Titer determination of Ad5 in blood: a cautionary note

Abstract

Recombinant adenoviruses are presently the most efficient in vivo gene transfer system available. Targeting single organs or large tumors by adenoviral vectors requires an intravascular route of application. During the first pass of viral particles through the vascular bed of the target tissue, virus uptake is not quantitative and undefinite amounts of particles leak into circulation. To determine the amount of leaking particles and to calculate organ-specific uptake (in-/outflow ratio), it is necessary to titrate virus particles directly in blood. In preclinical and clinical trials titration is currently mostly done with blood plasma instead of full blood. However, this technique provides valid results only as long as there is no affinity between adenovirus particles and erythrocytes. In this study we demonstrate that Ad5 particles, as mostly employed for gene therapy, have a strong affinity to human erythrocytes. At 60 min after coincubation of human erythrocytes and Ad5 particles, more than 98% of the particles are attached to the surface of erythrocytes. Therefore, ignoring the amount of red cell bound particles by performing titration in plasma leads to severe miscalculation of organ-specific transfer rates or virus circulation half-life. The biological impact of an increased affinity between virus particles and erythrocytes will be discussed.

Characterisation of systemic dissemination of nonreplicating adenoviral vectors from tumours in local gene delivery

“…First, the amount of adenoviral vectors associated with cells in blood samples shown in Figure 2B was extremely low compared with that in the plasma. Even the cellassociated vectors could be originally in the plasma and then nonspecifically bind to erythrocytes (Cichon et al, 2003) or be co-precipitated with blood cells during the centrifuging process. Second, most tumour cells injected intravenously accumulated in the lung (Figure 3), which was consistent with data in the literature that lung is the primary target of metastatic tumour cells in mice (Lee, 1983).…”

Extended plasma circulation time and decreased toxicity of polymer-coated adenovirus

“…Plasma circulation time, toxicity of polymer-coated adenovirus NK Green et al adenovirus binds extensively to human erythrocytes, 19 a phenomenon not seen in mice, raises an important issue for extravasation of adenovirus following intravenous administration in clinical trials. Our studies have shown that over 90% of a therapeutic dose of type 5 adenovirus is sequestered by human erythrocytes under physiological conditions ex vivo, but that pc-virus shows 10-fold decreased binding (Lyons et al, submitted for publication), which may improve bioavailability of the virus following intravenous injection to patients.…”

RAD001 (Everolimus) Improves the Efficacy of Replicating Adenoviruses that Target Colon Cancer

“…The majority of i.v. administered virus is either sequestered on red cells or cleared by macrophages within minutes of injection (4,5). This can be circumvented by depletion of Kupffer cells and coating the virus with hydrophilic polymers like hydroxypropyl methacrylamide (6), but delivery of the virus to the tumor in large amounts remains a major problem.…”