- 1 Vectors with modified initiation codon for the translation of AAV-Rep78 useful for production of AAV in insect cells The present application is a divisional application of Australian Application No. 2013254897, which is incorporated in its entirety herein by reference. 5 Field of the invention The present invention relates to the production of adeno-associated virus in insect cells and to adeno-associated virus with improvements in expression and stability of the viral rep proteins that increase the productivity of adeno-associated viral vectors in insect cells. 10 Background of the invention Adeno-associated virus (AAV) may be considered as one of the most promising viral vectors for human gene therapy. AAV has the ability to efficiently infect dividing as well as non-dividing human cells, the AAV viral genome integrates into a single chromosomal site in the host cell's genome, and most importantly, even though AAV is 15 present in many humans it has never been associated with any disease. In view of these advantages, recombinant adeno-associated virus (rAAV) is being evaluated in gene therapy clinical trials for hemophilia B, malignant melanoma, cystic fibrosis, and other diseases. Host cells that sustain AAV replication in vitro are all derived from mammalian 20 cell types. Therefore, rAAV for use in gene therapy has thus far mainly been produced on mammalian cell lines such as e.g. 293 cells, COS cells, HeLa cells, KB cells, and other mammalian cell lines (see e.g. US 6,156,303, US 5,387,484, US 5,741,683, US 5,691,176, US 5,688,676, US 20020081721, WO 00/47757, WO 00/24916, and WO 96/17947). rAAV vectors are typically produced in such mammalian cell culture systems 25 by providing DNA plasmids that contain the therapeutic gene flanked by the origin of AAV replication (inverted terminal repeats or ITRs), genes for AAV replication proteins Rep78, Rep68, Rep52, and Rep40, and genes for virion or structural proteins VP1, VP2, and VP3. In addition, a plasmid containing early genes from adenovirus (E2A, E40RF6, VARNA) is provided to enhance the expression of the AAV genes and improve vector 30 yield (see e.g. Grimm et al, 1998, Hum. Gene Ther. 9: 2745-2760). However, in most of these mammalian cell culture systems, the number of AAV particles generated per cell is - 1 a in the order of 104 particles (reviewed in Clark, 2002, Kidney Int. 61_(Suppl. 1): 9-15). For a clinical study, more than 1015 particles of rAAV may be required. To Iroduce this number of rAAV particles, transfection ard culture with approxinately 10" cuhured huan 293 cells, the equivalent of 5,000 175-c' flasks of cells, would be required, which means transfecting up to 10 29)3 cell. Therefore large scade production of rA.AV using 5 mammalian cell culture systems to obtain material for clinical trials has already proven to be problenatic, production at commercnl scale may not even be feasible. F:urthermre there is always the risk, that a vector fkmr clinical use that is produced in a mammal ian cell culture will be contaminated with undesirable, perhaps pathogenic, material present in the marumahan host cell, I T0 overcome these problems of mammalian productions systems, recently, an AAV production system has been developed using sec cells (Urabe et a, 2002, Hum. Gene 'her. 13 1935-1943; US 20030148506 and 8 2004019789), For production of AAV in insect cells some nodhicaions were necessary in order to achieve the correct stoichiometry of the three AAV capsid proteins (VP1l VP2 and 15 VP3) which relies on a combinaion of alterate usage of two splice acceptor sites and the suboptimal utilization of an AX iniation codon for VP2 that is not accurately reprodcod by insect cells, To micn tHe correct stic-try f the proteins in insect cells Urabe et al (20supr) use a constuct that is transcribed info a sngle polyci stronic messenger that is able to press all three VP proteins without requiring 20 splicing and wherein the most upstream initiator codon is replaced by the suboptimal initiator codon AG. In corending application (PIT/N2005/050018) the present inventors have further improved the infectivity of baculovirus-produced rAAV vectors based production by fuheopt of the stoichiometry of AAV cansi in insect cells, 25 For expression of the AAV Rep proteins in the AAV insect cell expression sy stem as initially-, developed by lrabe et A (2,sp a recombinant baculovims construct is used hat harbors two independent Rep expression units (one for Rep7S and one for Rep2), each under the control of a separate insect eel promoter the AIE and Poll promoters, respectely In this system, the AEl promoter, a nuch weaker 30 promoter tharn the Pol promoter, was chosen for driving Rep78 expression since it is known that in mamnmalian cells a less abundant expression of Rep78 as compared to RepS2 favours high vector yields (Li et al 1997, J Virol .71; 5236-43; Grimn et al. 1998, supna).
3 More recendy however Kohlbrenner et al. (2005. Mol. Their. : 121725) reported that the bactlo virus construct tbr expression of the two Rep protein, as used by Urabe et a, suffers from an inherent instability. By splitting the palindromic orientation of the two Rep genies ini rabe's orinal vector and dgng o separate 5 baculovirus vectors for expressing RepS2 and Rep Kolbrenner et a M200, supr) increased the passaging stabilty of the vector However, ds pite the consistent expression of IRep and Rep$2 from the two independent bacidovirusRep constructs in insect cells over at least 5 passages. rAAV vector yield is 5 to 1fAld lower as compared to the original baculovirus-Rep conshatr designed by Urabe et al, (2002, 10 supra) There is thus still a need to overcome the above serious hruations of large scab' commerciall) production of AAV vectors in insect cells. Thus it is an object of the present invention tP provide for means and methods that allow for stable and high yield (large seale production of AAV ectorsin insect cells. 15 esrpion of r thenntn Definitions As used herein th t operaly inke refers to alinkage of polynuckotide (or polypeptide) elements in a funetonal relationship A nucleic acid is "opeably 20 line" when it is paced into a funcional relationship with another ucVe acid sequence, For instance a iras i relatory seuence s operabl ikd to a codng sequence if iacts the of the coding sequence, Operably linked means that the NA sequences being linked are typical contiguous and, where necessary to joinl two proin encoding regions, contiguous and in reading'frane 25 ' xpression control sequence" reers to a nucleic acid sequence that regulates the expressnof a nuclotide sque o which it is operably inked An expression control sequence is 'operably linked" to a nucleotide sequence when the expression control sequence controls and regulates the transcription an/or the translation of the nucleotide sequence. Thus, an expression control sequence cainclude promoters, 30 enhances, internal ribosone entry sites ( RESt I-ranscription terinators, a start codon in front of a protein-encoding gene, splicing signal for introns, and stop codons. The term "expression control sequence" is intended to include, at a mininrn, a sequence whose presence are designed to influence expressionand can also include additional 4 advantageous components, For example leader seqences and fusion partner sequences are expression control sequences. The term can also inclde the design of the nucleic acid sequence such that undesirable, potential initiation codons in and out of frame, are removed trom the sequence It Can also inl uIde the design of the nucleic acid sequence 5 such that undesirable potential spIce sites are removed i includes sequences or polyadenylation sequences (pA Ich direct the addition of a polyA tail, i e a string of adenine residues af the Mend of a nRNA, sequences referred to as polyA sequences It also can be designed to enhance mRNA stability; Expression control sequences which affect the transcription and transit aion stabilty, e.g, promoters, as well as 10 sequenes which effect the translation. e~g Kozak sequences are known in insect cells. Expression control sequences can be of such nature as to modulate the nucleotide sequence to which it is operably linked such that lower expression levels or higher express-ion levels are achieved. As used herein, the term "promoter or"transcription regulatory sequence"refers 13 to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction mf transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences including but not limited to transcription 20 factor binding Sites, repressor and activator protein binding sites, and any other sequences ofncdeotides known to one of skill in the art to act direct or indirectly to regulate the amount of trscription from the promoter. A "constitutive" promoter is a promoter that is active in most tissues under most physiological and declopmrnal conditions, An induciblee' promoter is a promoter that is physiologically or 25 developmentally regulated, e.g. by the application of a chemical induce A "tissue specific" promoter is only active in specific types of tissues or cells The terms "substantially identical" substantiall identity" or "essentially similar" or "essential similarity" means that two peptide or two nucteotide sequences, when optuimally aligned, such as by the programs GAP or BESTFIT- using defeat parameters. 30 share at least a. certain percentage of sequence identity as defined elsewhere herein. GAlP uses the Needleman and Wunsch global alignment algorithm to align two sequecesover their entire lenIgth, mximizing the .number of matches and mirizes the number of gaps, Generally, the GAP default parameters are used, with a gap creation penalty - 50 (nucleotides) /8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins), 1or nucleotides the default scoring mairix used is nwsgapdna and for proteins the default scoring matrix is 3Blosum62 {Henikoff & Henikoff; 1992. PNAS 89, 915~91 It is clear than when RNA sequences are said W 5be essential similar or have a certain degree of sequence identity with iDNA sequences. thyiine (T} in the DNA sekence is considered equal to tracil a) in the RNA sequence Seoquence alignments and scores for percnae squence identt' may be deternned using conipater programs, such as the 000 Wisconsin Package Version 10.3. available from Accelrys lIn, 9685 Scranxton Road, San Diego. CA 92121,752 10 USA or the open-sourcesoftwvare Emboss for Windowscurrent version 2,I07 Alteratively percent sir anty or idntiy a be deteranined by searching against databases such as FASTA, BLASTet. Nucleotide sequences encoding parvoviral. Rep proteins f the invention. may also be defined by tie r capability to hybidise with the nueleotide sequence of SEQ 1D 15 NO10, respectively, under moderate, or preferably under stringent hybridisation condtons. Stringent hybridisaton conditions are herein defined as conditions that allow a nucleic acid sequence of at least about 25, preferably about 50 nucleotides, 75 or 100 and most preferably of about 200 01 nore nuotdes to hybridise at a temperatue of about 65T in a. solution conrprising about I M salt, preferably 6 x SSC 20 or any other soluion having a comparable ionic strength, and washing. at 6500 in a solution comrising about (3 M salt or Ues, preferably 0.2 x SSC or any other solution having a. comparable ionic strength Preferably the hybridisation is performed overmht, ie. at least for 10 hours and preferably washing is yperfonned for at least one hour with at least wo changes of the washing solution.hese conditions wi l usually 25 allow the specitc hybr idisation of sequences having about 90% or more sequence identity Moderate conditions are herein defined as conditions that allow a nucleic acid sequences of at least 50 nucleotides preferably of about 200 or more nucleotides, to hybridseat a. teRperatre of about 45C in a solu tion Iomprisngabou ' M s't 30 preferably 6 x SSC or any other sdution having a covnpanible ionic strength, and washing at room emperature in a solution comprising about 1 M salt, preferably 6 N SSC or any other solution having a comparable ionic strength, Preferably. the hybridisa ion is perorNmed overnight, ie. at least for 1 hIours. and preferably washing 6 is perfortmned for at least one hour witha least two changes of the washing solution. These conditions wtil usually alkw the specific hybridisation of sequences having up to 50% seance identity. The person skilled in the art will be able to modify these hybridsation conditions in order to specifically kleni6 sequences varying in identity between 50% and 90% 'Det-ai leddeetinothinnin The present invention relates the use of animal parvoviruses, in particular dependoviruses such as infectious human or simiai AAV. and the components thereof 10 (eg. an animal parvovims genone) for use as vectors for introduction and/or expession of nuclic acids in mamnialian cells. In particular. the invention relates to improvements in productivity of such parvoviral vectors when produced in insect cells, Vimses of the Parvoviridae family are small DNA animal viruses h T family Parvoviridac may be divided between two subtlunilies; the Parovirinae, wi ch infect 15 vertebrates and the Densovirinae, which infect insects Members of the subfumly Parvovirinae are herein referred to as the parvoviruses and include the genus Dependovirtis. As may be deduced from the name of their genus, members of the Depenovirs are unique in that they usually require cornfetion with a helper virus such as adenovir us or herpes vius fcr productive infection in cel culturelhe genus 20 Dependovirus includes AAV which nonml t y inf ets humans (eg, srotypes I 2 3A, 3B, 4, 5. and 6) or primates (eg. serotypes 1. and 4), and related virses that infect other wammblooded animals (eg, bovine, canine, equine, and ovine adeno-associated viruses). Further itormation on parvoviwmses and other mnenibers of the Parvoviidac is described in Kenneth , Bems "Parvovirida; The Viruses and Their Repication 25 Chapter 69 in Fields Wrology 3d Ed. 1996) For convenience the present invention is further exemplified and described herein by reference to AAR It is however understood that the invention is not limited to AAV but may equally be applied to other parvoviruses The genomic organization of all known AAV serotypes is very similar, The 30 genome of AAV is a near, single-stranded DNA molecule that is less than about 5,000 nucleotides (mt in length. inverted terminal repeats (1E1s) thark the unique coding nucleotide sequences for the nonstructual replication (Rep) proteins and the stmtunal (VP) proteins, The V1 proteins (YPE, 2 and -3) form the capsid.he terminal 145 nI are self complementary and are organized so that an energetically stable intramoleeuiar duplex iorm.ning a ha hairphirnay be rmned, These h.a.. pin structures ituncton as an origin for viral DNA replication, serving as primers for the cellular DNA polymnerase complex Folowing wtAAV infection in narrnalian cells the Rep genrs 5 (i, Rep78 and Rep52) are expressed fron the P5 promoter and the P19 promotor, respectvely and both Rep proteins have a function in the repheanon of the xira.l eno me A splicing event in the Rep ORF results in the exOression of actually four Rep proteins (i.e Rep78, Rep65s, Rep52 and Rep40) Howevr it has been shown. that the unspced nRNA encoding Rep78 and Rep52 proteins, in mrnian cells are 10 sufficicnt for AAV vector production. Also in insect eells the Rep'78 and RepS2 proteins suffic, for ANV vector production. A "recombinant parvoviral or AAY vector" (or "'rAAV vector") herein refers to a vector comparing one or umore polymcleotide sequences of interest, genes of interest or "transguenes" that are flanked by parvoviral or AAV inverted terminal repeat 15 sequences (ITRs) Such rAAV vectors can be replcated and packaged into infectious lirai particles when present in an insect host cell that is expressing AAV rep and cap gene products (iie. AAV Rep and Cap Proteins) when an rAAY vector is incorporated into a larger nudeic aid construct(g. in a chromosome or in. another vector such as a plasmid or baculovirs used for clonina r ansfecion) then the rAAV vector is 20 tyilcaliy refer-red toas a "pro--vectol which c-an be rescued byepiato anld encapsidaticn in the presence of AAN packaging funcnons and necessary helper functions. In a first aspect the inventior relates to a nuceotide sequence comprising an open reading frame comprising xuleotide sequences encoding animal parvovirses Rep 25 proteins wherein the initiation codon for translation oyf the parvoviral Rep7S protein is a suboptirmal initiation codon. The suboptinial initiation codon preferably is an initiation codoio that effects partil exon. skipping, Partial exci skipping is herein understood to mean that at least part of the ribosons do riot initiate translation at the suboptimai initiation odon of the Rep78 protein but at an inniation eodorn further 30 downstram, whereby preferahiy bthe initiation codon further downstream is the initiation codon oF the Rep52 protein. The .suboptima1 itiation codon preferably effects partial exon skipping upon expression of the nucleotie sequence in annsect cell. Prerably, the suboptimat initiation codon effects partial exon skipping in an 8 insect cell so as to produce in the insect cl a nolar ratio of Rep78 to RepS2 in the range of 1: 10 to 10:1, 1: 5 to 5:1, or 1:3 to 3:1, prefeably at about 20 40 hours post infection, more preferably at about 30- 40 hours post nation using a baculovirus expression. The molar ration of the Rep87 and Rep52 may be detemtned by neans of 5 Wester blottingz as descnbed in Example 1.13, preferably using a monoclonal antibody that recognizes a common epitope of both Rep78 arnd Rep52, or using the antibody described in Eample 1 -3. The term "uboptinal initiation codon" herein not only refers to the tri nucleotide intitiat ion codon itself but alo to its context, Thus, a suboptiantiation 10 codon may consist of an "optimal" ATG codon in a suboptimal context. eg a. non Kozak cantexti However, more preferred are subopanal initiation codons wherein the tri-nucleotide intimation codon itself is suboptinal, ie is not A t.G Suboptinal is herein understood to mean. that the codon is less efficient in the inititiation of translation in an otherwise identical context as compared to the norma ATG codon. 15 Preferably, the eficiency of suboptimal codon is less than 90, 80. 60 40 or 20% of the efficiency of the normal AGl codon in an otherwiseidentical context Methods for comparing the relative efficiency of inititiation of translation are known per so to the skilled person Preferred suboptimal initiation codons may be selected from AGO, TTG CT. and GTG. More preferred is ACG 20 A nuodeotide sequence encoding animal parvoviruses Rep proteins, is herein understood as a nucieoide sequence encoding the non-structural Rep proteins that are required and sffielent for parvoviral vector production in insect cells such the Rep78 and Rep$2 proteins, The animal parvovirus nucleofide sequence prefrably is trom a dependovirus, more preferably froMn a human or simian adeno-associatW virus (AAV) 25 and most pretbrably from an AAV which normay infects humans (es. serotypes 1. 2 3A, 31B, I5 and 6) or primates (eIg s s and 4). An exaniple of a nucleotide seqtience encoding animal parvovirses Rep proteins is given in SEQ ID No,10, which depicts a part of the AAV serotype-2 sequence genome encoding the Rep proteins The Rep78 coding sequence comprises nucleotides 11 1876 and the Rep52 coding 30 sequence comprises nucleotides 683 - 1876, It is understood that the exact molecular weights of the Rep78 and Rep52 proteins as well as the exact positions of the translation initiation codons may differ between different parvoviruses, However the skilled person will know how to identify the corresponding position in nucleotide 9 sequence from other parvoviuses than AAW2 A nucleotide sequence encoding animal pa voviruses Rep proteinsrmay thus also be defined as a nucleotide sequence a) that encodes a polypeptide comprising an amino acid sequence that has at least 50 60, 70, 8088 89, 90, 95, 97, 9-8 or 99% sequence identty with the amino 5 aci'd', secquence of EQ ID N 11 b. that has at least 50, 60; 70. 80, 81, 82, 85, 90, 95, 97. 98. or 99% sequence identity with the nuceotide sequence ofpositions 11 - 1876 of SEQ D NO, 10; c) the compIemenary strand of which hybridises to a nucleic acid molecule sequence of (a) or (b) 10 d) nucleotide sequences the sequence of which differs fron the sequence of a nucleic acid n aecule of (c) due to the dgenery of the netic code. Preferably, the mleotide sequence encodes animal parvoviruses Rep proteins that are required and sufficient for parvovira vector production in insect cells. A further preferred nucleotide sequence of the invention comprises an expression 5 control sequence that comprising a nine nucleotide sequence of SEQ. ID NO: 7 or a n 'leotide sequence substantially homologous to SEQ. ID NO: 7. upstream of the initiation codon of the nucleoide sequence encoding the parvoviral Rep?8 protein, A sequence with substantial identity to the nucleotide sequence of SEQII NO: 7 and that wi help increase expression of the pavovmt Rep78 protein is eg a sequence 20 which has at least 60%, 70% 80% or 90% identity to the nine nucleotide sequence of SEQ ID NO; 7 Elimination of possible false translation initiation sites ini the Rep protein coding sequences, other than the Rep78 and Rp52 translation initiation sites, of other parvoviruses will be well understood by an arisan of skill in the art, as will be the 25 elimination of putative sphee sites tha a be recognised in insect cels. The various modifications of the wii-type parvovia sequences for proper expression rn insect cells is achieved by application of weilknown genetic engineering techniques such. as described e,g. in Sambrook and Russell (200 "Moecniar Cloning; A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory Cold Spring Harbor Laboratory 30 Press, New York. Various further modifcations of Rep protein coding regions are known to the skilled artisan which could increase yield of Rep protein. These modifications are within the scope of the present invention.
In a further aspect the invention relates to a nucleic acid construct comprising a nucleotide sequrice encodirng parvoviral Rep proteins as defined above, Preferably. in the construct. e nucleotide sequence encoding the parvoviral Rep proteins is operably hlked to expression control sequences fo expression in an insect celL These 5 expression control sequences will at least include a promoter that is active in insect cells. Techniques known to one skilled in the art expressing foreign genes in insect host cells can be used to practice the inventon. Methodology for molecular engnwering and expression of polypeptides in insect eel is described, for example, in Smnmners and Smith. 1986, A Manual of Methods fBr Baculovirus Vectors aind Insect Culture 10 Procedures, Texas Agricultural Experimental Station Bul N. 55 College Station, Tex,; Ickow 199. In Prokop et aL, Cloning and Expression of ileterologous Genes in Insect Cells with Baculovirus Vectors Recombinant DNA Technology and Appicatins 9-152; King, L A. and R. D Possee, 1992,The baculovirus expression system, Chapma and H1all United Kingdom; (Reily. DT I LA K. Miller, V A. 15 ILuckow, 1992. Baculovirus Expression Vectors; A Laborat ory Manual, New York; W. ,L- Freeman and Richardson, CD., 1995. Baculovirus Expression Protocols, Methods in Molecular Biology, volume 39; US 4,74511; US2003148506; nd WO 03/074714, A particularly suitable promoter for transcription of the nuc'eoide sequence of the invention encoding of the parvoviral Rep proteins is e.g. the polyhedron promoter, 20 However; other promoters hat are active in insect cells are known in the art, o g the p10 p3 1El I or Al . promoters and hrther promotes described in the above references Prefirab'v the nucleic acid construct for expression of the parvuviral Rep proteins in insect cells is an insect cell-compatible vector. An "insect celc.ompatible vector" or 25 "vector" is understood to a nucleic acid inolecule capable of productive transtornation or tnsfection of an insect or insect cell. Exemplary biological vectors ineliud pasmids linear nucleic acid molecules, and recombinant viruses- Any vector can he employed as long as it is inct el.-cormpatible. The vector may ntegrate into the insect cells genorne but the presence of the vector in the insect cell need not be 30 permanent and transient episomal vectors are also included, The vectors can be introduced by any means known, for example by chemical treatment of the cells, electroporation or infection. in a prefrred embodiment, the vector is a baculovrusa viral vector or a plasmid. In a more preferred embodiment, the vector is a baculovirus.
i.e. the construct is a baculoviral Vector. Baculoviral vectors and methods for their use are described in the above cited refer s on molecular engineering ofinsect cells, ha another aspect the invention relates to an insect cell t omprises no more than one type oT nucleotide sequencecomrisinpg a i open reading frame encoding 5 a parvoviral Rep protein Preferably the single open reading frame encodes one or more of the parvoviral Rep proteins, more prelerably the open reading frame encodes all of the parvoviral Rep proteins, most preferably the open reading frame encodes the lL~ length Rep 78 protein ton which preierably at least both Rep 52 and Rep 78 proteins may be expressed in the insect cel It is understood herein thai the insect cell may 10 comprise more than one copy of the single type of nueleotide sequence, eg. in a nmulticopy episode ector, but that hese are multiple copies of essentially one and the same nucleic acid molecule or at least nucleic acid molecules that encode one and the same Rep anino acid sequence e g nucleic acid moleules that only differ between each other due to the degeneracy of the genetic code. 'he presence of only a singe tpe 1$ of nucleic acid molecule encoding the parvo viral Rep proteins avoids recombination between homologous sequences as may be present in different types of vectors conprising Rep sequences. which may give rise to deective Rep expression constructs that affect (stability of) parvoviral production levels in insect cells.Preferably, in the insect cell, the nucleotide sequence comprising the singieopen reading frtme encoding 20 one or more parvoviral Rep proteins is part of a nucleic acid construt wherein the nucleotide sequence is operabiy inked to expresson control sequences for expression in an insect cell. A rather preferred insect cell comprises as a "first" nueleotide sentence a nueleotide sequence as defned above encoding parvoviral Rep protein preferably a coding sequence with a suboptimal iitiation codon as defined above, or a 25 nucleic acid construct as defined above orthe insect cell comprises as a "rsC" nucleic acid construct a nucleic acid construct as defined above conmprising such ntucleotide sequences. Any insect cell which allows fiar replication of, a recombinant pareoviral(rAAV) vector and which can be maintained in culture can be used in accordance withe 30 present invention, For example, the cell line used can be t&orn Spodoptera trgiperda drosophila cell lines, or mosquito cell lines. e g., Aedes albopictus derived cell lines Preferred insect cells or cell lines are cells fror the insect species which are susceptible to baculovirus infection, including e g 8e301, SeIZD2l09 SeUCRI Sf9, 3(900+ 12 SI, BTJTN5B 1 -4, M ., Tn368 HzAnE Ha2302, Hz2.E5. High Five (Invitrogen, CA, LISA) and expresSF S ( ,1 603,526; Prein Sciences Corp,, CT SA) A preferred insect cell according to the invention, in addition to the above described "first" nuleotide sequence ora nucleic acid construct, furher comprises: 5 a) a second nucleotide sequence cornprNsing at least one parvoviral inverted terminal repeat (TR) nucleotide sequence; and, b) a third nucleoide sequence comprising parvoviral Cap protein coding sequences operably linked to expression. control sequences for expression in an insect cell. 10 In the context of the invention "at least one parvoviral ITR nucleotide sequience" is understood to mean a paindromnic sequence, comtprising mostly complementary symmetrically arranged sequences also refered to as "A. B*," and "C" regions The ITR functions as an origin of replication, a site having a "cis" role inreplication ie being a, recognition site for ranks acting replication proteins such as eg. Rep 78 (or 15 Rep68 which recognize the palindrome and specific sequences internal to the palindrome. One exception to the symmetry of the [TR sequence is the "D" region of the ITMR. It is unique (not having a comnpement within one iTR.). Nicking of singer stranded DNA occurs at the junction between the A and D regions, It is the region where new DNA synthesis initiates The D region normatlv sits to one side of the 20 plindome and provides directionality to the nucleic acid replication step. An parvovirus replicating in a tnuammalian cell typicallyhas two ITR sequences i iS, howev, possible to enginer an ITR so tha binding sites are on both stlands of the A regions and D regions are located symmetricaly one on each side of the palindrome. On a doublestranded ircular DNA template (e a plasmidtu the R or Rep68S 25 assisted nucleic acid replication then proceeds in both directions and a single ITR sufnces for parvovraal replication of a circular vector. tius, one NTR nucleotide sequence can be used in the context of the present invention. Preferably, however, two or another even number of regular l FRs are used. Most preirably, two l1"R sequences are used. A preferred parvoviral ITR is an AAV ITR, For safety reasons it may be 30 desirable to construct a recombinant parvovira (rAAV) vector that is unable to further propagate after initial introduction into a ceil Such a safety mechanism for limiting undesirable vector propagation in a recipients may be provided by using rAAV with a chimeic UIR as described in 152003148506: 3 The number of nuclici acid constructs employed in the insect cell for the production of the recomubinant parvoviral (rAAV) vector is not limiing in the invention. For example, onetwo.three four fivC or more separate constructs can be empioved to produce rAV in insect cells in accodantce with the methods of the present invention. If five constructs are employed one construct encodes AAV VP I. another construct encodes AAV VP2, yet another constructencodes AAV VP3, still yet another construct encodes the Rep protein as defined above and a final construct comprises at least one AAV ITR I fewer than. five constructs are used, he constructs can comprise various combinations o the al least one A' ITR. and the VP , VP2, 10 VP3, and the Rep protein coding sequences, Preibly two constructs or three constructs are used, with two constructs being nore preferred as described above if two constructs are used, preferably he insect cell comprises (a) a first nucleic acid construct for expression of the Rep protein as defined above, which construct further comprises the third nucleotide sequences as defined in (b) above (comprising 15 parvoviral Cap protein codin nces operably linked to at least one expression control sequence for expression in an insect ceil; see also below); and (c) a second nucleic acid construct comprising the second nucleotide sequence as defined in (a) above (comprising at least one pRsequenc If three constructs are used, preferably the same confuration. as used for two construts is 20 used except that separate constructs are used for expression of the capsid proteins and tor expression of the Rep proteins. The sequences on each construct can be in any order relative to each other. For Oxample, if one construct comprises iTRs and an ORF comprig . V apsid proteins, the VP ORF can be located on the construct such that, upon replication of the DNA between ITR 25 sequences, the VP ORF is replicated or not repliatedFor another example the Rep coding sequences and/or the ORF comprising nucleotide sequences encoding VP capsid proteins can be in any order on a construct in is understood that also the second, third and further nucleic acid construt(s) preferably are an insect celbcompatible vectors, preferably a baculoviralvectors as described above, Alternatively in the insect 30 cellofhe invention, one or more of the frst nule0tde sequence, second nucleotide sequence third nucleotide sequence, and fourth nucleotide sequence and optional further nucleotide sequences may be stably integrated in the genoine of the insect cell One of ordinary skill in the art knows how to stable introduce a nucleotide sequence 14 into the insect genome and how to idettif a cell having such a nucleotide sequete in the geiomeh The incorporation into the genorme niay be aided by, fobr exam.nple, the use of a vetor comprising nucleotide sequences highly homologous to regions oftthe insect genome. The use of specific sequences such as transposons, is another way to introduce a nucleotide sequence into a genome. In the invention the third. nucleotide sequence comprsin parvvin eapsid (Cap) prtein coding sequences is herein understood to comprises sequences encoding each of the three parvoviral capsid proteins, VP1, -2 and -3. The third nucleotitde sequence comprising the capsid protein coding sequences may be present in various 10 forms. E'g. separate coding sequences for each of the capsid proteins V -P -2 and -3 nray use'd. whereby each codinig sequence is operably linked to expression control sequences for expression in an insect ecll. More preferably, however, the third nucleotide sequence comprises a single open reading frame encoding all three of the animal parvoviral (AAV) VPF1, VP2, and VP3 capsid proteins, herein the initiation 15 codon for translation of the VP I capsid protein isa suboptinmal initiation codon that is not AiTO as e.g. described Urabe et at (2002, ura). A suboptinal invitation codon fbr the VPI capsid protein may be as defimed above for the Rep78 protein- More preferred suboptimal inidation codons for the VP rapsid protein may he selected from ACG l."T'O CT and 1070 of which Ci and G(TO are most preferred A prefrrcd 20 third nucleotide sequence fA the expression of the capsid proteins iter comprises an expression control sequence comparing a nine ieotide sequence of SE 0 ID NO: 7 or a nucleotide sequence substantially homologous to SEQ iDNw: 1upstrean of the i.ntlion codon of the nucleotide sequence encoding the VP Iapsid protein. A sequence with substantial identity to the ncleotide sequence of SEQ I NO: 7 and 25 that will hep increase expression of VPl is e g a sequence which has at least 601%, 70%. 80% or 9{)%t identity to the nine nucleotide sequence of SEQ ID NO: 7, A further preferred third nucleotide sequence for expression of he capsd proteins iirper preferably comprises at least one modiWatn ofthe nucleotde sequence encoding the VP capsid protein selected from aiong a C atitcleotde position 12, an A at 30 nuceotide position 21, and a C at nucleotdc position 24(with reference to position I being the first nucleotide of the translation initiation codon2 see SEQ ID NO . himinat ion of possible fase i.nrtation codorns for transition of VP'1 of other serotypes will be well understood by an artisan of still in the art, as will be the elimination of 15 putative spHee sites that nay be recognized in insect cells, VWrious further miodificatons ofVP coding regions 'ire knwn lo skiled artisan which could either increase yield of VP and virion or have other desired effects such as altered tropism or reduce antigenicity of the virion, These modifications are within the scope of the 5 present invention. Prerably the nuceeottde sequence ol the invtion encoding the parvovia capsiproteins is ply lked to expression control sequences for expression in an insect cell wich will ai least include a promoter that is active in insect cells Such. control sequences and further techniques and nateriak (e g vectors) tr expressing parvovirt capsid proteins in insect host cells are already described 10 above for the Rep proteins. in a preferred embodinent of the invention, the second nudeotidc suence present in the insect cells of the invenon, ie, the sequence comprising alteast one parvoviral (AAV) ITR further comprises at least one nucleotide sequence encoding a gene product of interest, whereby preferably the at least one nuoleotide sequence 15 encoding a gene product of interest becomes incorporated into the genorne of a recombinant parvoviral (rA.)}ector produced in the insect celL Preferably, at least one nucleotide sequence encoding a gene product of interest is a sequence for expression in a mainnmalian cell Preferably, the second nucleotide sequence comprises two parvoviral (AAV) 1FR. nucleotide sequences and wherein the at least one 20 nucleotide sequence encoding a. gene product of interest is located between the two parvoviral (AAV) FIR UcMotide sequences, Preterably the nuclcotide sequence encoding a gene product of interest (for expression in the mammalian cell) will be incorporated into the recombinant parvovirai (rAAV) vector produced in the inect cell if it is located between two regular IRs, or is located on either side of an [FR 25 engineeredwth two D regions, The second macleotide sequence defined herein above nay thus comprise a nucleotide sequence encoding at least onegene product of interest Ibr expression in a mammalan cell, located such that it wil be incorporated into an reco.inant parvoviral (rAAV) vector replicated in the insect cell Any nucleotidesequence can be 30 incorporated for later expression in a imaunaian cell transfected with the recombinant veetr produced in accordance with the present invention. The nucleotide sequence may e g, encode a protein it may express an RNAi ageit, e an RNA molecule that is capable of RNA interference such as e.g. a shRNA (short hairpinRNA) or an siRNA (short interfering RNA). "iRNA" means a small intcrferinga RNA that is a shor.-length double-stranded RNA that are not toxic in matmnal an cells (Elbashir et ab, 2001, Nature 411t 49498; Capen et at 2001, Proc. Nail. Acad. Sdi USA 98: 9742-47), in a preferred embodiment the second rncleotide sequence may 5 comprise two nucleotide sequences and each encodes one gene product interestt fr expression in a maninialian cell. Each of he two nucleotide sequences encoding a product of interest is located such that it will be incorporated into a recombinant parvoviral (rAAV) vector replicated in the insect cl, The product of interest fr expression in a iamminan celmay bea feapeu 10 gene product A therapeutic gene product can be a polypieptide; or an RNA molecule (siRNAN) or other gen product hat, when expressed in a target cellrovides a desired therapeutic cffect such as e.g, ablation of an undesired activity. e, the ablation of an infeced ce or the complementation of a genetic detected g amusing a deficiency in an enzymatic act iivty Examples of therapeutic polypeptide gene products include (FTR, 1$ Factor IX, Lipoprotein ipase (LPi, preferably LPL S447X; see WO 0 1 002 2 Apolipoprotei AL Uridine Diphosphate icuronosytransferase (UG'I) Retinitis Pigmentosa iTlase Regulator Interacting Protein (RPGRI and cytokines or interleukins like esg. I Lt) Ateraivly tor in addition as a second gene product, second nucleotide 20 sequence dehned herein above may comprise a nucleotide sequence encoding a polypeptide itat seve as marker proteins to assess cell transfornatoin and expression. Suitable marker proteins fr this purpose are eg. the fluorescent protein GFP, and the selectable marker genes S V ihymidine kiase (for selecton on IIAT mediam'utf bacterial hygromycin B phosphotransferase (for selection on hygrornyein B3) Tn 25 aioglycoid phosphoirans se (r selection on 4), and dihydroh late reductase {DIFR) (for selection on nethotrexate)CD20 the lowaffinity nerve growth factor gene. Source for obtanng these marker genes and methods for their use are provided in Sambrook and Russel (2001) 'Molecular Cloning: A Laboratory Manual
(
3 edition), Cold Spring Harbor Laboratoiy Cold Spring Harbor Laboratory Press. 30 New York. Furthermore, second nucleotide sequence defined herein above may comprise a nucleotide sequence encoding a polypeptide that may serve as a faik-safe nhanisni that allows to cure a subject from cells trasduced with the recornmant parvoviral (raAV) vector of the invention, if deemed necessary; Such a ntleotide 17 sequence, often referred to as a suicide gene, encodes a protein that is capable of conve ing a prodrug into a toxic substance that is capable of killing the transgenic cells in which the protein is expressed Suiable examples of such suicide genes include e g the Ecoi cytosine deanase gene or one of the thymidine kinase gees from HIepes 5 Simplex irs, Cytomegalovirs and Varicella-Zoster virus, in which ase ganciclovir may be Used as prodrug to kill the transgenic celL in the suAect (see eg. Clair et al 7 Antimicrob Agents Chemother : 844 849 In another embodiment one of the gene products of interest can be an AAV protein, In particular. a Rep protein suh as Rep'78 or Rep68. or a functional fragment 10 thereof A nucleotide sequence encoding a Rep,8 and/or a Rep68, if present on the genomncof a recombinant parvoviral (rAAV) vector of the iidention and expressed in a mammal ian cell transduced with the vector allows for integralicm of the recombinard parvoviral (rAAV) vector into the genome of the transduced mammalian cell Express ion of Rep78 and/or Rep68 in an rAANtransduced or infected mammalian cell 15 can provide an advantage for cerian uses of the recomonbiant parvovira rAAV} vector, by allowing lng term or permanent expression of any other gene product of interest introduced in the cell by the vector, In the recombinant parvoviral (rAA) vectors of the invention. the at least one nucleotide sequences) encoding a gene product of interest for expression in a 20 mammalian cel preferably ishare operably linked to at least one mammalian cell compatible expression control sequence g. a promoter. Marny such promotes are known in the art jsee Sambrook and Russet, 2001, supra) Contitutive Promoters that are broadly expressed n many cel types such as the CMV promoter may be used, orerfered will be promoters theatre inducible tissucellype 25 specific, or cell cycle-specFi For example, fr iver-specie expression a promoter may be selected from an ,l-anti-trypsin promoter, a thyroid hormonebinding globulin promoter, an. albumin promoter LPS (tyroxine-binding gIoblin) promoter H.R ApoCiR hybrid promoter, HIR-hAAT hybrid promoter and an apolipoprotein E promoter. Other examples include the E2F promoter for tumorelective, and, in 30 particrar neurological celltumorselective expression (Parr c a, 1997 Nat, Med, 3:1145-9 or the IL-2 promoter for use in mononuclear blood cells (Hagenbaugh et at 1997, J Exp Med; 185: 210110), 18 AAV is able to inct a number of mammalian cells.See. eg. Tratschin et at. (1985, MoL Cell Bio :32513260) and GDrim et at. (1999 Hum Gene Ther 04442450,, However- AA transduction of human syno vial ibroblasts is sigificant yv more efficient than in siiilar marine celss ennnigs et al Arthritis Res. 5 31 i (20 , and the cellular tropicity of AAV differs among wrotypes. See eg, Eavidson ct al, (2000, Proc. NA. Acad, Sc USA, 97:34283432) who discus differences among AAV2. AAV4, and AAV5 with respect to mammalian CNS cell tropism and transduction efficiency. AAk sequences that may be used in the present invention for the production of 10 recombinant AAV vectors in insect cells can be derived fron the genomeof any AAV serotype. Generally, the AAV serotypes have genomic sequences of significant homology at the amino acid and the nucleic acid levels, provide an identical set of genetic functions, produce virons which are essentity physically and functionally equivalent, and replicate and assemble by pratically identical nechansms For the 15 genomi c sequence of the various AAV serotypes and an overview of the geinomic sinilarities seec.g. GenBank Accession number U89790; Gentank Accesson number JO1901; ieniBank Accession number AF943303; Genlank Accession number AF85716: Chlorini et at. (1997. J, Vi. 71682333); Srivastava i al. (1983,3 J Vir, 415555164 ;hrini et a (1999, 1 ir 731309-1319); Ruedge et at. (998, J. Vir 20 72:309319) and Wu et at (2000 Vir 74: 8635-47) AAV serotypes I 2, 3, 4 and 5 are preferred source of AAV nucleotide sequences f use in the context of te present invention. Preferably the AAV IR sequences for use in the context of the present invention are derived from AAV1, AAV2,and/or AAV4. Likewise the kep(RepT8 and Rep5} coding sequences a preferably derived from AAVI, AAV2. and/or 25 AAV4, The sequences coding for the VP I VP2, and VP3 capsid prote..ins for use in the context of the present invention may however be taken froni any of the known 42 serotyps mo referably fron AAVI, AAV5 AAV3, AAV4A AAV5, AAV6,.AAV7, AAVS w- A xV9 or newly developed AAV-like varticles obt-ained by ecc. caps-dd shufflintechniqees and AAV capsid libraries, 30 AAV Rep and ITR sequences are particutady conserved among most serotypes, The Rep?8 proteins of various AAV serotynes are etmore dn 89% identical and the total uieleotide sequence identtv at the genorme level beveen AAV2, AAV3A, AAV3iB, and AAV6 is around 82% BantehSchaal ct at, 1999. 1, Virol. 73(2A939- U9 947) Moreover the Rep sequences and ITRs of many AAV serotypes are known to cfiietly crosscomiplement (ieafutionally substitute) corresponin sequences forn other serotypes in production of AAV particles in mianuimlan cels. 1S2003148506 reports that AAN Rep and ITR sequcalso efficiently cross 5 complement other AAV Rep and IT R sequences in insect cells. The AAI VP proteins are knovn to determine the cellular tropicit of the AAV virion. The VP proteinoencodig sequences are significantly less conserved than Rep proteins and genes arnong diflereni. AAV serotypes. The abiliy of Rep and [TR sequences to crosscnompement coresponding seque ces of other sertypes allows for 10 the production of pseudotyped rAAV particles comprising the capsid proteins of a serotype (eg, AAV3) and the Rep and/ord TR sequences of another A.AV srotype (eig., AAV2). Such pseudotyped rAAV particles are a part of the present invention. Modified "AAV sequences also can be used in the context of the present ierntion, eg, for the production. of rAAV vectors in .nsect cells, Such modified 15 sequences e.g.include sequences having at east about 70% at least about 75%, at least about 80% atl least out , least abAou 90% at least about 95%, or niore nucleotide andior arnino acid sequence identity (g, a. sequence having about 99% nucleotide sequence identity) to an. AAV I, AAV2 AAX3 AAV4, AAV5, AAV6, AAV7 AAV8 or AAV9 ITRD Rep, or VP can be used in place of wild-type AAV ITR 20 Rep, or VP sequences Although similar to other .AAV serotypes in many respects, AAVS di h-rs fon other human and simian AAV sero types more than other known human and simian serotypes. In view thereof the production of rAAV5 can differ nom product oit other scrotypes in insect cells. Where methods of the invention are employed to 25 produce rAAV5, it is preferred that one or more constructs conp sing; collectively in the case of more than one construct a nmeleotIde sequence comprising an AAV IIR a nucleotide sequence compares an AAV5 Rep coding sequence (ie. a nucleotide sequence comprises an AAV5 Rep7) Such ITR and Rep sequences can be modified as desired to obtain efficient production of rAAVS or pseudotyped rAAV vectors in 30 insect cell, E the star codon of the Rep sequences can be modified, V.P splice sites can be modifted or eliminated, and/or the VPI start codon and nearby nucleotides can be modified to improve the production of rAAVS vectors in the insect cell 20 In another aspect the ivention thus relates to a method for producing a recombinant parvoviral (rAAVvmiin (comprising a recomrbi nant parvoviral (rAAV) vector as defined above) in an insect cli. Preferably he method comprises the steps of (a) culturing an insect cell as deined in herein above under condions such that 5 recombinant parvovind (rAAV) vector is produced a b recovery of the recombinant parvoviral trAAV) vector I is understood here tlm the recombinant parvovirai (rAAV) vector produced in the method preferably is an infectious parvoviral or AAV virion that cornprise the nsreo mnat parv,-oviral (rAAV ) vector nucleic aetds. rowing conditions br insect cells in culture, ad production of heteroogous products 10 in insect cells in culture are welknown in the art and described eit. in the above cited references on molecular nginwering of insects els. Preferably the method ftither comprises the step of affinitypurification of the (virions comprising the) recombinant parovira (rAAV) vector using an anti-AAV ttntihody, preferably an nmobilised antibody. The anti-AAV antibody prerably is an 15 monoclonal antibody. A particularly suitable antibody is a single chain camelid antibody or a fragment thereof as eog. obtainable f t rom r or lamas (see eg. Muyidermans. 2001 o Biotchnol. 74: 277-302), The antibody for affinity-purification of rAAV preferably is an antibody thaf specifically binds an epitope on a AAV capsid protein whereby preferably the epitope is an epitope that is present on eapsid protein of 20 more han one AAV serotype, Eg the antibody may be raised or selected on the basis of specific binding to AAV2 capsid but at the same time also it may also specifically hind to AAVI. AAV3 and AAVS eapsids; In a further aspect the invenon relates to a rAAV vision produced in the above described methods of the invention. using the nucleic acid construcs and cells as 25 defined above. Prefirably the rAAY virion comprises in its genone at east one nuckleode.- sezquence enoiga gene product of interest, wNhereb)y theI at least one1 nucleotide sequence is not a native AAV nucleotide sequence, and whercbyin the stoChiomety of the AAV VjPI., VIP2, and VP3 capsid proteins the amount of VP1 (a is at last 100, 105 1 0, 120, 150,200 or 400% of the amount of VP2: or (h is at least 30 8, 10, 10.5 11, 12, 15, 20 or 40% of the amount of VP3 or c) is at least as defined in both (a) and (b). Preferably the amount of VPI1 VP2 and VP3 is determined using an antibody recognising an epitope that is common to each of VPN) VP2 and VP3. Various immunoassays are available in the art that will allow quantify the relative amounts of VPV VP2 and/or VP3 (see e p Using Antibodies, E arlow and D, Lane 1999, Cold Spring -Harbor Laboratory Press, New York). An suitable anti.body reco'nising an epitope that is common to each of the three capsid poteins is eg. the mouse antiCap BR atibody (as is commercially available foi Progen, Germany) A prFerred rAAV 5 virion according to the inventiOn is a irion comprising in -as genome at least otne nucleotde sequence encodn a gene product of interest whereby the at least one mleotide sequence is not a native AAV nucleotide sequence. and whereby the AAV virion comrises a VP1 capsid protein comprises a leucine or a Valine at andno acid position I A miore preterred AAV irion according to the I.wenton has the .ratsiof 10 capsid proteins as defined above and comprises a VPI capsid protein comprises a leucine or a waline at arno acid position L In this document and in its claims, the verb "to comrorise" and its conjugations is used in its nondinutiung sense to mean that items following the word are included, but items not specificaly mentioned are not excluded In additionvreference to an element 15 by the indeaite article 'a" or "an" does not exclude the possibility that more than one of the cement is present unless the context clearly requires that there be one and only one ofthe elements The indefinite article "a" or "an" thus usually means atw least one". Decito PL-u a Iof t he .'hIuresz 20 Figure J A) Organisation of Rep expression in the wild type AAV genome. The Rep78 and Rep gens are expressed fi.n respectively the PS and P.19 promoter Epression of Rep68 and Rep4O (which are the spliced variants of resp rept8 and Rep2) are not shown, Both expression axits contain a ATG-hiitiation site. 13) The constmuc of the invention has the Rep ORF under the control of a single 25 promoter (e gthe polyhedron (PolH) promoter . This promoter drives the expression of both Rep78 and Rep52 because the Rep78 initiation codon Alt is converted to the aemate ACGO initiation codon and partially skipped by the ribosome C) The original constmut by Uabe et at (20302, sra drives Rep78 and RepS2 independently from two different promoters (respAl E1 and polH); 30 Piguye2:, Westen blot analy si s of Rep prteins expressed from recombinant baculovirus that was passaged 5 times on insect Cells. The original baeuo virus designed by Urabe et a, 2002 (original REP/Baeto-Bac) results ita slo decrease of Rep78/52 expression over 5 passages, The expression unit for Repl8 and 52 designed by Urabe e7 al 2002 insehe in baculovirus backbone PSC (original REP / PSC) also results in a decrease of Rep78/52 expression following passagiig on insect cells. lHowever the baculovirus vith the REP expression unit containing the AG initiation codon in the PEC backbone (REI>ACG /PSQC results in stable expressicof 5 Rep2/52 over at least 5 passages Western blot analysis was performed as described in Example I. 3. Eigre: Results of Tabie I plotted in a graph, *gg Comparison of the stabilities of various rAAV constructs in insect cells rAAV production SO cells was pertormted as described above in Example IUor all 10 productions tohe FIR containiing baculovirus and the capsid gene containing baculovirs were identical, the passage niber was the same as the Rep gene containing baculoviruses 4 different Rep gene containing baculoviruses were used: I) The REPS AGO / PSC, 2) SLR: the original construct by Urabe et al. (2002, pra) 3) Rep52+ Rep78(B2B): Iwo separate Bac-to-Bac baculoviruses, one containing the Rep 78 gene 15 and the other one containing the Rep 52 gene. 4)RepS2 Rep78(PSC) Two separate protein sciences baculoviruses one containing the Rep 7$ gene and the other Con containing the Rep 52 gene Finre 5: Stabilty of the REP-ACG / PSC baculovirus constructs up to passage '8 rAAV productions in SF+ cells were perormed as described n Example i 20 Figurej6: Comparison fl the eflct of passage effet on rep protein expression of the origil o mt rom Ure et A (002' supra) with a REPCG / PEG construct in accordance with the invention. The bacalovirus passages and"he westem blot were done as described in Examiplel I During a normal passage of the rep baculoviruses, samples were taken at 40 hours afer addition of the bacidoviruses to the SF cells and 25 western blot was performed.
ExNamples I aterials & Methods 5 In order to express Rep7S and Rep2 frou a sole bicistronic messenger RNA. the ATO inniadin codon o' Rep78 situated on the exprlssionector pFastfac{ualSLR (Urabe et aL 2002, supra) vas converted to ACO. The upstrean prmer used was; 10) 5'-&en - ;aag tcfittaagcGGK GGt sIt. A sA. aT'2SCATZ5AGTO SEQ 1D N PRIMER SEQUENCE forward The 3 primer that was used in the CR reaction was flanking t REl78 gcne 15 and comains a Xbal site TCTAGA) Xbal 51-AGGUTCTAGATTCGAAAGC COCG-3 HSEQ l NO.9) 20 PRIMER SEQUENCE reverse The seance between the above-mentioned primer set was ampliied by PaR (reaction voltume 50 p.; ix PI$ Amp Buffer 0TmM INTP 1mM MgSO4, 150mM primer fiw., 150mM prinr re' 2 enhancer solution template 50ng 25 (pFas3acDualSLR'i. U Platinum Pf (jnvitgern Crsbad, CA, USA) using the folo Wing protocol: i cycle of 95'C, 5 min; 35 cycles of 9VC. 15 se; 55CC 30 se; 72 2 Mn; 1 y of 7240, 10 min; 4C, fOr ever).The PCR prout was cloned in PCRtbunt TOPO using the Zero Blunt TOPO PCR clomrn kit (Invitroger). The Rep$ Was subecloned into pfastaacfuat n vitrogen using the restriction sites Spet 30 and Xbal, The intated Rep expeso a te was hnaii eioned (sig restriction enzyines BstZi171 and Avril) into the baculovirus expression constuct (cut open with [Beo R and Xabl) pPSC10 (Protein Science' CorporationN eriden, CT, USAt The sequence analysis of the constmut was verioed by Basecl ear Lciden, the Nethenkds, 24 L 2 Recombinant baculovirus production Reconibiruat baculoviruses derived forn the Autographa catifn'ica nuclear polyhydrosis virus (NP were produced using the GceneXpress Bacuk.K.iT (Protein Sciences Corporatin) isfCtion was performed as folkows:in a round bottont14ml 5 tube 200 I GRACE ium was mixed with 6 pi cellfectine (hintrej), and in a eppendorf tube 200 p! RACE medium was mixed with50 p virat DNA (protein sciences) and 2 pg transfer plasmid (REP). The contents from the eppendorf tube were added to the tube and mixed carefully After an incubation period of 30 ninuts at RT 1§00 p]tGlRACE was added to the transfection mi insect cel in a T25 flask were 10 washed with GR ACE medium and the transfection mixture was added dropwise to the cl layer Ater an incubation of 6 hours at 2817 SF900I serui supplemented with 10% FBS was added carefully and the 1'25 flask was put in a 28*C stove for 5 days after whic the recombinant baculovirus was harvested. 13 Western blot analysis i5 isec cells (SF+) were infected wiih baculovirustRE At 16 40, aid 64 hours post-infection eclis a sample was taken and cens were lysed by aIddin 011\ 10 x 'TRIS lysis bufer ti5M NaCL 0IESM TRIS 0 O0 M MgCL, 1% TRITON X 100, pHS5. filter steri"ised) and incubated a 28 0 for 0 minutes in a. shaker (Innova 44. New Brnswick) rie DNA and RNA was degraded by incubation with benzonase at 374C 20 for 30 mines. Cell lysate was centrifuged (,900 x g; 15 mi; 4C). NuPAGE L.DS sample bu f4tr 4x lnvtirozen) kwas added to a sample of the supernatant and was Ioaded onto a 4- 12% Bis Iris gel (120V), Proins were blotted onto a PYD membrane (BioRad) Wr 30 minutes, 10V (SemiTr biotingy Wester ununoemstry was performed by blocking the membrane with SuperblockPBS blocking buffer (PIERCE) 25 and subsequent incubation with mouse anti-Rep (30309 Progen, Germany; dilution 1:50) and rabbit antin-mouse.HRP (DAKO, dilution 1:5001) The Rep-proteins were visualized by chemoluminescent stain with Imi-light plus Westem-bloting substrate (Roche). 1.2 Results 30 The performance of the newly designed Rep-construct of the invention (REP ACG / PSC)*1 was compared with the original Rep constructs in both 1)I P7SC baculovirus backbone and in 2) Bacto~Bac baculovirus backbone (Urabe et aL,2002 All three constructs were serially passaged until passage 5, AAVI -LPLproduction experiments 25 were performed using the passage 34 and 5 Repeconstruets in combination with an AAW PL Pn. ad a.AA \V'ap recombinant bacuiovirus of respectively passage 2, .3 4 and 5 (AA LI and AAV-Cap recombinant Baculovirus used here are described below in Example 2). AVI L production yields were determined by qPCR arid are shovn in 5 Table L The original baculovirus designed by Urabe et a, 2002 originala REP/Bac-to Ba) resuls in a fast decrease of AAV production over 5 passages The expression unit for Rep designed by Urabe et At, 2002 inserted in baculov irus backbone PSC (original REP / PSC) also results in a decrease of AAV production following passaging on. insect cells. However the baculoviruswih the REP expression unit containing the ACG 10 initiation codon in the PSC backbone (REPtACG PSC) results in stable AAV production over at least 5 passages. Therefore, reproductie productionyields of AAV LPL over several passages (eg. 2 to 5) were oly obtained using bacuioviuses containing the REP~ACG constructs origial REP/ REP ACG orgmal REP PSC PSC Bac-toiBac passage vg vmg/m vg/il 2 538E+09 304E+09 362E+0 3 917E09 4 W77E09 28 E+09 4 1 A6B+09 7.81B+09 759E1+08 5 TE5 0 990+09 2ME5+08 15 Table 1 Production of rAAV virions using th. baculovirus constructs of several passages: Sf9 cel s were infeted with three recombinant bacidov irses encoding a L PL-etor unit of passage 2, 3, 4 or 5 a Rep-expression unit of passage 2 3, 4 or 5 and a Cap-expression unit of passage 2, 3. 4 or 5. Aer three days cells were harvested 20 and AAV yields (vector genomes per muygnn);were determined by qPCR 26 titer (geWs/n!) Ratio Rato OR] Rcp 8 Rep52_ 1 OR'' Rep (RF Rep orinalR E cAto P2 4E+09 2 2)E+ 0S 2, 4E-08 2 5.8 original RIly ac-tolBac P3 6 41+08 0 6 07 5 0E 4-07 1 -13 1 '3 oYnkIal REP/Ba-co~Bac P4 2 1E+09 I) E+ 7 ) 9 7E 7 2 i 32j2 2 original RiE PP BeBac P5 E4)9 3 2E07 25F 507 5F AS 6947 REP-ACG / PSC' (CA P2 310+09 2TE9 2E9 011 14 REPhAC( PSC (4) P3 23E+09 2L 0E+O 2B+ 1,11 os REIAG PSC(C4) 4 2509 2 2 09 20 E -09 1,13 1,08 REP-ACO !PSC(C P5 2P 7E49 2,1 E+09 2 5E+09 L,26 107 REP-AiCG / PSC (A) P 25E+i9 2 2E409 2 5+9 8 A140 RP PAC6 PSC (M)2P 4P 2 9 E0 9 00 8 1E+0 04 RE P j S1 CA 3)P4 2'Q4N 2 24 0 2 oE09 Ij0 105 REPA \CG PS(- (A) PS IE+09 E09 E+i09 103 0,98 origial REP/BacntoBac P2 1 4 IIE 09 1 MEAY9 0,95 087 original I PBac-to IBac P3 7 ME+ON 6 E 8 E+08 1 I07 0 88 original REPa 1c -o-Bac P>4 I W 13+ ,18 US E 8 0 1,8 103 orvinai RE~LA.3 to P -ac 215 1 '1 9 F,3,0 6 C 2 _3 74 V9T2 Table 2: QPCR perfonned on the varous Bac-Rep onmets allowing massaging on insect c-elk (Passage- 2-5) able 2 showsthe results of a quantitaive PCR (Q-CR) assa' that was desinecd for the Rep"expression unit in the recombinant baculoviruses and for a flanking baculovirus ORF (geno copies per' rl; geKmi The ratio between the (9CR values detcrinOs the presece of deletions in the Rep1haculovirus. A ratio of .1 theoretiai 10 means that all bacuvi Nses in he batch contain a recombinant RepY8 or 52-sequence. The original baculovirs desiugd by Umbe et -l 2002 origini RE1/3acfo-Bac) shows significant amounts of the reconibinant bauelonrovi ia e 5 tave de letions in the Rep sequences The expression unit for Rep7x and 52 designed by Uabe d A 2002 inserted in baculovirus backbone Sc (oi inal REP / PS() showsa vyi -ndy 15 and dramatic fs ot recombinant baculovirus However, the baulovirus wich the REP expression uit containing the AGG initiation eon in the PS(C lne (REP~ACG / PSC) (clone C4 and A3) show stable recombinant bacutoviruses over at least 5 passages, 27 .Exanwe 2I Cap constructed 2. 1 Baculovius plasmid constretion i order to xps \rora soe polycistronie mnessePger RNA, the 5 baculovirus-AAV ap constret was designed as desTibed by (rabe et af, 2002, suprat Briefly tthe ATG initiation codon of VP was mutated to ACG) A potential AT iitiatiore codon at postion 11i has been changed to A Ct. The spice arcceptor site downstream of the VPI initiation codon was destoyed (mutation at position 21 and 24 The mutated Cap expession cassette was loaded into a bacukloirus exrsdo 10 construct: pfastE acDual (pFBDAAV 1 mlu with BamH UStui restriction st\s TisM plasmid (pFBDAAVIVPm U 9l was the starting material f&r introduction of alterate initiation codans for ViI The forward primer used by Urabe et al, (2002. supra in order to it the mntioned mutations was: 15 m 21. 24 5f tgeggtoctg taagAC ICTGCGACGGT ATC§TACCCCA§G1C§1-*i ' (SEQ N0 NO ) The following forward priners were used to make the expression constructs using 20 pFBDAAVIVPmI (Drabe et al, 2002 supra)as starting maeria 5' cgggat c gtagTTGGCTGCCCGCCGTTATCTACCGATTGCtTC (SEQ MD NO,2) 25 5 -ogcggatec~gtatagATTGCTGCCGACGGTTATCTAeC GATTGGeTC-3 (SEQ MD NO. 5-ac Ggateovt taGTGGCTGCCGACGGTTATCTA7CGATTGGCTC-3 (SEQ D NO. 4) 30 'SEgegg tMt NaO 5CTG TCGPCCACGCT [ TACCCGAT1?G § C (SEQ 1 D o 5 The backward-primer that was used in the IR reactions with the above, forward prinTers was directed to position ~ 230 bp downstream of the VPIinitiation codon and ortamins a unique Stu I site (AGGCCT). .5 5-GTCGTAGGCTTGT CGTGCTGNAGGGCCrGC (SEQ ID NO. 6) Fragments were amplified with the above-naeentioned sets of forward amd backward primer pairs by PCR. Folwing digestion of PCR products with Dmn] and 10 Stuil the oR. products were subeloned into the Bamih / Std sites of pFBDAAVI vpmil resulting in the various to be tested bacuiovnus AAVCap constucts. DNA constructs were verified by sequence analysis at Baseclear Leiden, the Nethedands. 2 1.2 Recombinant baculovirust rodution 15 Reconibinant baculoviruses derved from the Autographa caf bricas mear polyhydrosis virus (AcNPV) were produced sing the Bae01o0Ba baculovirus expression system (InvirogerQ rBac-Cap was ampiied by ifeing 2xdl 0 Sf9 CelIs per Ui at an mo of 01 ihree days after ndetion the cells were spun down and the supernatant containing the virs recovered, 20 2.1.3 recomin n idii to rAAv batches were produced vsing three recombi nant bacutoviruses according to Urabe et aQ 2001 However for this study one baculovirus harbored an expression constrmt for the PL ' transgee The therapeutically active agent expressed from the transgene is a naturally occurringvariant of human ipoprotein ipase, a single chain 25 polypeptide of 448 amino a Tb eP15 variant has a deletioni ottwo amino acids at the (>terninus of the protein Ihe second bacdovirus harboured an expression construct for the AAV repication genesRep 7R and Rep 52. The third baculovirs harbored the AAV1 capsid squec with either an ACG or a ITG,TG GIG irmtiationi codon for VPLI 30 MabmaanrAAV batches produced wih the plasmidtransfection system were produced according to Grinm et al, 1998 (Novel tools for production and purification of recombinant adeno-associated virus vectors. Hetn Gene Tiher. 1998 Dee 1t0(l81274560).
29 23 Western blot apalsis insect cells were infected with bactiovirus-Cap. At three days post-infection cells were centrifuge (3.000 g; 15 ninta The supernatant was iltered through a 0,22um Mille filter. NuPAGE LDS sample bwter (invr..gen) was added to a sample of the 5 supernatant and was loaded onto a 4-i2% Bi-ris gel The get was run at iOO0. Proteins were blotted onto a nitrocellulose membrane (BioRad) forI hr. 1 00 350-mA Western immunochenisty was performed by blocking the membrane with % marvel, dried skimmed milk and subsequently incubation with mouse anti-Cap (131 from Progen Germany dilution I:5() and rabbit anti noue-lARP (DAKO, dilution 1A100), 10 VPI ,2 and 3 were visualized by chemoluninescei staining with lumi-light plus Western-blotin g substrate (Roche), 2.1.4 3ioc-hemical. measurements Human LP <hativity was assayed as previously described uing a radioactive trioleo villyerv envision substrae (Nilsson Ehie and Sehoflt 1976 . Human 15 LP ' immoreacive mass was assayed using a sandwich ELISA with chicken IlgY and mouse 'D2 anti-bUP antibodies (Lu et al, 2000. Pasma trigwe.-ride levels were measured by using commercial kits following manufacturer protocols (Bloehringer manhein #450032) 20 2.2, Reslt 2) 2) 2 Construction ot recombinant baeulo virus in order to introduce ditterent alternate intuation codons for VP1 expression in the baculovirus plasmid designed by Urabe et at (2002.supra) a series of upstream primers were designred containing a 13aH restriction site and either a TTlG, AT, (iTG or CTG codon in place of the ACG initiation codon of Vll P using these 25 primers in combination with a downstream primier containing a Sad site resulted in amplified fragments that were subeloned into the BamHI1/Stl site of p73DVPm1 I (Ba-Cap). The resulting baculovisni plasmids were used f(r the prepanaion of recombinant baculoviruses using the Bace-to -Ra baculovirus expression system. The prepared recombinant baculoviruses were infected on insect cells in order to produce 30 AAV capsids. At three days following infetion viral protei n. expression ofthe diterent baculovirus batches were determnned on Western blots. From the Western blots it became clear that the baculotirus construct containing the TUG initiation codon for VP1 expressed this protein to a higher level compared to the previously used ACG 30 initiation codon. The ratio between VPI and VP2 using the TTG codon was found to be 1 which is siitar to what is reported for wild type AAV tot shown 22. inecion orAVbatches on, cels in culture In order to nvestigate the infectivitv of the AAV capsids derived from 5 recombinant baculo viruses with the TIC iniiaton con n rAAV was generated. Also a rAA\V batch was generated by plasnid trunsfetionR on mammalian HEK293 cell A vector genome titer of both rAAV batches was dermined by qPICR. This titer was used to infet HEK 293 cells in a microtiter plate at an increasing rmoi. At two days blowing section an quantitative assay (lPR ' mass assay for the transgene 10 product (LP"") was performed on the medium of the infected cells. The assay showed that the amount of LPL [ X'" produced by bacuiovirustproduced rAAV was similar to dhe ,RL produced by the plasmid-produced rAAV (not shown). 2.2.3 section of rAAV batches in mice The rAAV batches produced with the baculoviru-prodaction systemand wih the 15 conventional mamnalian plasm production were injected intramscularly in mice to follow LPL 5 ' protein actvity and trigyceride tasting in vivo. At 3 days, 7 days and at 2 weeks following injection blood samples were taken and evaluated, Between. 3 and 7 days post virus administration blood-phsma sampled fro both mice injected with manimaiianrAAV and one mouse injected with baculo-rAAV was tuned 20 from milky to completely clear Blood plasia derived from one bacdo-rAAVlnjected mouse remained relatively rnky however fit level was clay reduced. Triglyceride levels were lowered respectively of all heated mice (not shown On day 14 TG levels in both mammianAAV and baculovi.rus-(TTo-AAV treated mice TG levels were reduced for %6% Plasma sampls taken at two weeks after virus administration showed 25 that the LPL. activity of the mice treated with baculovirusAAV ann mammalan AAV was similar (not shown). Exapl 3;StbiltyofrAA cnstucs ithmoifid ep78 Up ti iaton codon in insect cells 30 31 CmaiooftesaiiisovaiurAVcntctinnetcells rAAV productions in S cells were performed as described above in Example 1. Forall productions the TR conai.n.ing bacuovirus and the capsid gene contairnig baculovirus were identical, the passage number was the same as the Rep gene containing baculoviruses 4 different Rep gene containing baculoviruses were used 1) The REP~ACG I PSC 2) SLR: the origil construct by Urabe et al (2002 spra,3) RepS2 + Rep?8(B213)v To separate Bac-to-Bae baculoviruses, one containing the Rep 78 gene and te other one containing the Rep 52 gene. 4) Rep2 Rep78PSC) Two 5 separate protein sciences baculoviruses one containing the Rep 78 gene and the other one contains the Rep 52 gene. Results are shown in Figure 4. At fifth baculovirus passage rAAV production is already improved by more than a faetor 10 using a REP-ACG PSC in accordance with invention as conipared to the original Rep construt and compared to the s"pht Rep 10 3.2nstts rAAV productions in SF+ cells were perfbrned as deserted in xaple 1 For all prodictions the ITR containing baculovirus and the capsid gene containing baculovirus were identica, the passage number was the sam as the REP-ACG / PSC 15 bacutovirus Resihs are shown in Figure 5. The R.EPACU ! PSC baculovirus is stable to at least passage 8 rAAV production tigers of REP~A1-,($ / PSC are stable up to at least 8th passage of the baculovirus, 3.3 Passageefect on rep protein expression The et of passage number on the expression of Rep protein for the original 20 construct from Urabe e at (2002, siun) was compared to a REP-ACGi ' PSC construct 4 accorlance with the invention he baculovis passages and the western bot were done as described in Example 1. During a normal passage of the rep baculoviruses, were taken at 40 hours after addition of the baculo viruses to the SF eelis and western blot was performed. Figure 6 clearly shows diminished Rep expression in 25 g passages compard to eier passages for the original Utrabe construct (SLR., whil- th- Rep expression in the REP-AOG G PSC construct stays the same in the higher passages compared to the lower ones, 30