534. Development of Innovative Scalable Protocols for the Purification of Lentiviral Vectors Pseudotyped With GaLV-TR or Mutated Measles Virus Glycoproteins

Abstract

Lentiviral (LV) vectors are powerful tools for gene transfer and genome editing in dividing and non-dividing cells but their clinical application requires complex and scalable processes of production which are not yet developed for all possible LV pseudotypes used in research. The ability of pseudotyping LV by different glycoproteins (gp) allows targeting of different tissues and types of cells. Modified Gibbon Ape Leukaemia Virus (GaLV-TR) gp pseudotyped LV are interesting to transduce specifically hematopoietic stem cells (HSC). Mutated measles virus gp engineered with single chain Fv can be redirected to specific cell surface receptors such as MHC Class II antigens (MV-CMH-II) enabling the transduction of dendritic cells. However, reliable and efficient purification schemes for these pseudotypes have not yet been developed. Here we report the development of a new upstream process (USP) protocol and for the first time novel downstream process (DSP) protocols allowing high yield production of stable and infectious LV-GaLV-TR and LV-MV-CMH-II particles. We identified critical conditions in chromatographic and tangential flow filtration (TFF) steps for preserving the infectivity/functionality of lentiviral vectors during purification. This was done by identifying for each step, the critical parameters affecting LV infectivity, including pH, salinity, presence of stabilizers, temperature, and secondly by defining the optimal order of these steps. A three-step process was optimized for LV-GALV-TR purification consisting of one TFF, and two chromatographic steps (ion-exchange and size exclusion chromatography, permitting recoveries of >50% (ip – infectious particles). With this process, purified GaLV pseudotyped LV vectors enabled the transduction of 70% human CD34+ cells in the presence of the Vectofusin peptide, whereas in the same conditions non-purified vector transduced only 9% of the cells. A comparable, but simplified purification protocol was developed for LV-MV-CMH-II vector in a two step protocol (TFF, size exclusion chromatography) leading to yields of 60% (ip). Our protocols will allow for the first time the purification of various LV pseudotypes that are biologically-active, stable and with sufficient recovery in the perspective of preclinical studies and clinical applications.