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Recently, the first human medicine containing a genetically modified organism (GMO medicine) was authorized for use in the European market. Just as any medicinal product, the market authorization for a GMO medicine contains a precise description of the therapeutic use for which the medicinal product is intended. Within this use, the application of the GMO medicine is permitted, without the need for the institution to obtain a specific permit. In practice, however, medicinal products are also frequently prescribed for treatment outside the registered therapeutic use, a practice that is referred to as "off-label use." While off-label use of conventional medicines is permitted and has been very useful, the off-label use of GMO medicines is not covered in the European Union (EU) legislation or guidelines and falls under each member state's national environmental legislation. This implies that in the Netherlands and most other EU member states, an environmental permit will be required for any institution that uses the GMO medicine outside the registered application(s). In the Netherlands, this permit is identical to the permits required for the execution of clinical trials involving nonregistered GMOs. The application procedure for such permit is time-consuming. This process can therefore limit the therapeutic options for medical professionals. As a consequence, desired treatment regimens could be withheld for certain patient (groups). To make future off-label use of GMO medicines permissible in a way that is acceptable for all stakeholders, regulators should adopt a proactive attitude and formulate transparent legislative procedures for this. Only then the field can maintain the public acceptance of GMO medicines, while maintaining the freedom to operate of medical professionals.

The efficacy of adenovirus (Ad)-based gene therapy of solid tumors, such as prostate cancer, is limited. One of the many problems is that the virus infects many different cell types in the body, resulting in high toxicity, whereas the target cancer cells are often less prone to wild-type Ad infection. Our aim was to develop genetically de- and retargeted Ad vectors to reduce off-target effects and increase target infection for prostate cancer. We have previously reported an Ad5 vector specific for the cancer-associated receptor Her2/neu, created by inserting Her2/neu-reactive Affibody (R) molecules (ZH) into the HI loop of a coxsackievirus and adenovirus receptor binding-ablated fiber (Ad[ZH/1]). In addition to virus retargeting to Her2/neu, this virus was further modified from wild-type Ad by changing the RGD motif in the penton base to EGD and by substitution of the KKTK motif in the third shaft repeat to RKSK, resulting in the vector Ad[ZH/3]. The ZH-containing vectors could be produced to high titers and were specific for their target, resulting in efficient infection and killing of Her2/neu-positive androgen-dependent PC346C prostate cancer cells in vitro. Here we show that the oncolytic Ad[ZH/3] vector significantly prolonged survival time and reduced serum prostate-specific antigen levels in an orthotopic prostate tumor model in nude mice to the same extent as wild-type Ad5. Our results show that Her2/neu targeting using Ad-based vectors for prostate cancer is feasible and may serve as a basis for the development of gene therapy of human prostate cancer as well as other Her2/neu-expressing cancers.

Revision surgery for loosened hip prostheses is a heavy burden for elderly patients with comorbidity. As an alternative to surgery we performed a study to stabilize the prosthesis by percutaneous cement injection after removing inflammatory tissue with an intraarticular virus-directed enzyme prodrug approach. Twelve elderly patients with debilitating pain from a loosened hip prosthesis were included in a phase 1 dose-escalating clinical study. The patients were admitted to the hospital for 10 days for an intraarticular vector and prodrug injection, and subsequently for a percutaneous bone cement injection. This paper reports the adverse and serious adverse events of the study. After prodrug injection 9 of 12 patients had gastrointestinal adverse events (nausea, vomiting, and diarrhea), and 8 patients had hepatic adverse events (rise in aspartate aminotransferase and alanine aminotransferase). Five patients developed anemia (World Health Organization grade 1 or 2) from hematomas after cement injection. There were four serious adverse events in the first 6 months after vector injection, but these were not related to gene therapy as judged by an independent safety committee. There was no dose-limiting toxicity. However, the extensive comorbidity in these patients makes it difficult to fully establish the safety of the approach in this small and heterogeneous patient population.

Loosening of orthopedic hip prostheses is an increasing health problem. In elderly patients with comorbidity,revision surgery may lead to high mortality rates. A less invasive surgical technique is therefore required to reduce these patient risks. To this end a percutaneous gene therapy approach was designed to destroy the periprosthetic loosening membrane, and enable refixing of the hip prosthesis with percutaneous bone cement injections under radiological guidance. In this phase 1/2 dose-escalating gene therapy clinical trial, 12 patients were treated. Toxicity and hip function variables were monitored up to 6 months posttreatment. All patients completed the study and no dose-limiting toxicity was observed. Improvement in walking distance, independence,and pain was demonstrated particularly in patients receiving 3 x 10(10) and 1 x 10(11) viral particles. Taken together, these data show that this gene therapy approach targeted at the interface membrane around a loosened hip prosthesis is a feasible treatment option for elderly patients for whom surgical intervention is not appropriate.

The presence of replication-competent adenoviruses (RCAs) in batches of replication-defective adenovirus (Ad) vectors is a major problem for the application of these vectors in gene therapy. RCAs are generated by recombination between sequences in the Ad vector and homologous Ad sequences in the helper cells, resulting in the acquisition by the vector of early region 1. To prevent the formation of RCAs, we have developed helper cell lines, which we named PER, and matched Ad vectors that do not have sequence overlap. PER cells contain the Ad serotype 5 (Ad5) E1A- and E1B-encoding sequences (Ad5 nucleotides 459-3510) under the control of the human phosphoglycerate kinase (PGK) promoter. We demonstrate that PER cells synthesize high levels of the Ad5 E1A and E1B proteins. The yields from PER cells of E1-deleted Ads are similar to those obtained from earlier helper cells, such as 911 and 293 cells. Propagation of matched Ad vectors, which lack Ad5 nucleotides 459-3510, in one of the PER clones, PER.C6, does not result in the generation of RCAs, in contrast to propagation in 293 cells. We conclude that the combination of PER.C6 cells and nonoverlapping E1-deleted adenoviral vectors eliminates the problem of RCA generation by homologous recombination, and allows cost-effective production of safe, clinical-grade batches of recombinant Ad vectors.

Currently, the preferred host for the production of early region-1 (E1)-deleted recombinant adenoviruses (rAdV) is cell line 293, which was generated by transformation of human embryonic kidney cells by sheared adenovirus 5 (Ad5) DNA. To develop alternative hosts for the production of rAdV, we generated adenovirus-transformed human cell lines by transformation of human embryonic retinoblasts (HER) with a plasmid containing base pairs 79-5789 of the Ad5 genome. One of the established HER cell lines, which we called 911, exhibited favorable growth characteristics and was chosen for further study. This cell line is demonstrated to have several characteristics in common with the well-known 293 cell line: The 911 cell line is highly transfectable, and exhibits similar frequencies of homologous recombination. However, it has additional characteristics that make it a useful alternative for 293. The 911 cells perform particularly well in plaque assays. Upon infection with E1-deleted adenoviruses, plaques become apparent in monolayers of 911 cells already after 3-4 days versus 4-10 days in monolayers of 293 cells, thereby reducing the time required for quantitative plaque assays. Furthermore, yields of E1-deleted adenovirus vectors up to three times as high as those achieved with 293 cells can be obtained with 911 cells. Finally, the Ad5-DNA content of the 911 cell line is completely known. These features make the 911 cell line a useful alternative for the construction, propagation, and titration of E1-deleted recombinant adenoviruses.

Hemophilia A is caused by the lack of functional blood-clotting factor VIII. We have used retrovirus-mediated gene transfer to generate various cell lines, rodent as well as human, that secrete the human factor VIII protein. To study whether transplantation of genetically modified fibroblasts is a feasible approach for gene therapy of hemophilia A, we implanted the factor VIII-secreting cells into immune-deficient mice. Implantation of factor VIII-secreting primary human skin fibroblasts resulted in long-term persistence of the transplanted cells; cells recovered from the implants up to 2 months post-implantation still had the capacity to secrete factor VIII when regrown in tissue culture. However, we were unable to detect any human factor VIII in plasma samples of the recipient mice. The absence of human factor VIII in the recipients' plasma is shown to be due neither to (epigenetic) inactivation of the retroviral vector in vivo, nor to inability of the stationary cells to secrete factor VIII protein. However, we did note a rapid clearing of the human factor VIII: CAg from plasma upon intravenous injection of plasma-derived human factor VIII in mice (t1/260 min vs. 10 hr in humans). This phenomenon can fully explain the apparent absence of human factor VIII in the recipients' plasma.