457. A Characterization of Transgene-Specific “Proteomes” in Lentiviral Vector Clinical Production Lots Identifies Differences between Vectors with High and Low Infectious Titers

Gene delivery using viral vectorsis a powerful tool which can be used to correct genetic diseases and treat acquired illnesses. HIV-based lentiviral vectors (LVs) are advantageous in gene therapy applications for due to their genomic integration and ability to infect non- dividing cells. We have found that scale-up for is successful in retaining physical particle titers; however, the transduction efficiency of certain vectors, particularly in sensitive primary cells (e.g. human CD34+ cells), is significantly less than expected. Our working hypothesisis that current large-scale production methods may concentrate proteins that modulate transduction. To address this possibility, we characterized the “lentiviral vector proteome” of several different clinical vector lots using an OrbitrapVelos Pro Hybrid Mass Spectrophotometer. Since X-VIVO 10 medium was the final product medium in all LV lots, proteins identified in a sample of unused medium were eliminated from the analysis. We first identified a subset of 24 proteins common to ALL vector lots. Not surprisingly, further analysis showed that 6 of these proteins are both “top 25” exosomemarkers in the ExoCartadatabase and known gag-pol interacting proteins listed in the HIV-1 Human Interaction Database. Independent of production size and tangential flow filtration brand, we next identified 57 proteins unique to a low titer LV expressing human beta-globin (BG). We similarly identified 47 proteins unique to a high titer LV expressing adenosine deaminase(ADA). BG lots were enriched in proteasome subunit proteins and ADA lots in ribosomal subunit proteins. Interestingly, the ADA lots were also enriched in several 14-3-3 family proteins that are known to modulate intracellular signaling pathways. It is possible that the mere presence or relative levels of specific proteins, such as 14-3-3 proteins, can affect transduction. We can speculate that the mechanism involves indirect effects of the vector transgene on the producer cells which yields a particle that is more stable and/or has an increased capacity to transduce cells.