Gene therapy in VHL-null cells using human artificial chromosomes

Last month, our conference summary highlighted some of the work that is taking place in the field of gene therapy. Currently, many advanced gene therapy systems are derived from viruses, such as the adenovirus and lentivirus. However, these viral vectors are often held back by their limited cloning capacity and lack of copy number control, as well as their potential for deleterious integration into the host genome and unwanted immunological responses.

Human artificial chromosomes (HACs) offer an alternative system which does not suffer from these disadvantages. In particular, these vectors have a functional centromere which enables the HAC to be stably maintained as a single-copy episome, without the risk of integration into the host genome. They also have a large cloning capacity, which allows entire genomic loci (including all their regulatory elements) to be used. A recent paper by Kim et al. (2011) has now used such a vector in a VHL-null human kidney cancer cell line (786-O) to demonstrate that it can correct for its VHL deficiency.

The authors first isolated the VHL gene from human genomic DNA using transformation-associated recombination cloning. The vectors were then constructed and transfected into CHO cells, and RT-PCR and fluorescence in situ hybridisation (FISH) were used to confirm the presence of the VHL-containing HAC within these cells.

Using microcell-mediated chromosome transfer, the VHL-HAC was transferred to VHL-null 786-O cells. FISH showed that this construct propagated autonomously without any integration into host chromosomes, and RT-PCR confirmed the expression of wild-type VHL within these cells. Moreover, when compared to controls, this HAC downregulated the protein levels of HIF2α, Cyclin D1 and Cdk1, which are known targets of VHL. Additionally, VHL-deficient 786-O cells have a more branched and invasive phenotype when supplemented with either hepatocyte growth factor or foetal bovine serum, but these phenotypes and their migration were substantially suppressed with the VHL-HAC.

This study made use of a HAC in which the centrosome can be inactivated, resulting in the loss of the HAC. This feature provides a useful phenotypic control for any HAC-associated alterations within the cells. Accordingly, centrosome inactivation and HAC loss led to the increased expression of HIF2α, Cyclin D1 and Cdk1, back to their original levels. Altogether, these results suggest that the VHL gene expressed from the HAC produces a functional protein which can correct for the VHL deficiency within these 786-O cells.

Much more work is necessary before HACs can be developed as a treatment for VHL, but a similar system could be tested in FLCN-null cells. Nevertheless, a number of BHD-associated gene therapy projects are currently in progress – to learn more, please do read our articles about Dr Justin Roth, Dr Richard Harbottle, Dr Laura Denby and Dr Suet-Ping Wong. Alternatively, join us at 4th BHD Symposium in Cincinnati on 28th-30th March to hear more about the current state of BHD research in general. Registration and abstract submission are open, and further information about the Patient and Family sessions can also be found here.


  • Kim JH, Kononenko A, Erliandri I, Kim TA, Nakano M, Iida Y, Barrett JC, Oshimura M, Masumoto H, Earnshaw WC, Larionov V, & Kouprina N (2011). Human artificial chromosome (HAC) vector with a conditional centromere for correction of genetic deficiencies in human cells. Proceedings of the National Academy of Sciences of the United States of America, 108 (50), 20048-53 PMID: 22123967

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