Search

Background: Gene therapy of familial hypercholesterolemia (FH) requires successful transfer and lifelong expression of a functional low density lipoprotein receptor (LDLr) gene in the liver. Most of the vector systems currently employed for gene therapy use promoter elements which do not modulate transgene expression in a physiological manner., Methods: To study the in vivo effects of constitutive LDLr gene expression in the absence of interfering immunological reactions we established a new mouse model which combines homozygous LDLr deficiency and severe combined immune deficiency (SCID)., Results: Adenovirus-mediated transfer and expression of the LDLr gene under the control of a commonly used virus-derived promoter (minimal CMV promoter) leads to prolonged reduction of serum cholesterol levels in LDLr-deficient SCID mice. During the first 10 days after gene therapy serum cholesterol drops to about 10% of pretherapeutic values. Serum cholesterol persists on this level for 2 weeks and then slowly starts to rise again. Four months after vector application serum levels have reached about 40% of pretherapeutic values. However, as early as 5 days after gene transfer, the histological analysis of liver sections revealed the formation of crystalline lipid/cholesterol deposits in the cytosol of hepatocytes. During the following 8 weeks the amount of crystals increased in size and density. The intracellular storage of lipid and cholesterol reduced cell viability and induced an accelerated loss of therapeutic DNA from mice livers as was shown in a comparative expression study employing a transgene with a different metabolic function (human alpha 1-antitrypsin)., Conclusions: The non-physiological constitutive overexpression of an LDL receptor gene induces an imbalance between the speed of LDL uptake and metabolism which leads to pathological accumulation of lipids and cholesterol in hepatocytes. To protect cells from negative effects of LDLr overexpression, future vector design should consider the use of physiologically controlled expression elements., (Copyright 2004 John Wiley & Sons, Ltd.)

Background: Recombinant adenoviruses are highly efficient gene transfer vehicles but their administration to mammals is accompanied by a strong inflammatory response. The present study reports additional side effects observed during adenoviral gene transfer studies in rabbits., Methods: Hematological and serological parameters, the course of viremia and the organ distribution were analyzed after in vivo administration of E1-deleted adenoviruses in rabbits., Results: The systemic administration of a therapeutic dose of 5 x 10(11) infectious particles/kg (infusion time 20 min) led to an average reduction of 80-90% in the platelet count within 48 h. Full recovery took 10-14 days. Virus administration induced a strong but transient erythroblastosis (peaking 24 h after administration) which settled 48 h later. Normochromic anemia occurred over the next 10 days with hemoglobin levels dropping by about 40% to reach the lowest level 10 days after administration and taking two months for full recovery. Dose-dependent thrombocytopenia was also found in mice, but neither erythroblastosis nor anemia was observed (in equivalent doses). The hematological findings did not improve after local injection via the portal vein. Local and systemic administration led to a comparable course of viremia. Only minor differences were found in the biodistribution of viruses between local and systemic administration. Large amounts of viral DNA and transgene expression were found in the lungs, the kidneys and the ovaries, even after local administration via the portal vein., Conclusions: Local intravenous injection via the portal vein does not prevent systemic spread of viral vectors and the occurrence of vector-related side effects. The hematological changes observed in rabbits suggest the need for careful monitoring of hematological and rheological parameters in clinical trials.