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.
Highlights
The liver is the largest organ in the body performing essential functions in metabolism, detoxification, and production of plasma proteins [1]
To confirm that the reduced amount of vector genomes and the lower transgene expression levels observed in livers of rats injected at P4, as opposed to those injected at P30, were due to the proliferation-related dilution of episomal AAV vector genomes, we injected rats at P4 with AAV2/8-TBG-enhanced Green Fluorescent Protein (eGFP) and collected livers at P15, P30 and P90
The eGFP-positive cells in livers collected at P90 appeared in clusters when rats were injected at P4 but not at P30, possibly representing clones from a single cell containing integrated vector genomes, as previously described in mice injected at birth with AAV2/8 [30]
Summary
The liver is the largest organ in the body performing essential functions in metabolism, detoxification, and production of plasma proteins [1]. Given its central role in maintaining homeostasis and regulating metabolism, and the high accessibility of hepatocytes via the bloodstream through a fenestrated epithelium, the liver has been widely utilized for the expression of therapeutic transgenes via somatic gene transfer [4,5]. This approach has been used to treat several inherited and acquired disease models, either affecting the liver itself or requiring systemic delivery of a therapeutic protein, such as hemophilia, lysosomal storage disorders, and others [4,5]. While this is associated with a low risk of insertional mutagenesis, it may represent a limitation for the transduction of actively replicating cells as in the case of newborn tissues [4]
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