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WO2011112090A2 - Procédé d'identification d'acides nucléiques codant pour une protéine rep variante - Google Patents

Procédé d'identification d'acides nucléiques codant pour une protéine rep variante Download PDF

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WO2011112090A2
WO2011112090A2 PCT/NL2011/050171 NL2011050171W WO2011112090A2 WO 2011112090 A2 WO2011112090 A2 WO 2011112090A2 NL 2011050171 W NL2011050171 W NL 2011050171W WO 2011112090 A2 WO2011112090 A2 WO 2011112090A2
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parvoviral
rep
bac
mutant
sequence
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WO2011112090A3 (fr
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Yvet Noordman
Andrew Christian BAKKER
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Amsterdam Molecular Therapeutics AMT IP BV
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14311—Parvovirus, e.g. minute virus of mice
    • C12N2750/14341—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14344—Chimeric viral vector comprising heterologous viral elements for production of another viral vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • the present invention relates to a method for identifying a Parvoviral nucleotide sequence encoding a mutant Rep polypeptide having one or more improved properties.
  • the invention also relates to a nucleic acid construct which may be used in such a method and to a baculovirus such a nucleic acid construct and to a sequence encoding a mutant rep polypeptide identifiable by such a method.
  • the invention relates further to a method for the preparation of a library of nucleic acid sequences encoding mutant Parvoviral Rep proteins.
  • Adeno-associated virus is considered one of the most promising viral vectors for human gene therapy.
  • AAV has the ability to efficiently infect dividing as well as non-dividing human cells, the AAV viral genome integrates into a single chromosomal site in the host cell's genome, and most importantly, even though AAV is present in many humans, it has never been associated with any disease.
  • rAAV recombinant adeno-associated virus
  • rAAV for use in gene therapy has traditionally been produced on mammalian cell lines such as e.g. 293 cells, COS cells, HeLa cells, KB cells, and other mammalian cell lines.
  • mammalian cell lines such as e.g. 293 cells, COS cells, HeLa cells, KB cells, and other mammalian cell lines.
  • the number of AAV particles generated per cell is of the order of 10 4 particles (reviewed in Clark, 2002, Kidney Int. 61(Suppl. 1): 9-15). For a clinical study, more than 10 15 particles of rAAV may be required.
  • an AAV production system has been developed using insect cells (Urabe et al., 2002, Hum. Gene Ther. 13: 1935-1943; US 20030148506 and US 20040197895).
  • This baculovirus expression vector system (BEVS) is based on infection of insect cells with baculoviruses containing a gene to be expressed flanked by AAV ITRs, a baculovirus expressing the AAV rep gene and a baculovirus encoding the AAV cap gene leading to production of infectious rAAV particles.
  • the AAV rep and cap genes may be present on the same baculovirus.
  • This invention is based on the development of a new method for the identification of new, mutant/mutated Rep encoding sequences. Such sequences have improved properties as compared to their corresponding wild type sequences.
  • a mutant/mutated sequence is one which is different from its corresponding wild type sequence.
  • a mutant/mutated sequence may typically be one which does not exist in nature.
  • a providing a plurality of Parvoviral nucleotide constructs, each construct comprising: a Parvoviral nucleotide sequence encoding a mutant Rep polypeptide operably linked to a regulatory sequence; and at least one Parvoviral Inverted Terminal Repeat (ITR) flanking said mutant Rep encoding sequence and regulatory sequence; b. transferring the plurality of nucleotide constructs into host cells which are capable of expressing Paroviral cap polypeptides;
  • ITR Parvoviral Inverted Terminal Repeat
  • the improved properties may be determined in relation to a reference sequence which is typically the sequence used to generate the plurality of nucleic acid constructs in the method.
  • the reference sequence may be a wild type or may be a mutant sequence itself. That is to say, the method may be used to identify a Parvoviral nucleotide sequence encoding a mutant Rep polypeptide having one or more improved properties in comparison to a reference sequence. That reference sequence may be used to generate the plurality of Parvoviral nucleotide constructs, each construct comprising: a Parvoviral nucleotide sequence encoding a mutant Rep polypeptide operably linked to a regulatory sequence; and at least one Parvoviral Inverted Terminal Repeat (ITR) flanking said mutant Rep encoding sequence and regulatory sequence.
  • ITR Parvoviral Inverted Terminal Repeat
  • the invention also provides a nucleic acid construct which comprises two Parvoviral ITRs and, located between the two ITRs, a nucleotide sequence encoding a mutant Parvoviral Rep polypeptide and a nucleotide sequence encoding a reporter polypeptide.
  • a nucleic acid construct may be use in a method of the invention.
  • a nucleic acid construct of the invention may be comprised within a baculovirus.
  • the invention also provides a method for the preparation of a library of nucleic acid sequences encoding mutant Parvoviral Rep polypeptides, which method comprises carrying out random mutagenesis on a Parvoviral Rep encoding sequence, thereby to prepare a library of nucleic acids.
  • a sequence encoding a mutant Rep polypeptide identifiable by a method of the invention is also provided by the invention. Such a sequence may be used in the manufacture of a Parvoviral vector, for example for use in the manufacture of AAV.
  • Figure 1 shows a schematic representation of pVD142(lot#2).
  • Figure 2 shows a schematic representation of pVD156
  • Figure 3 shows a schematic representation of pVD143.
  • Figure 4 shows the restriction analysis of the two different pVD142-Rep-EP libraries
  • M Smartladder (Eurogentec). The lower panel is overexposed to detect the 175bp fragment.
  • Figure 5 shows the quality control of Bac.Rep-EP libraries.
  • Baculoviral DNA was isolated from pO and pi baculovirus stocks and used in an insert control PCR using primerset #349/350. As shown for all baculovirus stocks this resulted predominantly in a ⁇ 1.9kbp fragment.
  • Four different plasmids were used as positive controls (lane 2-4).
  • Figure 6 shows the amplified Rep-EP3 sequences selected after the first selection round, the introduction of new mutations and restriction analysis of the two new libraries.
  • A) Schematic overview of the viral DNA isolated after the first selection round. The primer binding sites that flank the selected Rep-EP3 sequences are indicated by pr321-pr324.
  • B) C) An agarose gel showing the amplified PCR products when using the AMT primer set #321/#322 and different amounts of viral DNA input isolated after the first selection round. The 1869bp fragments were isolated from gel and lOng was used as a template in the EP-PCR to introduce new mutations.
  • Figure 7 shows the quality control of the Rep-EP libraries second selection round used in the second selection round.
  • Baculoviral DNA isolated from pi baculovirus stocks was used in an insert control PCR using primerset #349/350. Except for Bac.VD142, all baculovirus stocks revealed the presence of the correct 1.9kbp fragment. Plasmids pVD142 and pVD143 were used as positive controls (lane 2-3).
  • Figure 8 shows the amplified selected Rep-EP-EP3 sequences after the second selection round when using the AMT primer set #323/#324 and different amounts of viral DNA template.
  • the 1915bp fragment obtained with the PCR in lane 5 was isolated from gel and cloned to pDONR221, resulting in the pDONR221-selectEP-EP3 library. Plasmids pVD142 and pVD143 are used as a negative control and positive control in the two different PCR reactions.
  • M smartladder (Eurogentec).
  • Figure 9 shows the virus titers of rAAV5 productions with Bac.Rep-select-EP- EP3 clones 1-20 and the control productions with Bac.VD142 and Bac.VD143.
  • Virus titers of the rAAV5 productions using plaque purified clones were determined in crude lysates using the Q-PCR method. Productions with clones 12, 13 and 16-20 gave virus titers that were in the range of l-2xlO n gc/ml, while the positive control production with Bac.VD143 (black bar) resulted in a titer of 4xl0 10 gc/ml. The negative control production with Bac.VD142 generated a value of 8.4xl0 7 gc/ml.
  • Figure 10 shows the Rep protein expression from Rep baculovirus constructs comprising the YF mutations.
  • X At the upper part a schematic overview of the Rep78 and Rep52 replication proteins is shown which are expressed by Bac.VD88. Functional domains present in the Rep proteins are indicated at the top (Chiorini, J. A., F. Kim, L. Yang, and R. M. Kotin.
  • Figure 11 shows the Rep protein expression from Rep baculovirus constructs comprising the GPR mutations.
  • X At the upper part a schematic overview of the Rep78 and Rep52 replication proteins is shown which are expressed by Bac.VD88. Functional domains present in the Rep proteins are indicated at the top (Chiorini, J. A., F. Kim, L. Yang, and R. M. Kotin.
  • Figure 12 shows the CMV-SEAP transgene replication during rAAV5 production using the different Rep baculovirus constructs.
  • Representative agarose gels showing the LMW-DNA isolated from rAAV5 productions using the Rep baculovirus constructs comprising the YF (A) or GPR (B) mutations and the control constructs.
  • LMW-DNA was isolated from cell pellets that were harvested 1, 2 and 3 days p.i.
  • the monomeric and dimeric replicative forms of the transgene are indicated with RFm and RFd, respectively. Higher order forms are only indicated with arrows.
  • Figure 13 shows the virus titers of rAAV5 productions performed with the different Rep baculovirus constructs.
  • Virus titers were determined in clarified crude lysates using the CMV Q-PCR method.
  • Productions with Bac.VD216 and Bac.VD217 significantly improved the virus titers, while Bac.VD228 reduced the virus titer.
  • Figure 15 shows the amount of residual baculovirus DNA in rAAV5 batch purified samples as ratio from the transgene.
  • Primer set pr59/60 targets the CMV promoter and is used to determine the ratio between transgene and residual DNA present in the batches.
  • the HR3 primer set is not shown.
  • B) The amount of residual left ORF DNA determined with pr406/407 is shown as the CMV/left ORF ratio.
  • Figure 16 shows a sequence comparison of VD88, VD210, VD211, VD212, VD214, VD215, VD216, VD217, VD218 and VD220.
  • the present invention relates to animal parvoviruses, in particular dependoviruses such as infectious human or simian AAV, and to the generation of novel components thereof (e.g., an animal parvovirus genome).
  • Such parvoviruses may be used as vectors for introduction and/or expression of nucleic acids in mammalian cells.
  • the invention relates to improvements in Rep proteins (and nucleotides encoding such proteins) which may be used to increase productivity of such parvoviral vectors and to create parvoviral vectors of increased quality, for example when such vectors are produced in insect cells.
  • Viruses of the Parvoviridae family are small DNA animal viruses.
  • the family Parvoviridae may be divided between two subfamilies: the Parvovirinae, which infect vertebrates, and the Densovirinae, which infect insects.
  • Members of the subfamily Parvovirinae are herein referred to as the parvoviruses and include the genus Dependovirus.
  • members of the Dependovirus are unique in that they usually require coinfection with a helper virus such as adenovirus or herpes virus for productive infection in cell culture.
  • the genus Dependovirus includes AAV, which normally infects humans (e.g., serotypes 1, 2, 3 A, 3B, 4, 5, and 6) or primates (e.g., serotypes 1 and 4), and related viruses that infect other warm-blooded animals (e.g., bovine, canine, equine, and ovine adeno-associated viruses). Further information on parvoviruses and other members of the Parvoviridae is described in Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication," Chapter 69 in Fields Virology (3d Ed. 1996). For convenience the present invention is further exemplified and described herein by reference to AAV. It is however understood that the invention is not limited to AAV but may equally be applied to other parvoviruses.
  • the genomic organization of all known AAV serotypes is very similar.
  • the genome of AAV is a linear, single-stranded DNA molecule that is less than about 5,000 nucleotides (nt) in length.
  • Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences for the non- structural replication (Rep) proteins and the structural (VP) proteins.
  • the VP proteins (VP1, -2 and -3) form the capsid.
  • the terminal 145 nt are self-complementary and are organized so that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. These hairpin structures function as an origin for viral DNA replication, serving as primers for the cellular DNA polymerase complex.
  • Rep78 and Rep52 are expressed from the P5 promoter and the P19 promotor, respectively and both Rep proteins have a function in the replication of the viral genome.
  • a splicing event in the Rep ORF results in the expression of actually four Rep proteins (i.e. Rep78, Rep68, Rep52 and Rep40).
  • Rep78, Rep68, Rep52 and Rep40 Rep proteins
  • the invention concerns a selection procedure for the laboratory directed protein evolution of the nucleic acids encoding new Rep proteins.
  • the Rep mutant proteins that are selected using this procedure may be used to improve Parvoviral production methods, in particular baculovirus-based production processes and especially in the use of such production processes to manufacture AAV.
  • Parvoviruses for example recombinant adeno-associated viruses (AAVs), the baculovirus expression vector system (BEVS) is well known.
  • AAVs adeno-associated viruses
  • BEVS baculovirus expression vector system
  • the non- structural AAV Rep78 and Rep52 proteins the therapeutic gene flanked by ITRs are replicated and packaged into the preformed empty capsids.
  • the mutant Rep proteins that are generated may possess improved ssDNA encapsidation properties as compared to the wild type Rep protein and, at the same time, the Rep proteins will be evolved to function better in host cells, such as insect cells, in general.
  • Creating molecular diversity is the first step in the method of the invention.
  • a plurality of nucleic acids encoding mutant Rep sequences i.e. a library of mutant nucleic acids encoding mutant Rep proteins may be created.
  • the frequencies of mutations that are introduced into the Rep gene may be changed by varying the amount of template and PCR cycles.
  • the resulting plurality of potential Rep encoding sequences are consecutively cloned into a vector containing one cassette such that each Rep encoding sequence is under the control of the a promoter, such as a baculovirus polyhedrin promoter, and a second reporter gene expression cassette, such as a green fluorescent protein (GFP) expression cassette, under the control of a promoter, such as the CMV and the baculovirus plO promoter, which in total is flanked by one or more Parvoviral ITRs (typically flanked by two AAV ITRs).
  • the library may be transferred into host cells. Typically, this may be carried out by way of a baculovirus library, with each baculovirus comprising at least one mutant Rep encoding sequence. This system is advantageous since it only produces recombinant
  • baculoviruses because baculovirus DNA that is not homologously recombined with a transfer vector is unable to generate replicative baculoviruses.
  • the selection procedure may consist of rAAV productions (if the mutant Rep encoding sequences are from AAV) that are performed with the different Rep mutant libraries.
  • the mutant Rep proteins with a higher selection advantage are capable of packaging their own encoding ssDNA into a higher number of capsids.
  • Rep mutant sequences which are encapsidated in the rAAV particles
  • these may be re-cloned and subjected to a new selection round.
  • additional variation may be introduced by subjecting these libraries to another round of mutation induction.
  • Rep mutants with beneficial properties will be enriched in the library after each selection round and non-functional or Rep mutants without improved functions will disappear from the library.
  • Parvoviral nucleotide sequence encoding a mutant Rep polypeptide having one or more improved properties which method comprises:
  • each construct comprising: a Parvoviral nucleotide sequence encoding a mutant rep polypeptide operably linked to a regulatory sequence; and at least one Parvoviral Inverted
  • Terminal Repeat flanking said mutant rep encoding sequence and regulatory sequence
  • the parvoviral sequences will be AAV sequences incorporated into baculo viral vector constructs and the host cells will be insect cells.
  • a plurality of mutant nucleic acids is provided.
  • the plurality of nucleic acids encodes a plurality of different Parvoviral Rep proteins so that specific sequences may be selected. It is not the case that each of the nucleic acids is distinct, i.e. different from all others. It may be that one or more nucleotide sequences is present two or more times within the plurality of sequences. This will almost certainly be the case if the plurality of sequences is amplified, for example by PCR.
  • a plurality of nucleic acids encoding a plurality of different Parvoviral Rep proteins may be generated according to methods well known to those skilled in the art.
  • mutagenesis is carried out on an initial sequence.
  • the initial sequence may be a nucleotide sequence encoding an animal parvovirus Rep protein.
  • An animal parvovirus Rep protein is herein understood as a nucleotide sequence encoding a non- structural Rep protein that is typically required for parvoviral vector production in insect cells such the Rep78 and Rep52 proteins.
  • the animal parvovirus nucleotide sequence preferably is from a dependovirus, more preferably from a human or simian adeno-associated virus (AAV) and most preferably from an AAV which normally infects humans (e.g., serotypes 1, 2, 3A, 3B, 4, 5, and 6) or primates (e.g., serotypes 1 and 4).
  • AAV adeno-associated virus
  • An example of a nucleotide sequence encoding an animal parvoviruse Rep protein is given in SEQ ID No. 1 and 3, which depict a wild type Rep sequence (Rep78 from AAV2) and a variant thereof (the so-called VD88 variant; see WO2007/148971) respectively.
  • SEQ ID NOs: 1 and 3 both encode Rep78 and Rep 52.
  • the exact molecular weights of the Rep78 and Rep52 proteins, as well as the exact positions of the translation initiation codons may differ between different parvoviruses.
  • the nucleotide sequence used as a starting encodes animal parvovirus Rep proteins that are required and sufficient for parvoviral vector production in insect cells (typically a Rep78 and a Rep52).
  • the nucleic acid which is used to generate a plurality of mutant sequences may comprising an open reading frame comprising nucleotide sequences encoding animal parvovirus Rep proteins (i.e. more than one Rep protein), wherein the initiation codon for translation of the parvoviral Rep78 protein is a suboptimal initiation codon.
  • the suboptimal initiation codon preferably is an initiation codon that effects partial exon skipping. Partial exon skipping is herein understood to mean that at least part of the ribosomes do not initiate translation at the suboptimal initiation codon of the Rep78 protein but at an initiation codon further downstream, whereby preferably the initiation codon further downstream is the initiation codon of the Rep52 protein.
  • the suboptimal initiation codon preferably effects partial exon skipping upon expression of the nucleotide sequence in an insect cell.
  • the suboptimal initiation codon effects partial exon skipping in an insect cell so as to produce in the insect cell a molar ratio of Rep78 to Rep52 in the range of 1:10 to 10: 1, 1:5 to 5:1, or 1:3 to 3:1, preferably at about 20 - 40 hours post infection, more preferably at about 30 - 40 hours post infection, using a baculovirus expression.
  • the molar ratio of the Rep78 and Rep52 may be determined by means of Western blotting as described in Example 2.1.5, preferably using a monoclonal antibody that recognizes a common epitope of both Rep78 and Rep52, or using the antibody described in Example 2.1.5.
  • suboptimal initiation codon not only refers to the trinucleotide intitiation codon itself but also to its context.
  • a suboptimal initiation codon may consist of an "optimal" ATG codon in a suboptimal context, e.g. a non- Kozak context.
  • suboptimal initiation codons wherein the tri-nucleotide intitiation codon itself is suboptimal, i.e. is not ATG.
  • Suboptimal is herein understood to mean that the codon is less efficient in the inititiation of translation in an otherwise identical context as compared to the normal ATG codon.
  • the efficiency of suboptimal codon is less than 90, 80, 60, 40 or 20% of the efficiency of the normal ATG codon in an otherwise identical context.
  • Methods for comparing the relative efficiency of inititiation of translation are known per se to the skilled person.
  • Preferred suboptimal initiation codons may be selected from ACG, TTG, CTG, and GTG. More preferred is ACG.
  • the starting sequence used for mutagenesis may also be one in which possible false translation initiation sites in the Rep protein coding sequences, other than the Rep78 and Rep52 translation initiation sites, are eliminated. Also, putative splice sites that may be recognised in insect cells may be eliminated.
  • the various modifications of the wild-type parvoviral sequences for proper expression in insect cells is achieved by application of well-known genetic engineering techniques such as described e.g. in Sambrook and Russell (2001) "Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York.
  • Various further modifications of Rep protein coding regions are known to the skilled artisan which could increase yield of Rep protein. These modifications are within the scope of the initial sequence used to generate a plurality of mutants in the present invention.
  • the starting sequence for mutagensis comprises no more than one type of nucleotide sequence comprising a single open reading frame encoding a parvoviral Rep protein.
  • the single open reading frame encodes one or more of the parvoviral Rep proteins, more preferably the open reading frame encodes all of the parvoviral Rep proteins, most preferably the open reading frame encodes the full-length Rep 78 protein from which preferably at least both Rep 52 and Rep 78 proteins may be expressed in the insect cell.
  • the insect cell may comprise more than one copy of the single type of nucleotide sequence, e.g.
  • nucleic acid molecules that encode one and the same Rep amino acid sequence, e.g. nucleic acid molecules that only differ between each other due to the degeneracy of the genetic code.
  • the presence of only a single type of nucleic acid molecule encoding the parvoviral Rep proteins avoids recombination between homologous sequences as may be present in different types of vectors comprising Rep sequences, which may give rise to defective Rep expression constructs that affect (stability of) parvoviral production levels in insect cells.
  • the nucleotide sequence comprising the single open reading frame encoding one or more parvoviral Rep proteins is part of a nucleic acid construct wherein the nucleotide sequence is operably linked to expression control sequences for expression in an insect cell.
  • Mutagenesis may be carried out in any manner known to the skilled person. For example, such mutagenesis could be random, although such mutagenesis could be directed (i.e. for example, to target specific sequences/structures within a Rep protein). Random mutagenesis may be carried out to achieve low mutation rates, for example to provide sequences which encode a Rep protein having one, two, three, four, five, six, seven, eight, nine or ten or more amino acid changes (as compared with the starting sequence on which mutagenesis is carried out).
  • Techniques which may be used to carry out random mutagenesis include E. coli
  • Error prone PCR is a modification of standard PCR protocols, designed to alter and enhance the natural error rate of the polymerase.
  • Taq polymerase may be used because of its naturally high error rate, with errors biased toward AT to GC changes.
  • alternative forms of polymerase whose biases allow for increased variation in mutation type (i.e. more GC to AT changes).
  • Error-prone PCR reactions typically contain higher concentrations of MgCl 2 compared to basic PCR reactions, in order to stabilize non-complementary pairs. MgCl 2 can also be added to increase the error-rate.
  • Other ways of modifying mutation rates include varying the rations of nucleotides in the reaction, or including a nucleotide analog such as 8-oxo-GTP or dITP. Mutation rates may also be modified by changing the number of effective doublings by increasing/decreasing the number of cycles or by changing the initial template concentration.
  • Each construct typically comprises: a Parvoviral nucleotide sequence encoding a mutant rep polypeptide operably linked to a regulatory sequence (or expression control sequence); and at least one Parvoviral Inverted Terminal Repeat (ITR) flanking said mutant rep encoding sequence and regulatory sequence;
  • TTR Inverted Terminal Repeat
  • operably linked refers to a linkage of polynucleotide (or polypeptide) elements in a functional relationship.
  • a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in the same reading frame.
  • regulatory sequence refers to a nucleic acid sequence that regulates the expression of a nucleotide sequence to which it is operably linked.
  • An expression control sequence is “operably linked” to a nucleotide sequence when the expression control sequence controls and regulates the transcription and/or the translation of the nucleotide sequence.
  • an expression control sequence can include promoters, enhancers, internal ribosome entry sites (IRES), transcription terminators, a start codon in front of a protein-encoding gene, splicing signal for introns, and stop codons.
  • the term “expression control sequence” is intended to include, at a minimum, a sequence whose presence are designed to influence expression, and can also include additional advantageous components.
  • leader sequences and fusion partner sequences are expression control sequences.
  • the term can also include the design of the nucleic acid sequence such that undesirable, potential initiation codons in and out of frame, are removed from the sequence. It can also include the design of the nucleic acid sequence such that undesirable potential splice sites are removed. It includes sequences or polyadenylation sequences (pA) which direct the addition of a polyA tail, i.e., a string of adenine residues at the 3'-end of a mRNA, sequences referred to as polyA sequences. It also can be designed to enhance mRNA stability.
  • pA polyadenylation sequences
  • Expression control sequences which affect the transcription and translation stability e.g., promoters, as well as sequences which effect the translation, e.g., Kozak sequences, are known in insect cells.
  • Expression control sequences can be of such nature as to modulate the nucleotide sequence to which it is operably linked such that lower expression levels or higher expression levels are achieved.
  • promoter or “transcription regulatory sequence” refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter.
  • a “constitutive” promoter is a promoter that is active in most tissues under most physiological and developmental conditions.
  • An “inducible” promoter is a promoter that is physiologically or developmentally regulated, e.g. by the application of a chemical inducer.
  • a "tissue specific” promoter is only active in specific types of tissues or cells.
  • At least one parvoviral ITR nucleotide sequence is understood to mean a palindromic sequence, comprising mostly complementary, symmetrically arranged sequences also referred to as "A,” "B,” and “C” regions.
  • the ITR functions as an origin of replication, a site having a "cis” role in replication, i.e., being a recognition site for trans acting replication proteins such as e.g. Rep 78 (or Rep68) which recognize the palindrome and specific sequences internal to the palindrome.
  • Rep 78 or Rep68
  • One exception to the symmetry of the ITR sequence is the "D" region of the ITR. It is unique (not having a complement within one ITR).
  • Nicking of single- stranded DNA occurs at the junction between the A and D regions. It is the region where new DNA synthesis initiates. The D region normally sits to one side of the palindrome and provides directionality to the nucleic acid replication step.
  • An parvovirus replicating in a mammalian cell typically has two ITR sequences. It is, however, possible to engineer an ITR so that binding sites are on both strands of the A regions and D regions are located symmetrically, one on each side of the palindrome.
  • the Rep78- or Rep68- assisted nucleic acid replication then proceeds in both directions and a single ITR suffices for parvoviral replication of a circular vector.
  • one ITR nucleotide sequence can be used in the context of the present invention.
  • two or another even number of regular ITRs are used.
  • Most preferably, two ITR sequences are used.
  • a preferred parvoviral ITR is an AAV ITR.
  • rAAV recombinant parvoviral
  • Such a safety mechanism for limiting undesirable vector propagation in a recipient may be provided by using rAAV with a chimeric ITR as described in US2003148506.
  • the plurality of nucleic acids encoding mutant Rep proteins are comprised within nucleic acid constructs.
  • the nucleotide sequence encoding the parvoviral Rep proteins is operably linked to expression control sequences for expression in an insect cell.
  • expression control sequences will at least include a promoter that is active in insect cells.
  • Techniques known to one skilled in the art for expressing foreign genes in insect host cells can be used to practice the invention. Methodology for molecular engineering and expression of polypeptides in insect cells is described, for example, in Summers and Smith. 1986. A Manual of Methods for Baculo virus Vectors and Insect Culture Procedures, Texas Agricultural Experimental Station Bull. No. 7555, College Station, Tex.; Luckow. 1991.
  • a particularly suitable promoter for transcription of the nucleotide sequence of the invention encoding of the parvoviral Rep proteins is e.g. the polyhedron promoter.
  • other promoters that are active in insect cells are known in the art, e.g. the plO, p35, IE-1 or ⁇ IE-1 promoters and further promoters described in the above references.
  • a further preferred nucleic acid construct of the invention comprises an expression control sequence that comprising a nine nucleotide sequence of the sequence disclosed at page 9, lines 14 to 21 of WO2007/148971 or a nucleotide sequence substantially homologous thereto, upstream of the initiation codon of the nucleotide sequence encoding the parvoviral Rep78 protein.
  • a sequence with substantial identity to the nucleotide sequence of the sequence referred to above and that will help increase expression of the parvoviral Rep78 protein is e.g. a sequence which has at least 60%, 70%, 80% or 90% identity to the nine nucleotide sequence referred to.
  • the nucleic acid construct for expression of the parvoviral Rep proteins in insect cells is an insect cell-compatible vector.
  • An "insect cell-compatible vector” or “vector” is understood to a nucleic acid molecule capable of productive transformation or transfection of an insect or insect cell.
  • Exemplary biological vectors include plasmids, linear nucleic acid molecules, and recombinant viruses. Any vector can be employed as long as it is insect cell-compatible.
  • the vector may integrate into the insect cells genome but the presence of the vector in the insect cell need not be permanent and transient episomal vectors are also included.
  • the vectors can be introduced by any means known, for example by chemical treatment of the cells, electroporation, or infection.
  • the vector is a baculovirus, a viral vector, or a plasmid.
  • the vector is a baculovirus, i.e. the construct may be a baculoviral vector.
  • Baculoviral vectors and methods for their use are described in the above cited references on molecular engineering of insect cells.
  • the plurality of nucleotide constructs is transferred into host cells which are capable of expressing Parvoviral cap polypeptides.
  • Such transfer may be carried out according to any suitable transformation or transfection technique (the terms “transformation” and “transfection” may be used interchangeably herein to indicate any method by which a nucleic acid is transferred into a cell). These transfer techniques are well known to those skilled in the art.
  • the host cell may be any cell in which it is possible for parvoviral virions to be generated.
  • the host cell will typically be capable of expressing Parvoviral cap proteins.
  • a host cell for use in the invention comprises parvoviral capsid
  • Cap protein coding sequences herein understood to comprise sequences encoding each of the three parvoviral capsid proteins, VP1, -2 and -3.
  • the sequence comprising the capsid protein coding sequences may be present in various forms, e.g. separate coding sequences for each of the capsid proteins VP1, -2 and -3 may used, whereby each coding sequence is operably linked to expression control sequences for expression in the host cell.
  • the Cap protein encoding sequences comprise a single open reading frame encoding all three of the animal parvoviral (AAV) VP1, VP2, and VP3 capsid proteins, wherein the initiation codon for translation of the VP1 capsid protein is a suboptimal initiation codon that is not ATG as e.g. described by Urabe et al. (2002, supra).
  • a suboptimal initiation codon for the VP1 capsid protein may be as defined above for the Rep78 protein. More preferred suboptimal initiation codons for the VP1 capsid protein may be selected from ACG, TTG, CTG and GTG, of which CTG and GTG are most preferred.
  • a preferred nucleotide sequence for the expression of the capsid proteins further comprises an expression control sequence comprising a nine nucleotide sequence referred to at page 9, lines 14 to 21 of WO2007/148971 or a nucleotide sequence substantially homologous thereto, upstream of the initiation codon of the nucleotide sequence encoding the VP1 capsid protein.
  • a sequence with substantial identity to the said nucleotide sequence of WO2007/148971 and that will help increase expression of VP1 is e.g. a sequence which has at least 60%, 70%, 80% or 90% identity to the nine nucleotide sequence of WO2007/148971.
  • a further preferred nucleotide sequence for expression of the capsid proteins further preferably comprises at least one modification of the nucleotide sequence encoding the VP1 capsid protein selected from among a C at nucleotide position 12, an A at nucleotide position 21, and a C at nucleotide position 24 (with reference to position 1 being the first nucleotide of the translation initiation codon; see see the VP capsid sequence as disclosed in WO2007/148971).
  • nucleotide sequence of the invention encoding the parvoviral capsid proteins is operably linked to expression control sequences for expression in an insect cell, which will at least include a promoter that is active in insect cells.
  • control sequences and further techniques and materials for expressing parvoviral capsid proteins in insect host cells are already described above for the Rep proteins.
  • a cell for use in the invention in addition to the above described mutant sequence, further comprises nucleotide sequences comprising parvoviral Cap protein coding sequences operably linked to expression control sequences for expression in an insect cell.
  • nucleic acid constructs employed in an insect cell for the production of the recombinant parvoviral (such as rAAV) virions in the method of the invention is not limiting in the invention.
  • one, two, three, four or more separate constructs can be employed to produce rAAV in insect cells in accordance with the methods of the present invention. If four constructs are employed, one construct encodes AAV VP 1, another construct encodes AAV VP2, yet another construct encodes AAV VP3, still yet another construct encodes the AAV ITR/Rep protein as defined above. If fewer than four constructs are used, the constructs can comprise various combinations of the AAV ITR/Rep protein coding sequences and the VP1, VP2, VP3 coding sequences.
  • the insect cell comprises: (a) a first nucleic acid construct for expression of the Rep proteins as defined above; and (b) a second construct which comprises the parvoviral Cap protein coding sequences operably linked to at least one expression control sequence for expression in an insect cell; see also below.
  • the sequences on each construct can be in any appropriate order relative to each other.
  • one construct comprises AAV ITR/Rep protein coding sequences and an ORF comprising nucleotide sequences encoding VP capsid proteins
  • the VP ORF can be located on the construct such that, upon replication of the DNA between ITR sequences, the VP ORF is replicated or not replicated.
  • the Rep coding sequences and/or the ORF comprising nucleotide sequences encoding VP capsid proteins can be in any order on a construct.
  • the nucleic acid construct(s) used in the invention preferably are an insect cell- compatible vectors, preferably a baculo viral vectors as described above.
  • one or more nucleotide sequence may be stably integrated in the genome of the insect cell.
  • One of ordinary skill in the art knows how to stably introduce a nucleotide sequence into the insect genome and how to identify a cell having such a nucleotide sequence in the genome.
  • the incorporation into the genome may be aided by, for example, the use of a vector comprising nucleotide sequences highly homologous to regions of the insect genome.
  • the use of specific sequences, such as transposons, is another way to introduce a nucleotide sequence into a genome.
  • the nucleotide sequence present in the host cells, such as insect cells, in the method of invention i.e. the sequence comprising at least one parvoviral (AAV) ITR, further comprises at least one nucleotide sequence encoding a gene product of interest, whereby preferably the at least one nucleotide sequence encoding a gene product of interest becomes incorporated into the genome of a recombinant parvoviral (rAAV) vector produced in the insect cell.
  • at least one nucleotide sequence encoding a gene product of interest is a sequence for expression in a mammalian cell.
  • the nucleic acid comprising Rep coding sequences flanked by at least on parvoviral (AAV) ITR nucleotide sequence also comprises a nucleotide sequence encoding a gene product which is a reporter polypeptide, also flanked by the at least one parvoviral (AAV) ITR nucleotide sequences.
  • the Rep encoding cassette and a reporter gene product encoding sequence will be incorporated into the recombinant parvoviral (rAAV) vector produced in the insect cell if the two cassettes are located between two regular ITRs.
  • Any cell which allows for replication of a recombinant parvoviral (for example rAAV) vector and which can be maintained in culture can be used in accordance with the present invention.
  • AAV for example is able to infect a number of mammalian cells. See, e.g., Tratschin et al. (1985, Mol. Cell Biol. 5:3251-3260) and Grimm et al. (1999, Hum. Gene Ther. 10:2445-2450).
  • AAV transduction of human synovial fibroblasts is significantly more efficient than in similar murine cells, Jennings et al., Arthritis Res, 3: 1 (2001), and the cellular tropicity of AAV differs among serotypes.
  • the host cell used in the invention may be a mammalian cell line.
  • cells from Spodoptera frugiperda, drosophila cell lines, or mosquito cell lines, e.g., Aedes albopictus derived cell lines are preferred.
  • Preferred insect cells or cell lines are cells from the insect species which are susceptible to baculovirus infection, including e.g.
  • the host cells are exposed to conditions (i.e. grown/cultured under those conditions) which allow for expression of Parvoviral cap polypeptides and the expression of a Parvoviral mutant rep polypeptide so that the Parvoviral nucleotide constructs (comprising the mutant Rep encoding nucleotide sequence flanked by one or more ITR) may be packaged into said cap polypeptides giving rise to Parvoviral virions.
  • a nucleotide construct used in the invention may also be referred to as a "recombinant parvo viral or AAV vector" (or "rAAV vector”), i.e.
  • rAAV vectors comprising one or more polynucleotide sequences of interest, genes of interest or "trans genes" (here mutant Rep protein encoding sequences) that are flanked by parvoviral or AAV inverted terminal repeat sequences (ITRs).
  • TTRs parvoviral or AAV inverted terminal repeat sequences
  • rAAV vectors can be replicated and packaged into infectious viral particles when present in an insect host cell that is expressing AAV rep and cap gene products (i.e. AAV Rep and Cap proteins).
  • AAV Rep and Cap proteins i.e. AAV Rep and Cap proteins
  • the rAAV vector in a chromosome or in another vector such as a plasmid or baculovirus used for cloning or transfection), then the rAAV vector is typically referred to as a "pro-vector" which can be "rescued” by replication and encapsidation in the presence of AAV packaging functions and necessary helper functions.
  • Parvoviral virions are then isolated from the host cell culture and/or supernatant.
  • the selection procedure effectively consists of a parvoviral (for example rAAV if the mutant Rep encoding sequences are from AAV) production performed with the Rep mutant library.
  • those mutant Rep proteins with a higher selection advantage are capable of packaging their own encoding ssDNA into a higher number of capsids and should be preferentially represented in the host cell culture and/or supernatant.
  • This may involve a method further comprises the step of affinity-purification of the (virions comprising the) recombinant parvoviral (rAAV) vector using an anti-AAV antibody, preferably an immobilised antibody.
  • the anti-AAV antibody preferably is an monoclonal antibody.
  • a particularly suitable antibody is a single chain camelid antibody or a fragment thereof as e.g. obtainable from camels or llamas (see e.g. Muyldermans, 2001, Biotechnol. 74: 277-302).
  • the antibody may be used for affinity-purification of rAAV, preferably the antibody is one that specifically binds an epitope on a AAV capsid protein, whereby preferably the epitope is an epitope that is present on capsid protein of more than one AAV serotype.
  • the antibody may be raised or selected on the basis of specific binding to AAV2 capsid but at the same time also it may also specifically bind to AAV1, AAV3 and AAV5 capsids.
  • the improved properties may be improved in comparison with the sequence that was initially used to generate the plurality of Rep mutant encoding nucleotides.
  • improved properties may be in comparison with the corresponding wild type sequence for example a wild Rep protein from a parvovirus, such as AAV.
  • a nucleic acid of the invention will typically display improved properties as compared to its corresponding wild type sequence or the sequence used to generate the plurality of sequences for screening. For example, it may lead to improved Parvoviral product (i.e. higher virus titre) when used to produce recombinant Parvovirus, such as rAAV, as compared to the corresponding wild type. It may lead to a better quality product in terms of having fewer empty virions in comparison to full virions (i.e. virions filled with vector) or to put it another way, a lower (i.e. improved) total/full particle ratio. It may also lead to the accumulation of less residual DNA than a corresponding wild type sequence.
  • a nucleic acid of the invention may be one which produces a measurable improvement in any such relevant property, for example one of those mentioned above, in comparison to the corresponding wild type sequence.
  • Preferred nucleic acids are those which show an improvement as compared to the wild type in any relevant property of at least about 10%, at least about 25%, at least about 50%, at least about 100%, at least about 200%, at least about 500% or at least about 1000% or more.
  • step (e) the Parvoviral nucleotide constructs recovered from the Parvoviral virions isolated in step (d) may be used as a second library of Parvoviral nucleotide constructs which is then subjected again to steps (b) to (e), optionally further repeating step (b) to (e).
  • a method of the invention may comprise only one round of selection. However, more typically, the method may be carried out using two, three, four, five or more rounds of selection.
  • the library of Parvoviral nucleotide constructs may be subjected to mutagenesis between each round of selection.
  • the improved property may be an increased ability to package Parvoviral genomes within Parvoviral cap polypeptides as compared with the corresponding wild-type Parvoviral rep polypeptide or the ability to be expressed more highly.
  • AAV sequences may be used in the invention.
  • AAV sequences that may be used in the present invention for the production of recombinant AAV vectors in insect cells can be derived from the genome of any AAV serotype.
  • the AAV serotypes have genomic sequences of significant homology at the amino acid and the nucleic acid levels, provide an identical set of genetic functions, produce virions which are essentially physically and functionally equivalent, and replicate and assemble by practically identical mechanisms.
  • genomic sequence of the various AAV serotypes and an overview of the genomic similarities see e.g.
  • AAV serotypes 1, 2, 3, 4 and 5 are preferred source of AAV nucleotide sequences for use in the context of the present invention.
  • the AAV ITR sequences for use in the context of the present invention are derived from AAVl, AAV2, and/or AAV4.
  • the Rep (Rep78 and Rep52) coding sequences are preferably derived from AAVl, AAV2, and/or AAV4, although in principle may be derived from any AAV serotype.
  • sequences coding for the VP1, VP2, and VP3 capsid proteins for use in the context of the present invention may however be taken from any of the known 42 serotypes, more preferably from AAVl, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 or newly developed AAV-like particles obtained by e.g. capsid shuffling techniques and AAV capsid libraries.
  • AAV Rep and ITR sequences are particularly conserved among most serotypes.
  • the Rep78 proteins of various AAV serotypes are e.g. more than 89% identical and the total nucleotide sequence identity at the genome level between AAV2, AAV3A, AAV3B, and AAV6 is around 82% (Bantel-Schaal et al., 1999, J. Virol., 73(2):939- 947).
  • the Rep sequences and ITRs of many AAV serotypes are known to efficiently cross-complement (i.e., functionally substitute) corresponding sequences from other serotypes in production of AAV particles in mammalian cells.
  • US2003148506 reports that AAV Rep and ITR sequences also efficiently cross- complement other AAV Rep and ITR sequences in insect cells.
  • the AAV VP proteins are known to determine the cellular tropicity of the AAV virion.
  • the VP protein-encoding sequences are significantly less conserved than Rep proteins and genes among different AAV serotypes.
  • the ability of Rep and ITR sequences to cross-complement corresponding sequences of other serotypes allows for the production of pseudotyped rAAV particles comprising the capsid proteins of a serotype (e.g., AAV3) and the Rep and/or ITR sequences of another AAV serotype (e.g., AAV2).
  • pseudotyped rAAV particles are a part of the present invention.
  • Modified "AAV" sequences also can be used in the context of the present invention, e.g. for the production of rAAV vectors in insect cells.
  • Such modified sequences e.g. include sequences having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more nucleotide and/or amino acid sequence identity (e.g., a sequence having about 75-99% nucleotide sequence identity) to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 ITR, Rep, or VP can be used in place of wild-type AAV ITR, Rep, or VP sequences.
  • AAV5 differs from other human and simian AAV serotypes more than other known human and simian serotypes.
  • the production of rAAV5 can differ from production of other serotypes in insect cells.
  • one or more constructs comprising, collectively in the case of more than one construct, a nucleotide sequence comprising an AAV5 ITR, a nucleotide sequence comprises an AAV5 Rep coding sequence (i.e. a nucleotide sequence comprises an AAV5 Rep78).
  • ITR and Rep sequences can be modified as desired to obtain efficient production of rAAV5 or pseudotyped rAAV5 vectors in insect cells.
  • the start codon of the Rep sequences can be modified, VP splice sites can be modified or eliminated, and/or the VP1 start codon and nearby nucleotides can be modified to improve the production of rAAV5 vectors in the insect cell.
  • the invention also provides a nucleic acid construct which comprises two Parvoviral ITRs and, located between the two ITRs, a nucleotide sequence encoding a mutant Parvoviral Rep polypeptide and a nucleotide sequence encoding a reporter polypeptide.
  • a reporter polypeptide is one that may be used to serve as a marker protein to assess cell transformation and expression.
  • Suitable marker proteins for this purpose are e.g. the fluorescent protein GFP, and the selectable marker genes HSV thymidine kinase (for selection on HAT medium), bacterial hygromycin B phosphotransferase (for selection on hygromycin B), Tn5 aminoglycoside phosphotransferase (for selection on G418), and dihydrofolate reductase (DHFR) (for selection on methotrexate), CD20, the low affinity nerve growth factor gene.
  • HSV thymidine kinase for selection on HAT medium
  • bacterial hygromycin B phosphotransferase for selection on hygromycin B
  • Tn5 aminoglycoside phosphotransferase for selection on G418)
  • DHFR dihydrofolate reductase
  • second nucleotide sequence defined herein above may comprise a nucleotide sequence encoding a polypeptide that may serve as a fail-safe mechanism that allows curing from cells transduced with the recombinant parvoviral (rAAV) vector of the invention, if deemed necessary.
  • rAAV parvoviral
  • Such a nucleotide sequence often referred to as a suicide gene, encodes a protein that is capable of converting a prodrug into a toxic substance that is capable of killing the transgenic cells in which the protein is expressed.
  • Suitable examples of such suicide genes include e.g. the E.coli cytosine deaminase gene or one of the thymidine kinase genes from Herpes Simplex Virus, Cytomegalovirus and Varicella-Zoster virus, in which case ganciclovir may be used as prodrug to kill the transgenic cells in the subject (see e.g. Clair et al., 1987, Antimicrob. Agents Chemother. 31 : 844-849).
  • the nucleotide sequence(s) encoding a reporter polypeptide is/are typically operably linked to at least one host cell-compatible expression control sequence, e.g., a promoter (as described above).
  • nucleic acid construct may be comprised with a vector, such as a viral vector, for example a baculovirus comprising a nucleic acid construct as described above.
  • a vector such as a viral vector, for example a baculovirus comprising a nucleic acid construct as described above.
  • the invention further provides a method for the preparation of a library of nucleic acid sequences encoding mutant Parvoviral Rep polypeptides which method comprises carrying out random mutagenesis on a Parvoviral Rep encoding sequence, in particular on an AAV Rep encoding sequence, such as a sequence encoding the Rep78 protein. Random mutagenesis techniques are well-known to those skilled in the art and some of these techniques are outlined above. The resulting mutant nucleic acids may be pooled, thereby to prepare a library of nucleic acid sequences encoding mutant Parvoviral Rep polypeptides.
  • the resulting nucleic acids may be incorporated into nucleic acid constructs, for example in which the Rep encoding sequences are operably linked to expression control sequences.
  • the invention also provides a sequence encoding a mutant rep polypeptide identifiable by a method according to the invention. Such a mutant rep polypeptide may be used in the manufacture of a Parvoviral vector.
  • This type of method for producing a recombinant parvoviral (rAAV) virion (comprising a recombinant parvoviral (rAAV) vector as defined above) in an insect cell.
  • the method comprises the steps of: (a) culturing an insect cell comprising a Parvoviral mutant rep polypeptide as identified by the method of the invention under conditions such that recombinant parvoviral (rAAV) vector is produced; and, (b) recovery of the recombinant parvoviral (rAAV) vector.
  • the recombinant parvoviral (rAAV) vector produced in the method preferably is an infectious parvoviral or AAV virion that comprise the recombinant parvoviral (rAAV) vector nucleic acids.
  • Growing conditions for insect cells in culture, and production of heterologous products in insect cells in culture are well-known in the art and described e.g. in the above cited references on molecular engineering of insects cells.
  • the resulting rAAV virion produced in the above described methods preferably comprises in its genome at least one nucleotide sequence encoding a gene product of interest, whereby the at least one nucleotide sequence is not a native AAV nucleotide sequence, and whereby in the stoichiometry of the AAV VP1, VP2, and VP3 capsid proteins the amount of VP1: (a) is at least 100, 105, 110, 120, 150, 200 or 400% of the amount of VP2; or (b) is at least 8, 10, 10.5, 11, 12, 15, 20 or 40% of the amount of VP3; or (c) is at least as defined in both (a) and (b).
  • the amount of VP1, VP2 and VP3 is determined using an antibody recognising an epitope that is common to each of VP1, VP2 and VP3.
  • an antibody recognising an epitope that is common to each of the three capsid proteins is e.g. the mouse anti-Cap Bl antibody (as is commercially available from Progen, Germany).
  • a preferred rAAV virion according to the invention is a virion comprising in its genome at least one nucleotide sequence encoding a gene product of interest, whereby the at least one nucleotide sequence is not a native AAV nucleotide sequence, and whereby the AAV virion comprises a VP1 capsid protein comprises a leucine or a valine at amino acid position 1.
  • a more preferred AAV virion according to the invention has the ratio's of capsid proteins as defined above and comprises a VP1 capsid protein comprises a leucine or a valine at amino acid position 1.
  • Plasmid pVD142 is constructed by inserting the baculovirus plO promoter in front of the GFP expression cassette in pVD 111. Plasmid pVD 111 is constructed in several consecutive steps. First, an EcoRV-SapI linker was ligated into SnaBI linearized pDEST8 plasmid (Invitrogen) which comprises the Gateway destination cassette under control of the polyhedrin promoter resulting in pDEST8-linker plasmid.
  • SnaBI linearized pDEST8 plasmid Invitrogen
  • the BBsIxAvrll Gateway destination fragment was isolated from this plasmid, blunted and cloned in between AAV2 ITR sequences by ligating it into pTRCGW that was digested with KpnlxSphl, blunted and dephosphorylated. Subsequently, the ITR-Gateway destination cassette-ITR fragment was obtained by digesting the plasmid with
  • the CMV-GFP expression cassette was isolated from pFBGFPR using SspIxBstAPI, blunted and cloned into the EcoRV linearized pAcDB3 plasmid comprising the Gateway cassette flanked by ITRs resulting in pVDll l.
  • the forward primer (AMT primer #327) sequence contains a Xbal site (underlined) 5'- TCCGGACTCTAGAGGACCTTTAATTCAACCCAACAC -3'
  • the reverse primer (AMT primer #332) sequence contains a Xhol site (underlined) 5'- GCCTTCGCTCGAGCTCCTTTGATTGTAAATAAAATG -3'
  • the vector pVDl ll was digested with XhoI*NotI and the 9517bp fragment was purified from agarose gel using the QIAquick Gel Extraction Kit (Qiagen).
  • the plasmid pVD142 was digested with XhoI*NotI and the 1038bp fragment was isolated from agarose gel, purified and ligated into the XhoI*NotI digested vector pVDl ll. Subsequently, the ligation mix was transformed into chemically competent One Shot CcdB survival cells (Invitrogen) plated onto LB plates containing ampicilin and chloramphenicol and grown overnight at 30 °C.
  • the forward primer (AMT primer #321) sequence contains a part of the AttB 1 site (underlined):
  • the reverse primer (AMT primer #322) sequence contains a part of the AttB2 site (underlined)
  • the PCR protocol and program are described in attachment 3.1 and 3.2, respectively (A-0181 p 005).
  • the PCR product of 1869bp was purified from gel and used in a second PCR to add the complete AttB sites on each end of the PCR product. This PCR was performed with the following primers:
  • the forward primer (AMT primer #323) sequence contains the AttBl site (underlined): 5'- GGGGACAAGTTTGTACAAAAAAGCAGGCTCCTGTTA -3'
  • the reverse primer (AMT primer #324) sequence contains the AttB2 site (underlined) 5 ' - GGGGACCA CTTTGTACAAGAAAGCTGGGTTTATTG -3 '
  • the 1915bp PCR product was purified from gel and a BP Clonase reaction was performed with the Gateway BP Clonase II enzyme mix.
  • the pDONR221 plasmid (Invitrogen) was used as the entry vector and the reaction was performed according to the Gateway® Technology with ClonaseTM II manual Version A.
  • the BP clonase mixture was transformed into chemically competent TOP 10 cells (Invitrogen) and plated onto LB plates containing kanamycine.
  • the baculovirus transfer vector pVD143 contains the pPolH-AAV2 Rep78/ACG and the pCMV-plO-GFP expression cassette between ITRs.
  • This plasmid is constructed according to the Gateway® Technology using pVD142 (lot #2) as the destination vector and pVD156 as the entry clone.
  • the LR reaction was performed as described in the Gateway® Technology with ClonaseTM II manual Version A, but incubated o/n instead of lh.
  • the LR clonase mixture was transformed into one vial of chemically competent TOP10 cells (Invitrogen) and added to 300ml LB medium containing ampicilin.
  • the forward primer (AMT primer #321) sequence contains a part of the AttBl site (underlined):
  • the reverse primer (AMT primer #322) sequence contains a part of the AttB2 site (underlined)
  • the PCR product of 1869bp was purified from gel and used in the EP-PCR to introduce the random point mutations.
  • Rep-EP Rep error prone
  • the initial amount of target DNA was ranging from O.lng to 250ng, resulting in the libraries Rep-EPl to Rep-EP5 (Table 1).
  • Rep-EP PCR products were purified from gel and to add the complete AttB sites a PCR was performed on lOOng of each Rep-EP library with the following primers:
  • the forward primer (AMT primer #323) sequence contains the AttBl site (underlined): 5'- GGGGACAAGTTTGTACAAAAAAGCAGGCTCCTGTTA -3'
  • the reverse primer (AMT primer #324) sequence contains the AttB2 site (underlined) 5 ' - GGGGACCA CTTTGTACAAGAAAGCTGGGTTTATTG -3 '
  • the pDONR221 plasmid (Invitrogen) was used as the entry vector and the reactions were performed according to the Gateway® Technology with ClonaseTM II manual Version A, with the exception that the reactions were done o/n instead of lh. Thereafter, lul of the BP Clonase reaction mixture was transformed into chemically competent TOPIO cells (Invitrogen) and plated onto LB plates containing kanamycine. After o/n culturing of several different clones of each library miniprep DNA was isolated and checked with restriction analysis using NcoI*KpnI. The mutation frequency of each library was estimated by sequencing of two clones of each library using AMT primer #210 :
  • BP reaction mixture of the Rep-EPl and 3 was transformed into two vials of chemically competent TOPIO cells (Invitrogen), added to 500ml LB medium containing kanamycine and grown o/n at 30°C.
  • Maxiprep DNAs were isolated using the QIAGEN Plasmid maxiprep kit (Qiagen), checked by restriction analysis using NcoI*KpnI and are named pDONR221 -Rep-EPl and pDONR221-Rep-EP3, respectively.
  • the two generated pDONR221-Rep-EP libraries were cloned into the destination vector pVD142 with the LR recombination reaction and according to the Gateway® Technology with ClonaseTM II manual Version A. For each library the reactions were performed in triplo and incubated for 2h. Each LR reaction mixture was transformed into one vial of chemically competent TOP10 cells (Invitrogen) and after that the three transformations were combined. To determine the EP library size 30 ⁇ of each transformed library was plated onto LB plates containing ampicilin and the remaining amount was added to 500ml LB medium.
  • baculovirus Rep-EP libraries (Bac.Rep-EP) Sf9 cells were co-transfected with one of the different pVD142-Rep-EP libraries and the/Zas/zBAC viral DNA (Oxford Expression Technologies) according to the FlashBAC one-step baculovirus protein expression User Guide, but with the exception of some small changes.
  • lxlO 6 Sf9 cells were seeded in a well of 6-wells plate and incubated at 28°C for lh to attach.
  • the co-transfection mix was prepared by diluting 20ng of flash AC DNA and 2 ⁇ g pVD142-Rep-EPl or -EP3 in 500 ⁇ 1 of SF-900II medium (Invitrogen).
  • the transfection reagent Cellfectin from Invitrogen (5 ⁇ 1) was diluted separately in 500 ⁇ 1 SF-900II medium, added to the DNA mixture and incubated for 15 min at RT.
  • the attached cells were washed once with SF-900II medium and incubated for 5h with the DNA/Cellfectin mixture.
  • 1ml of fresh SF900II medium supplemented with 20% FBS
  • wash added to the cells and 5 days after transfection the Bac.Rep-EPl and EP-3 pO were harvested by centrifugation the culture medium at
  • Bac.Rep-EP libraries 1900g for 15min.
  • the supernatant containing the baculoviruses was transferred to new tubes and stored at 4°C in the dark.
  • the amplification of the Bac.Rep-EP libraries to pi was performed in SF+ cells under serum free conditions. Briefly, the Bac.Rep-EP pO libraries were diluted 1 : 100 in shaker flasks containing 50ml SF+ cells at a density of ⁇ 2.0xl0 6 c/ml and harvested three days p.i. as described above.
  • the baculovirus stocks Bac.VD142 and Bac.VD143 were generated simultaneously and in the same way as the Bac.Rep-EP libraries.
  • Bac.select-EP3 pi and Bac.EP- EP3 pi were made in the same way.
  • Rep baculovirus libraries The insert of the different Rep baculovirus libraries was checked by performing a PCR on baculoviral DNA isolated from these stocks using the forward primer (AMT primer #349): 5'- GCGGATCATCACAAGTTTGTAC -3'
  • Baculovirus stocks with the correct insert should generate a 1935bp fragment. 1.1.1.7 Generation of the baculovirus select- EP-EP3 clones
  • the Bac.select-EP-EP3 clones were generated in the same manner as the baculovirus libraries.
  • the ORF/Rep ratios of the Bac.select-EP-EP3 clones 1-20 pi were determined by Q-PCR analysis using the primer sets prl80/181 and pr209/210.
  • Prl80 5' CGAACCGATGGCTGGACTATC 3'
  • Prl81 5' TGCTGCTACAAGATTTGGCAAGT 3'
  • Pr209 5' CTAAACGGGTACGATCCCCAAT 3'
  • rAAV5 productions were performed using the Rep libraries Bac.Rep-EPl or Bac.Rep-EP3 and Bac.VD92 p5 in a 1:1 ratio. Productions with Bac.VD142 and Bac.VD143 were taken along as control productions.
  • the different baculovirus stocks were diluted 1:100 in shaker flasks containing in log-phase growing SF+ cells at a density of ⁇ 2.0xl0 6 c/ml.
  • Three days p.i. the rAAV5 productions were harvested by adding lOx lysisbuffer and after a lh incubation a benzonase treatment was performed at 37°C. Crude lysates were clarified by 1900g centrifugation and the virus titers were determined using a Q-PCR method with the CMV promoter as target.
  • rAAV5 productions were performed using the selected Rep-EP3 library (Bac.selectEP3 pi) or the selected Rep-EP3 library that was subjected to a new mutation round (Bac.EP-EP3 pi) and Bac.VD92 p5 in a 1:1 ratio. Productions with Bac.VD142 and Bac.VD143 were taken along as control productions. Productions were performed as in the first selection round and repeated twice.
  • the rAAV5 virus titers were determined using a Q-PCR method with the CMV promoter as target.
  • Crude lysates from the rAAV5 productions with Bac.Rep-EP3 pi or Bac.EP-EP3 pi were clarified by 1900g centrifugation and subsequent filtration using a 0.45 ⁇
  • Millipak 20 (Millipore) filter. Further purification of the different rAAV5 particles was performed by affinity chromatography AVB sepharose column (GE). The purified rAAV5 batches were stored at -20°C. Viral titers of crude lysates and affinity eluates were determined using a Q-PCR method with the CMV promoter as target.
  • Viral DNA was isolated from the purified rAAV5 samples.
  • 1/50 volume of DNAse I (Roche) was added to the purified rAAV5 sample and incubated for 20min at 37 °C.
  • DNAse I (Roche) was added to the purified rAAV5 sample and incubated for 20min at 37 °C.
  • the end concentrations in the sample were next changed to lOmM Tris-HCl (pH 8.0), lOOmM NaCl, lOmM EDTA and 0.5% SDS.
  • Proteinase K (Roche) was added to a final concentration of 2mg/ml and the proteinase K treatment was performed for lh at 37°C.
  • RNA lysis buffer Promega
  • poly(A) poly(A) to a final concentration of 24ng/ml
  • RNA lysis buffer Promega
  • MagneSil BLUE Promega
  • MagneSil beads were pelleted by 2800g centrifugation for 5min at RT, washed once with RNA lysis buffer and two times with 80% ethanol. The MagneSil beads were then transferred to an eppendorf tube and washed twice with 80% ethanol.
  • the MagneSil beads were not pelleted by centrifugation, but separated by using the magnetic separation device for eppendorf tubes (Chemagen). The MagneSil beads were incubated at 65 °C until all ethanol was evaporated. Viral DNA was first eluted in 200 ⁇ 1 MQ by incubating for 5 min at 65°C followed by thoroughly vortexing of the beads and then the supernatant was transferred to a new eppendorf tube. The second elution in 200 ⁇ 1 MQ was performed o/n at 4°C. The two eluates were combined and used in a PCR reaction to amplify and clone the selected Rep library.
  • a PCR using AMT primerset #321/#322 was performed on different amounts (2.5, 10 or 25 ⁇ 1) of isolated viral DNA.
  • the amplified fragments of ⁇ 1869bp were purified from gel and lOng was used in the EP-PCR with AMT primerset #323/#324 to introduce new mutations.
  • the ⁇ 1915bp PCR products were purified from gel and cloned to pDONR221 as described in section 1.1.1.5, resulting in the pDONR221-EP-EP3 library.
  • the pDONR221-select-EP3 library contains only the Rep-EP3 sequences that were selected in the first round and that were not subjected to a new mutation round.
  • This library was made by performing a PCR on different amounts (2.5 or ⁇ ) of isolated viral DNA using the AMT primerset #323/#324 and cloning the amplified products to pDONR221 as described above. Maxiprep DNA isolates from the different libraries were checked with restriction analysis using Pstl. The mutation frequency was estimated by sequencing six clones of each library. This sequencing was performed using AMT primer #210.
  • the pDONR221-select-EP3 and pDONR221-EP-EP3 libraries were cloned into the destination vector pVD142 (lot#2) as described in section 1.1.1.5 and are named pVD142-selectEP3 and pVD142-EP-EP3, respectively.
  • pVD142-selectEP3 and pVD142-EP-EP3 were cloned into the destination vector pVD142 (lot#2) as described in section 1.1.1.5 and are named pVD142-selectEP3 and pVD142-EP-EP3, respectively.
  • pVD142-selectEP3 and pVD142-EP-EP3 libraries were cloned into the destination vector pVD142 (lot#2) as described in section 1.1.1.5 and are named Bac.select-EP3 and Bac.EP- EP3, respectively.
  • viral DNA was isolated from the purified rAAV5 sample that was produced with Bac.EP-EP3 pi.
  • the viral DNA isolation, amplification and cloning of the selected EP-EP3 library was performed as described above. This results in the new library named pDONR221-selectEP-EP3. From this library 20 clones were randomly picked and checked with restriction analysis using BstXI.
  • the pDONR221-select-EP-EP3 clones 1-20 were cloned into the destination vector pVD142 (lot#2) as described in section 1.1.1.5 and are named pVD142-select-EP-EP3 clones 1-20.
  • Bac.select-EP-EP3 clones 1-20 in their ability to produce rAAV5 30ml productions were performed.
  • the Bac. select- EP-EP3 was added in 5:1:1 ratio compared to Bac.VD92 and Bac.VD43.
  • Rep-EP libraries were generated by performing an error prone PCR (EP-PCR) on the 1869bp fragment that comprises the AAV2 Rep78/ACG expression cassette. By changing the amount of initial target DNA in the different EP-PCRs the number of introduced mutations will alter. After cloning the Rep-EP libraries into the Gateway entry vector pDONR221 the mutation frequencies were determined by partly sequencing of two clones of each library. The estimated amount of mutations per kbp in each library is shown in Table 1. As expected, the library made out of a low initial target DNA has a high mutation frequency and the one made out of a high initial target DNA has a low mutation frequency.
  • the sizes of the five libraries ranged from -0.7- 1.3xl0 4 clones and were estimated by counting the amount of bacterial colonies that were formed after plating a small percentage of the transformed cells, while the rest of the library was grown in a shaker flask.
  • Rep-EP 1 and EP3 libraries were cloned to the baculovirus transfer plasmid
  • the baculovirus libraries used were checked by performing a PCR on baculoviral DNA isolated from these stocks. This PCR specifically amplifies the DNA that is present between the two Att recombination sites, the part where the Rep-EP library is located.
  • the PCR on viral DNA from Bac.VD143, Bac.Rep-EPl and 3 amplified a DNA fragment from the expected size demonstrating that the baculoviruses contain the Rep libraries ( Figure 5).
  • rAAV5 productions were performed using the Rep libraries Bac.Rep-EPl or 3 pi and Bac.VD92 p5 in a 1:1 ratio.
  • the virus titers of the different rAAV5 productions in two independent experiments are shown in Table 2.
  • the production with Bac.VD143 gave comparable virus titers.
  • the production with Bac.Rep-EPl gave a similar virus titer as the negative control production with Bac.VD142. Thus, this is background level.
  • the production with Bac.Rep-EP3 resulted twice in a very low virus titer.
  • this virus titer is slightly above background level and indicates that there is probably specific packaging of ssDNA encoding Rep-EP3 sequences. Therefore only the selected Rep-EP3 library was isolated, amplified and re-cloned to the entry vector.
  • Table 2 Virus titers of rAAV5 productions after first selection round. Virus titers of the rAAV5 productions performed in two independent experiments determined with Q-
  • the rAAV5 particles that were produced during the first selection round with the Rep- EPS library were purified using affinity chromatography and the viral DNA, presumably containing the selected Rep-EP3 library, was isolated. Subsequently, this selected Rep-EP3 library was amplified by PCR and different amounts of initial target DNA ( Figure 6B). After performing an additional PCR on selected Rep-EP3 library using primerset #323/324 the correct products ( ⁇ 1915bp) were purified and cloned into the entry vector pDONR221, resulting in the new libraries pDONR221-select- ⁇ 3(2.5 ⁇ 1) and pDONR221-select-EP3( ⁇ l).
  • the selected Rep-EP3 library was also subjected to a new mutation round using the GeneMorph II Random Mutagenesis Kit.
  • the 1869bp fragments were used as a template in the error prone PCR using #323/324.
  • the obtained fragments were purified and cloned to the entry vector resulting in pDONR221-EP-EP3(2 ⁇ 1) and ⁇ 221- ⁇ - ⁇ 3(10 ⁇ 1).
  • the mutation frequency of the different libraries was also determined again by partly sequencing of three clones of each library. As shown in Table 3 the average mutation frequency for the pDONR221- select-EP3 library is -5.9 mutations/kbp and after a new mutation round the frequency was increased to -8.2 mutations/kbp.
  • Table 3 Mutation frequency in the select-EP3 libraries. Three clones of each select- EP or EP-EP3 library were sequenced using the Rep specific AMT primer #210 and the number of mutations was determined by sequence analysis. The mutation frequency for each library was estimated and is represented as mutations/kbp.
  • Bac.select-EP3 and Bac.EP-EP3 were generated by recombination of the plasmids libraries with the baculo viral flashB AC DNA and amplified to pi.
  • Bac.VD142, Bac.VD143 and Bac.Rep-EP3 were made at the same moment and also amplified to pi.
  • the new baculovirus libraries and control baculoviruses were also checked by PCR analysis.
  • Bac.VD143 and the three Rep baculovirus libraries all revealed the presence of the correct fragment ( Figure 7).
  • 400ml rAAV5 productions were performed using the baculovirus libraries and Bac.VD92 p5 in a 1 : 1 ratio and three days p.i. the productions were harvested virus titers were determined in crude lysates using the CMV Q-PCR method.
  • the control productions were performed in 25ml volumes.
  • the virus titers of the different rAAV5 productions are shown in Table 4.
  • the control productions with Bac.VD143 and Bac.VD142 gave virus titers of 2.3xl0 9 and 7.4xl0 4 gc/ml, respectively.
  • the production with the selected Rep-EP3 library (Bac.select-EP3) was slightly higher than the production with the Rep-EP3 library used in the first selection round (Bac.Rep-EP3), but both were still very low. Remarkable, the production with Bac.EP-EP3 resulted in 3-fold higher virus titer as compared to Bac.VD143, which could indicate that this library contains more improved Rep proteins than the Bac.select-EP3 library.
  • Table 4 Virus titers of rAAV5 productions after second selection round. Virus titers of the rAAV5 productions were determined with Q-CPR analysis.
  • the rAAV5 particles that were produced during the second selection round of the Rep- EP-EP3 library were purified using affinity chromatography and the viral DNA (i.e. the selected Rep-EP-EP3 library) was isolated. Subsequently, this selected Rep-EP-EP3 library was amplified by PCR using primerset #323/324 ( Figure 8) and different amounts of initial target DNA.
  • the ⁇ 1915bp product obtained with 5 ⁇ 1 initial target DNA was purified and cloned into the entry vector pDONR221, resulting in the new library pDONR221-select-EP-EP3. From this new library 20 clones were randomly picked and checked by restriction analysis.
  • the restriction analysis on the different clones and the pDONR221-select-EP-EP3 library revealed the same restriction fragments as the positive control pVD156. Thereafter, the 20 clones were transferred to the plasmid pVD142 using the LR recombination reaction, resulting in pVD142-select-EP-EP3 clones 1-20. Miniprep DNA isolates of these 20 clones were checked with restriction analysis using Mscl. The obtained restriction fragments had the expected sizes and were comparable to the restriction pattern of pVD143. The destination plasmid pVD142 was used as the negative control and was shown to have a different restriction pattern.
  • Bac.select-EP-EP3 clones 1-20 were tested in rAAV5 productions and virus titers were compared to productions using Bac.VD143 or Bac.VD142.
  • Figure 9 A productions with clones 4, 6, 7, 9 and 15 generated virus titers that were comparable to background level (Bac.VD142).
  • Clones 1 and 3 gave comparable titers as the positive control Bac.VD183 ( Figure 9A, black bar), while clones 8, 12 and 16-18 generated higher virus titers. Productions with all other clones resulted in lower virus titers as the Bac.VD143, but were above background level.
  • the virus titers in the control production is ⁇ 3xl0 10 gc/ml and the productions with clones 1, 3 and 14 gave virus titers of 6-7xl0 10 gc/ml, while the productions with clones 2, 5 and 10 were lower than the control productions.
  • Productions with clones 12, 13 and 16-20 resulted in virus titers of l-2xlO n gc/ml, which is 3-5 fold higher as compared to the control productions.
  • the Rep sequences from REP-ACG/PSC (patent application WO2007148971; herein also referred to as pVD88), pVD142-selectEP-EP3 clone 13, 16-18 and 20 were amplified by PCR using primerset pr460/pr461, digested with PpuMI and Xbal and cloned into the vector pVD88 which was digested with PpuMI*XbaI resulting in the constructs pVD210, pVD215-218 and pVD220, respectively.
  • the forward primer pr460 sequence contains the PpuMI restriction site (underlined): 5'- TACGAGATTGTGATTAAGGTCCCCAG-3 '
  • the reverse primer pr461 sequence contains the Xbal restriction site (underlined) 5 ' - CATCACTCTAGACTTACTTGGCTCCACCCTTTTTG -3 '
  • pVD211 and pVD212 The plasmids pVD211 and pVD212 were generated by performing a PCR using primerset pr460/pr462 on pVD142-selectEP-EP3 clone 19 and 20, PCR fragments were digested with PpuMI and SexAI and by cloning these inserts into pVD88 which was digested with PpuMI*SexAI.
  • the reverse primer pr462 sequence contains the SexAI restriction site (underlined) : 5 ' - GCTGCTGG CCCACCAGGTAG -3 '
  • pVD214 the Rep sequence was amplified from pVD142-selectEP-EP3 clone 12 (described in example 1) using primerset pr460/pr463. Plasmid pVD88 digested with PpuMI*XbaI was used as the vector. The reverse primer pr463 sequence contains the Xbal restriction site (underlined): 5 ' -CATCACTCTAGAATCACT CTAAACAGTCTTTCTGTC -3'
  • the Rep68/ACG sequence that is present in pVD228 was generated using primerset pr460/pr487 and pVD88 as template.
  • the PCR fragment was digested with PpuMI and Xbal and cloned in pVD88 digested with the same enzymes resulting in pVD228.
  • Primer pr487 consists of three parts, i.e. an additional sequence which contains an Xbal restriction site (underlined), the 25bp unique sequence for Rep68 (bold), and the sequence which is homologous with Rep78 (italic) : 5 ' - CATCACTCTAGATTATCAG AGAGAGTGTCCTCGAGCCAATCrGTCTGC GTAGTTGATCG -3'
  • Recombinant baculoviruses were generated by co-transfecting Sf9 with one of the different transfer plasmids (i.e. pVD210-212, pVD214-220 and pVD228) and BacPSCl viral DNA (Protein Sciences). Five days after transfection, the culture medium was harvested and a plaque assay was performed. After 10 days of incubation, recombinant plaques were amplified to pi and ORF/Rep ratios were determined with Q-PCR method. Correct clones which have an ORF/Rep around 1 were amplified to p2. The amplification of recombinant baculoviruses (p2, p3 and p4) were performed in expresSF+ cells (Protein Sciences, cat no 1000) under serum free conditions.
  • rAAV5 productions were performed in expresSF+ cells using the baculoviruses Bac.Rep:Bac.VD179:Bac.VD92.
  • the baculovirus stock Bac.VD179 contains the SEAP reporter gene under control of the CMV promoter and is flanked by viral AAV2 ITRs.
  • Bac.VD92 contains the AAV5 capsid gene coding for VP1, VP2 and VP3, under control of the Polh promoter. Each production is performed in duplo, repeated three times and compared to the control production with Bac.VD88.
  • the rAAV5 virus titers were measured in the clarified crude lysate using a CMV-Q- PCR method. To isolate intact rAAV5 particles from the crude lysate batch affinity purifications were performed using the AVB Sepharose HP resin. Finally, the eluate was aliquoted and stored at -20°C.
  • Resuspension Solution Cells were lysed in 200 ⁇ 1 Lysis Solution and incubated at RT for 5 min. Subsequently, 35 ⁇ 1 Proteinase K solution (20mg/ml) was added and incubated at 55°C for 30 min. Samples were neutralized by adding 380 ⁇ 1
  • Residual baculovirus DNA impurities present in the rAAV5 batch affinity purified samples were analyzed using the Q-PCR method. Total DNA was isolated from the rAAV5 particles and analysed using Q-PCR. Bac.VD43 baculovirus DNA was used as a standard line instead.
  • the CMV primerset to quantify the amount of transgene and the different primersets used to determine the amount of residual baculovirus DNA are the following:
  • Prl80 5' CGAACCGATGGCTGGACTATC 3'
  • Pr 181 5 ' TGCTGCTAC AAGATTTGGC AAGT 3 '
  • Pr406 5' ACAGCCATTGTAATGAGACGCACAA 3'
  • Pr407 5' CCTAGCGCCCGATCAGCAACTATAT 3'
  • Baculovirus HR3 region primerset (65kbp downstream of R-ITR):
  • Pr341 5' CGGGACACGCCATGTATT 3'
  • the transgene/residual DNA ratios measured in the different rAAV5 samples were compared to rAAV5 particles produced with Bac.VD88.
  • the amount of total rAAV5 particles present in the purified batches was determined SyproRuby staining. In brief, equal amounts of each sample were mixed with 4x LDS sample buffer containing 200mM DTT and heated for 5 min at 90°C. Total protein was stained using SyproRuby and VP3 bands were quantified with the ImageQuantTl software ID analysis version 7.0 (GE Healthcare). A rAAV5 control sample with known total particle concentration was taken along on each gel and used to determine to total particle concentration in each sample.
  • the Rep protein expression from the different Rep baculovirus constructs was determined by western blot analysis on lysates obtained 24h p.i. ( Figure 10B and 11B).
  • the control construct Bac.VD88 expresses the Rep78 and Rep52 proteins and Bac.VD228 the two shorter Rep proteins Rep68 and Rep40, which lack the C-terminal Zinc-finger domain.
  • the baculoviruses Bac.VD210 and Bac.VD215-218 express shortened Rep78 and Rep52 mutant forms that have a molecular weight of ⁇ 60kDa and ⁇ 30kDa and are indicated with Reppy78 and Reppy52 ( Figure 10B), respectively.
  • Bac.VD211 encodes for the full length Rep proteins and the expression was shown to be comparable to Bac.VD88. Remarkably, all constructs except for Bac.VD215 and Bac.VD216 showed a cross reactive band which is migrating somewhat faster than the full length Rep78 or Reppy78. This could be the result of an alternative translation start site, because in the Rep sequence in Bac.VD215 and
  • Bac.VD216 an ATG (M) was mutated to a GTG (V) and this constructs does not show this cross reactive band. Interestingly, Bac.VD216 expresses more Reppy52 as compared to the other constructs.
  • the Rep expression pattern of Bac.VD212 which encodes for the full length Rep is comparable to Bac.VD88, but the expression levels are lower ( Figure 11B).
  • Bac.VD220 expresses Reppy78 and Reppy52 mutant forms that have the same molecular weight as the ones expressed by Bac.VD210 and -215- 218 (see Figure 10B), while the shortened Rep proteins expressed by Bac.VD214 are larger and migrate at the expected sizes of ⁇ 65kDa and ⁇ 39kDa.
  • Rep78 is required for replicating the therapeutic gene that is flanked by viral ITRs and has to be packaged into the preformed capsids.
  • the baculoviruses Bac.VD215-218 express the shortened Rep78 proteins which also have 1-3 point mutations in their N-terminal domain.
  • This Rep78-specific part comprises the DNA binding domain and mutations in it could affect the binding to the viral ITRs maybe resulting in altered replication and/or packaging of the transgene.
  • Replicative forms of the transgene can be detected relatively easily in insect cells (Urabe, M., T. Nakakura, K. Q. Xin, Y. Obara, H.
  • Bac.VD179 that is used in the different experiments comprises the CMV-SEAP transgene and is ⁇ 3.1kbp.
  • To get rid off host cell and baculovirus genomic DNA only low-molecular weight DNA was isolated from cell pellets 1, 2 and 3 days p.i. using a plasmid miniprep kit (Ziegler, K., T. Bui, R. J. Frisque, A. Grandinetti, and V. R. Nerurkar. (2004) J. Virol. Methods 122:123-127).
  • Bac.VD88, -211 and -212 the RFd is more abundant as compared to the productions in which Reppy78 is expressed (i.e Bac.VD210 and Bac.VD214-218).
  • the pattern of the replicated DNA at day 3 p.i. is comparable to that at day 2. Remarkably, in the productions with Bac.VD88,
  • Bac.VD211 and Bac.VD212 much more additional bands and smears are present.
  • Bac.VD228 and -218 only the RFm is present, but hardly detectable as compared to other productions.
  • much more higher order RFs are detectable suggesting that the full length protein replicates the rAAV genome different than the shortened Reppy78 proteins.
  • the most interesting Rep mutant constructs increase the vector yield and at the same moment also improve the product quality by reducing the amount of residual baculovirus DNA and the total/full particle ratio.
  • these parameters can only accurately be determined in purified batches and therefore the most interesting productions (i.e. Bac.VD88, -210, -216, -217 and -220) were purified using AVB
  • Primer set prl80/181 targets a region 595bp downstream of the R-ITR which is located in ORF1629, the so-called right ORF.
  • the third primer set (not depicted in Figure 15 A) pr340/341 targets a region near HR3 which is located 65kbp downstream from the R-ITR. This target is hypothesized to be a representative for the complete baculovirus genome that is present as residual DNA in the batches.
  • the amount of residual DNA present in the purified rAAV5 batches are represented as transgene to residual DNA ratios and are shown in Figure 15B-D.

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Abstract

La présente invention concerne un procédé de sélection d'une séquence nucléotidique de parvovirus codant pour un polypeptide Rep mutant présentant une ou plusieurs propriétés améliorées, ledit procédé consistant: a. à prendre une pluralité de constructions nucléotidiques de parvovirus, chaque construction comprenant: une séquence nucléotidique de parvovirus codant pour un polypeptide Rep mutant lié de manière fonctionnelle à une séquence régulatrice; et au moins une répétition terminale inversée (ITR) de parvovirus flanquant ladite séquence de codage Rep mutante et ladite séquence régulatrice; b. à transférer la pluralité de constructions nucléotidiques dans des cellules hôtes qui peuvent exprimer des polypeptides Cap de parvovirus; c. à exposer les cellules hôtes à des conditions qui permettent l'expression de polypeptides Cap de parvovirus et l'expression d'un polypeptide Rep mutant de parvovirus de sorte que les constructions nucléotidiques de parvovirus peuvent être encapsulées dans lesdits polypeptides Cap, ce qui donne naissance à des virions de parvovirus; d. à isoler des virions de parvovirus à partir de la culture de cellules hôtes et/ou de surnageant; et e. à isoler une séquence nucléotidique de codage Rep de parvovirus mutant à partir d'un desdits virions, pour ainsi sélectionner une séquence nucléotidique de parvovirus codant pour un polypeptide Rep mutant présentant une ou plusieurs propriétés améliorées.
PCT/NL2011/050171 2010-03-11 2011-03-11 Procédé d'identification d'acides nucléiques codant pour une protéine rep variante Ceased WO2011112090A2 (fr)

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CN112041487A (zh) * 2018-04-25 2020-12-04 拜耳公司 鉴定抗体轻链和重链可变结构域的配对
US12168776B2 (en) 2017-08-09 2024-12-17 Bioverativ Therapeutics Inc. Nucleic acid molecules and uses thereof
US12364774B2 (en) 2018-08-09 2025-07-22 Bioverativ Therapeutics Inc. Nucleic acid molecules and uses thereof for non-viral gene therapy

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4745051A (en) 1983-05-27 1988-05-17 The Texas A&M University System Method for producing a recombinant baculovirus expression vector
US6103526A (en) 1998-10-08 2000-08-15 Protein Sciences Corporation Spodoptera frugiperda single cell suspension cell line in serum-free media, methods of producing and using
US20030148506A1 (en) 2001-11-09 2003-08-07 The Government Of The United States Of America, Department Of Health And Human Services Production of adeno-associated virus in insect cells
WO2003074714A1 (fr) 2002-03-05 2003-09-12 Stichting Voor De Technische Wetenschappen Systeme d'expression de baculovirus
US20040197895A1 (en) 2001-11-09 2004-10-07 Kotin Robert M. Packaging lines for generation of high titers or recombinant aav vectors
WO2007148971A2 (fr) 2006-06-21 2007-12-27 Amsterdam Molecular Therapeutics B.V. Vecteurs aav avec séquences de codage rep ameliorées pour une production dans des cellules d'insecte

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060166363A1 (en) * 2004-01-27 2006-07-27 Sergei Zolotukhin Modified baculovirus expression system for production of pseudotyped rAAV vector
WO2008036146A2 (fr) * 2006-07-14 2008-03-27 Sanofi Pasteur Biologics Co. Construction de vaccins antiviraux de recombinaison par insertion directe à médiation par transposon de déterminants immunologiques étrangers dans des protéines de virus vecteur
EP2250256B1 (fr) * 2008-02-19 2017-08-02 uniQure IP B.V. Optimisation de l'expression de protéines rep et cap de parvovirus dans des cellules d'insectes

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4745051A (en) 1983-05-27 1988-05-17 The Texas A&M University System Method for producing a recombinant baculovirus expression vector
US6103526A (en) 1998-10-08 2000-08-15 Protein Sciences Corporation Spodoptera frugiperda single cell suspension cell line in serum-free media, methods of producing and using
US20030148506A1 (en) 2001-11-09 2003-08-07 The Government Of The United States Of America, Department Of Health And Human Services Production of adeno-associated virus in insect cells
US20040197895A1 (en) 2001-11-09 2004-10-07 Kotin Robert M. Packaging lines for generation of high titers or recombinant aav vectors
WO2003074714A1 (fr) 2002-03-05 2003-09-12 Stichting Voor De Technische Wetenschappen Systeme d'expression de baculovirus
WO2007148971A2 (fr) 2006-06-21 2007-12-27 Amsterdam Molecular Therapeutics B.V. Vecteurs aav avec séquences de codage rep ameliorées pour une production dans des cellules d'insecte

Non-Patent Citations (27)

* Cited by examiner, † Cited by third party
Title
BANTEL-SCHAAL ET AL., J. VIROL., vol. 73, no. 2, 1999, pages 939 - 947
CHIORINI, J. A., F. KIM, L. YANG, R. M. KOTIN., J. VIROL., vol. 73, 1999, pages 1309 - 1319
CHLORINI ET AL., J. VIR., vol. 71, 1997, pages 6823 - 33
CHLORINI ET AL., J. VIR., vol. 73, 1999, pages 1309 - 1319
CLAIR ET AL., ANTIMICROB. AGENTS CHEMOTHER., vol. 31, pages 844 - 849
CLARK, KIDNEY INT., vol. 61, no. 1, 2002, pages 9 - 15
DAVIDSON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 97, 2000, pages 3428 - 3432
E. HARLOW, D. LANE: "Using Antibodies", 1999, COLD SPRING HARBOR LABORATORY PRESS
GRIMM ET AL., HUM. GENE THER., vol. 10, 1999, pages 2445 - 2450
JENNINGS ET AL., ARTHRITIS RES, vol. 3, 2001, pages 1
KENNETH I. BERNS: "Fields Virology", 1996, article "Parvoviridae: The Viruses and Their Replication"
KING, L. A., R. D. POSSEE: "The baculovirus expression system", 1992, CHAPMAN AND HALL
MUYLDERMANS, BIOTECHNOL., vol. 74, 2001, pages 277 - 302
O'REILLY, D. R., L. K. MILLER, V. A. LUCKOW: "Baculovirus Expression Vectors: A Laboratory Manual", 1992
PROKOP ET AL.: "Cloning and Expression of Heterologous Genes in Insect Cells with Baculovirus Vectors", RECOMBINANT DNA TECHNOLOGY AND APPLICATIONS, pages 97 - 152
RUTLEDGE ET AL., J. VIR., vol. 72, 1998, pages 309 - 319
SAMBROOK, RUSSEL: "Molecular Cloning: A Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORY PRESS
SAMBROOK, RUSSELL: "Molecular Cloning: A Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORY PRESS
SRIVASTAVA ET AL., J. VIR., vol. 45, 1983, pages 555 - 64
SUMMERS, SMITH: "A Manual of Methods for Baculovirus Vectors and Insect Culture Procedures, Texas Agricultural Experimental Station Bull. No. 7555", 1991, COLLEGE STATION
TRATSCHIN ET AL., MOL. CELL BIOL., vol. 5, 1985, pages 3251 - 3260
URABE ET AL., HUM. GENE THER., vol. 13, 2002, pages 1935 - 1943
URABE, M., T. NAKAKURA, K. Q. XIN, Y. OBARA, H. MIZUKAMI, A. KUME, R. M. KOTIN, K. OZAWA, J. VIROL., vol. 80, 2006, pages 1874 - 1885
W. H. FREEMAN, RICHARDSON, C. D., BACULOVIRUS EXPRESSION PROTOCOLS, METHODS IN MOLECULAR BIOLOGY, vol. 39, 1995
WU ET AL., J. VIR., vol. 74, 2000, pages 8635 - 47
ZIEGLER, K., T. BUI, R. J. FRISQUE, A. GRANDINETTI, V. R. NERURKAR, J. VIROL. METHODS, vol. 122, 2004, pages 123 - 127
ZIEGLER, K., T. BUI, R. J. FRISQUE, A. GRANDINETTI, V. R. NERURKAR: "A rapid in vitro polyomavirus DNA replication assay", J. VIROL. METHODS, vol. 122, 2004, pages 123 - 127, XP004603083, DOI: doi:10.1016/j.jviromet.2004.08.012

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12168776B2 (en) 2017-08-09 2024-12-17 Bioverativ Therapeutics Inc. Nucleic acid molecules and uses thereof
CN112041487A (zh) * 2018-04-25 2020-12-04 拜耳公司 鉴定抗体轻链和重链可变结构域的配对
US12364774B2 (en) 2018-08-09 2025-07-22 Bioverativ Therapeutics Inc. Nucleic acid molecules and uses thereof for non-viral gene therapy

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