A Dual-Targeted Lentiviral Vector Homing in on Prostate Bone Metastases

The field of gene therapy has held great promise for the treatment of hereditary and acquired diseases. In the past decade, much effort has been focused on the construction of safe and efficient viral vectors.1–4 However, the field has suffered major setbacks due to questions about the safety of the viral vectors used for transgene delivery.1 In light of the safety concerns of retroviral vectors, much effort has been made toward improving the safety of lentiviral vectors via the incorporation of specific targeting mechanisms, with the aim of restricting transgene expression to only the cells of interest. Broadly defined, these targeting schemes fall into two classes: transductional and transcriptional. In this issue of Molecular Therapy, Pariente et al.5 report the construction of a dual-targeted lentiviral vector, combining both transductional and transcriptional taregeting, that they use to treat prostate bone metastases following systematic injection. This work marks a successful attempt at a dual-targeted lentiviral vector that may have significant impact on the development of in vivo lentiviral gene therapy. One of the primary goals of vector design is a vector that can home in on specific cell types to deliver transgenes efficiently while introducing few side effects. This goal is particularly relevant to cancer gene therapy, in which the delivery of transgenes should usually be restricted to tumor cells. A secondary goal is to create vectors that can be produced on a large scale at a sufficient titer for clinical applications. Among the choices of viral vector currently being used in gene therapy clinical trails worldwide, lentiviral vectors constitute about 0.7% of the total as reported in 2007 by the Journal of Gene Medicine database. The main advantages of lentiviral vectors are the ability to transduce both dividing and nondividing cells and the ability to be integrated into the host genome for sustained transgene expression. Transcriptional targeting usually involves the incorporation of a tissue-specific promoter into the lentiviral genome to restrict the transgene expression to only the selected cell type.6,7 Expansion of lentiviral tropism via transductional targeting is commonly achieved through modification of the viral envelope protein by pseudotyping of the viral vector with glycoproteins from other viruses (for an updated list, see Table 1 of ref. 4). Among the expanding list of proteins used for pseudotyping, the vesicular stomatitis virus G protein (VSV-G) has been the most widely used.8 However, VSV-G pseudotyped lentiviral vector suffers from potential shortcomings such as broad tissue tropism and neutralization by human serum.3 In principle, a lentiviral vector that combines both transductional and transcriptional targeting mechanisms could offer improved tissue selectivity over a vector containing either transductional or transcriptional targeting alone.9,10 In the new work reported in this issue of Molecular Therapy, transcriptional targeting was achieved by cloning of an enhanced prostate-specific promoter, PSE-BC,11 into a third-generation self-inactivating lenti-viral vector that drives the expression of a humanized luciferase gene. The vector was pseudotyped with a modified Sindbis virus envelope, which was created by the insertion of the IgG-binding region of protein A from Staphylococcus aureus into the Sindbis envelope protein E1 at its receptor-binding domain.12,13 Modification of viral tropism was achieved by incubation of the lentiviral vector with a monoclonal antibody, 1G8, that binds prostate stem cell antigen (PSCA).14 PSCA is a cell-surface antigen that has been found to be overexpressed in 87% of clinical samples of bone metastases.14 A clear advantage of the reported approach is the relative ease and flexibility of using the engineered protein A binding domain in the Sindbis glycoprotein to pseudotype via incubation with an antibody. Hence, it is easy to envision that the versatility of the two-tier targeting mechanism should allow targeting of a wide variety of tissue types by pairing appropriate antibodies with tissue-specific promoters. Of particular interest to cancer gene therapy, the authors found that luciferase transgene expression using either the VSV-G pseudotyped, transcriptionally targeted vector PSE-BC/VSV-G or the dual-targeted vector PSE-BC/1G8 was largely confined to the grafted tumor cells after systematic injection. However, upon closer inspection, vector DNA was found in virtually all organs except the brain with the VSV-G singly targeted lentivirus. In comparison, when using the dual-targeted vector, the vector DNA was found predominantly in the grafted tumor, with diminished levels in the liver and spleen and negligible amounts in all other organs. These data highlight the advantage of applying transductional targeting in selective gene transfer into targeted tumor that can lead to a reduction in the risk of insertional mutagenesis in normal, nontargeted organs. Of significant impact is the system in which the authors demonstrated, as a proof of principle, targeting of prostate bone metastases. Prostate cancer is the second leading cause of cancer-related death among U.S. men, and bony metastases are developed at the end stage of the disease that contribute to serious clinical complications and morbidity.15 Figure 1 illustrates some of the aggressive features of prostate cancer bone lesion. Bone metastases are extremely hard to treat because of variables such as the structural barrier of bone, the unique microenvironment of tumor cells and bone interphase, and the existence of multiple metastatic lesions. Hence, the dual-targeting technology developed by Pariente et al. that can achieve gene delivery to the bony lesions holds promising prospects to improve the clinical outcome.16 Figure 1 The aggressive behavior of prostate cancer bone lesion Eventual implementation of this technology in the clinic requires further improvement of the vector in several areas. For example, the envelope modifications of the dual-targeted vector may have slightly diminished its infectivity in general, resulting in a lower luciferase expression level as compared with the VSV-G pseudotyped control vector. As the authors noted, boosting the activity of the tissue-specific promoter could be one way to compensate for the lowered infectivity. Additionally, the transductional modification scheme utilized in this model relies heavily on the incorporation of a single antibody; therefore, caution should be applied in the selection of the antibody candidate. One must take into account the heterogeneous nature of the diseased cells as well as the specificity of the antibody. This is particularly relevant to cancer treatment because many types of cancer lack a universally expressed surface biomarker.17 Perhaps a viral vector mixture containing a cocktail of several surface and genetic modifications is needed to address the heterogeneity of the disease. A third issue that should be addressed in future studies is the impact of the host immune system on the dual-targeted vector. Additional experimentation in immune-competent animal models will be critical for further advancement of this technology. Collectively, the work by Pariente et al. marks an important step in the right direction toward improving the safety of lentiviral vectors and their application in targeted gene medicine.