Streptavidin Paramagnetic Particles Provide a Choice of Three Affinity-Based Capture and Magnetic Concentration Strategies for Retroviral Vectors

Abstract

Three strategies have been designed to concentrate infectious retroviral vectors from the supernatants of human- (HT1080) and murine- (NIH 3T3) based packaging cells. Streptavidin-conjugated paramagnetic particles in conjunction with (i) antibodies directed against murine fibronectin, (ii) biotinylated lectins, or (iii) biotin-modified packaging cell-surface proteins allow affinity-mediated magnetic concentration of retroviral vectors. Retroviral titers (assayed by colony formation of human myeloid K562 cells) are increased by 1-4 x 10(3)-fold after volume reductions of only 125-fold. Using these procedures, preparations of 5 x 10(8) cfu/ml are routinely made from relatively low-titer (2-5 x 10(5) cfu/ml) starting material. High-titer (paramagnetic) retroviral vector preparations can be used for magnetic field-dependent retroviral infection in vitro. Magnetic field-dependent localization such as this may enable the in vivo administration of formulations that concentrate retroviral infection to the required target tissues and organs.

Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo

“…The size and surface chemistry of magnetic particles can be tailored to meet specific demands on physical and biological characteristics, also the linkage between vector and magnetic particle can be designed accordingly. This conclusion is in accordance with parallel work by Hughes et al, 19 who most recently attached retroviral vectors via biologic linkage to commercially available magnetic microbeads. These authors demonstrated a similarly encouraging improvement in vector targeting and efficacy by magnetic force as we found for any vector type in a less sophisticated approach.…”

Hyaluronan treatment of biotinylated packaging cells increases titre of progeny retrovirus affinity-captured onto paramagnetic particles.

“…The procedure reported by Hughes et al (2001) was followed as summarised in Figure 1. Briefly, on Day 1 of a given experiment 1x10 6 PG13.pBabe.puro cells were seeded onto a 9cm diameter round plate in 15mL Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal calf serum (FCS), 2 mM L-glutamine, 100 µg/mL streptomycin, and 100 U/mL penicillin.…”

“…However, in other therapeutic contexts, complexing viral vectors within polysaccharide scaffold mimics of the extracellular matrix has proven advantageous for transduction of target cells (Thomas andShea 2013, Sun et al 2014) Le Doux et al (1999) previously reported that hyaluronan inhibits retroviral transduction. However, Hughes et al (2001) showed that paramagnetic particles coated with antibodies to the extracellular matrix component, fibronectin, could capture retrovirus particles secreted from the fibroblast-derived packaging cell line, PG13.pBabe.puro (Miller et al 1991). Fibronectin and hyaluronan are believed to interact extensively to define extracellular matrix behaviour in normal and cancerous tissues (Evanko et al 2015).…”

“…Fibronectin and hyaluronan are believed to interact extensively to define extracellular matrix behaviour in normal and cancerous tissues (Evanko et al 2015). Hughes et al (2001) also reported that paramagnetic particles coated with the lectin Concanavalin A, which is known to bind the carbohydrate component of cell surface glycoproteins (Pratt et al 2012), could efficiently capture retroviral particles. However, chemical biotinylation of PG13.pBabe.puro cells allowed capture of progeny virus particles by streptavidin-coated paramagnetic particles (SPMPs) and resulted in the highest retroviral titre increase in the Hughes et al (2001) study.…”

“…In light of observations by Hughes et al (2001), that the extracellular matrix components of packaging cell appear to co-associate with their progeny retroviruses, we sought to test the hypothesis that the presence of an additional, exogenous extracellular matrix component, hyaluronan, could enhance the capture of biotinylated progeny virus from PG13.pBabe.puro cells. Possible mechanisms for such an enhancement of capture include cross-linking of streptavidin-bound virus particles to non-bound particles via hyaluronan-based interactions.…”