The liver sieve and gene therapy.

We were intrigued by the article by Mount et al1 on the reversal of hemophilia B following the intraportal injection of an adenoassociated viral vector encoded with the coagulation factor IX gene into hemophiliac dogs. Mount et al reported substantial correction of hemophilia B in 3 of the 4 animals. However, we think that the fourth dog, Beech, is more interesting. He obtained only temporary reversal and died. Necropsy revealed early cirrhosis thought to be associated with iron overload secondary to concurrent pyruvate kinase deficiency.1 Furthermore, at 12 years of age, Beech was the oldest dog studied. Cirrhosis and old age both are associated with loss of the liver sieve.2,3 The “liver sieve” is a term used to describe the endothelial cells that line the hepatic sinusoids. These cells are perforated by multiple fenestrae of approximately 100-nm diameters, within very thin cytoplasmic extensions (Figure 1). Lacking a basal lamina, sinusoidal fenestrae are truly discontinuous and thus allow unimpeded passage of macromolecules up to those with a diameter of about 100 nm including, potentially, viral vectors such as those involved in gene therapy. Indeed, we suggested 25 years ago that the normal liver sieve allows circulating viruses smaller than 100 nm to contact hepatocytes,4 and this has largely been substantiated since.5,6 The adeno-associated virus used by Mount et al has a diameter of about 20 nm.7 Mount and colleagues suggested that in Beech’s case the usual hepatic tolerance of the viral vector and gene was decreased secondary to a change of the normal tolerance-inducing liver endothelial cell phenotype that might occur in cirrhosis. We agree, and posit that loss of fenestrations hinders contact between hepatocytes and circulating lymphocytes, which also is known to induce tolerance.8 We therefore suggest that loss of fenestrations in the hepatic sinusoids, by hindering interactions between both the lymphocytes and the viral vector in the portal blood with the hepatocytes, decreased the uptake, expression, and tolerance of the introduced viral vector and its gene in Beech. Conversely, it is of interest that interventions undertaken to improve success of gene therapy, such as partial hepatectomy,9 are associated with increased fenestration of the liver sieve.10 Therefore, modulation of the liver sieve may prove to have an important role in the outcome of gene therapy, either through influencing hepatocyte-lymphocyte interactions or, alternatively, the physical uptake of the viral vector.