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(12) United States Patent (10) Patent No.: US 8,697.417 B2 Bakker Et Al

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(12) United States Patent (10) Patent No.: US 8,697.417 B2 Bakker Et Al USOO8697417B2 (12) United States Patent (10) Patent No.: US 8,697.417 B2 Bakker et al. (45) Date of Patent: Apr. 15, 2014 (54) BACULOVIRAL VECTORSCOMPRISING FOREIGN PATENT DOCUMENTS REPEATED CODING SEQUENCES WITH DIFFERENTIAL CODON BASES WO 9636712 11, 1996 WO WOO114539 A2 * 3, 2001 ............. C12N 15.00 WO WOO168888 A2 * 9, 2001 ........... C12N 15/864 (75) Inventors: Andrew Christian Bakker, Almere WO WO O3018820 A2 * 3, 2003 ........... C12N 15/864 (NL); Wilhelmus Theodorus Johannes WO WO O3061582 A2 * 7, 2003 Maria Christiaan Hermens, Almere WO WO 2005035,743 A2 * 4, 2005 ............... C12N 7/O1 WO 2007046703 A2 4/2007 (NL) WO 2007084773 A2 7/2007 (73) Assignee: Uniqure IP B.V., Amsterdam (NL) OTHER PUBLICATIONS (*) Notice: Subject to any disclaimer, the term of this Urabe et al. Insect Cells as a Factory to Produce Adeno-Associated patent is extended or adjusted under 35 Virus Type 2 Vectors. Human Gene Therapy 2002, vol. 13, pp. 1935 U.S.C. 154(b) by 725 days. 1943. Jayaraj et al. GeMS: an advanced software package for designing (21) Appl. No.: 12/670,780 synthetic genes. Nucleic Acids Research 2005, vol. 33, No. 9, pp. 3O11-3016. (22) PCT Filed: Jul. 25, 2008 Hunter et al. Colocalization of adeno-associated virus Rep and capsid proteins in the nuclei of infected cells. Journal of Virology 1992, vol. (86). PCT No.: PCT/NL2008/050512 66, No. 1, pp. 317-324.* Urabe et al., “Insect cells as a factory to produce adeno-associated S371 (c)(1), virus type 2 vectors.” Human Gene Therapy, 2002, 13:1935-1943. (2), (4) Date: Jun. 30, 2010 Kohlbrenner et al., “Successful production of pseudotyped ra AV Vectors using a modified baculovirus expression system. Molecular (87) PCT Pub. No.: WO2009/014445 Therapy, 2005, 12:1217-1225. PCT Pub. Date: Jan. 29, 2009 Office Action Israel (translated to English) in Corresponding Patent Application (203535) dated Feb. 12, 2012. (65) Prior Publication Data A. Villalobos et al., “Gene Designer: a synthetic biology tool for constructing artificial DNA segments”. BMC Bioinformatics 7:285 US 2010/0261254A1 Oct. 14, 2010 (Jun. 6, 2006). J. Meghrous et al., “Production of Recombinant Adeno-Associated Related U.S. Application Data Viral Vectors Using a Baculovirus/Insect Cell Suspension Culture (60) Provisional application No. 60/952,081, filed on Jul. System: From Shake Flasks to a 20-L Bioreactor.” Biotechnol. Prog. 26, 2007. 21:154-160 (2005). D. Hills et al., “Baculovirus expression of human basic fibroblast (30) Foreign Application Priority Data growth factor from a synthetic gene: role of the Kozak consensus and comparison with bacterial expression' Biochimica et Biophysica Jul. 26, 2007 (EP) ..................................... O7113257 Acta, 1260:14-28 (1995). S. Cecchini et al., “Toward exascale production of recombinant (51) Int. Cl. adeno-associated virus for gene transfer applications. Gene Therapy CI2N 7/00 (2006.01) 15:823-830 (2008). CI2N 7/01 (2006.01) CI2N 7/02 (2006.01) * cited by examiner CI2N 5/10 (2006.01) (52) U.S. Cl. Primary Examiner — Louise Humphrey USPC ...... 435/235.1; 435/348; 435/239; 536/23.72 (74) Attorney, Agent, or Firm — Browdy and Neimark, (58) Field of Classification Search PLLC None See application file for complete search history. (57) ABSTRACT The present invention relates to production of proteins in (56) References Cited insect cells whereby repeated coding sequences are used in baculoviral vectors. In particular the invention relates to the U.S. PATENT DOCUMENTS production of parvoviral vectors that may be used in gene 5,871,974 A * 2/1999 Huse ............................ 435/69.7 therapy and to improvements in expression of the viral rep 2003/0228696 A1 12/2003 Robinson et al. proteins that increase the productivity of parvoviral vectors. 2006/0166363 A1* 7/2006 Zolotukhin et al. .......... 435/456 2009,019 1597 A1 7/2009 Samulski et al. 30 Claims, 5 Drawing Sheets U.S. Patent Apr. 15, 2014 Sheet 1 of 5 US 8,697.417 B2 Fig 1 Fig 2 Ef Osas S. 3.90 : 0.50 0.30 O3) O. O. 83 assage U.S. Patent Apr. 15, 2014 Sheet 2 of 5 US 8,697.417 B2 Fig 3 Rep i ORF Ratio Bac.VD 83 O90 0.80 0.70 0.60 0.50 or 0.40 0.30 3.2 3. Q00 is Passage * AAW production with Bac.VD 83 1.E+08 p2 33 p4 p8 p8 p7 p8 p9 p Passage U.S. Patent Apr. 15, 2014 Sheet 3 of 5 US 8,697.417 B2 Fig 5 ORF CCK B.W83 1.00E+1 sco 100E-10 m - 1.00E+09 {{--08 1,00E+07 100E+06 p3 p4 p5 p6 p7 p8 assage Fig 6 P3 P4. P5 P8 P7 P8 P9 PO P 88 U.S. Patent Apr. 15, 2014 Sheet 4 of 5 US 8,697.417 B2 Fig 7 Q-PCR (Cilis) - ** S: 38 s S&S& r. 8:8 S: 38.8 &m ... 8 Šs W Y-w sea & S$8 & s went sen s E: so es s & 8:3 Fig 8 Tota:fui particie ratio i wda w wa : &S cy: 3. vey wass s f rew w www. were s xico S. s: ages 38 U.S. Patent Apr. 15, 2014 Sheet 5 of 5 US 8,697.417 B2 Fi9. 9 Bac. W88 Bag. Vi89 M P3 P4 P5 P3 P4 P5 Fig 10 R3; W 33 3 24 S US 8,697,417 B2 1. 2 BACULOVIRAL VECTORS COMPRISING For expression of the AAV Rep proteins in the AAV insect REPEATED CODING SEQUENCES WITH cell expression system as initially developed by Urabe et al. DIFFERENTIAL CODON BASES (2002, Supra), a recombinant baculovirus construct is used that harbours two independent Rep expression units (one for FIELD OF THE INVENTION Rep78 and one for Rep52), each under the control of a sepa rate insect cell promoter, the AIE1 and PolH promoters, The present invention relates to the fields of medicine, respectively. molecular biology, and gene therapy. The invention relates to However, Kohlbrenner et al. (2005, Mol. Ther. 12: 1217 production of proteins in insect cells whereby repeated cod 25: WO 2005/072364) reported that the baculovirus construct ing sequences are used in baculoviral vectors. In particular 10 for expression of the two Rep protein, as used by Urabe et al., the invention relates to the production of parvoviral vectors suffers from an inherent instability. By splitting the palindro that may be used in gene therapy and to improvements in mic orientation of the two Rep genes in Urabe's original expression of the viral rep proteins that increase the produc vector and designing two separate baculovirus vectors for tivity of parvoviral vectors. expressing Rep52 and Rep78, Kohlbrenner et al. (2005, 15 Supra) increased the passaging stability of the vector. How BACKGROUND OF THE INVENTION ever, despite the consistent expression of Rep78 and Rep52 from the two independent baculovirus-Rep constructs in The baculovirus expression system is well known for its insect cells over at least 5 passages, ra AV vectoryield is 5 to use as eukaryotic cloning and expression vector (King, L.A., 10-fold lower as compared to the original baculovirus-Rep and R. D. Possee, 1992, “The baculovirus expression sys construct designed by Urabe et al. (2002, supra). tem'. Chapman and Hall, United Kingdom; O'Reilly, D. R. In WO2007/148971 the present inventors have signifi et al., 1992. Baculovirus Expression Vectors: A Laboratory cantly improved the stability of ra AV vector production in Manual. New York: W.H. Freeman.). Advantages of the bacu insect cells by using a single coding sequence for the Rep78 lovirus expression system are among others that the and Rep52 proteins wherein a suboptimal initiator codon is expressed proteins are almost always soluble, correctly 25 used for the Rep78 protein that is partially skipped by the folded and biologically active. Further advantages include scanning ribosomes to allow for initiation of translation to high protein expression levels, faster production, Suitability also occur further downstream at the initiation codon of the for expression of large proteins and Suitability for large-scale Rep52 protein. production. However, in large-scale or continuous production There is however still a need for further improvements in of heterologous proteins using the baculovirus expression 30 large scale (commercial) production of heterologous pro system in insect cell bioreactors, the instability of production teins, including ra AV vectors, in insect cells. Thus it is an levels, also known as the passage effect, is a major obstacle. object of the present invention to provide for means and This effect is at least in part due to recombination between methods that allow for stable and high yield (large scale) repeated homologous sequences in the baculoviral DNA. production of heterologous proteins and parvoviral vectors. The baculovirus expression system has also successfully 35 been used for the production of recombinant Adeno-associ DESCRIPTION OF THE INVENTION ated virus (AAV) vectors (Urabe et al., 2002, Hum. Gene Ther. 13: 1935-1943: U.S. Pat. No. 6,723,551 and US Definitions 20040197895). AAV may be considered as one of the most As used herein, the term “operably linked’ refers to a promising viral vectors for human gene therapy. AAV has the 40 linkage of polynucleotide (or polypeptide) elements in a ability to efficiently infect dividing as well as non-dividing functional relationship. A nucleic acid is “operably linked' human cells, the AAV viral genome integrates into a single when it is placed into a functional relationship with another chromosomal site in the host cells genome, and most impor nucleic acid sequence. For instance, a transcription regula tantly, even though AAV is present in many humans it has tory sequence is operably linked to a coding sequence if it never been associated with any disease.
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