813. Cell Entry Targeting of Lentiviral Vectors through Pseudotyping with the Measles Virus H and F Proteins

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 2 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

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Sabrina Funke,' Andrea Maisner,' Roberto Cattaneo.' Klaus Cichutck, I Christian 1. 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

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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