AAV vectors transduce hepatocytes in vivo as efficiently in cirrhotic as in healthy rat livers

Abstract

In liver cirrhosis, abnormal liver architecture impairs efficient transduction of hepatocytes with large viral vectors such as adenoviruses. Here we evaluated the ability of adeno-associated virus (AAV) vectors, small viral vectors, to transduce normal and cirrhotic rat livers. Using AAV serotype-1 (AAV1) encoding luciferase (AAV1Luc) we analyzed luciferase expression with a CCD camera. AAV1Luc was injected through the hepatic artery (intra-arterial (IA)), the portal vein (intra-portal (IP)), directly into the liver (intra-hepatic (IH)) or infused into the biliary tree (intra-biliar). We found that AAV1Luc allows long-term and constant luciferase expression in rat livers. Interestingly, IP administration leads to higher expression levels in healthy than in cirrhotic livers, whereas the opposite occurs when using IA injection. IH administration leads to similar transgene expression in cirrhotic and healthy rats, whereas intra-biliar infusion is the least effective route. After 70% partial hepatectomy, luciferase expression decreased in the regenerating liver, suggesting lack of efficient integration of AAV1 DNA into the host genome. AAV1Luc transduced mainly the liver but also the testes and spleen. Within the liver, transgene expression was found mainly in hepatocytes. Using a liver-specific promoter, transgene expression was detected in hepatocytes but not in other organs. Our results indicate that AAVs are convenient vectors for the treatment of liver cirrhosis.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a <em>Gaussia</em> Luciferase SERCaMP

“…GLuc-SERCaMP release can be detected from a viral range of 7.6 x 10 7 vg to 7.6 x 10 9 vg (Figure 4). This range is 4-400 times lower than reported in previous work utilizing firefly luciferasebased viral injections of the liver 19 . At higher concentrations, we observed a loss of detectable expression over time, which is possibly due to an immune response of the animal to the transgene 22 .…”

“…To circumvent the conversion of AAV single-stranded genome to double-stranded DNA, AAV-SERCaMP was packaged as an AAV serotype-1 double-stranded vector 20 . AAV serotype-1 has been shown to effectively transduce rat livers 19,21 ; however, the caveat of our injection technique is the potential for virus to travel to other tissues throughout the body. Although the vector was directly injected into the liver, our methodology as presented cannot discern the source of SERCaMP release, and therefore it is possible that tissues other than the liver may contribute to the GLuc-SERCaMP signal.…”

“…We have detected steady levels of GLuc-SERCaMP in circulation of unchallenged animals for 56 days post-injection (latest timepoint tested thus far) and predict longer experiments are possible 12 . AAV vectors are small in size, measuring approximately 20 nm in diameter, and pose minimal biosafety requirements 19 . To circumvent the conversion of AAV single-stranded genome to double-stranded DNA, AAV-SERCaMP was packaged as an AAV serotype-1 double-stranded vector 20 .…”

MicroRNA 122, Regulated by GRLH2, Protects Livers of Mice and Patients From Ethanol-Induced Liver Disease

“…1E). AAV8 vectors have previously been shown to be equally effective in transducing both healthy and cirrhotic livers 23 . Indeed, we noted equivalent activity of co-expressed Gaussia Luciferase (GLuc) in the serum CCl4 treated mice 2 weeks following injection in both Oil and CCl4-treated mice (Supplementary Fig.…”

Regulation of hepatocyte identity and quiescence

“…However, efficient transduction of the cirrhotic liver with adenoviral viral vectors may also be hampered by collagen deposits [205]. This situation might be circumvented using other types of viral vectors such as adeno-associated viruses (AAV) [206], albeit delivery of the therapeutic factor to a significant proportion of the parenchyma would still be needed to influence overall liver function and even more to prevent HCC. In this regard, an interesting study recently showed that AAV vector-delivered HNF4a to rats with decompensated cirrhosis reversed terminal chronic hepatic failure.…”