Transient expression of genes delivered to newborn rat liver using recombinant adeno-associated virus 2/8 vectors.
ABSTRACT In vivo adeno-associated virus (AAV) delivery to adult liver results in sustained expression of the transgene. However, it has been suggested that AAV delivery to the newborn liver may result in transient expression. In the present study, we analysed transgene expression after AAV8 delivery of a therapeutic or a marker gene to newborn rat liver.
Recombinant AAV 8 vectors carrying either the human UGT1A1 cDNA or the lacZ gene were injected intravenously in 2-day-old Gunn or Wistar rats. Serum bilirubin level was recorded in Gunn rats and beta-galactosidase expression was monitored by immunohistochemistry or enzyme activity. The molecular forms of AAV genome were analysed by the polymerase chain reaction and Southern blotting in whole liver and by the quantitative polymerase chain reaction in macroscopically dissected beta-galactosidase clusters.
In Gunn rat, complete serum bilirubin normalization occurred after AAV delivery but hyperbilirubinemia resumed thereafter. Similarly, beta-galactosidase expression was maximum at day 7, but only a few (less than 1%) beta-galactosidase positive cells were recorded at 1 or 3 months. These cells gathered in small clusters and the AAV copy number was 75-fold higher in positive cell clusters than in the surrounding parenchyma.
The results obtained in the present study show that in vivo AAV delivery to newborn rats results in transient expression in most hepatocytes. Expression of the trangene was persistent in small clusters of cells and preliminary data support the hypothesis that integration of the viral genome occurs in these clusters. Altogether, our data confirm the low efficiency of AAV vectors for gene therapy of liver diseases when delivered in the newborn period.
Article: Targeted therapies to treat non-AIDS-defining cancers in patients with HIV on HAART therapy: treatment considerations and research outlook.[show abstract] [hide abstract]
ABSTRACT: Highly active antiretroviral therapy has led to a dramatic improvement in the prognosis of patients diagnosed with HIV and AIDS. This includes a significant decline in the rates of AIDS-related cancers, including Kaposi's sarcoma and non-Hodgkin's lymphoma. Unfortunately, rates of non-AIDS-defining cancers are on the rise, and now exceed the rates of AIDS-related cancers in patients with HIV. Treating non-AIDS-defining cancers in patients who are on highly active antiretroviral therapy is an open and complicated clinical question. Newer targeted therapies are now available to treat cancers which were historically refractory to traditional cytotoxic chemotherapy. Highly active antiretroviral therapy agents are notorious for causing drug-drug interactions. The co-administration of targeted chemotherapies with highly active antiretroviral therapy could well impede the efficacy or increase the toxicity of these targeted therapies. Unfortunately little is known about possible drug-drug interactions because HIV patients are typically excluded from clinical trials. We highlight what is known about how and why highly active antiretroviral therapy agents can affect drug metabolism. We then present the clinical and pharmacological data for nine recently approved targeted therapies - imatinib, dasatinib, nilotinib, erlotinib, sunitinib, lapatinib, bortezomib, sorafenib, and temsirolimus. We conclude with considerations on how to use these new agents to treat non-AIDS-defining cancers, and discuss a future research agenda to better understand and predict potential highly active antiretroviral therapy-targeted therapy interactions.Current opinion in oncology 10/2009; 21(5):445-54. · 4.09 Impact Factor
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ABSTRACT: Fetal gene therapy is one of the possible new therapeutic strategies for congenital or perinatal diseases with high mortality or morbidity. We developed a novel delivery strategy to inject directly into the fetal mouse trachea. Intratracheal (i.t.) injection at embryonic day 18 (E18) was more efficient in targeting the fetal lung than conventional intra-amniotic (i.a.) delivery. Viral vectors derived from adeno-associated virus serotype 6.2, with tropism for the airway epithelium and not earlier tested in the fetal mouse lung, were injected into the fetal trachea. Bioluminescence (BL) imaging (BLI) was combined with magnetic resonance (MR) imaging (MRI) for noninvasive and accurate localization of transgene expression in vivo. Histological analysis for β-galactosidase (β-gal) revealed 17.5% of epithelial cells transduced in the conducting airways and 1.5% in the alveolar cells. Stable gene expression was observed up to 1 month after injection. This study demonstrates that direct injection of rAAV2/6.2 in the fetal mouse trachea is superior to i.a. delivery for transducing the lung. Second, as stable gene transfer was detected up to 1 postnatal month, this approach may be useful to evaluate fetal gene therapy for pulmonary diseases such as cystic fibrosis, requiring both substantial numbers of transduced cells as well as prolonged gene expression to obtain a stable phenotypic effect.Molecular Therapy 12/2010; 18(12):2130-8. · 6.87 Impact Factor
Article: Impact of age at administration, lysosomal storage, and transgene regulatory elements on AAV2/8-mediated rat liver transduction.[show abstract] [hide abstract]
ABSTRACT: Liver-directed gene transfer is being investigated for the treatment of systemic or liver-specific diseases. Recombinant vectors based on adeno-associated virus serotype 8 (AAV2/8) efficiently transduce liver cells allowing long term transgene expression after a single administration in animal models and in patients.We evaluated the impact on AAV2/8-mediated rat liver transduction of the following variables: i) age at vector administration, ii) presence of lysosomal storage in liver cells, and iii) regulatory elements included in the transgene expression cassette. We found that systemic administration of AAV2/8 to newborn rats results in vector genome dilution and reduced transduction efficacy when compared to adult injected animals, presumably due to hepatocyte proliferation. Accumulation of glycosaminoglycans in lysosomes does not impact on levels and distribution of AAV2/8-mediated liver transduction. Transgene expression occurs in hepatocytes but not in Kupffer or liver endothelial cells when the liver-specific thyroxine-binding-globulin promoter is used. However, extra-hepatic transduction is observed in the spleen and kidney of animals injected at birth. The use of target sequences for the hematopoietic-specific microRNA miR142-3p does not improve liver transduction efficacy neither reduce immune responses to the lysosomal enzyme arylsulfatase B. The inclusion of a variant of the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE-m) decreases AAV2/8-mediated liver transduction levels.As AAV2/8-mediated liver gene transfer is entering in the clinical arena, these data will provide relevant information to the design of efficient AAV2/8-based therapeutic strategies.PLoS ONE 01/2012; 7(3):e33286. · 4.09 Impact Factor