USOO8697417B2

(12) United States Patent (10) Patent No.: US 8,697.417 B2 Bakker et al. (45) Date of Patent: Apr. 15, 2014

(54) BACULOVIRAL VECTORSCOMPRISING FOREIGN PATENT DOCUMENTS REPEATED CODING SEQUENCES WITH DIFFERENTIAL CODON BASES WO 9636712 11, 1996 WO WOO114539 A2 * 3, 2001 ...... C12N 15.00 WO WOO168888 A2 * 9, 2001 ...... C12N 15/864 (75) Inventors: Andrew Christian Bakker, Almere WO WO O3018820 A2 * 3, 2003 ...... C12N 15/864 (NL); Wilhelmus Theodorus Johannes WO WO O3061582 A2 * 7, 2003 Maria Christiaan Hermens, Almere WO WO 2005035,743 A2 * 4, 2005 ...... C12N 7/O1 WO 2007046703 A2 4/2007 (NL) WO 2007084773 A2 7/2007 (73) Assignee: Uniqure IP B.V., Amsterdam (NL) OTHER PUBLICATIONS (*) Notice: Subject to any disclaimer, the term of this Urabe et al. Insect Cells as a Factory to Produce Adeno-Associated patent is extended or adjusted under 35 Virus Type 2 Vectors. Human Gene Therapy 2002, vol. 13, pp. 1935 U.S.C. 154(b) by 725 days. 1943. Jayaraj et al. GeMS: an advanced software package for designing (21) Appl. No.: 12/670,780 synthetic genes. Nucleic Acids Research 2005, vol. 33, No. 9, pp. 3O11-3016. (22) PCT Filed: Jul. 25, 2008 Hunter et al. Colocalization of adeno-associated virus Rep and capsid proteins in the nuclei of infected cells. Journal of Virology 1992, vol. (86). PCT No.: PCT/NL2008/050512 66, No. 1, pp. 317-324.* Urabe et al., “Insect cells as a factory to produce adeno-associated S371 (c)(1), virus type 2 vectors.” Human Gene Therapy, 2002, 13:1935-1943. (2), (4) Date: Jun. 30, 2010 Kohlbrenner et al., “Successful production of pseudotyped ra AV Vectors using a modified baculovirus expression system. Molecular (87) PCT Pub. No.: WO2009/014445 Therapy, 2005, 12:1217-1225. PCT Pub. Date: Jan. 29, 2009 Office Action Israel (translated to English) in Corresponding Patent Application (203535) dated Feb. 12, 2012. (65) Prior Publication Data A. Villalobos et al., “Gene Designer: a synthetic biology tool for constructing artificial DNA segments”. BMC Bioinformatics 7:285 US 2010/0261254A1 Oct. 14, 2010 (Jun. 6, 2006). J. Meghrous et al., “Production of Recombinant Adeno-Associated Related U.S. Application Data Viral Vectors Using a Baculovirus/Insect Cell Suspension Culture (60) Provisional application No. 60/952,081, filed on Jul. System: From Shake Flasks to a 20-L Bioreactor.” Biotechnol. Prog. 26, 2007. 21:154-160 (2005). D. Hills et al., “Baculovirus expression of human basic fibroblast (30) Foreign Application Priority Data growth factor from a synthetic gene: role of the Kozak consensus and comparison with bacterial expression' Biochimica et Biophysica Jul. 26, 2007 (EP) ...... O7113257 Acta, 1260:14-28 (1995). S. Cecchini et al., “Toward exascale production of recombinant (51) Int. Cl. adeno-associated virus for gene transfer applications. Gene Therapy CI2N 7/00 (2006.01) 15:823-830 (2008). CI2N 7/01 (2006.01) CI2N 7/02 (2006.01) * cited by examiner CI2N 5/10 (2006.01) (52) U.S. Cl. Primary Examiner — Louise Humphrey USPC ...... 435/235.1; 435/348; 435/239; 536/23.72 (74) Attorney, Agent, or Firm — Browdy and Neimark, (58) Field of Classification Search PLLC None See application file for complete search history. (57) ABSTRACT The present invention relates to production of proteins in (56) References Cited insect cells whereby repeated coding sequences are used in baculoviral vectors. In particular the invention relates to the U.S. PATENT DOCUMENTS production of parvoviral vectors that may be used in gene 5,871,974 A * 2/1999 Huse ...... 435/69.7 therapy and to improvements in expression of the viral rep 2003/0228696 A1 12/2003 Robinson et al. proteins that increase the productivity of parvoviral vectors. 2006/0166363 A1* 7/2006 Zolotukhin et al...... 435/456 2009,019 1597 A1 7/2009 Samulski et al. 30 Claims, 5 Drawing Sheets U.S. Patent Apr. 15, 2014 Sheet 1 of 5 US 8,697.417 B2

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U.S. Patent Apr. 15, 2014 Sheet 4 of 5 US 8,697.417 B2

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3 24 S US 8,697,417 B2 1. 2 BACULOVIRAL VECTORS COMPRISING For expression of the AAV Rep proteins in the AAV insect REPEATED CODING SEQUENCES WITH cell expression system as initially developed by Urabe et al. DIFFERENTIAL CODON BASES (2002, Supra), a recombinant baculovirus construct is used that harbours two independent Rep expression units (one for FIELD OF THE INVENTION Rep78 and one for Rep52), each under the control of a sepa rate insect cell promoter, the AIE1 and PolH promoters, The present invention relates to the fields of medicine, respectively. molecular biology, and gene therapy. The invention relates to However, Kohlbrenner et al. (2005, Mol. Ther. 12: 1217 production of proteins in insect cells whereby repeated cod 25: WO 2005/072364) reported that the baculovirus construct ing sequences are used in baculoviral vectors. In particular 10 for expression of the two Rep protein, as used by Urabe et al., the invention relates to the production of parvoviral vectors suffers from an inherent instability. By splitting the palindro that may be used in gene therapy and to improvements in mic orientation of the two Rep genes in Urabe's original expression of the viral rep proteins that increase the produc vector and designing two separate baculovirus vectors for tivity of parvoviral vectors. expressing Rep52 and Rep78, Kohlbrenner et al. (2005, 15 Supra) increased the passaging stability of the vector. How BACKGROUND OF THE INVENTION ever, despite the consistent expression of Rep78 and Rep52 from the two independent baculovirus-Rep constructs in The baculovirus expression system is well known for its insect cells over at least 5 passages, ra AV vectoryield is 5 to use as eukaryotic cloning and expression vector (King, L.A., 10-fold lower as compared to the original baculovirus-Rep and R. D. Possee, 1992, “The baculovirus expression sys construct designed by Urabe et al. (2002, supra). tem'. Chapman and Hall, United Kingdom; O'Reilly, D. R. In WO2007/148971 the present inventors have signifi et al., 1992. Baculovirus Expression Vectors: A Laboratory cantly improved the stability of ra AV vector production in Manual. New York: W.H. Freeman.). Advantages of the bacu insect cells by using a single coding sequence for the Rep78 lovirus expression system are among others that the and Rep52 proteins wherein a suboptimal initiator codon is expressed proteins are almost always soluble, correctly 25 used for the Rep78 protein that is partially skipped by the folded and biologically active. Further advantages include scanning ribosomes to allow for initiation of translation to high protein expression levels, faster production, Suitability also occur further downstream at the initiation codon of the for expression of large proteins and Suitability for large-scale Rep52 protein. production. However, in large-scale or continuous production There is however still a need for further improvements in of heterologous proteins using the baculovirus expression 30 large scale (commercial) production of heterologous pro system in insect cell bioreactors, the instability of production teins, including ra AV vectors, in insect cells. Thus it is an levels, also known as the passage effect, is a major obstacle. object of the present invention to provide for means and This effect is at least in part due to recombination between methods that allow for stable and high yield (large scale) repeated homologous sequences in the baculoviral DNA. production of heterologous proteins and parvoviral vectors. The baculovirus expression system has also successfully 35 been used for the production of recombinant Adeno-associ DESCRIPTION OF THE INVENTION ated virus (AAV) vectors (Urabe et al., 2002, Hum. Gene Ther. 13: 1935-1943: U.S. Pat. No. 6,723,551 and US Definitions 20040197895). AAV may be considered as one of the most As used herein, the term “operably linked’ refers to a promising viral vectors for human gene therapy. AAV has the 40 linkage of polynucleotide (or polypeptide) elements in a ability to efficiently infect dividing as well as non-dividing functional relationship. A nucleic acid is “operably linked' human cells, the AAV viral genome integrates into a single when it is placed into a functional relationship with another chromosomal site in the host cells genome, and most impor nucleic acid sequence. For instance, a transcription regula tantly, even though AAV is present in many humans it has tory sequence is operably linked to a coding sequence if it never been associated with any disease. In view of these 45 affects the transcription of the coding sequence. Operably advantages, recombinant adeno-associated virus (ra AV) is linked means, that the DNA sequences being linked are typi being evaluated in gene therapy clinical trials for hemophilia cally contiguous and, where necessary to join two protein B, malignant melanoma, cystic fibrosis, hyperlipoproteine encoding regions, contiguous and in reading frame. mia type I and other diseases. "Expression control sequence” refers to a nucleic acid To overcome problems with mammalian productions sys 50 sequence that regulates the expression of a nucleotide tems for AAV Urabe et al. (2002, supra) developed an AAV sequence to which it is operably linked. An expression control production system in insect cells. For production of AAV in sequence is "operably linked to a nucleotide sequence when insect cells some modifications were necessary in order to the expression control sequence controls and regulates the achieve the correct stoichiometry of the three AAV capsid transcription and/or the translation of the nucleotide proteins (VP1,VP2 and VP3), which relies on a combination 55 sequence. Thus, an expression control sequence can include of alternate usage of two splice acceptor sites and the Subop promoters, enhancers, internal ribosome entry sites (IRES), timal utilization of an ACG initiation codon for VP2 that is not transcription terminators, a start codon in front of a protein accurately reproduced by insect cells. To mimic the correct encoding gene, splicing signal for introns, and stop codons. stoichiometry of the capsid proteins in insect cells Urabeet al. The term “expression control sequence' is intended to (2002, Supra) use a construct that is transcribed into a single 60 include, at a minimum, a sequence whose presence are polycistronic messenger that is able to express all three VP designed to influence expression, and can also include addi proteins without requiring splicing and wherein the most tional advantageous components. For example, leader upstream initiator codon is replaced by the Suboptimal initia sequences and fusion partner sequences are expression con tor codon ACG. WO2007/046703 discloses further improve trol sequences. The term can also include the design of the ment of the infectivity ofbaculovirus-producedra AV vectors 65 nucleic acid sequence Such that undesirable, potential initia based production by optimisation of the stoichiometry of tion codons in and out of frame, are removed from the AAV capsid proteins as produced in insect cells. sequence. It can also include the design of the nucleic acid US 8,697,417 B2 3 4 sequence Such that undesirable potential splice sites are other solution having a comparable ionic strength, and wash removed. It includes sequences or polyadenylation sequences ing at 65°C. in a solution comprising about 0.1 M salt, or less, (pA) which direct the addition of a polyA tail, i.e., a string of preferably 0.2xSSC or any other solution having a compa adenine residues at the 3'-end of a mRNA, sequences referred rable ionic strength. Preferably, the hybridisation is per to as polyA sequences. It also can be designed to enhance formed overnight, i.e. at least for 10 hours and preferably mRNA stability. Expression control sequences which affect washing is performed for at least one hour with at least two the transcription and translation stability, e.g., promoters, as changes of the washing solution. These conditions will usu well as sequences which effect the translation, e.g., Kozak ally allow the specific hybridisation of sequences having sequences, are known in insect cells. Expression control about 90% or more sequence identity. sequences can be of such nature as to modulate the nucleotide 10 Moderate conditions are herein defined as conditions that sequence to which it is operably linked such that lower allow a nucleic acid sequences of at least 50 nucleotides, expression levels or higher expression levels are achieved. preferably of about 200 or more nucleotides, to hybridise at a As used herein, the term “promoter” or “transcription regu temperature of about 45°C. in a solution comprising about 1 latory sequence” refers to a nucleic acid fragment that func M salt, preferably 6xSSC or any other solution having a tions to control the transcription of one or more coding 15 comparable ionic strength, and washing at room temperature sequences, and is located upstream with respect to the direc in a solution comprising about 1 M salt, preferably 6xSSC or tion of transcription of the transcription initiation site of the any other solution having a comparable ionic strength. Pref coding sequence, and is structurally identified by the presence erably, the hybridisation is performed overnight, i.e. at least of a binding site for DNA-dependent RNA polymerase, tran for 10 hours, and preferably washing is performed for at least Scription initiation sites and any other DNA sequences, one hour with at least two changes of the washing solution. including, but not limited to transcription factor binding sites, These conditions will usually allow the specific hybridisation repressor and activator protein binding sites, and any other of sequences having up to 50% sequence identity. The person sequences of nucleotides known to one of skill in the art to act skilled in the art will be able to modify these hybridisation directly or indirectly to regulate the amount of transcription conditions in order to specifically identify sequences varying from the promoter. A “constitutive' promoter is a promoter 25 in identity between 50% and 90%. that is active in most tissues under most physiological and developmental conditions. An “inducible' promoter is a pro DETAILED DESCRIPTION OF THE INVENTION moter that is physiologically or developmentally regulated, e.g. by the application of a chemical inducer. A “tissue spe In some aspects the present invention relates the use of cific' promoter is only active in specific types of tissues or 30 animal parvoviruses, in particular dependoviruses such as cells. infectious human or simian AAV, and the components thereof The terms “substantially identical”, “substantial identity” (e.g., an animal parvovirus genome) for use as vectors for or “essentially similar or “essential similarity” means that introduction and/or expression of nucleic acids in mamma two peptide or two nucleotide sequences, when optimally lian cells. In particular, the invention relates to improvements aligned, such as by the programs GAP or BESTFIT using 35 in productivity of such parvoviral vectors when produced in default parameters, share at least a certain percentage of insect cells. sequence identity as defined elsewhere herein. GAP uses the Viruses of the Parvoviridae family are small DNA animal Needleman and Wunsch global alignment algorithm to align viruses. The family Parvoviridae may be divided between two two sequences over their entire length, maximizing the num subfamilies: the Parvovirinae, which infect vertebrates, and ber of matches and minimizes the number of gaps. Generally, 40 the Densovirinae, which infect insects. Members of the sub the GAP default parameters are used, with a gap creation family Parvovirinae are herein referred to as the parvoviruses penalty 50 (nucleotides)/8 (proteins) and gap extension pen and include the genus Dependovirus. As may be deduced alty-3 (nucleotides)/2 (proteins). For nucleotides the default from the name of their genus, members of the Dependovirus scoring matrix used is nwsgapdna and for proteins the default are unique in that they usually require coinfection with a scoring matrix is Blosumó2 (Henikoff & Henikoff, 1992, 45 helper virus such as adenovirus or herpesvirus for productive PNAS 89,915-919). It is clear than when RNA sequences are infection in cell culture. The genus Dependovirus includes said to be essentially similar or have a certain degree of AAV, which normally infects humans (e.g., serotypes 1, 2, sequence identity with DNA sequences, thymine (T) in the 3A, 3B, 4, 5, and 6) or primates (e.g., serotypes 1 and 4), and DNA sequence is considered equal to uracil (U) in the RNA related viruses that infect other warm-blooded animals (e.g., sequence. Sequence alignments and scores for percentage 50 bovine, canine, equine, and Ovine adeno-associated viruses). sequence identity may be determined using computer pro Further information on parvoviruses and other members of grams, such as the GCG Wisconsin Package, Version 10.3, the Parvoviridae is described in Kenneth I. Berns, "Parvoviri available from Accelrys Inc., 9685 Scranton Road, San dae: The Viruses and Their Replication. Chapter 69 in Fields Diego, Calif. 92121-3752 USA or the open-source software Virology (3d Ed. 1996). For convenience the present inven Emboss for Windows (current version 2.7.1-07). Alterna 55 tion is further exemplified and described herein by reference tively percent similarity or identity may be determined by to AAV. It is however understood that the invention is not searching against databases Such as FASTA, BLAST, etc. limited to AAV but may equally be applied to other parvovi Nucleotide sequences encoding parvoviral Rep proteins of USS. the invention may also be defined by their capability to hybri The genomic organization of all known AAV serotypes is dise with the nucleotide sequences of SEQ ID NO.'s 1-4, 60 very similar. The genome of AAV is a linear, single-stranded respectively, under moderate, or preferably under stringent DNA molecule that is less than about 5,000 nucleotides (nt) in hybridisation conditions. Stringent hybridisation conditions length. Inverted terminal repeats (ITRs) flank the unique cod are herein defined as conditions that allow a nucleic acid ing nucleotide sequences for the non-structural replication sequence of at least about 25, preferably about 50 nucle (Rep) proteins and the structural (VP) proteins. The VP pro otides, 75 or 100 and most preferably of about 200 or more 65 teins (VP1, -2 and -3) form the capsid. The terminal 145 nt are nucleotides, to hybridise at a temperature of about 65°C. in a self-complementary and are organized so that an energeti solution comprising about 1 M salt, preferably 6xSSC or any cally stable intramolecular duplex forming a T-shaped hairpin US 8,697,417 B2 5 6 may beformed. These hairpin structures function as an origin this will be a cell of the genus Spodoptera, more preferably a for viral DNA replication, serving as primers for the cellular Spodoptera frugiperda cell. The codon usage is thus prefer DNA polymerase complex. Following witAAV infection in ably adapted to Spodoptera frugiperda or to an Autographa mammalian cells the Rep genes (i.e. Rep78 and Rep52) are californica nucleopolyhedrovirus (AcMNPV) infected cell. expressed from the P5 promoter and the P19 promoter, A codon adaptation index is herein defined as a measurement respectively and both Rep proteins have a function in the of the relative adaptiveness of the codon usage of a gene replication of the viral genome. A splicing event in the Rep towards the codon usage of highly expressed genes. The ORF results in the expression of actually four Rep proteins relative adaptiveness (w) of each codon is the ratio of the (i.e. Rep78, Rep68, Rep52 and Rep40). However, it has been usage of each codon, to that of the most abundant codon for shown that the unspliced mRNA, encoding Rep78 and Rep52 10 the same amino acid. The CAI index is defined as the geo proteins, in mammalian cells are sufficient for AAV vector metric mean of these relative adaptiveness values. Non-syn production. Also in insect cells the Rep78 and Rep52 proteins onymous codons and termination codons (dependent on suffice for AAV vector production. genetic code) are excluded. CAI values range from 0 to 1, A “recombinant parvoviral or AAV vector' (or “ra AV with higher values indicating a higher proportion of the most vector') herein refers to a vector comprising one or more 15 abundant codons (see Sharp and Li, 1987, Nucleic Acids polynucleotide sequences of interest, genes of interest or Research 15: 1281-1295; also see: Kim et al., Gene. 1997, “transgenes” that are flanked by parvoviral or AAV inverted 199:293-301; Zur Megede et al., Journal of Virology, 2000, terminal repeat sequences (ITRs). Such ra AV vectors can be 74: 2628-2635). In a preferred insect cell the difference in replicated and packaged into infectious viral particles when CAI between the nucleotide sequence coding for the common present in an insect host cell that is expressing AAV rep and amino acid sequence in the first and second nucleotide cap gene products (i.e. AAV Rep and Cap proteins). When an sequence at least 0.1, 0.2,0.3, 0.4,0.5,0.6, 0.7, 0.8. Prefer rAAV vector is incorporated into a larger nucleic acid con ably, in addition the CAI of the nucleotide sequence coding struct (e.g. in a chromosome or in another vector Such as a for the common amino acid sequence in the first nucleotide plasmid or baculovirus used for cloning or transfection), then sequence is at least 0.5,0.6, 0.7, 0.8, 0.9 or 1.0. the rAAV vector is typically referred to as a “pro-vector 25 A preferred nucleotide sequence coding for the common which can be “rescued by replication and encapsidation in amino acid sequence in the first nucleotide sequence is a the presence of AAV packaging functions and necessary coding sequence wherein at least 50, 75,90, 95, 98 or 99%, helper functions. and preferably all of the non-common codons or less-com In a first aspect the invention relates to an insect cell. The mon codons are replaced with a common codon encoding the insect cell comprises at least a first nucleotide sequence cod 30 same amino acid as outlined in Table 1 or in Table 2. A ing for a first amino acid sequence and a second nucleotide common codon is herein understood to be the most frequently sequence coding for a second amino acid sequence. Prefer used codon encoding each particular amino acid residue in ably, the first and secondamino acid sequences each comprise highly expressed Spodoptera frugiperda genes as shown in a common amino acid sequence of at least 50, 80, 100, 200, Table 1 or in highly expressed genes Autographa Californica 300,350 or 398 amino acids with at least 80, 85,90, 95, 98, 99 35 MNPV infected cells as shown in Table 2. All codons other or 100% amino acid identity between the first and second than common codons and less-common codons are “non amino acid sequences. In contrast, the nucleotide sequences common codons'. The non-common codons include the 'sec that encode the common amino acid sequences in the first and ond most frequent codons', which are understood as codons second amino acid sequences (as present in the first and having the one but highest frequency in Table 1 or Table 2. second nucleotide sequences, respectively) are less than 95. 40 Preferably the nucleotide sequence coding for the common 90, 89, 88.4, 85, 80, 75, 70, 65, 60, or 55% identical. amino acid sequence in the first nucleotide sequence has a Usually the first and second amino acid sequences will be continuous stretch of at least 25, 50, 100, 200 or 300 codons heterologous to the insect cell. Preferably at least one of the all of which are common codons. The coding sequence may common amino acid sequences in the first and second amino further be adapted for improved expression in the insect host acid sequences is a naturally occurring amino acid sequence. 45 cell by methods described in WO 2004/059556, and by modi More preferably, at least one of the first and second amino fying the CpG content of the coding sequence as described in acid sequences is naturally occurring amino acid sequences. WO 2006/015789. It is understood that such further adapta Most preferably, both of the first and second amino acid tions may cause that not all codons in the nucleotide sequence sequences are naturally occurring amino acid sequences. coding for the common amino acid sequence in the first In a preferred embodiment, the nucleotide sequence cod 50 nucleotide sequence are common codons. ing for the common amino acid sequence in the first nucle In a preferred embodiment of the insect cell all codons in otide sequence has an improved codon usage bias for the the nucleotide sequence coding for the common amino acid insect cell as compared to the nucleotide sequence coding for sequence in the first nucleotide sequence are common codons the common amino acid sequence in the second nucleotide in accordance with (either one of) Tables 1 or 2. More pref sequence. It is understood herein that whenever reference is 55 erably in Such an insect cell, all codons in the nucleotide made to codon usage bias for an insect cell, this includes sequence coding for the common amino acid sequence in the codon usage bias for a baculovirus infected insect cell, second nucleotide sequence are second most frequent codons including in particular codon usage bias for an Autographa in accordance with (either one of) Tables 1 or 2, whereby it is californica multiple nucleopolyhedrovirus (AcMNPV) understood that if in the first nucleotide sequence the com infected cell. The codon usage of the first nucleotide sequence 60 mon codons are in accordance with Table 1, the second most encoding the common amino acid sequence preferably is frequent codons in the second nucleotide sequence are also in adapted or optimized to the codon usage of the insect host accordance with Table 1, or that if in the first nucleotide cell. The adaptiveness of a nucleotide sequence encoding the sequence the common codons are in accordance with Table 2. common amino acid sequence to the codon usage of the host the second most frequent codons in the second nucleotide cell may be expressed as codon adaptation index (CAI). Pref 65 sequence are also in accordance with Table 2. erably the codon usage is adapted to the insect cell in which Codon optimization may be performed on the basis of the the first and second nucleotide sequence are present. Usually codon usage of the Spodoptera frugiperda organism as may US 8,697,417 B2 7 8 be found in a codon usage database (see e.g., WorldWideWeb ated virus (AAV) and most preferably from an AAV which URL. kazusa.or.jp/codon/). Suitable computer programs for normally infects humans (e.g., serotypes 1, 2, 3A, 3B, 4, 5, codon optimization are available to the skilled person (see, and 6) or primates (e.g., serotypes 1 and 4). An example of a e.g., Jayaraj et al., 2005, Nucl. Acids Res. 33(9):3011-3016: nucleotide sequence encoding animal parvoviruses Rep pro and on the internet). Alternatively the optimizations can be teins is given in SEQ ID No. 7, which depicts a part of the done by hand, using the same codon usage database. AAV serotype-2 sequence genome encoding the Rep pro In one embodiment of the insect cell of the invention, at teins. The Rep78 coding sequence comprises nucleotides least 50, 60, 80, 90 or 100% of the codons in the nucleotide 11-1876 and the Rep52 coding sequence comprises nucle sequence coding for the common amino acid sequence in the otides 683-1876, also depicted separately in SEQ ID No. 1 second nucleotide sequence are altered compared to the cor 10 and 5. It is understood that the exact molecular weights of the responding codon in the first nucleotide sequence to maxi Rep78 and Rep52 proteins, as well as the exact positions of mise the AT- or GC-content of the second nucleotide the translation initiation codons may differ between different Sequence. parvoviruses. However, the skilled person will know how to Thus, in a preferred embodiment of the invention, the dif identify the corresponding position in nucleotide sequence ference in nucleotide sequence between the first and second 15 from other parvoviruses than AAV-2. nucleotide sequence coding for the common amino acid A (first) nucleotide sequence encoding a parvoviral Rep52 sequences is maximised (i.e. the nucleotide identity is mini protein may thus also be defined as a nucleotide sequence: mised) by one or more of: a) changing the codon bias of the a) that encodes a polypeptide comprising an amino acid first nucleotide sequence coding for the common amino acid sequence that has at least 50, 60, 70, 80, 88, 89,90, 95.97, sequence; b) changing the codon bias of the second nucle 98, or 99% sequence identity with the amino acid sequence otide sequence coding for the common amino acid sequence; of SEQID NO. 6; c) changing the GC-content of the first nucleotide sequence b) that has at least 50, 60, 70, 80, 81, 82, 85,90, 95, 97,98, or coding for the common amino acid sequence; and d) chang 99% sequence identity with the nucleotide sequence of any ing the GC-content of the second nucleotide sequence coding one of SEQID NO.'s 1-5 and 10; for the common amino acid sequence. 25 c) the complementary strand of which hybridises to a nucleic A preferred embodiment of the invention of the insect cell, acid molecule sequence of (a) or (b): relates to the production of parvoviral proteins in the insect d) nucleotide sequences the sequence of which differs from cells of the invention. In particular the parvoviral proteins are the sequence of a nucleic acid molecule of (c) due to the produced in the insect cells in the context of producing degeneracy of the genetic code. recombinant parvoviral vectors, more preferably recombi 30 A (second) nucleotide sequence encoding a parvoviral nant animal parvoviral vectors, and most preferably recom Rep78 protein may thus also be defined as a nucleotide binant AAV vectors. Therefore, in this preferred embodiment sequence: of the insect cells of the invention, the first nucleotide a) that encodes a polypeptide comprising an amino acid sequence encodes an amino acid sequence of a parvoviral sequence that has at least 50, 60, 70, 80, 88, 89,90, 95.97, Rep52 or 40 protein and the second nucleotide sequence 35 98, or 99% sequence identity with the amino acid sequence encodes an amino acid sequence of a parvoviral Rep78 or 68 of SEQID NO. 8: protein. It is understood however that embodiments wherein b) that has at least 50, 60, 70, 80, 81, 82, 85,90, 95, 97,98, or the first nucleotide sequence encodes an amino acid sequence 99% sequence identity with the nucleotide sequence of of a parvoviral Rep78 or 68 protein and the second nucleotide positions 11-1876 of SEQID NO. 7: sequence encodes an amino acid sequence of a parvoviral 40 c) the complementary strand of which hybridises to a nucleic Rep52 or 40 protein are expressly included in the invention. acid molecule sequence of (a) or (b): For convenience in the embodiments we shall use the nucle d) nucleotide sequences the sequence of which differs from otide sequence encoding a parvoviral Rep52 or 40 protein as the sequence of a nucleic acid molecule of (c) due to the first nucleotide sequence and the nucleotide sequence encod degeneracy of the genetic code. ing a parvoviral Rep78 or 68 protein as second nucleotide 45 Preferably, the nucleotide sequence encodes animal parvovi sequence but the reverse of these embodiments is expressly ruses Rep proteins that are required and Sufficient for parvovi included in the invention. The common amino acid sequence ral vector production in insect cells. encoded by the first and second nucleotide sequences com The various modifications of the first and second coding prise or consists of the amino acid sequences from at least the nucleotide sequence as defined above, including e.g. the wild second amino acid to the most C-terminal amino acid of a 50 type parvoviral sequences, for proper expression in insect parvoviral Rep52 or 40 protein. Preferably the common cells is achieved by application of well-known genetic engi amino acid sequences comprise or consist of the first amino neering techniques such as described e.g. in Sambrook and acid to the most C-terminal amino acid of the parvoviral Russell (2001) “Molecular Cloning: A Laboratory Manual Rep52 or 40 protein. The amino acid identities between the (3rd edition), Cold Spring Harbor Laboratory, Cold Spring parvoviral common amino acid sequences are as defined 55 Harbor Laboratory Press, New York. Various further modifi above for the common amino acid sequences. Preferably, in cations of coding regions are known to the skilled artisan the insect cell, the parvoviral Rep proteins are adeno-associ which could increase yield of the encode proteins. These ated virus (AAV) Rep proteins. More preferably, the parvovi modifications are within the scope of the present invention. ral Rep proteins encoded in the first and second nucleotide In the insect cells of the invention the first and second sequences are of the same serotype. 60 nucleotide sequences are preferably part of a nucleic acid A nucleotide sequence encoding parvoviral Rep proteins, construct. The insect cell may comprise two separate nucleic is herein understood as a nucleotide sequence encoding the acid constructs, one for each of the first and second nucleotide non-structural Rep proteins that are required and Sufficient for sequences, or the insect cell may comprise a single type of parvoviral vector production in insect cells such the Rep78 or nucleic acid construct comprising both the first and second Rep68, and the Rep52 or Rep40 proteins. The animal par 65 nucleotide sequences. vovirus nucleotide sequence preferably is from a dependovi In a further aspect the invention relates to a nucleic acid rus, more preferably from a human or simian adeno-associ construct comprising a first and/or a second nucleotide US 8,697,417 B2 9 10 sequence coding for a first and a second amino acid sequence, lovirus, a viral vector, or a plasmid. In a more preferred respectively, that comprise a common amino acid sequence as embodiment, the vector is abaculovirus, i.e. the construct is a defined above. Preferably first and/or a second nucleotide baculoviral vector. Baculoviral vectors and methods for their sequences in the construct encode parvoviral Rep proteins as use are described in the above cited references on molecular defined above. Preferably, in the construct, the nucleotide engineering of insect cells. sequence encoding the first and second amino acid sequences The nucleic acid constructs of the invention may further are operably linked to expression control sequences for comprise an expression control sequence comprising a nine expression in an insect cell. These expression control nucleotide sequence of SEQ. ID NO: 9 or a nucleotide sequences will at least include a promoter that is active in sequence substantially homologous to SEQ. ID NO: 9. insect cells. Techniques known to one skilled in the art for 10 upstream of the initiation codons of the nucleotide sequence expressing foreign genes in insect host cells can be used to encoding the first and/or second amino acid sequences. A practice the invention. Methodology for molecular engineer sequence with Substantial identity to the nucleotide sequence ing and expression of polypeptides in insect cells is described, of SEQ.IDNO:9 and that will help increase expression of the for example, in Summers and Smith. 1986. A Manual of first and/or second amino acid sequences is e.g. a sequence Methods for Baculovirus Vectors and Insect Culture Proce 15 which has at least 60%, 70%, 80% or 90% identity to the nine dures, Texas Agricultural Experimental Station Bull. No. nucleotide sequence of SEQID NO: 9. 7555, College Station, Tex.; Luckow. 1991. In Prokop et al., The insect cell may be any cell that is suitable for the Cloning and Expression of Heterologous Genes in Insect production of heterologous proteins. Preferably the insect Cells with Baculovirus Vectors’ Recombinant DNA Technol cell allows for replication of baculoviral vectors and can be ogy and Applications, 97-152; King, L.A. and R. D. Possee, maintained in culture. More preferably the insect cell also 1992. The baculovirus expression system, Chapman and allows for replication of recombinant parvoviral vectors, Hall, United Kingdom; O'Reilly, D. R. L. K. Miller, V. A. including ra AV vectors. For example, the cell line used can Luckow, 1992, Baculovirus Expression Vectors: A Labora be from Spodoptera frugiperda, Drosophila cell lines, or tory Manual, New York; W.H. Freeman and Richardson, C. mosquito cell lines, e.g., Aedes albOpictus derived cell lines. D., 1995, Baculovirus Expression Protocols, Methods in 25 Preferred insect cells or cell lines are cells from the insect Molecular Biology, volume 39; U.S. Pat. No. 4,745,051: species which are Susceptible to baculovirus infection, US2003148506; and WO 03/074714. Suitable promoters for including e.g. Se301, SelZD2109, SeUCR1, Sf9, S1900+, transcription of the first and second nucleotide sequences of Sf21, BTI-TN-5B1-4, MG-1, Tn368, HZAm1, Ha2302, the invention include e.g. the polyhedron (PolH), p.10, p35, HZ2E5, High Five (Invitrogen, CA, USA) and expressF+(R) IE-1 or AIE-1 promoters and further promoters described in 30 (U.S. Pat. No. 6,103,526: Protein Sciences Corp., CT, USA). the above references. Since it is known that in mammalian A preferred insect cell according to the invention is an cells a less abundant expression of Rep78 as compared to insect cell for production of recombinant parvoviral vectors. Rep52 favours high vector yields (Liet al., 1997, J. Virol. 71: This insect cell further comprises, in addition to the above 5236-43; Grimm et al., 1998, Hum Gene Ther. 9, 2745-2760), described “first and “second nucleotide sequences or a preferably a weaker promoter is used for driving expression 35 nucleic acid constructs the first and second nucleotide of the Rep78 or 68 protein than the promoter used for expres Sequences: sion of the Rep52 or 40 protein. E.g. the stronger polyhedron a) a third nucleotide sequence comprising at least one par promoter may be used for expression of the Rep52 or 40 voviral inverted terminal repeat (ITR) nucleotide protein, the AIE1 promoter, a much weaker promoter than the sequence; and, PolH promoter, may be chosen for driving expression of the 40 b) a fourth nucleotide sequence comprising parvoviral Cap Rep78 or 68 protein. Preferably, the choice of promoters for protein coding sequences operably linked to expression the Rep52 or 40 protein and Rep78 or 68 protein, respectively, control sequences for expression in an insect cell. is such that in an insect cell so as to produce in the insect cell In the context of the invention “at least one parvoviral ITR a molar ratio of Rep78/68 to Rep52/40 in the range of 1:10 to nucleotide sequence' is understood to mean a palindromic 10:1, 1:5 to 5:1, or 1:3 to 3:1, preferably at about 20-40 hours 45 sequence, comprising mostly complementary, symmetrically post infection, more preferably at about 30-40 hours post arranged sequences also referred to as “A.” “B,” and “C” infection, using a baculovirus expression. The molar ratio of regions. The ITR functions as an origin of replication, a site the Rep78 and Rep52 may be determined by means of West having a “cis' role in replication, i.e., being a recognition site ern blotting, preferably using a monoclonal antibody that for trans acting replication proteins such as e.g. Rep. 78 (or recognizes a common epitope of both Rep78/68 and Rep52/ 50 Rep68) which recognize the palindrome and specific 40, or using e.g. a mouse anti-Rep antibody (303.9. Progen, sequences internal to the palindrome. One exception to the Germany; dilution 1:50). symmetry of the ITR sequence is the "D' region of the ITR. Preferably the nucleic acid construct for expression of the It is unique (not having a complement within one ITR). Nick first and second nucleotide sequences of the invention in ing of single-stranded DNA occurs at the junction between insect cells is an insect cell-compatible vector. An "insect 55 the A and Dregions. It is the region where new DNA synthesis cell-compatible vector' or “vector” is understood to be a initiates. The D region normally sits to one side of the palin nucleic acid molecule capable of productive transformation drome and provides directionality to the nucleic acid replica or transfection of an insector insect cell. Exemplary biologi tion step. An parvovirus replicating in a mammalian cell cal vectors include plasmids, linear nucleic acid molecules, typically has two ITR sequences. It is, however, possible to and recombinant viruses. Any vector can be employed as long 60 engineer an ITR so that binding sites are on both strands of the as it is insect cell-compatible. The vector may integrate into A regions and D regions are located symmetrically, one on the insect cells genome but the vector may also be episomal. each side of the palindrome. On a double-stranded circular The presence of the vector in the insect cell need not be DNA template (e.g., a plasmid), the Rep78- or Rep68-as permanent and transient episomal vectors are also included. sisted nucleic acid replication then proceeds in both direc The vectors can be introduced by any means known, for 65 tions and a single ITR suffices for parvoviral replication of a example by chemical treatment of the cells, electroporation, circular vector. Thus, one ITR nucleotide sequence can be or infection. In a preferred embodiment, the vector is a bacu used in the context of the present invention. Preferably, how US 8,697,417 B2 11 12 ever, two or another even number of regular ITRs are used. herein understood to comprises sequences encoding each of Most preferably, two ITR sequences are used. A preferred the three parvoviral capsid proteins, VP1, -2 and -3. The parvoviral ITR is an AAV ITR. For safety reasons it may be fourth nucleotide sequence comprising the capsid protein desirable to construct a recombinant parvoviral (ra AV) vec coding sequences may be present in various forms. E.g. sepa tor that is unable to further propagate after initial introduction 5 rate coding sequences for each of the capsid proteins VP1, -2 into a cell in the presence of a second AAV. Such a safety and -3 may used, whereby each coding sequence is operably mechanism for limiting undesirable vector propagation in a linked to expression control sequences for expression in an recipient may be provided by using ra AV with a chimeric insect cell. More preferably, however, the fourth nucleotide ITR as described in US2003148506. sequence comprises a single open reading frame encoding all The number of nucleic acid constructs employed in the 10 three of the animal parvoviral (AAV) VP1,VP2, and VP3 insect cell for the production of the recombinant parvoviral capsid proteins, wherein the initiation codon for translation of (ra AV) vector is not limiting in the invention. For example, the VP1 capsid protein is a suboptimal initiation codon that is one, two, three, four, five, or more separate constructs can be not ATG as e.g. described by Urabe et al. (2002, supra) and in employed to producer AAV in insect cells inaccordance with WO2007/046703. The suboptimal initiation codon for the the methods of the present invention. If five constructs are 15 VP1 capsid protein may be selected from ACG, TTG, CTG employed, one construct encodes AAV VP1, another con and GTG, of which CTG and GTG are most preferred. The struct encodes AAVVP2, yet another construct encodes AAV fourth nucleotide sequence for expression of the capsid pro VP3, still yet another construct encodes the Rep protein as teins may further comprises at one or modifications as defined above and a final construct comprises at least one described in WO2007/046703. AAV ITR. If fewer than five constructs are used, the con Various further modifications of VP coding regions are structs can comprise various combinations of the at least one known to the skilled artisan which could either increase yield AAVITR and the VP1,VP2, VP3, and the Rep protein coding of VP and virion or have other desired effects, such as altered Sequences. tropism or reduce antigenicity of the virion. These modifica Preferably, two, three or four constructs are used. If two tions are within the scope of the present invention. Preferably constructs are used, preferably the insect cell comprises: (A) 25 the nucleotide sequence of the invention encoding the par a first nucleic acid construct for expression of the Rep pro voviral capsid proteins is operably linked to expression con teins as defined above, which construct further comprises the trol sequences for expression in an insect cell, which will at fourth nucleotide sequences as defined in (b) above (compris least include a promoter that is active in insect cells. Such ing parvoviral Cap protein coding sequences operably linked control sequences and further techniques and materials (e.g. to at least one expression control sequence for expression in 30 vectors) for expressing parvoviral capsid proteins in insect an insect cell; see also below); and (B) a third nucleic acid host cells are already described above for the Rep proteins. construct comprising the third nucleotide sequence as defined In a preferred embodiment of the invention, the third nucle in (a) above (comprising at least one parvoviral/AAV, ITR otide sequence present in the insect cells of the invention, i.e. nucleotide sequence). If three constructs are used, preferably the sequence comprising at least one parvoviral (AAV) ITR. the same configuration as used for two constructs is used 35 further comprises at least one nucleotide sequence encoding except that separate constructs are used for expression of the a gene product of interest, whereby preferably the at least one capsid proteins and for expression of the Rep proteins. If four nucleotide sequence encoding a gene product of interest constructs are used, preferably the same configuration as used becomes incorporated into the genome of a recombinant par for three constructs is used except that separate constructs are voviral (ra AV) vector produced in the insect cell. Preferably, used for expression of the Rep78/68 proteins and for expres 40 at least one nucleotide sequence encoding a gene product of sion of the Rep 52/40 proteins. The sequences on each con interest is a sequence for expression in a mammalian cell. struct can be in any order relative to each other. For example, Preferably, the third nucleotide sequence comprises two par if one construct comprises ITRS and an ORF comprising voviral (AAV) ITR nucleotide sequences and wherein the at nucleotide sequences encoding VP capsid proteins, the VP least one nucleotide sequence encoding a gene product of ORF can be located on the construct such that, upon replica 45 interest is located between the two parvoviral (AAV) ITR tion of the DNA between ITR sequences, the VP ORF is nucleotide sequences. Preferably, the nucleotide sequence replicated or not replicated. For another example, the Rep encoding a gene product of interest (for expression in the coding sequences and/or the ORF comprising nucleotide mammalian cell) will be incorporated into the recombinant sequences encoding VP capsid proteins can be in any order on parvoviral (ra AV) vector produced in the insect cell if it is a construct. It is understood that also the third, fourth and 50 located between two regular ITRs, or is located on either side further nucleic acid construct(s) preferably are an insect cell of an ITR engineered with two D regions. compatible vectors, preferably a baculoviral vectors as The third nucleotide sequence defined herein above may described above. Alternatively, in the insect cell of the inven thus comprise a nucleotide sequence encoding at least one tion, one or more of the first nucleotide sequence, third nucle 'gene product of interest' for expression in a mammalian otide sequence, fourth nucleotide sequence, and fifth nucle 55 cell, located such that it will be incorporated into an recom otide sequence and optional further nucleotide sequences binant parvoviral (ra AV) vector replicated in the insect cell. may be stably integrated in the genome of the insect cell. One Any nucleotide sequence can be incorporated for later expres of ordinary skill in the art knows how to stably introduce a sion in a mammalian cell transfected with the recombinant nucleotide sequence into the insect genome and how to iden parvoviral (ra AV) vector produced in accordance with the tify a cell having Such a nucleotide sequence in the genome. 60 present invention. The nucleotide sequence may e.g. encode a The incorporation into the genome may be aided by, for protein it may express an RNAi agent, i.e. an RNA molecule example, the use of a vector comprising nucleotide sequences that is capable of RNA interference such as e.g. a shRNA highly homologous to regions of the insect genome. The use (short hairpinRNA) or an siRNA (short interfering RNA). of specific sequences, such as transposons, is another way to “siRNA means a small interfering RNA that is a short-length introduce a nucleotide sequence into a genome. 65 double-stranded RNA that are not toxic in mammalian cells In the invention, the fourth nucleotide sequence compris (Elbashiret al., 2001, Nature 411: 494-98: Caplenet al., 2001, ing parvoviral capsid (Cap) protein coding sequences is Proc. Natl. Acad. Sci. USA 98: 9742-47). In a preferred US 8,697,417 B2 13 14 embodiment, the third nucleotide sequence may comprise In the recombinant parvoviral (ra AV) vectors of the inven two nucleotide sequences and each encodes one gene product tion the at least one nucleotide sequence(s) encoding a gene of interest for expression in a mammalian cell. Each of the product of interest for expression in a mammalian cell, pref two nucleotide sequences encoding a product of interest is erably is/are operably linked to at least one mammalian cell located such that it will be incorporated into a recombinant compatible expression control sequence, e.g., a promoter. parvoviral (ra AV) vector replicated in the insect cell. Many such promoters are known in the art (see Sambrook and The product of interest for expression in a mammalian cell Russel, 2001, supra). Constitutive promoters that are broadly may be a therapeutic gene product. A therapeutic gene prod expressed in many cell-types. Such as the CMV promoter may uct can be a polypeptide, or an RNA molecule (siRNA), or be used. However, more preferred will be promoters that are 10 inducible, tissue-specific, cell-type-specific, or cell cycle other gene product that, when expressed in a target cell, specific. For example, for liver-specific expression a pro provides a desired therapeutic effect such as e.g. ablation of moter may be selected from an O. 1-anti-trypsin (AAT) pro an undesired activity, e.g. the ablation of an infected cell, or moter, a thyroid hormone-binding globulin promoter, an the complementation of a genetic defect, e.g. causing a defi albumin promoter, a LPS (thyroxine-binding globlin) pro ciency in an enzymatic activity. Examples of therapeutic 15 moter, an HCR-ApoCII hybrid promoter, an HCR-haAT polypeptide gene products include CFTR, Factor IX, Lipo hybrid promoter, an AAT promoter combined with the mouse protein lipase (LPL, preferably LPL S447X; see WO albumin gene enhancer (Ealb) element and an apolipoprotein 01/00220), Apollipoprotein A1, Uridine Diphosphate Glucu E promoter. Other examples include the E2F promoter for ronosyltransferase (UGT). Retinitis Pigmentosa GTPase tumour-selective, and, in particular, neurological cell tumour Regulator Interacting Protein (RP-GRIP), and cytokines or selective expression (Parret al., 1997, Nat. Med. 3:1145-9) or interleukins like e.g. IL-10, porphobilinogen deaminase the IL-2 promoter for use in mononuclear blood cells (Hagen (PBGD), and alanine:glyoxylate aminotransferase. baugh et al., 1997, J Exp Med; 185: 2101-10). Alternatively, or in addition as a third gene product, third AAV is able to infect a number of mammalian cells. See, nucleotide sequence defined herein above may comprise a e.g., Tratschinet al. (1985, Mol. Cell. Biol. 5:3251-3260) and nucleotide sequence encoding a polypeptide that serve as 25 Grimm et al. (1999, Hum. Gene Ther. 10:2445-2450). How marker proteins to assess cell transformation and expression. ever, AAV transduction of human synovial fibroblasts is sig Suitable marker proteins for this purpose are e.g. the fluores nificantly more efficient than in similar murine cells, Jennings cent protein GFP, and the selectable marker genes HSV thy et al., Arthritis Res, 3:1 (2001), and the cellular tropicity of midine kinase (for selection on HAT medium), bacterial AAV differs among serotypes. See, e.g., Davidson et al. hygromycin B phosphotransferase (for selection on hygro 30 (2000, Proc. Natl. Acad. Sci. USA,97:3428-3432), who dis mycin B), Tn5 aminoglycoside phosphotransferase (for cuss differences among AAV2, AAV4, and AAV5 with selection on G418), and dihydrofolate reductase (DHFR) (for respect to mammalian CNS cell tropism and transduction selection on methotrexate), CD20, the low affinity nerve efficiency. growth factor gene. Sources for obtaining these marker genes AAV sequences that may be used in the present invention and methods for their use are provided in Sambrook and 35 for the production of recombinant AAV vectors in insect cells Russel (2001) “Molecular Cloning: A Laboratory Manual can be derived from the genome of any AAV serotype. Gen (3" edition), Cold Spring Harbor Laboratory, Cold Spring erally, the AAV serotypes have genomic sequences of signifi Harbor Laboratory Press, New York. Furthermore, third cant homology at the amino acid and the nucleic acid levels, nucleotide sequence defined herein above may comprise a provide an identical set of genetic functions, produce virions nucleotide sequence encoding a polypeptide that may serve 40 which are essentially physically and functionally equivalent, as a fail-safe mechanism that allows to cure a subject from and replicate and assemble by practically identical mecha cells transduced with the recombinant parvoviral (ra AV) nisms. For the genomic sequence of the various AAV sero vector of the invention, if deemed necessary. Such a nucle types and an overview of the genomic similarities see e.g. otide sequence, often referred to as a Suicide gene, encodes a GenBank Accession number U89790; GenBank Accession protein that is capable of converting a prodrug into a toxic 45 number JO1901; GenBank Accession number AF043303: Substance that is capable of killing the transgenic cells in GenBank Accession number AF085716; Chlorini et al. which the protein is expressed. Suitable examples of such (1997, J. Vir. 71: 6823-33); Srivastava et al. (1983, J. Vir. Suicide genes include e.g. the E. coli cytosine deaminase gene 45:555-64); Chlorini et al. (1999, J. Vir. 73:1309-1319); Rut or one of the thymidine kinase genes from Herpes Simplex ledge et al. (1998, J. Vir. 72:309-319); and Wu et al. (2000, J. Virus, Cytomegalovirus and Varicella-Zoster virus, in which 50 Vir. 74: 8635-47). AAVserotypes 1,2,3,4 and 5 are preferred case ganciclovir may be used as prodrug to kill the transgenic source of AAV nucleotide sequences for use in the context of cells in the subject (see e.g. Clair et al., 1987, Antimicrob. the present invention. Preferably the AAV ITR sequences for Agents Chemother. 31: 844-849). use in the context of the present invention are derived from In another embodiment one of the gene products of interest AAV1, AAV2, and/or AAV4. Likewise, the Rep (Rep78/68 can be an AAV protein. In particular, a Rep protein, such as 55 and Rep52/40) coding sequences are preferably derived from Rep78 or Rep68, or a functional fragment thereof. A nucle AAV1, AAV2, and/or AAV4. The sequences coding for the otide sequence encoding a Rep78 and/or a Rep68, if present VP1,VP2, and VP3 capsid proteins for use in the context of on the genome of a recombinant parvoviral (ra AV) vector of the present invention may however be taken from any of the the invention and expressed in a mammalian cell transduced known 42 serotypes, more preferably from AAV1, AAV2. with the vector, allows for integration of the recombinant 60 AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 or parvoviral (ra AV) vector into the genome of the transduced newly developed AAV-like particles obtained by e.g. capsid mammalian cell. Expression of Rep78 and/or Rep68 in an shuffling techniques and AAV capsid libraries. rAAV-transduced or infected mammalian cell can provide an AAV Rep and ITR sequences are particularly conserved advantage for certain uses of the recombinant parvoviral among most serotypes. The Rep78 proteins of various AAV (ra AV) vector, by allowing long term or permanent expres 65 serotypes are e.g. more than 89% identical and the total nucle sion of any other gene product of interest introduced in the otide sequence identity at the genome level between AAV2. cell by the vector. AAV3A, AAV3B, and AAV6 is around 82% (Bantel-Schaalet US 8,697,417 B2 15 16 al., 1999, J. Virol. 73(2):939-947). Moreover, the Rep antibody is a single chain camelid antibody or a fragment sequences and ITRS of many AAV serotypes are known to thereof as e.g. obtainable from camels or llamas (see e.g. efficiently cross-complement (i.e., functionally substitute) Muyldermans, 2001, Biotechnol. 74:277-302). The antibody corresponding sequences from other serotypes in production for affinity-purification of ra AV preferably is an antibody of AAV particles in mammalian cells. US2003148506 reports 5 that specifically binds an epitope on an AAV capsid protein, that AAV Rep and ITR sequences also efficiently cross whereby preferably the epitope is an epitope that is present on complement other AAV Rep and ITR sequences in insect capsid protein of more than one AAV serotype. E.g. the anti cells. body may be raised or selected on the basis of specific binding The AAVVP proteins are known to determine the cellular to AAV2 capsid but at the same time also it may also specifi tropicity of the AAV virion. The VP protein-encoding 10 cally bind to AAV1, AAV3 and AAV5 capsids. sequences are significantly less conserved than Rep proteins In yet another aspect the invention relates a nucleic acid and genes among different AAV serotypes. The ability of Rep construct comprising a first and a second nucleotide sequence and ITR sequences to cross-complement corresponding as defined herein defined above. sequences of other serotypes allows for the production of In a different aspect the invention relates to a method for pseudotyped ra AV particles comprising the capsid proteins 15 producing a recombinant parvoviral (ra AV) virion (compris of a serotype (e.g., AAV3) and the Rep and/or ITR sequences ing a recombinant parvoviral (ra AV) vector as defined above) of another AAV serotype (e.g., AAV2). Such pseudotyped in an insect cell. Preferably, the method comprises the steps rAAV particles are a part of the present invention. of: (a) culturing an insect cell as defined inherein above under Modified AAV' sequences also can be used in the context conditions such that recombinant parvoviral (ra AV) vector is of the present invention, e.g. for the production of ra AV produced, wherein the insect cell comprises at least one vectors in insect cells. Such modified sequences e.g. include nucleic acid construct for expression of parvoviral Rep78/68 sequences having at least about 70%, at least about 75%, at and Rep52/40 proteins (such as e.g. a nucleic acid construct least about 80%, at least about 85%, at least about 90%, at comprising the first and second nucleotide sequences as least about 95%, or more nucleotide and/or amino acid defined herein above) and further comprises a third and a sequence identity (e.g., a sequence having about 75-99% 25 fourth nucleotide sequence as herein defined above, and nucleotide sequence identity) to an AAV1, AAV2, AAV3, wherein the nucleic acid construct(s) for expression of par AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 ITR, Rep, or voviral Rep78/68 and Rep52/40 proteins produces a Rep52/ VP can be used in place of wild-type AAV ITR, Rep, or VP 40 expression level in the insect cell that is higher than the Sequences. Rep78/68 expression level on a molar basis; and, (b) recovery Although similar to other AAV serotypes in many respects, 30 of the recombinant parvoviral (ra AV) vector. Preferably in AAV5 differs from other human and simian AAV serotypes the method the molar ratio of Rep52/40 to Rep78/68 protein more than other known human and simian serotypes. In view in the insect cell is higher than 10:1, preferably at least 11:1, thereof, the production of ra AV5 can differ from production 15:1, 20:1,30:1, 40:1, 50:1 or 60:1. A molar ratio of Rep52/40 of other serotypes in insect cells. Where methods of the inven to Rep78/68 protein in the insect cell higher than 10:1 advan tion are employed to produce rAAV5, it is preferred that one 35 tageously results in a better ration of full virions (i.e. com or more constructs comprising, collectively in the case of prising an raAV genome) to empty virions (see e.g. FIG. 8). more than one construct, a nucleotide sequence comprising However, a too high molar ratio of Rep52/40 to Rep78/68 an AAV5 ITR, a nucleotide sequence comprises an AAV5 Rep protein may result in a lower titer of the rAAV produced as coding sequence (i.e. a nucleotide sequence comprises an determined by number of gene copies. In one embodiment AAV5 Rep78). Such ITR and Rep sequences can be modified 40 therefore the molar ratio of Rep52/40 to Rep78/68 protein is as desired to obtain efficient production of ra AV5 or pseudot less than 100:1, 8.0:1,70:1, 60:1, 50:1, 40:1, 30:1, or 20:1. The ypedra AV5 vectors in insect cells. E.g., the start codon of the molar ratio of the Rep78/68 and Rep52/40 proteins may be Rep sequences can be modified, VP splice sites can be modi determined by means of Western blotting as described in fied or eliminated, and/or the VP1 start codon and nearby WO2007/148971, preferably using a monoclonal antibody nucleotides can be modified to improve the production of 45 that recognizes a common epitope of both Rep78/68 and rAAV5 vectors in the insect cell. Rep52/40, or using the antibody described in WO2007/ In another aspect the invention relates to a method for 148971. Preferably, the minimal molar ratio’s of the Rep52/ producing a recombinant parvoviral (ra AV) virion (compris 40 and Rep78/68 proteins as indicated above are achieved at ing a recombinant parvoviral (ra AV) vector as defined above) about 20-40 hours post infection, more preferably at about in an insect cell. Preferably, the method comprises the steps 50 30-40 hours post infection, using a baculovirus or similar of: (a) culturing an insect cell as defined inherein above under expression system. conditions such that recombinant parvoviral (ra AV) vector is Various means exist for increasing the relative expression produced; and, (b) recovery of the recombinant parvoviral level of the Rep52/40 proteins as compared to that of the (ra AV) vector. It is understood here that the recombinant Rep78/68 protein. In case that a single transcription unit for parvoviral (ra AV) vector produced in the method preferably 55 expression of both Rep78/68 and Rep52/40 proteins is used, is an infectious parvoviral or AAV virion that comprise the the coding sequence of the Rep78/68 and Rep52/40 proteins recombinant parvoviral (ra AV) vector nucleic acids. Grow may be adapted as follows to obtain a molar ratio of Rep52/40 ing conditions for insect cells in culture, and production of to Rep78/68 protein in the insect cell higher than 10:1: heterologous products in insect cells in culture are well a) the translation initiation codon of the Rep78/68 protein known in the art and described e.g. in the above cited refer 60 may be changed into a suboptimimal initiation codon and/or ences on molecular engineering of insects cells (see also suboptimal context thereofas described in WO2007/148971; WO2007/046703). b) elimination of one or more or all (9) ATG sequences that Preferably the method further comprises the step of affin occur between the translation starts of the Rep78/68 and Rep ity-purification of the (virions comprising the) recombinant 52/40 genes, respectively, preferably by isocoding changes in parvoviral (ra AV) vector using an anti-AAV antibody, pref 65 the nucleotide sequence. This is e.g. achieved in the pVD189 erably an immobilised antibody. The anti-AAV antibody Rep coding sequence described in Example 3 and in SEQID preferably is a monoclonal antibody. A particularly suitable NO: 11: US 8,697,417 B2 17 18 c) optimisation of the context of the translation initiation group consisting of a Pol promoter, p10 promoter, basic codon of the Rep52/40 protein inaccordance with the optimal protein promoter, an inducible promoter or a deltaE1 pro initiator context of 5'-N N N N N N AUGA a?c/g N-3' for moter or a E1 promoter, or any other late or very late bacu efficient translation initiation in lepidopteran cells (as lovirus gene promoter. More preferably, the first promoter is described in Changet al., 1999, Virology 259:369-383); selected from the group consisting of a PolH promoter, p 10 d) by incorporating an expression control sequence com promoter or basic protein promoter and wherein the second prising a nine nucleotide sequence of SEQ. ID NO: 9 or a promoter is a deltaE1 promoter or a E1 promoter, or any other nucleotide sequence Substantially homologous to SEQ. ID early or late baculovirus gene promoter. Preferably, the first NO: 9, upstream of the initiation codons of the Rep52/40 promoter in the nucleic acid construct of the invention is ap10 protein; 10 e) improving the codon usage bias of the part of the coding promoter and the second promoter is a PolH promoter or a sequence that codes for the Rep52/40 protein for expression 4xHsp27 EcRE+minimal Hsp70 promoter. In another in insect cells (as described above); and, embodiment, the first promoter in the nucleic acid construct f) changing the codon usage of the part of the coding of the invention is a 4xHsp27 EcRE+minimal Hsp70 pro sequence between the translation starts of the Rep78/68 and 15 moter and the second promoter is a PolH promoter. In yet Rep 52/40 proteins so that it is less adapted to expression in another embodiment, the first promoter in the nucleic acid insect cells (as described above). Combination of a) to f) are construct of the invention is a PolH promoter and the second included in the invention and in a preferred a) is combined promoter is a p10, a deltaE1 or an E1 promoter. In yet another with at least one of b) to f). embodiment, the first promoter in the nucleic acid construct Alternatively, and/or in addition a second trancription unit of the invention is a Poll promoter and the second promoter may be used for expression of the Rep52/40 protein. Expres is a deltaE1 or an E1 promoter. In yet another embodiment, sion of the Rep52/40 protein from this second trancription the first promoter in the nucleic acid construct of the invention unit may be increased by one or more of is a p10 promoter and the second promoter is a deltaE1 or an a) using a stronger promoter for the Rep52/40 trancription E1 promoter. In yet another embodiment, the first promoter in unit as compared to the promoter for the Rep78/68 unit (see 25 the nucleic acid construct of the invention is a PolH promoter below); and the second promoter is a PolH promoter. Most preferably, b) increasing the copy number of the Rep52/40 trancription the first promoter in the nucleic acid construct op the inven unit as compared to that of the Rep78/68 unit; tion is a PolH promoter and the second promoter is a deltaE1 c) improving the codon usage bias of the coding sequence promoter. that codes for the Rep52/40 protein for expression in insect 30 An "enhancer element' or "enhancer is meant to define a cells (as described above; e.g. SEQID NO: 2 or 10); d) optimisation of the context of the translation initiation sequence which enhances the activity of a promoter (i.e. codon of the Rep52/40 protein inaccordance with the optimal increases the rate of transcription of a sequence downstream initiator context of 5'-N N N N N N AUGA a?c/g N-3' for of the promoter) which, as opposed to a promoter, does not efficient translation initiation in lepidopteran cells (as 35 possess promoter activity, and which can usually function described in Chang et al., Supra); and, irrespective of its location with respect to the promoter (i.e. e) by incorporating an expression control sequence com upstream, or downstream of the promoter). Enhancer ele prising a nine nucleotide sequence of SEQ. ID NO: 9 or a ments are well-known in the art. Non-limiting examples of nucleotide sequence Substantially homologous to SEQ. ID enhancer elements (or parts thereof) which could be used in NO: 9, upstream of the initiation codons of the Rep52/40 40 the present invention include baculovirus enhancers and protein. enhancer elements found in insect cells. It is preferred that the An example of a construct wherein two separate transcrip enhancer element increases in a cell the mRNA expression of tion units are used for expression of the Rep78/68 and Rep a gene, to which the promoter it is operably linked, by at least 52/40 proteins is the pVD183 construct as described in 25%, more preferably at least 50%, even more preferably at Examples 2 and 3 herein. The nucleic acid constructs for use 45 least 100%, and most preferably at least 200% as compared to in the method for producing a recombinant parvoviral virion the mRNA expression of the gene in the absence of the (and that produce a Rep52/40 expression level in the insect enhancer element. mRNA expression may be determined for cell that is higher than the Rep78/68 expression level on a example by quantitative RT-PCR. molar basis) are a further aspect of the present invention. Herein it is preferred to use an enhancer element to enhance It is understood herein that the recombinant parvoviral 50 the expression of parvoviral Rep protein. Thus, in a further (ra AV) vector produced in the method preferably is an infec preferred embodiment, the first expression cassette com tious parvoviral or AAV virion that comprise the recombinant prises at least one baculovirus enhancer element and/or at parvoviral (ra AV) vector nucleic acids. Growing conditions least one ecdysone responsive element. Preferably the for insect cells in culture, and production of heterologous enhancer element is selected from the group consisting of hrl, products in insect cells in culture are well-known in the art 55 hr2, hr3, hr4 and hr5. and described e.g. in the above cited references on molecular In this document and in its claims, the verb “to comprise' engineering of insects cells (see also WO2007/046703). Pref. and its conjugations is used in its non-limiting sense to mean erably the method further comprises the step of affinity-pu that items following the word are included, but items not rification of the (virions comprising the) recombinant par specifically mentioned are not excluded. In addition, refer voviral (ra AV) vector using an anti-AAV antibody as 60 ence to an element by the indefinite article “a” or “an does described above. not exclude the possibility that more than one of the element A first promoter being equally strong or stronger than a is present, unless the context clearly requires that there be one second promoter for use in the invention may be defined as and only one of the elements. The indefinite article “a” or “an follows. The strength of the promoter may be determined by thus usually means “at least one'. the expression that is obtained under conditions that are used 65 All patent and literature references cited in the present in the method of the invention. In a preferred embodiment, the specification are hereby incorporated by reference in their first promoter or the second promoter is selected from the entirety. US 8,697,417 B2 19 20 The following examples are offered for illustrative pur The wild type Rep52 coding sequence in the Rep 52 poses only, and are not intended to limit the scope of the expression cassette is replaced with the codon optimized present invention in any way. Rep52 coding sequence of SEQ ID NO. 2 to produce pPSC10Rep-52CD. DESCRIPTION OF THE FIGURES The wildtype Rep52 coding sequence in the Rep78 expres sion cassette of pPSC10Rep-52CD is replaced with the AT FIG. 1 Physical map of pVD183. optimized Rep52 coding sequence of SEQID NO. 3 to pro FIG. 2 Ratios of the genomic copies of the ORF 1629 gene duce pPSC10Rep-52CD/78AT. and the Rep gene in the baculovirus samples taken at different The wildtype Rep52 coding sequence in the Rep78 expres passages of the baculovirus Bac.FBDSLR construct (Urabeet 10 sion cassette of pPSC10Rep-52CD is replaced with the GC al., 2002, Hum Gene Ther. 13(16):1935-43). Genomic copies optimized Rep52 coding sequence of SEQID NO. 4 to pro were measured by QPCR. duce pPSC10Rep-52CD/78GC.. FIG.3 Ratios of the genomic copies of the ORF 1629 gene 1.1.2 Recombinant Baculovirus Production and the Rep gene in the baculovirus samples taken at different Recombinant baculoviruses derived from the Autographa passages of the baculovirus pVD183 construct of the inven 15 californica multiple nuclear polyhydrosis virus (AcMNPV) tion. Genomic copies were measured by QPCR. are produced using the GeneXpress BaculoKIT (Protein Sci FIG. 4 raAV production with BacVD183. The dip in the ences Corporation). Transfection is performed as follows: in production is caused by a reduction in the amount of bacu a round bottom 14 ml tube 200 ul GRACE medium is mixed loviruses present. with 6 ulcellfectine (Invitrogen), and in aeppendorf tube 200 FIG.5 ORF QPCR on the passages of Bac.VD183. ul GRACE medium is mixed with 50 ul viral DNA (protein FIG. 6 Western blot Rep expression for several passages of sciences) and 2 ug transfer plasmid (REP). The contents from Bac.VD183. “88 indicates the Bac.VD88 construct, which is the eppendorf tube are added to the tube and mixed carefully. referred to as REP-ACG/PSC in WO2007/148971, which is After an incubation period of 30 minutes at RT 1,300 ul used here as a control. The amount of Rep expression is GRACE is added to the transfection mix. Insect cells in a T25 related to the concentration of Bac.VD183. 25 flask are washed with GRACE medium and the transfection FIG. 7 Q-PCR on crude cell bulk (CLB) from ra AV1 mixture is added drop wise to the cell layer. After an incuba productions using three different constructs for the Rep pro tion of 6 hours at 28°C. SF900II serum supplemented with teins: VD88, VD183, and VD189.5:1:1 refers to the ration of 10% FBS is added carefully and the T25 flask was put in a 28° the different baculoviruses used in the production, 5 refers to C. stove for 5 days after which the recombinant baculovirus is the Bac.VD88, Bac.VD183, or Bac.VD189, the first 1 refers 30 harvested. to the Bac.VD84 (containing the AAV1 capsid gene) and the 1.2 Results second 1 refers to the baculovirus containing the ITR con The performance of the newly designed pPSC10Rep struct, Bac.VD43. 52CD, pPSC10Rep-52CD/78AT and pPSC10Rep-52CD/ FIG.8 The CLB's from the three different raAV 1 produc 78GC plpSC10Rep is compared with the original Rep con tion were purified in a Llama column specific for the AAV 35 structs pFBDSLR of Urabe et al. (2002, supra). All four capsid and in the purified batches the genomic copies and the constructs are serially passaged until passage 5. Recombinant total ra AV particles were measured. Dividing the total ra AV AAV1 production experiments are performed using the pas particles by the Q-PCR number results in the total:full ratio sage 2, 3, 4, and 5 Rep-constructs in combination with a mentioned here. 5:1:1 refers to the ratio of the different bacu baculovirus containing an mammalian expression cassette of loviruses used in the production, 5 refers to the Bac.VD88, 40 a reporter gene between AAVITRs (AAV-LPL) and a bacu Bac.VD183, or Bac.VD189, the first 1 refers to the Bac.VD84 lovirus containing an insect cell expression cassette for the (containing the AAV1 capsid gene) and the second one refers AAV1-Cap (AAV-cap) of respectively passage 2, 3, 4 and 5. to the baculovirus containing the ITR construct, Bac.VD43. AAV-LPL and AAV-Cap recombinant Baculovirusses as used FIG. 9 Rep western blot. Samples were taken at several here are described in WO2007/046703. AAV1-LPL produc passages of the Bac.VD88 or Bac.VD189 baculovirus and a 45 tion yields are determined by QPCR. The original baculovi western blot was performed. The Rep52 amount relative to rus designed by Urabe et al., 2002 (original REP/Bac-to-Bac) the Rep78 amount is consistently higher for Bac.VD189. results in a fast decrease of AAV production over 5 passages. FIG. 10 Rep western blot. Samples were taken at several However, the baculovirus with the REP expression units of passages of the Bac.VD183 baculovirus amplification and a pPSC10Rep-52CD, pPSC10Rep-52CD/78AT and Rep western blot was performed. The Rep52 amount relative 50 pPSC10Rep-52CD/78GC results in stable AAV production to the Rep78 amount is much higher for Bac.VD183 then for over at least 5 passages. Therefore, reproducible production Bac.VD189 and Bac.VD88. yields of AAV-LPL over several passages (e.g. 2 to 5) are only obtained using baculoviruses containing the pPSC10Rep EXAMPLES 52CD, pPSC10Rep-52CD/78AT and pPSC10Rep-52CD/ 55 78GC constructs. 1. Example 1 2. Example 2 1.1. Materials & Methods 1.1.1 Baculovirus Plasmid Construction It has previously been described that baculovirus expres pFBDSLR (Urabe et al., 2002, supra) is a pFastEacDual 60 sion vectors containing 2 separate expression cassettes for the expression vector (Invitrogen) comprising 2 separate expres AAV Rep78 and Rep52 proteins are genetically unstable in sion cassettes for the AAV2 Rep78 and Rep52 proteins, baculoviruses (see e.g. WO2007/148971 and Kohlbrenner et whereby the expression of the Rep52 proteins is driven by the al., 2005, Mol. Ther. 12(6):1217-25). We have now set out to poll promoter and expression of the Rep78 protein from the apply codon usage optimization (with respect to autographa DIE promoter. This construct has been subcloned to pPSC10, 65 californica multiple nucleopolyhedrovirus (AcMNPV) a plasmid that is compatible with the GeneXpress BaculoKIT codon usage) of only the Rep52 coding sequence and not the (Protein Sciences Corporation). Rep78 coding sequence so as to introduce Sufficient changes US 8,697,417 B2 21 22 between the previously identical parts of the Rep52 and FIG. 4 shows production of ra AV with the stable Rep78 coding sequences to reduce the recombination events. Bac.VD183 construct. The dip in the production at the higher We now show that this is indeed the case. passages is caused by a reduction in the amount of baculovi 2.1 Cloning ruses used in the rAAV production (see FIG. 5). FIG. 5 shows A plasmid containing the original double rep expression the QPCR on the ORF from Bac.VD183, which is directly related to the amount of baculoviruses present in the sample. cassettes in the Protein Sciences Corporation plasmid The amount of baculoviruses used in the rAAV production pPSC10, pVD42 was modified. pVD42 contains the rep78 correlate with the amount of ra AV produced. gene driven by the deltaE1 promoter, and the rep52 gene 2.2.3 Rep Western Blot driven by the Po1H promoter, as in the original pFBDSLR FIG. 6 shows rep protein expression during the passages of construct (Urabe et al., 2002, Hum Gene Ther. 13(16):1935 10 Bac.VD183 as analysed by Western blot. 43). The rep52 coding sequence in pVD42 was replaced by a synthetic rep52 coding sequence the codon usage of which 3. Example 3 was adapted to Autographa Californica multiple nucleopoly hedrovirus (AcMNPV) codon usage (see Table 2; and World The effect of Rep52 expression level on two ra AV produc Wide Web URL kazusa.or.jp/codon/cgi-bin/showcodon 15 tion parameters was determined. In particular the effect of the .cgi?species=46015). This AcMNPV codon optimized AAV2 relative expression level Rep52 compared to the expression rep52 coding sequence is depicted in SEQ ID NO:-10. A level of Rep78 on 1) ra AV production level as expressed in physical map of the resulting plasmid pVD183, comprising genome copies per ml crude cell bulk (gc/mL CLB); and 2) the AcMNPV codon optimized AAV2 rep52 coding sequence the ratio of total ra AV virions to full ra AV virions (full ra AV driven from the Po 1H promoter and the wild type AAV2 virions are virions comprising a ra AV genome copy). These rep78 coding sequence driven from the deltaE1 promoter, is parameters were compared for three different ra AV Rep shown in FIG. 1. constructs that each result in different Rep52 expression lev 2.2 Results els and in different ratio’s between Rep52 and rep78 levels. We have made a recombinant baculovirus clone of the The three constructs were pVD88 (referred to as REP-ACG/ pVD183 plasmid and passaged the baculovirus 10 times to 25 PSC in WO2007/148971), pVD183 (described in Example 2 analyse its genetic stability. We analyzed the genetic stability herein above), and pVD189 (see below). of the construct by QPCR on the genome of the baculovirus 3.1 Construction of pVD189 and the Rep52 gene, by western blot, and by ra AV production The pVD88 construct was redesigned by eliminating 9 efficiency of the baculovirus. At the same time the original ATG sequences between the translation start of the Rep78 and Bac.VD42 baculovirus was passaged to passage 7 for com 30 Rep 52 genes, and by changing the Rep78 ACG translation parison. Earlier data about the stability of the Bac.VD42 (or initiation codon to CTG. See the sequence below. Baseclear Bac.FBDSLR) are also mentioned in WO2007/148971 (re (Leiden, The Netherlands) synthesized the new gene and ferred to as original REP/Bac-to-Bac). cloned it in pVD88 replacing the existing Rep gene to obtain 2.2.1 QPCR pVD189. The nucleotide sequence of the Rep coding Stability measured by QPCR on the baculovirus genomes. 35 sequence in pVD189 is depicted in SEQID NO: 11. The copy number of a gene that is essential for baculovirus 3.2 Production of ra AV replication and that is used for production of the BacVD183 Baculoviruses were made with the VD88, VD183, and from pVD183 by recombination at ORF1629 and ORF603 VD189 constructs, and these were used for production of between the pVD183 and the baculovirus backbone from rAAV1. Comparison of the VD88, VD183, and VD189 con Protein Sciences. ORF 1629 (ORF), has been measured by 40 structs in ra AV production resulted in betterra AV produc QPCR, and the copy number of the Rep genes have also been tion (genome copies) as measured by Q-PCR in the crude cell measured by QPCR. The ratio between these 2 genes should bulk (CLB). FIG. 7 shows that the standard Rep construct stay the same during Subsequent passages of the baculovirus. VD88 which results in the lowest amount of Rep52 (FIG.9) FIG. 2 shows for comparison that Bac.FBDSLR is rather results in approximately 4x10'GC/ml measured in the CLB. unstable. FIG.3 shows that Bac.VD183 is significantly more 45 VD189 which leads to a slightly higher Rep. 52 amount (FIG. stable. We note that the efficiency of the 2 primer sets used in 9) resulted in an ira AV production measured in CLB of the QPCR is not necessarily equal, therefore a ratio different approximately 9.5x10' GC/ml. VD183 which leads to a from 1 can be obtained. A more important indicator of stabil clearly higher Rep52 amount (FIG. 10) and resulted a ra AV ity is however that the ratio should stay relatively constant production measured in CLB of approximately 6x10' during multiple passages. Passage 3 from Bac. FBDSLR is 50 GC/ml. already Suboptimal, as the ratio is around 0.25 and only gets A very important quality parameter is the total:full ratio of worse. Bac.VD183 also starts around 0.3 but fluctuates the rAAV batch. FIG. 8 shows that the best ratio of total around that ratio, indicating that there is a stable situation. (virions): full (virions) is obtained with the VD183 construct Deletions in the baculovirus genome results in a baculovirus that shows the highest Rep52 amount relative to the Rep78 that grows faster then the baculovirus that has a full length 55 amount as compared to the Bac.VD189 and Bac.VD88 con genome, therefore when a deletion occurs, those clones will Structs in FIG. 9. overgrow the others. Variations in the QPCR method can 3.2 Additional Constructs result in the fluctuations seen in FIG. 3. The following constructs are constructed, tested and part of 2.2.2 ra AV Production the invention:

Constructs promoter (s) initation codons and coding sequences

1) WD88 PoH ACG-78 ------ATG-52 ------k

2) WD189 PoH CTG-78-atg's removed-ATG-52 ------k US 8,697,417 B2

- Continued Constructs promoter (s) initation codons and coding sequences

3) WD183 delta E1 ATG-78 ------k . PoH ATG- - - 52- - - SEQ ID NO : 10 ------k

4) WD196 PoH CTG-78 ------ATG-52 ------k

5) WD1.97 PoH ACG-78-atg' s removed-ATG-52------k 6) WD197/52 P10 ACG-78-atg' s removed-ATG-52------k -- PoH ATG--52- - - SEQ ID NO: 1.O------k 7) WD189/52 P10 CTG-- 78-atg's removed-ATG-52 ------k -- PoH ATG--52- - - SEQ ID NO: 1.O------k

8) WD183/10 p.10 ATG-- 78 ------PoH ATG--52- - - SEQ ID NO: 1.O------k 9) WD197/52cd PolH ACG-78-atg' s removed-ATG-52 - SEQ ID NO : 10 *

2O 1, 2, 4, 5, 8, and 9 have 1 trancription unit for expression Rep - Continued 78 and 52 proteins. 3, 6, and 7 have 2 trancription units for expression Rep. 78 and 52 proteins. 78 52 A rough estimate of the rep 78 and rep 52 proteins amounts and ratios for the different constructs during ra AV produc- 25 4) 5 : O.25 tion (rep78:rep52): 5) 1. 5

6) O 5 :30 78 52 30 7) Ofs :30 1) 1. 1. 8) 5 2O 2) 1.5 2 9) 1. 1O 3) 1. 2O

TABLE 1. Spodopterafiugiperda codon frequencies based on 127 coding sequences (33098 codons) fields: triplet frequency: per thousand (number) TTT 9.7(320) TCT 0.5(347) TAT 10.1(334) TGT 6.9(227) TTC 26.9(889) TCC 3.0(430) TAC 24.4(807) TGC 12.4(409) TTA 7.0(233) TCA 9.9(329) TAA 2.5(83) TGA 0.6(21) TTG 16.2(536) TCG 7.2(237) TAG 0.7(23) TGG 12.7(420) CTT 9.9(327) CCT 4.3(472) CAT 8.7 (289) CGT 15.9(525) CTC 17.0(564) CCC 3.7(453) CAC 16.2(535) CGC 15.1 (500) CTA 6.8(226) CCA 3.4(445) CAA 16.2(535) CGA 5.3(175) CTG 24.5(810) CCG 7.7(255) CAG 21.8(723) CGG 3.6(118) ATT 15.5(512) ACT 3.6(451) AAT 12.8(424) AGT 8.1(267) ATC 28.9(958) ACC 7.2(569) AAC 27.8(921) AGC 10.7(354) ATA 7.6(253) ACA 1.9(393) AAA 26.7(883) AGA 11.8(392) ATG 27.3(902) ACG 8.8(290) AAG 53.1 (1757) AGG 13.5(446) GTT 14.7(488) GCT 26.3(872) GAT 21.8(723) GGT 22.0(728) GTC 20.4(676) GCC 21.1 (697) GAC 32.3(1070) GGC 19.9(659) GTA 12.3(406) GCA 2.4(411) GAA 27.2(901) GGA 18.2(603) GTG 24.8(822) GCG 2.2(404) GAG 34.1 (1128) GGG 4.3(141)

Coding GC 50.58% 1st letter GC 53.42% 2nd letter GC 39.40%. 3rd letter GC 58.93% TABLE 2 Codon usage table Autographa Californica multiple nucleopolyhedrovirus (AcMNPV) based on 277 coding sequences (77487 codons) fields: triplet frequency: per thousand (number) UUU 37.6(2916) UCU 10.3(799) UAU 22.2(1721) UGU 11.2(865) UUC 11.3(879) UCC 7.2(556) UAC 26.1(2019) UGC 12.5 (967)

US 8,697,417 B2 33 34 - Continued gcg cag cca tog acg tca gac gog gala gct tcg atc aac tac go a gac 912 Ala Glin Pro Ser Thr Ser Asp Ala Glu Ala Ser Ile Asn Tyr Ala Asp 29 O 295 3 OO cgc tac caa aac aaa t t t ct cqt cac gtg ggc atgaat Ctg atg Ctg 96.O Arg Tyr Glin Asn Lys Cys Ser Arg His Val Gly Met Asn Lieu Met Lieu 3. OS 310 315 32O titt coc togc aga caa tdc gag aga atgaat cag aat tca aat atc tigc OO8 Phe Pro Cys Arg Glin Cys Glu Arg Met Asn Glin Asn. Ser Asn. Ile Cys 3.25 330 335 ttic act cac gga cag aaa gac tit tta gag tec titt CCC gtg tca gala O56 Phe Thr His Gly Gln Lys Asp Cys Lieu. Glu. Cys Phe Pro Val Ser Glu 34 O 345 35. O t ct caa ccc gtt tot gtc gtc. aaa aag gog tat cag aaa citg togc tac 104 Ser Glin Pro Val Ser Val Val Lys Lys Ala Tyr Glin Llys Lieu. Cys Tyr 355 360 365 att cat cat at C atg gga aag gtg cca gac got to act gcc tic gat 152 Ile His His Ile Met Gly Llys Val Pro Asp Ala Cys Thr Ala Cys Asp 37 O 375 38O

Ctg gtc. aat gtg gat ttg gat gac to at C titt gaa caa taa 194 Lieu Val Asn. Wall Asp Lieu. Asp Asp Cys Ile Phe Glu Glin 385 390 395

<210s, SEQ ID NO 6 &211s LENGTH: 397 212. TYPE: PRT <213> ORGANISM; adeno-associated virus 2

<4 OOs, SEQUENCE: 6 Met Glu Lieu Val Gly Trp Lieu Val Asp Lys Gly Ile Thr Ser Glu Lys 1. 5 1O 15 Glin Trp Ile Glin Glu Asp Glin Ala Ser Tyr Ile Ser Phe Asn Ala Ala 2O 25 3O Ser Asn. Ser Arg Ser Glin Ile Lys Ala Ala Lieu. Asp Asn Ala Gly Lys 35 4 O 45 Ile Met Ser Lieu. Thir Lys Thr Ala Pro Asp Tyr Lieu Val Gly Glin Glin SO 55 6 O Pro Val Glu Asp Ile Ser Ser Asn Arg Ile Tyr Lys Ile Lieu. Glu Lieu. 65 70 7s 8O Asn Gly Tyr Asp Pro Glin Tyr Ala Ala Ser Val Phe Lieu. Gly Trp Ala 85 90 95 Thir Lys Llys Phe Gly Lys Arg Asn. Thir Ile Trp Lieu. Phe Gly Pro Ala 1OO 105 11 O Thir Thr Gly Lys Thr Asn Ile Ala Glu Ala Ile Ala His Thr Val Pro 115 12 O 125 Phe Tyr Gly Cys Val Asn Trp Thr Asn Glu Asn Phe Pro Phe Asn Asp 13 O 135 14 O Cys Val Asp Llys Met Val Ile Trp Trp Glu Glu Gly Lys Met Thr Ala 145 150 155 160 Llys Val Val Glu Ser Ala Lys Ala Ile Lieu. Gly Gly Ser Llys Val Arg 1.65 17O 17s Val Asp Gln Lys Cys Llys Ser Ser Ala Glin Ile Asp Pro Thr Pro Val 18O 185 19 O Ile Val Thr Ser Asn Thr Asn Met Cys Ala Val Ile Asp Gly Asn Ser 195 2OO 2O5 Thir Thr Phe Glu. His Glin Gln Pro Leu Glin Asp Arg Met Phe Llys Phe 21 O 215 22O Glu Lieu. Thir Arg Arg Lieu. Asp His Asp Phe Gly Llys Val Thir Lys Glin 225 23 O 235 24 O US 8,697,417 B2 35 36 - Continued

Glu Val Lys Asp Phe Phe Arg Trp Ala Lys Asp His Val Val Glu Val 245 250 255 Glu. His Glu Phe Tyr Val Llys Lys Gly Gly Ala Lys Lys Arg Pro Ala 26 O 265 27 O Pro Ser Asp Ala Asp Ile Ser Glu Pro Lys Arg Val Arg Glu Ser Val 27s 28O 285 Ala Glin Pro Ser Thr Ser Asp Ala Glu Ala Ser Ile Asn Tyr Ala Asp 29 O 295 3 OO Arg Tyr Glin Asn Lys Cys Ser Arg His Val Gly Met Asn Lieu Met Lieu 3. OS 310 315 32O Phe Pro Cys Arg Glin Cys Glu Arg Met Asn Glin Asn. Ser Asn. Ile Cys 3.25 330 335 Phe Thr His Gly Gln Lys Asp Cys Lieu. Glu. Cys Phe Pro Val Ser Glu 34 O 345 35. O Ser Glin Pro Val Ser Val Val Lys Lys Ala Tyr Glin Llys Lieu. Cys Tyr 355 360 365 Ile His His Ile Met Gly Llys Val Pro Asp Ala Cys Thr Ala Cys Asp 37 O 375 38O Lieu Val Asn. Wall Asp Lieu. Asp Asp Cys Ile Phe Glu Glin 385 390 395

<210s, SEQ ID NO 7 &211s LENGTH: 1876 &212s. TYPE: DNA <213> ORGANISM; adeno-associated virus 2 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (11) . . (1876) <223> OTHER INFORMATION: Rep78 <4 OO > SEQUENCE: 7 cgcagcc.gcc atg ccg ggg ttt tac gag att gtg att aag gtc. CCC agc 49 Met Pro Gly Phe Tyr Glu Ile Val Ile Llys Val Pro Ser 1. 5 1O gac Ctt gac gag cat Ctg C cc ggc att tot gac agc titt gtgaac tig 97 Asp Lieu. Asp Glu. His Lieu Pro Gly Ile Ser Asp Ser Phe Val Asn Trp 15 2O 25 gtg gCC gag aag gala tig gag titg ccg cca gat tict gac atg gat ctg 145 Val Ala Glu Lys Glu Trp Glu Lieu Pro Pro Asp Ser Asp Met Asp Lieu 3 O 35 4 O 45 aat Ctg att gag cag gca CCC ctg acc gtg gCC gag aag Ctg cag cc 193 Asn Lieu. Ile Glu Glin Ala Pro Lieu. Thr Val Ala Glu Lys Lieu. Glin Arg SO 55 60 gac titt Ctg acg gala tig cqc cqt gtg agt aag goc cc.g gag gCC citt 241 Asp Phe Lieu. Thr Glu Trp Arg Arg Val Ser Lys Ala Pro Glu Ala Lieu 65 70 7s ttic titt gtg caa titt gag aag gga gag agc tac ttic cac atg cac gtg 289 Phe Phe Val Glin Phe Glu Lys Gly Glu Ser Tyr Phe His Met His Val 8O 85 90

Ctc gtg gala acc acc ggg gtg aaa to C atg gtt ttg gga cqt tt C Ctg 337 Lieu Val Glu Thir Thr Gly Val Lys Ser Met Val Lieu. Gly Arg Phe Leu 95 1 OO 105 agt cag att cqc gala aaa citg att cag aga att tac cqc ggg at C gag 385 Ser Glin Ile Arg Glu Lys Lieu. Ile Glin Arg Ile Tyr Arg Gly Ile Glu 11O 115 12O 125 ccg act ttg cca aac t t t c gog gtc. aca aag acc aga aat ggc gcc 433 Pro Thr Lieu Pro Asn Trp Phe Ala Val Thr Lys Thr Arg Asin Gly Ala 13 O 135 14 O gga ggc ggg aac aag gtg gtg gat gag to tac at C C cc aat tac ttg 481

US 8,697,417 B2 39 40 - Continued Thir Lys Glin Glu Wall Lys Asp Phe Phe Arg Trp Ala Lys Asp His Val 465 47 O 47s gtt gag gtg gag cat gaa titc tac git C aaa aag ggit gga gcc aag aaa. 489 Val Glu Val Glu. His Glu Phe Tyr Val Lys Lys Gly Gly Ala Lys Llys 48O 485 490 aga ccc gcc ccc agt gac gca gat at a agt gag ccc aaa cqg gtg cc s37 Arg Pro Ala Pro Ser Asp Ala Asp Ile Ser Glu Pro Lys Arg Val Arg 495 5 OO 5 OS gag to a gtt gcg cag cca t c acg to a gac gog gala gct tcg at C aac 585 Glu Ser Val Ala Glin Pro Ser Thir Ser Asp Ala Glu Ala Ser Ile Asn 510 515 52O 525 tac gca gac agg tac caa aac aaa tot tot cqt cac gtg ggc atgaat 633 Tyr Ala Asp Arg Tyr Glin Asn Lys Cys Ser Arg His Val Gly Met Asn 53 O 535 54 O

Ctg atg Ctg titt CCC to aga caa to gag aga atgaat cag aat to a 681 Lieu Met Lieu. Phe Pro Cys Arg Glin Cys Glu Arg Met Asn Glin Asn. Ser 545 550 555 aat at c togc titc act cac giga cag aaa gac tdt tta gag tigc titt coc 729 Asn Ile Cys Phe Thr His Gly Gln Lys Asp Cys Lieu. Glu. Cys Phe Pro 560 565 st O gtg to a gaa tot caa ccc gtt tot gtc gtc. aaa aag gog tat cag aaa 777 Val Ser Glu Ser Gln Pro Val Ser Val Val Lys Lys Ala Tyr Glin Lys sts 58O 585

Ctg to tac att cat cat atc atg gga aag gtg cca gac got to act 825 Lieu. Cys Tyr Ile His His Ile Met Gly Llys Val Pro Asp Ala Cys Thr 590 595 6 OO 605 gcc tic gat ctg gtc. aat gtg gat ttg gat gac tec atc titt gala cala 873 Ala Cys Asp Lieu Val ASn Val Asp Lieu. Asp Asp Cys Ile Phe Glu Gln 610 615 62O taa 876

<210s, SEQ ID NO 8 &211s LENGTH: 621 212. TYPE: PRT <213> ORGANISM; adeno-associated virus 2

<4 OOs, SEQUENCE: 8 Met Pro Gly Phe Tyr Glu Ile Val Ile Llys Val Pro Ser Asp Leu Asp 1. 5 1O 15 Glu. His Lieu Pro Gly Ile Ser Asp Ser Phe Val Asn Trp Val Ala Glu 2O 25 3O Lys Glu Trp Glu Lieu Pro Pro Asp Ser Asp Met Asp Lieu. Asn Lieu. Ile 35 4 O 45 Glu Glin Ala Pro Lieu. Thr Val Ala Glu Lys Lieu. Glin Arg Asp Phe Lieu. SO 55 6 O Thr Glu Trp Arg Arg Val Ser Lys Ala Pro Glu Ala Lieu. Phe Phe Val 65 70 7s 8O Glin Phe Glu Lys Gly Glu Ser Tyr Phe His Met His Val Lieu Val Glu 85 90 95 Thir Thr Gly Val Lys Ser Met Val Lieu. Gly Arg Phe Leu Ser Glin Ile 1OO 105 11 O Arg Glu Lys Lieu. Ile Glin Arg Ile Tyr Arg Gly Ile Glu Pro Thir Lieu 115 12 O 125 Pro Asn Trp Phe Ala Val Thir Lys Thr Arg Asn Gly Ala Gly Gly Gly 13 O 135 14 O Asn Llys Val Val Asp Glu. Cys Tyr Ile Pro Asn Tyr Lieu. Lieu Pro Llys 145 150 155 160

Thr Glin Pro Glu Lieu Gln Trp Ala Trp Thr Asn Met Glu Glin Tyr Lieu. US 8,697,417 B2 41 42 - Continued

1.65 17O 17s

Ser Ala Luell Asn Lell Thir Glu Arg Arg Lell Wall Ala Glin His 18O 185 19 O

Lell Thir His Wall Ser Glin Thir Glin Glu Glin ASn Glu Asn Glin Asn 195

Pro Asn Ser Asp Ala Pro Wall Ile Arg Ser Thir Ser Ala Arg 21 O 215 22O

Met Glu Luell Wall Gly Trp Lell Wall Asp Gly Ile Thir Ser Glu Lys 225 23 O 235 24 O

Glin Trp Ile Glin Glu Asp Glin Ala Ser Tyr Ile Ser Phe Asn Ala Ala 245 250 255

Ser Asn Ser Arg Ser Glin Ile Ala Ala Luell Asp Asn Ala Gly 26 O 265 27 O

Ile Met Ser Luell Thir Thir Ala Pro Asp Lell Wall Gly Glin Glin 27s 285

Pro Wall Glu Asp Ile Ser Ser Asn Arg Ile Lys Ile Luell Glu Luell 29 O 295 3 OO

Asn Gly Asp Pro Glin Ala Ala Ser Wall Phe Lell Gly Trp Ala 3. OS 310 315

Thir Phe Gly Arg Asn Thir Ile Trp Lell Phe Gly Pro Ala 3.25 330 335

Thir Thir Gly Lys Thir Asn Ile Ala Glu Ala Ile Ala His Thir Wall Pro 34 O 345 35. O

Phe Gly Wall Asn Trp Thir Asn Glu ASn Phe Pro Phe Asn Asp 355 360 365

Wall Asp Met Wall Ile Trp Trp Glu Glu Gly Met Thir Ala 37 O 375

Lys Wall Wall Glu Ser Ala Ala Ile Luell Gly Gly Ser Wall Arg 385 390 395 4 OO

Wall Asp Glin Cys Ser Ser Ala Glin Ile Asp Pro Thir Pro Wall 4 OS 415

Ile Wall Thir Ser Asn Thir Asn Met Cys Ala Wall Ile Asp Gly Asn Ser 425 43 O

Thir Thir Phe Glu His Glin Glin Pro Luell Glin Asp Arg Met Phe Phe 435 44 O 445

Glu Luell Thir Arg Arg Lell Asp His Asp Phe Gly Lys Wall Thir Glin 450 45.5 460

Glu Wall Asp Phe Phe Arg Trp Ala Asp His Wall Wall Glu Wall 465 470

Glu His Glu Phe Tyr Wall Gly Gly Ala Arg Pro Ala 485 490 495

Pro Ser Asp Ala Asp Ile Ser Glu Pro Arg Wall Arg Glu Ser Wall SOO 505

Ala Glin Pro Ser Thir Ser Asp Ala Glu Ala Ser Ile Asn Ala Asp 515 52O 525

Arg Tyr Glin Asn Lys Ser Arg His Wall Gly Met Asn Luell Met Luell 53 O 535 54 O

Phe Pro Arg Glin Cys Glu Arg Met Asn Glin Asn Ser Asn Ile Cys 5.45 550 555 560

Phe Thir His Gly Glin Asp Luell Glu Cys Phe Pro Wall Ser Glu 565 st O sts

Ser Glin Pro Wall Ser Wall Wall Lys Ala Tyr Glin Luell Tyr 58O 585 59 O

US 8,697,417 B2 45 46 - Continued tctgacttgg atctgaatct gattgagcag gcaccCCtga ccgtggc.cga gaagctgcag 18O cgcgactitt C tacggagtg gcgc.cgtgtgagtaaggc.cc cq9aggc cct titt Ctttgtg 24 O

Caatttgaga agggagagag ctact tccac ttacacgtgc ticgtggaaac Caccggggtg 3OO aaatcc titag titttgggacg titt cotgagt cagatt.cgcg aaaaactgat t cagaga att 360 taccgcggga t cagc.cgac tittgccaaac ttt cqcgg to acaaaga C Cagaaacggc 42O gccggaggcg ggaacaaggt ggtggacgag tectacatcc cca attactt gct coccaaa 48O acccagcctg agctc.cagtg ggcgtggact aatttagaac agt atttaag cqcctgtttg 54 O aatctgacgg agcgtaaacg gttggtggcg cagcatctga cqcacgtgtc. gcagacgcag 6OO gaggagaa.ca aagagaatca gaatcc.ca at tctgacgc.gc C9gtgat cag atcaaaaact 660 t cagcc aggt acatggagct ggtcgggtgg Ctcgtggaca aggggattac Ctcggagaag 72 O

Cagtggat.cc aggaggacca ggcct catac atct cct tca atgcggcct C Caact cqcgg 78O t cccaaatca aggctgcctt gga caatgcg ggaaagatta tagcctgac taaaaccgc.c 84 O cc.cgact acc tigtgggcca gcago'ccgtg gagga cattt C cagcaatcg gatttataaa 9 OO attittggaac taaacgggta catc.cccaa tatgcggctt CC9tctttct gggatgggcc 96.O acgaaaaagt toggcaa.gag galacaccatc. tctgtttg ggcctgcaac taccgggaag O2O accalacatc. c99aggcc at agcc.cacact gtgcc cttct acgggtgcgt aaactggacc O8O aatgagaact tt CCCttcaa cactgtgtc. gaCaagatgg tdatctggtg ggaggagggg 14 O aagatgaccg C calaggtogt ggagt cqgcc aaa.gc.cattc. tcggaggaag Caaggtgcgc 2OO gtggaccaga aatgcaagtic Ctcggcc.cag at aga.ccc.ga Citc.ccgtgat cqt cacct CC 26 O alacaccaa.ca ttgcgc.cgt gattgacggg aact caacga cct tcgalaca C cagcagcc.g 32O ttgcaaga cc ggatgttcaa atttgaactic accc.gc.cgt.c tigat catga Ctttgggaag 38O gtcaccalagc aggaagt caa agaCtttitt C. c99tgggcaa aggat cacgt ggttgaggtg 44 O gaggatgaat t ctacgtcaa aaagggtgga gccaagaaaa gaccc.gc.ccc cagtgacgca SOO gatataagtg agcc.caaacg ggtgcgcgag tdagttgcgc agc catcgac git cagacgc.g 560 gaagct tcga t caact acgc agaCagg tac Caaaacaaat gttct cqtca C9tgggcatg 62O aatctgatgc tigttitc cctd cagacaatgc gagagaatga at cagaattic aaatat citgc 68O ttcact cacg gacagaaaga citgtttagag togctitt.cccg togt cagaatc. tca accc.gtt 74 O tctgtcgt.ca aaaaggcgta t cagaaactg. tctacatt C at catat cat gggaaaggtg 8OO ccagacgctt gcactgcctg cgatctggtc. aatgtggatt tatgactg. Catctttgaa 86 O

Caataa. 866

The invention claimed is: (f) A sequence of at least 85% identity with SEQ ID 1. An insect cell comprising a baculoviral vector compris NO:10; 1ng: 55 (g) A nucleotide sequence complementary to the full (i) a first nucleotide sequence encoding a first amino acid length sequence of any of (a)-(f); and sequence of a parvoviral Rep52 protein and is selected (h) A nucleotide sequence that differs from the sequence from the group consisting of of any of (c)-(f) due to degeneracy of the genetic code: and (a) SEQID NO:1; 60 (ii) a second nucleotide sequence encoding a second amino (b) SEQID NO:5; acid sequence of a parvoviral Rep78 protein or a Rep68 (c) A sequence of at least 75% identity with SEQ ID protein, NO:2: wherein the first and the second amino acid sequences (d) A sequence of at least 60% identity with SEQ ID comprise a homologous region of at least 100 amino acid NO:3: 65 residues with at least 90% identity beginning at the (e) A sequence of at least 70% identity with SEQ ID second residue and ending at the C-terminal residue of NO:4: the parvoviral Rep 52, and US 8,697,417 B2 47 48 wherein the portion of the first nucleotide sequence that sequences and wherein the fifth nucleotide sequence is encodes the homologous region and the portion of the located between the two parvoviral ITR sequences. second nucleotide sequence that encodes the homolo 15. The insect cell according to claim 12, wherein at least gous region are less than 90% identical. one of the first, second, third and fourth nucleotide sequences 2. The insect cell according to claim 1, wherein the are stably integrated in the genome of the insect cell. homologous regions in the first and second amino acid 16. The insect cell according to claim 12, wherein the sequences share at least 99% amino acid sequence identity. parvovirus is AAV. 3. The insect cell according to claim 1, wherein 17. A method for producing a recombinant AAV virion in (i) the portion of the first nucleotide sequence that encodes an insect cell comprising the steps of the homologous region has an improved codon usage 10 (a) culturing the insect cell according to claim 12 under bias for the insect cell as compared to the portion of the conditions such that recombinant AAV virions are pro second nucleotide sequence that encode the homolo duced; and, gous region, or (b) recovering the recombinant AAV virions. (ii) the portion of the second nucleotide sequence that 18. The method according to claim 17, further comprising encodes the homologous region has an improved codon 15 the step of affinity-purification of the virion using an immo usage bias for the insect cell as compared to the portion bilized anti-AAV antibody. of the first nucleotide sequence that encodes the homolo 19. An isolated nucleic acid construct comprising the first gous region. and the second nucleotide sequence according to claim 1. 4. The insect cell according to claim 1, wherein the differ 20. A method for producing a recombinant AAV virion in ence in codon adaptation index between the nucleotides of the an insect cell comprising the steps of first and the second nucleotide sequences that encode the (a) culturing the insect cell of claim 1 in which all of said homologous region is at least 0.2. Rep proteins are AAV Rep proteins, which cell further 5. The insect cell according to claim 3, wherein the nucle comprises otide sequence with the improved codon usage bias com 25 (i) a third nucleotide sequence comprising at least one prises a continuous stretch of at least 25codons all of which AAV inverted terminal repeat (ITR) sequence; and, are common codons as listed in Table 1 or Table 2. (ii) a fourth nucleotide sequence comprising AAV 6. The insect cell according to claim 5, wherein all the capsid protein-coding sequences operably linked to codons in the nucleotide sequence with the improved usage expression control sequences for expression in said bias are common codons as listed in Table 1 or Table 2. 30 insect cell 7. The insect cell according to claim 1, wherein at least 50% of codons in the portion of the second nucleotide under conditions such that a recombinant AAV vector is sequence that encodes the homologous region are altered produced, and, compared to the corresponding codons in the first nucleotide (b) recovering the recombinant AAV vector. sequence to maximize AT-or GC-content of the second nucle 35 21. The method according to claim 20, wherein the nucleic otide sequence. acid construct for expression of the Rep78 or Rep68 protein 8. The insect cell according to claim 1, wherein the first and and the Rep52 protein comprises a single coding sequence for second nucleotide sequences are part of two separate nucleic expression of both the Rep78 or Rep68 protein and the Rep52 acid constructs wherein each of the first and the second nucle protein, and wherein the coding sequence further comprises otide sequence are operably linked to an expression control 40 one or more of the following characteristics: sequence for expression in said insect cell. (a) one, more or all ATG sequences that occur between the 9. The insect cell according to claim 1, wherein the first and translation starts of the Rep78 and Rep68 protein and second nucleotide sequences are part of a single nucleic acid between the translation starts of the Rep52 protein are construct wherein each of said sequences is operably linked to eliminated; a respective expression control sequence for expression in 45 (b) context of the translation initiation codon of the Rep52 said insect cell. protein is optimized in accordance with the optimal ini 10. The insect cell according to claim 1, wherein the par tiator context of voviral Rep proteins are adeno-associated virus (AAV) Rep proteins. 11. The insect cell according to claim 10, wherein the 50 parvoviral Rep proteins encoded by the first and second for efficient translation initiation in said insect cell; nucleotide sequences are of the same serotype. (c) an expression control sequence comprising a nine 12. The insect cell according to claim 1, wherein the insect nucleotide sequence of cell further comprises: (i) SEQID NO:9 or (a) a third nucleotide sequence comprising at least one 55 (ii) a nucleotide sequence Substantially homologous to parvoviral inverted terminal repeat (ITR) sequence; and, SEQID NO:9, (b) a fourth nucleotide sequence comprising parvoviral is present upstream of the initiation codon for the Rep52 capsid protein-coding sequences operably linked to protein; and, expression control sequences for expression in an insect (d) the portion of the sequence that encodes the Rep52 cell. 60 protein has an improved codon usage bias for the insect 13. The insect cell according to claim 12, wherein the cell cell as compared to the portion of the coding sequence further comprises at least a fifth nucleotide sequence encod between the translation starts of the Rep78/68 and ing a gene product of interest and which fifth nucleotide Rep52 proteins. sequence becomes incorporated into the genome of aparvovi 22. The method according to claim 20, wherein the insect ral vector produced in the insect cell. 65 cell comprises an additional coding sequence for the Rep52 14. The insect cell according to claim 13, wherein the third or Rep40 protein that is operably linked to a promoter that nucleotide sequence comprises two parvoviral ITR drives expression of the coding sequence in the insect cell, US 8,697,417 B2 49 50 and wherein the coding sequence for Rep52 or Rep40 protein 25. The method according to claim 18 wherein the anti comprises one or more of the following characteristics: body is a single chain camelid antibody oran antigen-binding (a) a stronger promoter for the Rep52 or Rep40 protein fragment thereof. coding sequence as compared to the promoter for the 26. The insect cell according to claim 1, wherein at least Rep78 or Rep68 protein-coding sequence; 300 amino acids of the homologous region have at least 90% sequence identity between the first and second amino acid (b) a higher copy number of the Rep52 or Rep40 protein Sequences. coding sequence as compared to that of the Rep78 or 27. The insect cell according to claim 1, wherein at least Rep68 protein-coding sequence; 350 amino acids of the homologous region have at least 90% (c) an improved codon usage bias of the Rep52 or Rep40 10 sequence identity between the first and second amino acid protein coding sequence for expression in insect cells; Sequences. (d) context of the translation initiation codon of the Rep52 28. The insect cell according to claim 1, wherein the entire or Rep40 protein is optimized in accordance with the homologous region has at least 90% sequence identity optimal initiator context of between the first and second amino acid sequences. 29. The cell of claim 1 wherein 15 (i) the insect cell is a Spodoptera frugiperda cell, (ii) the baculoviral vector is an Autographa Californica nucleopolyhedrovirus (AcMNPV) vector. for efficient translation initiation in said insect cell; and 30. The cell of claim 29, wherein either (e) present upstream of the initiation codon for the Rep52 (i) the portion of the first nucleotide sequence that encodes or Rep40 protein is an expression control sequence com the homologous region has improved codon usage bias prising a nine nucleotide sequence of for the insect cell as compared to the portion of the (i) SEQID NO:9, or second nucleotide sequence that encodes the homolo (ii) a nucleotide sequence Substantially homologous to gous region, or SEQID NO:9. (ii) the portion of the second nucleotide that encodes the 23. The insect cell according to claim 2 wherein the 25 homologous region has improved codon usage bias for homologous regions in the first and second amino acid the insect cell as compared to the portion of the first sequences share 100% amino acid sequence identity. nucleotide sequence that encodes the homologous 24. The insect cell according to claim 6, wherein all the region, codons in the nucleotide sequence coding for the homologous wherein the nucleotide sequence with the improved codon region in the nucleotide sequence without the common 30 usage bias comprises a continuous stretch of at least 25 com codons are second most frequent codons as listed in Table 1 or mon codons as listed in Table 1 or Table 2. Table 2. k k k k k UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION

PATENT NO. : 8,697,417 B2 Page 1 of 1 APPLICATION NO. : 12/670780 DATED : April 15, 2014 INVENTOR(S) : Bakker et al.

It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

On the Title Page:

The first or sole Notice should read --

Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 883 days.

Signed and Sealed this Twenty-ninth Day of September, 2015 74-4-04- 2% 4 Michelle K. Lee Director of the United States Patent and Trademark Office