Hepatic gene therapy using lentiviral vectors: Has safety been established?


  • Potential conflict of interest: Nothing to report.

  • See Article on Page 397

The goal of gene therapy is to deliver genetic material to target cells in a safe, effective manner to provide a therapeutic outcome to the cell or host. The field has generally relied on reengineered viruses to increase the efficiency of this process. Viruses such as adenovirus, adeno-associated virus, gamma-retroviruses (γ-RVs), and lentiviruses (LVs) have been modified to remove much of their genetic material to create vectors for reporter or therapeutic genes of interest. Because viruses have evolved to deliver a genetic payload to cells, they are an obvious platform. This strategy comes with some inherent risks, and investigators have continued to learn from both successes and setbacks in pursuit of developing safe, effective viral vectors.


fumarylacetoacetate hydrolase





Liver-directed gene therapy holds great promise for the treatment of monogenetic disorders as well as some acquired hepatic diseases because of its accessibility and the prominent role of the organ in a variety of diseases. A number of vectors have been evaluated in various preclinical and clinical studies of hepatic gene therapy.1–3

Retroviral vectors offer several distinct advantages, including long-term gene expression, low immunogenicity, and a reasonably large packaging capacity (□8 kb). Because of these characteristics, a number of clinical trials have used γ-RV or LV with promising results.4–9 Although the early safety measures of these trials were lauded for minimal side effects, enthusiasm for gene therapy was weakened by the development of lymphoproliferative disorders in several of the patients.

Because retroviruses normally integrate their proviral DNA in a semirandom manner and virologists have long known the ability of oncoretroviruses to induce tumor formation, it is not surprising that follow-up studies to the early trials established that the observed tumorigenesis was caused by the insertional mutagenesis of γ-RV.10–14 Although γ-RV has been demonstrated to prefer integration near transcription start sites, LV has been shown to integrate into active genes.10–16 Because of this difference in integration and the relative stability of lentiviral genetic material, LV has been considered less likely to cause insertional mutagenesis and clonal expansion.17 In addition, retroviral vectors have undergone successive rounds of refinement, and current γ-RV and LV vectors have incorporated many features to reduce the risk of replication-competent vectors and insertional mutagenesis while maintaining robust gene expression.18

In this issue, Rittelmeyer et al. investigated the ability of a latest-generation therapeutic LV to induce tumorigenicity and clonal expansion in a mouse model of chronic hepatic disease.19 The researchers first performed in vitro experiments that showed that murine hepatocytes transduced with their LV exhibit a similar integration profile that has been reported for other cell types.20 Because LV in these cells preferred intragenic regions and certain “hot spots” that have also been noted for other cell types, it suggests that inherent integration bias may be more responsible for the integration patterns than enhanced cell proliferation. Further studies that compare a broad range of transduced cell types and incorporate novel statistic methods may be able to clarify this issue.

In theory, there is a greater risk of LV-induced tumor formation in hepatic gene therapy, because the liver has the unusual property of self-renewal and gene transfer in itself may offer a selective advantage to treated cells. To test this, the investigators designed a comprehensive study to evaluate both integration and clonal expansion. To mimic a disease model and purposefully skew their system toward the induction of genotoxicity, they performed serial transplantation of LV-infected liver cells in fumarylacetoacetate hydrolase (Fah)-deficient mice (Fah(−/−) mice). The researchers also used a potentially genotoxic spleen focus-forming virus promoter to drive Fah gene expression to further increase the possibility of genetic damage. As anticipated, a substantial therapeutic effect was observed for the first generation of treated mice, as reflected by an increase in long-term survival. Although these mice still formed tumors, the nodules did not appear to be caused by insertional mutagenesis. Because Fah(−/−) mice are predisposed to tumor formation even with treatment, it is difficult to conclusively exclude the role of LV in transduced animals. It will be critical to see whether forthcoming treatments of non-tumor-prone adult animals with liver-directed LV-mediated therapies demonstrate a lack of oncogenesis in the liver.

The investigators further analyzed hepatocytes from up to four generations of serially transplanted mice. Importantly, they found no evidence of insertional mutagenesis in these animals, with only slight clonal expansion of cells transduced with LV. The researchers performed a thorough evaluation of the integration events from 38 mice that were serially transplanted. The data obtained by ligation-mediated polymerase chain reaction and 454 Life Sciences pyrosequencing of repopulated livers implies a polyclonal distribution, not clonal dominance, of LV-transduced hepatocytes. The researchers also noted that approximately 4% of the integration events were located next to genes with a potential cancer risk; these clonal events could provide a rich source of data for future investigations. It would also be interesting to examine the integration profile and clonality in Fah(−/−) mice that are repopulated with LV-transduced human hepatocytes to determine whether any species differences are observed.

Overall, this study nicely complements a growing body of work that indicates that gene therapy in adult animals with LV is not genotoxic, even in disease models. Furthermore, these data suggest that the liver is a safe target organ for gene therapy, because treatment with the latest-generation LV has a low risk of inducing tumor formation through insertional mutagenesis. Although each disease and therapeutic vector is different and every treatment option will need to be independently evaluated for safety and efficacy, it appears that hepatic gene therapy is, once more, a promising possibility.