Introduction Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and is currently one of the most difficult to treat. Surgical resection and liver transplantation are considered curative therapies but they are feasible for only a small number of patients. Other therapeutic options, such as radiofrequency ablation and arterial chemoembolization, are effective only in small tumours. Recombinant adeno-associated virus (rAAV) vectors are ideally suited for gene transfer-based therapeutic approaches for HCC because of their safety profile and remarkable tropism for the liver, with >90% of the vector particles being detectable in hepatocytes following a single systemic administration in non-human primates.
Methods Sequence complementarity to miR-122a (122aT) was cloned downstream of luciferase under the control of a new liver specific promoter/enhancer element, HLP, and flanked by the AAV-inverted-terminal-repeats (ITRs). AAV8 capsid-pseudotyped AAV8-HLP-LUC±122aT vector stocks were prepared by 293T-based three-plasmid transient transfection, and used for: in vitro transduction of HCC cell lines HUH-7 (miR-122a-positive) and SNU-387 (miR-122a-negative); in vivo tail vein injection of tumour-bearing SCID and B6 mice.
Results A single tail-vein injection of rAAV8 in tumour-bearing mice resulted in selective transduction of the liver as well as of HCC, but not neuroblastoma xenografts. We cloned the 122aT into our expression cassette to improve the transgene expression for HCC, taking advantage of the differential expression of miR-122a (abundant in normal liver but down regulated in most HCC). Normalised transgene expression with AAV8-HLP-LUC-122aT was at least 26.5-fold higher in SNU-387 when compared to HUH-7 cell lines. Tail-vein injection of rAAV8-HLP-LUC resulted in high transgene expression in liver, but mice transduced with rAAV8-HLP-Luc-122aT had little or no luciferase expression despite the proviral DNA presence. Subcutaneous HCC xenografts in mice showed a strong luciferase signal following tail-vein injection of both of these vectors.
Conclusion Inclusion of 122aT in the expression cassette allows regulation of rAAV mediated transgene expression in HCC cell lines and normal liver, and reduces expression in miR-122a positive cells (eg, normal hepatocytes). This novel vector, therefore, has the potential to deliver therapeutic transgenes to HCC in preference to normal hepatocytes in the liver, thus limiting toxicity.
Competing interests None declared.