Transient expression of genes delivered to newborn rat liver using recombinant adeno‐associated virus 2/8 vectors

Flageul, Maude; Aubert, Dominique; Pichard, Virginie; Nguyen, Tuan Huy; Nowrouzi, Ali; Schmidt, Manfred; Ferry, Nicolas

doi:10.1002/jgm.1343

Cited by 24 publications

(15 citation statements)

References 24 publications

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“…We previously reported peak-and-drop kinetics of transgene expression after AAV2/8-mediated liver neonatal gene transfer in MPS VI rats and cats [15], [34]. The results described here suggest that AAV vector dilution occurs in the first weeks after gene delivery in newborn rats and is associated with strong reduction of transgene expression levels, as previously reported [4], [30], [31]. This is expected given the high rate of hepatocyte proliferation in the neonatal period [29] and the episomal status of recombinant vector genomes in cells transduced with AAV [8], [9].…”

Section: Discussionsupporting

confidence: 85%

“…In addition, the eGFP-positive cells in livers collected at P90 appeared in clusters when rats were injected at P4 but not at P30 (data not shown), possibly representing clones from a single cell containing integrated vector genomes, as previously described in mice injected at birth with AAV2/8 [30]. Thus, as previously reported [9], [30], [31], hepatocyte proliferation results in dilution of AAV vector genomes in transduced cells and reduced efficacy of long-term liver transduction in newborn rats.…”

Section: Resultssupporting

confidence: 79%

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Impact of Age at Administration, Lysosomal Storage, and Transgene Regulatory Elements on AAV2/8-Mediated Rat Liver Transduction

et al. 2012

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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.

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Section: Discussionsupporting

confidence: 85%

Section: Resultssupporting

confidence: 79%

Impact of Age at Administration, Lysosomal Storage, and Transgene Regulatory Elements on AAV2/8-Mediated Rat Liver Transduction

et al. 2012

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“…Transduction with a nonintegrating rAAV vector results in transient gene expression in actively dividing cells and remains a challenge in many gene therapy studies (Flageul et al, 2009;Wang et al, 2012). At present, many methods are being evaluated for rAAV readministration, for example, by serotype switching (Weinstein et al, 2010;Wang et al, 2012), capsid engineering , or immune modulation and tolerance induction to prevent immune responses to the vector and/or transgene (Goudy et al, 2011;Wang et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

Immunological Ignorance Allows Long-Term Gene Expression After Perinatal Recombinant Adeno-Associated Virus-Mediated Gene Transfer to Murine Airways

et al. 2014

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“…A study by Mayra et al described AAV9-mediated knockdown in cardiac and skeletal muscle after intraperitoneal delivery into neonatal mice, with no apparent shRNA production in the liver [18]. This preference for muscle over liver could be related to significant decreases in viral genomes per liver cell reported within 1-2 weeks after AAV8 delivery to neonatal mice, likely due to rapid division of hepatocytes in neonates [19,20]. Similarly, Wang et al detected viral genomes in the heart but not in the liver 2 months after intraperitoneal administration of AAV8 to neonates [21].…”

Section: Discussionmentioning

confidence: 99%

Systemic Delivery of shRNA by AAV9 Provides Highly Efficient Knockdown of Ubiquitously Expressed GFP in Mouse Heart, but Not Liver

2013

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AAV9 is a powerful gene delivery vehicle capable of providing long-term gene expression in a variety of cell types, particularly cardiomyocytes. The use of AAV-delivery for RNA interference is an intense area of research, but a comprehensive analysis of knockdown in cardiac and liver tissues after systemic delivery of AAV9 has yet to be reported. We sought to address this question by using AAV9 to deliver a short-hairpin RNA targeting the enhanced green fluorescent protein (GFP) in transgenic mice that constitutively overexpress GFP in all tissues. The expression cassette was initially tested in vitro and we demonstrated a 61% reduction in mRNA and a 90% reduction in GFP protein in dual-transfected 293 cells. Next, the expression cassette was packaged as single-stranded genomes in AAV9 capsids to test cardiac GFP knockdown with several doses ranging from 1.8×1010 to 1.8×1011 viral genomes per mouse and a dose-dependent response was obtained. We then analyzed GFP expression in both heart and liver after delivery of 4.4×1011 viral genomes per mouse. We found that while cardiac knockdown was highly efficient, with a 77% reduction in GFP mRNA and a 71% reduction in protein versus control-treated mice, there was no change in liver expression. This was despite a 4.5-fold greater number of viral genomes in the liver than in the heart. This study demonstrates that single-stranded AAV9 vectors expressing shRNA can be used to achieve highly efficient cardiac-selective knockdown of GFP expression that is sustained for at least 7 weeks after the systemic injection of 8 day old mice, with no change in liver expression and no evidence of liver damage despite high viral genome presence in the liver.

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Transient expression of genes delivered to newborn rat liver using recombinant adeno‐associated virus 2/8 vectors

Cited by 24 publications

References 24 publications

Impact of Age at Administration, Lysosomal Storage, and Transgene Regulatory Elements on AAV2/8-Mediated Rat Liver Transduction

Impact of Age at Administration, Lysosomal Storage, and Transgene Regulatory Elements on AAV2/8-Mediated Rat Liver Transduction

Immunological Ignorance Allows Long-Term Gene Expression After Perinatal Recombinant Adeno-Associated Virus-Mediated Gene Transfer to Murine Airways

Systemic Delivery of shRNA by AAV9 Provides Highly Efficient Knockdown of Ubiquitously Expressed GFP in Mouse Heart, but Not Liver

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