gag cac cag cag ccg ttg cag gac cgg atg ttc aaa ttt 672 thr thr phe glu his gln gln pro leu gln asp arg met phe lys phe gaa ctc acc cgc cgt ctg gag cat gac ttt ggc aag gtg aca aag cag 720 glu leu thr arg arg leu glu his asp phe gly lys val thr lys gln gaa gtc aaa gag ttc ttc cgc tgg gcg cag gat cac gtg acc gag gtg 768 glu val lys glu phe phe arg trp ala gln asp his val thr glu val gcg cat gag ttc tac gtc aga aag ggt gga gcc aac aaa aga ccc gcc 816 ala his glu phe tyr val arg lys gly gly ala asn lys arg pro ala ccc gat gac gcg gat aaa agc gag ccc aag cgg gcc tgc ccc tca gtc 864 gcg gat cca tcg acg tca gac gcg gaa gga gct ccg gtg gac ttt gcc 912 gac agg tat ggc tgc cga tgg tta tct tcc aga ttg gct cga gga caa 960 met glu leu val gly trp leu val asp arg gly ile thr ser glu lys ile met ala leu thr lys ser ala pro asp tyr leu val gly pro ala pro pro ala asp ile lys thr asn arg ile tyr arg ile leu glu leu asn gly tyr glu pro ala tyr ala gly ser val phe leu gly trp ala gln lys arg phe gly lys arg asn thr ile trp leu phe gly pro ala phe tyr gly cys val asn trp thr asn glu asn phe pro phe asn asp cys val asp lys met val ile trp trp glu glu gly lys met thr ala thr thr phe glu his gln gln pro leu gln asp arg met phe lys phe glu leu thr arg arg leu glu his asp phe gly lys val thr lys gln glu val lys glu phe phe arg trp ala gln asp his val thr glu val ala his glu phe tyr val arg lys gly gly ala asn lys arg pro ala atg gct gcc gat ggt tat ctt cca gat tgg ctc gag gac aac ctc tct 48 met ala ala asp gly tyr leu pro asp trp leu glu asp asn leu ser gag ggc att cgc gag tgg tgg gac ttg aaa cct gga gcc ccg aag ccc 96 aaa gcc aac cag caa aag cag gac gac ggc cgg ggt ctg gtg ctt cct 144 ggc tac aag tac ctc gga ccc ttc aac gga ctc gac aag ggg gag ccc 192 gtc aac gcg gcg gac gca gcg gcc ctc gag cac gac aag gcc tac gac 240 cag cag ctc aaa gcg ggt gac aat ccg tac ctg cgg tat aac cac gcc 288 gln gln leu lys ala gly asp asn pro tyr leu arg tyr asn his ala gac gcc gag ttt cag gag cgt ctg caa gaa gat acg tct ttt ggg ggc 336 aac ctc ggg cga gca gtc ttc 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pro arg asp trp gln arg leu ile asn asn asn tgg gga ttc cgg ccc aag aga ctc aac ttc aaa ctc ttc aac atc caa 960 gtc aag gag gtc acg acg aat gat ggc gtc aca acc atc gct aat aac 1008 ctt acc agc acg gtt caa gtc ttc tcg gac tcg gag tac cag ctt ccg 1056 tac gtc ctc ggc tct gcg cac cag ggc tgc ctc cct ccg ttc ccg gcg 1104 tyr val leu gly ser ala his gln gly cys leu pro pro phe pro ala gac gtg ttc atg att ccg caa tac ggc tac ctg acg ctc aac aat ggc 1152 asp val phe met ile pro gln tyr gly tyr leu thr leu asn asn gly agc caa gcc gtg gga cgt tca tcc ttt tac tgc ctg gaa tat ttc cct 1200 ser gln ala val gly arg ser ser phe tyr cys leu glu tyr phe pro tct cag atg ctg aga acg ggc aac aac ttt acc ttc agc tac acc ttt 1248 gag gaa gtg cct ttc cac agc agc tac gcg cac agc cag agc ctg gac 1296 glu glu val pro phe his ser ser tyr ala his ser gln ser leu asp cgg ctg atg aat cct ctc atc gac caa tac ctg tat tac ctg aac aga 1344 act caa aat cag tcc gga agt gcc caa aac aag gac ttg ctg ttt agc 1392 cgt ggg tct 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asp arg asp val tyr leu gln gly pro ile trp ala lys ile pro his aca gat gga cac ttt cac ccg tct cct ctt atg ggc ggc ttt gga ctc 1920 aag aac ccg cct cct cag atc ctc atc aaa aac acg cct gtt cct gcg 1968 aat cct ccg gcg gag ttt tca gct aca aag ttt gct tca ttc atc acc 2016 caa tac tcc aca gga caa gtg agt gtg gaa att gaa tgg gag ctg cag 2064 aaa gaa aac agc aag cgc tgg aat ccc gaa gtg cag tac aca tcc aat 2112 lys glu asn ser lys arg trp asn pro glu val gln tyr thr ser asn tat gca aaa tct gcc aac gtt gat ttt act gtg gac aac aat gga ctt 2160 tyr ala lys ser ala asn val asp phe thr val asp asn asn gly leu tat act gag cct cgc ccc att ggc acc cgt tac ctt acc cgt ccc ctg 2208 met ala ala asp gly tyr leu pro asp trp leu glu asp asn leu ser gln gln leu lys ala gly asp asn pro tyr leu arg tyr asn his ala asn leu gly arg ala val phe gln ala lys lys arg val leu glu pro ser gly asn trp his cys asp ser thr trp leu gly asp arg val ile tyr lys gln ile ser ser ala ser thr gly ala ser asn asp asn his his 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