![]() expression of shRNAs or ectopic proteins) to hepatocytes using this approach for example, administration of the AAV8- TBG-P21 vector results in P21 overexpression in hepatocytes, inhibiting their ability to proliferate ( Raven et al., 2017). Alternatively, instead of the Cre recombinase, it is possible to deliver other constructs as cargo (e.g. This allows the study of deleting/overexpressing a gene in the whole liver parenchyma ( Bird et al., 2018) or in a small number of hepatocytes using comparatively fewer genetic copies of vector. genetic copies) of AAV8- TBG vector that are administered the higher the dose of the vector, the more hepatocytes will be transduced. The number of transduced hepatocytes is proportional to the dose (i.e. In particular, the Cre recombinase together with a hepatocyte-specific promoter like the Thyroxin Binding Globulin ( TBG) promoter can be incorporated into the AAV8 genome and this is reported to be a specific means of Cre recombinase expression in hepatocytes, while avoiding undesired expression in extrahepatic cells ( Nakai et al., 2005 Malato et al., 2011 Lee et al., 2020a, b). Through the insertion of tissue-specific promoters, expression of the vector's ‘cargo’ can be further cell type-restricted. rAAV8-mediated hepatocellular gene editing has multiple applications including gene therapy ( Nathwani et al., 2011), lineage tracing experiments, gene deletion or gene overexpression in all or specific populations of the hepatocytes. rAAV8 is a commonly used AAV serotype due to its strong propensity to transduce hepatocytes ( Nakai et al., 2005). Currently, a widely used approach is to target hepatocytes with an AAV-based vector. ![]() There are a number of ways to manipulate hepatocellular gene expression ( Kellendonk et al., 2000). In mice, after transducing their target cells, AAVs enter the cell nucleus where they persist in an episomal form and only rarely integrate into the host genome ( Duan et al., 1999 Miller et al., 2004). There are different serotypes of AAV (AAV1, 2, 4, 5, 6, 7, 8, and 9), each of which exhibits a various transduction efficiencies in the different target tissues ( Zincarelli et al., 2008). They elicit a very mild immune response, especially the recombinant AAV vectors (rAAVs) that have undergone modifications to partly evade the immune system ( Rogers et al., 2011 Rabinowitz et al., 2019). AAVs are small (20 nm), single-stranded DNA viruses that belong to the family of Parvoviridae. As AAVs are replication deficient, they are a relatively safe and efficient way to express the Cre recombinase, overexpress specific proteins or introduce shRNA into in vivo model systems. Taking advantage of the Cre-Lox system, Adeno-associated viruses (AAVs) are an important vector system for gene expression manipulation and their use has risen dramatically in the last 20 years. These data advance the technique of hepatocellular genome editing through Cre-Lox recombination using Cre expressing AAV vectors, demonstrating their minimal effects on murine physiology and highlight the more subtle off target effects of these systems. Overall, there was no evidence of liver injury, and only mild T-cell infiltration was observed 14 days following AAV8 infection. Notably, there were no additional transcriptomic changes upon expression of Cre recombinase by the AAV8 vector. Furthermore, we observed transcriptional changes in genes involved in circadian rhythm and response to infection. ![]() The latter was enhanced upon Cre recombinase expression by the vector. We observed an acute and transient trend for reduction in homeostatic liver proliferation together with induction of the DNA damage marker γH2AX following AAV8 administration. To do this, we injected C57BL/6J wild-type mice with either recombinant AAV8 vectors expressing Cre recombinase or control AAV8 vectors and characterised the changes in general health and in liver physiology, histology and transcriptomics compared to uninjected controls. Here, we sought to identify the short-term off-target effects of AAV8 administration in mice. However, their off-target effects in mice have not been thoroughly explored. Adeno-associated virus 8 (AAV8) vectors are valuable instruments for the manipulation of hepatocellular gene expression. The liver is central to metabolic homeostasis and a site of many diseases, making the targeting of hepatocytes attractive. The ability to manipulate gene expression in specific cells under temporal control is a powerful experimental tool. Mice are a widely used pre-clinical model system in large part due to their potential for genetic manipulation.
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