Reducing Immune Response against Lentiviral Vectors: Lentiviral Vector Presentation of CD47, The ‘Marker of Self’

Abstract

Immune response to viral gene therapy vectors and their transgene products is a significant problem in the field of gene therapy. Viral vectors, because they are derived from viruses, can induce an immune response. This makes gene delivery inefficient and can pose a significant danger to patients. Macrophages act as immunological gatekeepers at the interface of tissue and lymph. They take up antigens from the extracellular environment and then present them to the immune system. Macrophage uptake has been shown to be inhibited by CD47 interaction with SIRP alpha. Viral vectors presenting CD47 on their surface should show reduced levels of phagocytosis by macrophages, and thus reduced presentation and clearance by the immune system. In this work, HEK 293T cells were transduced, using a lentiviral vector, to over-express CD47 with green fluorescent protein (GFP) at the C-terminus. These transduced cells were then transfected to produce a second set of lentiviral vectors. Since the lentivirus takes a piece of the cell membrane to make its envelope when it buds from the cell, these vectors express CD47 on their envelope. The main goal of this work is to qualitatively and quantitatively characterize the presentation of CD47 by these lentiviral vectors. Fluorescent microscopy of equilibrium density gradient fractions indicates that these lentiviral vectors present CD47-GFP. The fluorescence intensity of individual and aggregated viral vectors was quantified. This will be the first step in using CD47 expression as a method to reduce immune response to lentiviral vectors in order to increase the efficacy and safety of lentiviral vector mediated gene therapy.