86 Generation of More Infectious Type 1 AAV Vectors in Insect Cells Hoechst dramatically augments and accelerates rAAV mediated gene expression in human airway epithelia The mechanism appears to be tr[.]
Hoechst dramatically augments and accelerates rAAV-mediated gene expression in human airway epithelia The mechanism appears to be transcriptional upregulation 84 High Titer Adeno-Associated Viral Vector Production by a Vero-Derived Producer Cell Line Clifford J Beall ,' Philip R Johnson.! K Reed Clark.' [Center/or Gene Therapy; Columbus Children s Research Institute, Columbus, OH; 2Joseph J Stokes , J/: Research Institute, Children s Hospital 0/Pennsylvania, Philadelphia, PA Recombinant adeno-associated virus (rAAV) vectors arc being proposed and utilized as gene transfer vehicles for an ever-widening array of human applications The intense interest in rAAV has been fueled by the finding that these simple vectors can efficiently transduce a variety ofpost-mitotie cells when administered in vivo Promising data from animal models has resulted in the initiation of several ongoing human clinical trials While these advances are encouraging, obstacles remain for the general implementation of rAAV vectors as a universal gene transfer vehicle One lingering concern is that rAAV vectors have been difficult to produce at a level that makes them commercially viable We addressed this issue several years ago when we pioneered the use ofgenetically engineered Hel.a based cell lines for rAAV production Although this production method produces an extremely pure product, it seems prudent and logical to pursue identification of more "regulatory friendly" approved cell line substrates for large-scale rAAV synthesis We have identified Vero cells (African green monkey derived) as a promising alternative cell line substrate for rAAV vector production Vero cells have been used as a cell line substrate for the production of numerous human vaccines, including poliovirus (both oral and inactivated) and rabies The safety of the cell line is attested to by phannaeovigilance ofmore than 20 million doses of rabies vaccine and more than I billion ofOPV We stably transfccted Vero cells with a plasmid encoding G418 resistance, an rAAV genome containing enhanced green fluorescent protein, and the AAV2 rep and cap genes From this, we selected lines that produced rAAV2.eGFP upon infection with human Adenovirus type 5, though the yield with the human helper virus was lower than from comparable HeLa based lines We then investigated the ability of simian adenoviruses to provide optimal help for rAAV production We screened a panel of20 monkey adenoviruses and found that one, Simian Adenovirus 13, stimulated high-titer production ofrAAV The numbers of viral particles produced per cell (75,000 vg) were similar to HeLa based producer lines Vero producer cell lines could be an especially valuable production alternative for human applications requiring high safety margins such as genetic vaccines widely administered to healthy subjects 85 Insect Cell Production System: Alternative Startcodon Use for Improved AAV Infectivity Andrew Bakker; Masashi Urabe.! Saskia Haast ,' Janneke Meu lenberg,' Jaap Twisk ,' Lizzy Cornijn,' Keiya Ozawa.! Sander van Deventer, I Wim Hermens.' /Process Development, Amsterdan Molecular Therapeutics (AMT), Amsterdam, Netherlands; lDivisioll a/Genetic Therapeutics, Center/or Molecular Medicine, Jichi Medical University; Tochigi Japan Introduction: The baculovirus production system for AAV seems the most straightforward system for the large scale production of AAV needed for human application The original baculovirus production system was developed usingAAV serotype (Urabe et al , 2002) In insect cells splicing docs not occur therefore in this system the ACG starteodon is used for VP I translation ribosome scanning to the second ACG for VP2 translation and subsequent S34 scanning to the ATG for VP3 We observed that for AAV I vectors containing the ACG initiation codon, a different stoichiometry of the VP proteins in the AAV caps ids was obtained compared to AAV I vectors produced with the plasmid transfection production platform (Grimm et al., 1998) that we previously used The VPI amount in the AAV capsid is one of the main factors determining the infectivity (Girod et al.) We investigated whether inclusion of alternative startcodons for VPI translation could influence the stoichiometry ofVPI VP2 and VP3 in the final AAV capsids, and the infectivity of the resulting particles ill vitro Methods: The startcodon ofVPI of the original system was changed to ITG, GTG , and CTG For recombinant baeulovirus preparation a commercial kit purchased from Protein Sciences (PSC) was used Results: We found that the VPI amount in the AAV capsid is equal to the wiJdtype amount when the GTG codon is used, and is higher when the CTG codon is used Caps ids produced using the CTG startcodon contained VP I levels nearing the level of the plasmid production system (FSB), but the yield of capsids was times reduced compared to the ACG and GTG startcodons (figure I) M CTG GTG 85 84 ACG TTG O)) b~ • - - - - - 41 83 FSB M' The CTG startcodon in conjunction with a putative kozak sequence resulted in higher expression of the capsid proteins The resulting AAV capsids have higher VP I concentration and demonstrate better infectivity in vitro compared to the AAV capsids with lower VPI amounts, but production is still times lower than the ACG and GTG codons Conclusion: The stoichiometry of the VP proteins in the AAV capsids seems variable and can be influenced by the production system The infectivity of capsids produced with the GTG startcodon was as high as the AAV's obtained with the plasmid transfection system The CTG startcodon seems best, but production ofthese capsids remains challenging Grimm et al., Hum Gene Ther 1998, Dec I0;9( 18):2745-60 Urabe et al., Hum Gene Ther 2002, Nov I; 13(16):1935-43 Girodetal., J Gen Virol 2002, May; 83(Pt 5):973-8 86 Generation of More Infectious Type AAV Vectors in Insect Cells Masashi Urabe ,' Hiroaki Mizukarni,' Andrew Bakken? Akihiro Kume, I Wim Hermens," Kciya Ozawa.' 'Div: Genet Then, Jichi Med Univ., Shimotsuke, Japan; l Vector Development, Amersham Molecular Therapeutics Amsterdam, Netherlands Adeno-associated virus (AAV) vector is a promising gene transfer vector and is being evaluated for some human applications, such as coagulation factor IX deficiency, lipoprotein lipase (LPL) deficiency, and Parkinson's disease, Serotype I AAV vector is able to transduce muscles most efficiently among serotypes identified However, supplemental therapy for a defective protein with type I AAV vector would still require huge quantities ofvector particles The production of AAV vectors in insect cells with recombinant baculoviruses is able to easily generate large quantities ofAAV vector particles and is suitable for studies with large animals and human applications One ofthe problems in the insect ccIl-bascd rAAV production system is the low infectivity ofAAV vector particles, which is attributed to Molecular The 4lpy Volume 15.Supplement tã ã\ br 2007 Co pyright â "111e American SOI;ic;ty o f Gene "l1u:mpr low VPl content in AAV capsids The AAV capsid proteins , VPI, VP2, and VP3 are synthesized from two spliced mRNAs One message is translated into VPI while another transcript codes for VP2 and VP3 The initiation codon for VP2 is a non-A UG codon, ACG, which is poorly utilized , resulting in ribosome scanning through to the VP3 initiation codon (AUG) Since in insect cells splicing of the AAV mRNA docs not occur, the baculovirus-inscct cell system for rAAV production utilizes a modified VP gene on which the initiation codon for VPI is mutated to an ACG codon, enabling synthesis of all three VP proteins from one mRNA However, the use ofthe ACG codon for VP I failed to produce rAAV1 comparable to HEK293 cell-produced counterpart in infectivity In an attempt to confer the infectivity to insect cell-produced rAAV I, we tested other non-AUG codons for VPI and found one non-AUG codon significant ly generated rAAVI particles into which a larger quantity ofVPI molecules was incorporated Infection ofHEK293 cells with rAAVI expressing secreted form of alkaline phosphatase (SEAP) with higher VPI content showed approximately 50% increase in the SEAP activity in culture supernatants These results indicate that the new version ofrAAV production system will contribute to a number of applications of type I AAV vectors 87 Development and Characterization of a Relative Potency Assay for rAAV1 Vectors for Use as Vaccines Eric J Pastor,' Daryn Debelak,' Christopher J Gerard,' Richard Peluso.' 'Analytical Methods, Targeted Genetics Corporation, Seattle, IVA Vectors based on AAV arc being developed not only for gene transfer but also as candidate vaccine vectors due to their efficient transduction ofskelctal muscle and long-term transgene expression It is this transduction and long term expression ofthe transgene and the subsequent in vivo immune response that are assumed will be responsible for the clinical effectiveness (potency) of the product One of the requirements for characterization and release of vaccines is a potency assay which will be predictive of the clinical effectiveness However until clinical effectiveness is established, the mechanism of action clearly understood, and an immune correlate identified, it is only possible to have a surrogate measure ofpotency We have developed, characterized and qualified a relative potency assay for a candidate AAV I-based vector encoding genes from HIV This relative potency assay measures the transgene expression of the test sample compared to a standardized and characterized assay reference control vector Typical of most cell-based methods, there is considerable variation in assay read-out from assay to assay The relative potency assay we developed is an in vitro method that tests the capacity ofa recombinantAAV I vector to transduce permissive cells and express gag p24 The conditions ofthe infection were chosen so protein production is proportional to vector dose (multiplicity of infection) over several logs range After a suitable transduction period, p24 levels are measured in transduced cell lysates using a qualified, commercially avai lable ELISA kit The relative potency of the test article is determined based on comparison with a reference standard control of the same vector in the same assay using a 4-parameter curve fit model The goal in the development of the assay was to ensure that it is appropriate for the requirements of product eharactcrization in that it is sensitive to changes in the product that would be detected by changes in levels of p24 protein expression The dose response curves generated by the reference standard and test articles were semi-log transformed and statistically fit by employing a 4-parameter fit dose response model Suitability criteria for the four parameters were established for the reference standard , and similarity criteria (parallelism) were established for test article comparison Relative potency was determined by the offset of the ED50 of the transformed test article dose-response curve relative to Molecular Therapy Volume 15 Supplement Iã \1.,)"2007 O:lprnghf â TIl[: American $Ol;:it,:'t}, or (ir,.'f'U,; Therapy the ED l u ofthe transformed reference standard dose-response curve A 100% potent test article was determined to have an inter-assay relative potency value of 103.3% with a CV of9.8% (N=I assays) and can be measured within 77.1% to 138.5% at a 99% confidence level The assay was shown to be specific for the vector expressing p24 protein and linear with respect to pre-ELISA dilutions This assay can be used for testing ofvaccine products in stability studies and to compare relative potencies of different lots of vaccines 88 Equal In Vivo Efficacy of Recombinant AAV1 Vector Produced in Either Mammalian or Insect Cells Jaap Twisk,1Colin J D Ross,' Lizzy Comijn ,' Dennis Biesrnans,' Saskia Haast,1 Andrew Bakker; Edwin S van Arnersfoort,' Sander J H van Deventer,' Janneke J M Meulenberg,' Michael R Hayden,' Wim T Hermens.' 'Research & Development, Amsterdam Molecular Therapeutics, Amsterdam, Netherlands; :!MedicalGenetics, Centre lor Molecular Medicine and Therapeutics, Vancouver, BC, Canada Insect cells can be used to produce recombinant adeno-associated vector (AAV), using baculovirus-mediated infection ofSF+ cells to introduce components for AAV production The system was originally developed and described by Urabe et al., Hum Gen Thcr, 2002 Aim: Intramuscular (1M) injection of AAVI_LPLS4HX is currently being tested in a clinical trial, for treatment of LPL deficiency in man (as introduced in Ripetal., Hum Gen Ther2005) AAVI vector used in that study was produced in mammalian cells In an effort to optimize large-scale production for future clinical use, we have tested the baculovirus system for production ofAAVI, first in shaker flasks and subsequently large-scale, in WAVE bioreactors, Such batches were used to treat LPL deficient (LPL-/-) mice, to monitor efficacy Results: Production in a WAVEbiorcactor increased the viral yield in crude lysed bulk to 4-fold , compared with production in shaker flasks, Both batches were purified using affinity chromatography, with an overall yield of 40-60% A single production run in the WAVE bioreactor (25L), including purification , takes 10 days to complete, yielding >4x 1014 genome copies (gc's) of purified AAVI vector Batches of AAVI-LPLsWX were subsequently tested in LPL-/- mice LPL-/- mice demonstrate extreme levels of plasma triglycerides (TO: elevated 100 to 200-fold over normal; Ross et al., A:rVB 2005) Upon 1M injection of vector produced in either insect cells or mammal ian cells, a prolonged dose-dependent reduction of plasma TG, by -15-20% at 3xlO" gclkg, 75-90% at b;1012 ge/kg, and 97-99% at 3x I0 12 gc/kg, was observed in LPL-/- mice When comparing several batches of vector at a fixed dose (I xl 12 gc/kg), -90% reduction in plasma TG was observed in LPL-/- mice upon 1M injectionofAAV I_LPLS+l 7X, irrespective ofthe production system used Conclusion: Large-sealeAAVI production, within a short time frame and with good yield, was accomplished in SF+ insect cells using a WAVE bioreactor, Equal therapeutic potency in vivo was demonstrated, when comparing AAV I produced in insect cells and mammalian cells 89 Process Strategies for High Yield Production of Adeno-Associated Viruses in Insect Cells Marc G Aucoin.t-' Michel Perrier.? Amine A Kamen.' 'Animal Cell Technology, Biotechnology Research Institute, NRC, Montreal, QC, Canada; lChemical Engineering, Ecole Polytechnique de Montreal, Montreal, QC, Canada Adeno-associated virus production in insect cells using a triple baculovirus infection, as first described by Urabe et al (2002) can benefit from process optimization Briefly, the three baculoviruses used include: BaeRep, containing the genes for the replication S35 ... utilized , resulting in ribosome scanning through to the VP3 initiation codon (AUG) Since in insect cells splicing of the AAV mRNA docs not occur, the baculovirus-inscct cell system for rAAV production... used for testing ofvaccine products in stability studies and to compare relative potencies of different lots of vaccines 88 Equal In Vivo Efficacy of Recombinant AAV1 Vector Produced in Either Mammalian... of 40-60% A single production run in the WAVE bioreactor (25L), including purification , takes 10 days to complete, yielding >4x 10 14 genome copies (gc''s) of purified AAVI vector Batches of AAVI-LPLsWX