814 The Thermo Sensitivity of the Reverse Transcription Process Is a Mechanism of MLV Based Vectors Inactivation Selective transdu ction of CD20 positive Iymphocvte s 814 The Thermo Sensitivity of the[.]
812 Composition of Purified Lentiviral Vector Products Intended for Gene Therapy: Implications for Quality Control of Vector Production Kenneth Keating,' Carl Dolman,' Robin Thorpe,' Yuan Zhao.' NIBSC, Potters Ban Hertfordshire, United Kingdom I Biotherapeutics, Purification and characterisation of Lentiviral vector products still presents a challenge to their clinical applications Purification methods, including ultra-centrifugation, size exclusion and ion exchange chromatography, have recently been appliedto these vectors Ultra-centrifugation produced partially purified vectors carrying a significantamount of cellular proteins, Size exclusion chromatography has been reported to significantly reduce cellular contaminants resulting in a product with up to 97% visualised purity The SEC method has thus been accepted for recent clinical trials of a Lentiviral vector product However, contamination of empty particles lacking therapeutic function is another common problem in Lentiviral vector production For example current Lentiviral products may contain up to 100 fold empty particles to infectious/functional vector particles The significanceand further removal of impurities, e.g empty particles, in final products has yet to be addressed for future clinical application We reason that characterisation of SEC purifiedproducts will be beneficial for establishing standard criteria for quality assessment of future products as well as for modifying available purification methods Therefore, components of three purified samples, that is, expressed VSV-G protein, recombinant empty particles (VSV-GlEmpty) lacking vector genome and complete vector particles (VSV-G/SIN-GFP) expressing GFP, were compared after SEC Eight different antibodies were used in this study, including antibodies against viral proteins VSV-G and 1-1 IV-I Gag Five cellular proteins, e.g CyP-A, that have been previously reported to associate with HIV-I virus were also analysed Our results demonstratedthat in addition to empty particles the impurity in SEC purifiedproducts includedglycoproteinVSV-G aggregates and cellular proteins, Some cellular components that were co-purified with Lcntiviralvectorsshoweddistinctiveassociationwith complete vector particles (VSV-G/SlN-GFP), but not with empty particles The significance of identified contaminants should be considered for the future modification of purification methods The potential application of identified cellular proteins that distinguished empty particles from complete vector particles is under investigation in order to develop a testing system for quality control of lentiviral vector products gene, respectively, were not infectious, while particles pseudotyped with VSV-G reached titers of 109 t.u.lml However,screening of all combinations of 15 Hand F protein variants carrying different deletions and amino acid exchanges in their cytoplasmic tails allowed the identificationof three pairs ofH/F variants that allowed efficient pseudotyping of HIV-I vectors Using an optimal H to F ratio, high titers (101 t.u.lml) on different human cell lines e.g HT1080, 293 '1 ~ A431 and Raji were obtained Also SIV vectors could be pseudotyped with these H/F variants Gene transfer with these vectors was stable, as demonstrated by reverse transcriptase inhibition and longterm cultivation of transduced cells By using native rceeptor blind H proteins displaying EGF or a scFv directed against C020, cell targeting vectors were generated On a panel of four different CHO cell lines expressing either the retargeted receptors or the measles virus receptors SLAM and C046, respectively, first targeting experiments were performed While there was no transduction ofCD46 positive cells, a low background transduction of SLAM positive cells was detectable In contrast, CI-I0-C020 and CI-IO-EGFR cells were transduced at least 103 fold more efficiently by the respectivetargetingvectors On 1-11' I080-C020 cells, titers of 106 t.u.lml were reached Remarkably, C020 positive lymphocytes were selectively transduced by the C020-targeting vector, at a similar efficiency as with VSV-G pseudotyped vector particles (see figure).The data demonstratethat the targetingconcept designed for MV can be transferred to Ientiviral vectors.As MV enters cells thorugh pH independent direct fusion at the cell membrane, we expect that this novel targeting system will offer high flexibility allowing retageting to any cell surface molecule of interest Selective transdu ction of CD20 positive Iymphocvte s Daudi (CD20+) ; ~:;== , ;: V8V-G Sabrina Funke,' Andrea Maisner,' Roberto Cattaneo.' Klaus Cichutck, I Christian Buchholz.' I Division oJMedical Biotechnology, Paul-Ehrlich-Institut, Langen, Germany; 2Institute oj Virology; Philipps Universityoj Marburg Marburg, Germany; JMolecular Medicine Program, Mayo Clinic, Rochester Targeting cell entry of retroviral gene transfer vectors is still an unsolved problem in gene therapy, Measles virus, which has two types of'glycoproteins, the hemagglutinin (H) proteinresponsiblefor receptorrecognition,and the fusion(F) protein mediatingmembrane fusion, can be efficientlyretargeted, by mutating the H protein binding sites for its native receptors and fusing single-chain antibody fragments (scFvs) to its cctodomain (Nakamura et al 2005) Wc hypothesise that rctroviral vectors pseudotyped with the measles glycoproteins can also be retargeted Particles released from cells transfected with the plasmids coding for the unmodified measles glycoproteins, HlV-l or MLV gag/pol and a packagable reporter S312 K562 (CD20-) -:::;:=: - I § ;: I wi'h e ~ 1t=;;1::;;::;;:j 10' MV-H-antiCD20f MV-F ' 813 Cell Entry Targeting of Lentiviral Vectors through Pseudotyping with the Measles Virus H and F Proteins HIV-1 pseudolyped with " til ,oJ ta" 10' " 814 The Thermo-Sensitivity of the Reverse Transcription Process Is a Mechanism of MLVBased Vectors Inactivation Marlene Carrno,' Amos Panct.! Manuel J T Carrondo,'-' Paula M Alves,' Pedro E Cruz.I 'Animal Cell Technology Laboratory, IBETIITQB Oeiras, Portugal; 2Department cf Virology; Hebrew University, Hadassah MedicalSchool, Jerusalem, Israel; JBiochemical Engineering Laboratory, FCTIUNL Monte da Caparica, Portugal Retroviruses are amongst themost widely studiedvectors forgene therapy However, these vectors display a fast inactivation rate that complicates their clinical application Nevertheless, there are no studies demonstrating which viral components suffer inactivation! degradation at physiological conditions In this work the thermosensitivity of the reverse transcription process was studied as well as of several viral components involved in this process The results indicatethat the capacityof virus to performthe reversetranscription process decreases during incubation at 37°C, both in endogenous Molecular Therapy Yofume 15 Supplement I, \by 2007 Copyright © '111C American Societyo f Gene TIICr.lpr reactions and intraccllularly The results have also shown that ribonuclease I-I inactivation and RNA degradation have lillie effect on the loss of the virus ability to perform reverse transcription In contrast, reverse transcriptase (RT) DNA polymerase activity showed fast inactivation at 37°C, explaining in part the loss of the virus capacity to synthesize full DNA Furthermore, two real-time RT-PCR methods were used to quantify the viral RNA degraded and intact inside the cells immediately after infection with samples pre-incubated at 37°C The results have shown that the quantity of RNA that enters the cells stays almost unaltered along incubation time and the intracellular RNA degradation is very similar to the one outside the cell Also, it was possible to verify that the amount ofviral RNA that is transcribed into DNA inside the cells decreases during incubation of the viral samples at 37°C, confirming that the reverse transcription process is affected by incubation ofthe viral samples at 37°C Based on these results and on the high correlation between the virus loss ofcapacity to perform reverse transcription and the loss of infectivity it was possible to conclude that the thermo-sensitivity of the reverse transcription process is a mechanism ofret roviraI vectors inactivation Strategies to stabilize the DNA polymerase activity of RT, which was thc viral component most affected by temperature, may now be pursued in order to improve the production and the applications of rctroviral vectors 815 Abstract Withdrawn 816 Improved Production Protocols for HIV-1Based Lentiviral Vectors Robert H Kutner; Jakob Reiser" 'Gene Therapy; Louisiana Health Sciences Center; New Orleans, LA Current issues that limit the general usefulness oflcntiviral (LV) vectors for preclinical gene therapy applications include low titers , the lack of a standardized method for titration, as well as problems related to their large-scale production To optimize LV vector production, we compared the performance of various second- and third-generation packaging constructs Subsequently, vector titers were boosted by including Lipolvlate" (I-1yClone), cholesterol, or Chemically Defined Lipid Concentrate (Gibco) as cell culture additives during vector production These production variables were evaluated by comparing the relationship between p24 concentrations, transduction units determined by now cytometry, virus particle titers based on viral RNA, together with integration units scored by qPCR using DNA extracted from transduced cells With vector production conditions optimized and a standardized titration method at hand , of a high-throughput technique for LV vector concentration was explored We developed a scaleablc protocol based on Mustang® Q (PALL) chromatography technology to isolate and concentrate the virus from crude cell culture supernatants, Mustang® Q ionexchange chromatography membranes have a large binding capacity for LV vector particles and are relatively insensitive to the effects of high now rates Our results indicate that the third-generation packaging constructs resulted in 2-10 x 107 transducing units/ml (unconcentrated virus) compared with 1-8 x 107 transducing units/ml using a second-generation packaging system The addition of cell culture additives boosted titers up to 1O-fold using LipoMate™ , and up to fold with the usc of Chemically Defined Lipid Concentrate For the scaleable purification protocol based on Mustang® Q ionexchange chromatography, we measured the potential to capture lentivirus at various now rates, the results of nuclease treatment on column capacity, and the effect of pH on binding The results obtained showed that times more virus bound at a now rate of 10 ml/min than at 0.3 ml/min , a higher capacity with DNase treatment than without, and an increase in binding at a pH of 8.0 compared to pH 7.0 or pH 6.0 We believe that these improved production Molecular Therapy Volume15 Supplement ~ "'r Copyright © The American Soci ety o r Gene Therapy 2007 conditions will ultimately simply the production of clinical-grade LV vectors 817 A Quantitative Kinetic Analysis of Centrifugation-Based Delivery of Retroviral Vectors into Mammalian Cells Venkata S Tayi,' ·2 Pascal R Beauchesne,'> Bruce D Bowen,' James M Piret.l-' 'Michael Smith Laboratories University ofBritish Columbia VCll/couvel; BC, Canada; 2Chemical and Biological Engineering, University ofBritish Columbia l't7ncouvel; BC Canada Recombinant retroviruses have a broad host range and can efficiently integrate into the host chromosome However, because of limitations such as low diffusivity (-6 x 10.8 cmvs), short halflife (-6 h) and the requirement of target cell division for genome integration, retroviruses typically transduce low fractions of target cells in conventional transduction protocols The transduction efficiency is governed by the kinetics of several processes that occur in series A model was formulated that includes the time-dependent elements of extracellular diffusion, convection and decay, adsorption and entry into the target cell, and intracellular kinetics coupled with cell cycle dynamics The model also predicts the average copy number of integrated genes along with transduction efficiency, by tracking the populations of extracellular virus , viral intermediates in cell cytoplasms, integrated viral genomes, and the corresponding populations of target cells This information is particularly useful from a clinical viewpoint as higher copy numbers can have a toxic effect on target cells The initial active retrovirus concentration in the extracellular medium, which cannot be directly quantified by current experimental measurements, was estimated by comparing experimental data obtained under static conditions with the model predictions Centrifugation for up to h was then used to concentrate the MSCV-IRES-GFP retroviral vectors near the K-562 target cells The model-predicted centrifugal transduction efficiencies were found to be in good agreement with the experimental results at low retrovirus concentrations, but deviated at high concentrations A saturation of transduction efficiency at 75% was observed in these latter experiments though the model did not predict saturation By introducing a susceptibility factor, which distinguishes the fraction of cells that are susceptible to retroviruses, the model was ab le to predict all of the experimental transduction data, obtained for centrifugations up to h reaching a 20-fold enhancement This model should prove useful for accelerating the development and optimization of viral transduction processes 818 Development of Scaleable Retroviral Transduction Strategies Utilizing Whole Cell Lysate and Acoustic Technology Pascal R Beauchcsnc.P Venkata S Tayi,1.2 Bruce D Bowen ,' James M PiretY 'Michael Smith Laboratories University ofBritish Columbia, Vancollvel; BC Canada; /Chemtcal and Biological Engineering University ofBritish Columbia, l't7ncouvel; BC, Canada Recombinant retroviruses provide efficient gene delivery systems that allow stable integration of specific transgenes into the genome of target cells Despite the availability of high-titer vector preparations, multiple retroviral exposures of patient cells arc used in order to achieve an acceptable transduction efficiency This is due in part to the low encounter frequency of vectors and cells as a result of the short half-life (-6 h) and limited diffusivity (-6 x 10-8 cmvs) of retroviruses Various strategies, including the use of fibronectin or protamine sulfate, have enhanced transduction efficiency by 3- to 10fold However, most ofthese techniques involve significant surface interactions and , therefore, arc less readily scaleable to handle the S313 ... thermo- sensitivity of the reverse transcription process is a mechanism ofret roviraI vectors inactivation Strategies to stabilize the DNA polymerase activity of RT, which was thc viral component most affected... stays almost unaltered along incubation time and the intracellular RNA degradation is very similar to the one outside the cell Also, it was possible to verify that the amount ofviral RNA that is. .. clinical-grade LV vectors 817 A Quantitative Kinetic Analysis of Centrifugation -Based Delivery of Retroviral Vectors into Mammalian Cells Venkata S Tayi,'' ·2 Pascal R Beauchesne,''> Bruce D Bowen,'' James