Off-label prescription of genetically modified organism medicines in europe: emerging conflicts of interest?

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.

Replacement of native adenovirus receptor-binding sites with a new attachment moiety diminishes hepatic tropism and enhances bioavailability in mice.

The in vivo efficacy of adenoviral vectors (AdVs) in gene delivery strategies is hampered by the broad tissue tropism of the virus and its efficient binding to human erythrocytes. To circumvent these limitations, we developed a prototype AdV lacking native binding sites. We replaced the adenoviral fiber with a chimeric molecule consisting of the fiber tail domain, the reovirus sigma1 oligomerization domain, and a polyhistidine tag as model targeting moiety. We also abolished the integrin-binding motif in the penton base protein. The chimeric attachment molecule was efficiently incorporated onto AdV capsids, allowed efficient propagation of AdV without requirement for complementing fiber and conferred highly specific tropism to the AdV. Importantly, the targeted AdV exhibited markedly reduced tropism for liver cells. In comparison with control AdV with native tropism, the targeted AdV showed 1000-fold reduced transduction of HepG2 cells and 10,000-fold reduced transduction of mouse liver cells in freshly isolated liver slices. After intravenous inoculation of C57BL/6 mice, the targeted AdV exhibited delayed clearance in comparison with the native AdV, leaving approximately 10-fold greater levels in the blood 2 hr after inoculation. For all tissues analyzed, the targeted AdV displayed significantly reduced in vivo transduction in comparison with the native vector. Furthermore, in contrast to the native AdV, the targeted AdV did not bind human erythrocytes. Together, our findings suggest that the targeted AdV design described here provides a promising platform for systemic in vivo gene delivery.

Intravenous administration of the conditionally replicative adenovirus Ad5-Delta24RGD induces regression of osteosarcoma lung metastases.

Metastatic osteosarcoma (OS) has a very poor prognosis. New treatments are therefore wanted. The conditionally replicative adenovirus Ad5-Delta24RGD has shown promising anti-tumor effects on local cancers, including OS. The purpose of this study was to determine whether intravenous administration of Ad5-Delta24RGD could suppress growth of human OS lung metastases. Mice bearing SaOs-lm7 OS lung metastases were treated with Ad5-Delta24RGD at weeks 1, 2 and 3 or weeks 5, 6 and 7 after tumor cell injection. Virus treatment at weeks 1-3 did not cause a statistically significant effect on lung weight and total body weight. However, the number of macroscopic lung tumor nodules was reduced from a median of >158 in PBS-treated control mice to 58 in Ad5-Delta24RGD-treated mice (p = 0.15). Moreover, mice treated at weeks 5-7 showed a significantly reduced lung weight (decrease of tumor mass, p < 0.05), a significantly increased body weight gain (decrease of disease symptoms, p < 0.005) and a reduced number of macroscopic lung tumor nodules (median 60 versus > 149, p = 0.12) compared to PBS treated control animals. Adenovirus hexon expression was detected in lung tumor nodules at sacrifice three weeks after the last intravenous adenovirus administration, suggesting ongoing viral infection. These findings suggest that systemic administration of Ad5-Delta24RGD might be a promising new treatment strategy for metastatic osteosarcoma.

Enhanced tumor cell kill by combined treatment with a small-molecule antagonist of mouse double minute 2 and adenoviruses encoding p53.

Strategies to treat cancer by restoring p53 tumor suppressor functions are being actively investigated. These approaches range from expressing an exogenous p53 gene in p53 mutant cancers to antagonizing a p53 inhibitor in p53 wild-type (WT) cancer cells. In addition, exogenous p53 is used to strengthen the anticancer efficacy of oncolytic adenoviruses. Many cancers express high levels of the major negative regulator of p53, mouse double minute 2 (MDM2) protein. Recently, a novel class of highly potent and specific MDM2 antagonists, the Nutlins, was identified. We envisioned that Nutlins could protect both endogenous and exogenous p53 from MDM2-mediated inactivation. We therefore investigated treating human cancer cells with a combination of adenovirus-mediated p53 gene therapy and Nutlin. Combination treatment resulted in broadly effective cell kill of p53 WT and p53-negative cancer cells. Cytotoxicity was associated with profound cell cycle checkpoint activation and apoptosis induction. We also tested Nutlin in combination with oncolytic adenoviruses. Nutlin treatment accelerated viral progeny burst from oncolytic adenovirus-infected cancer cells and caused an estimated 10- to 1,000-fold augmented eradication of p53 WT cancer cells. These findings suggest that Nutlins are promising compounds to be combined with p53 gene therapy and oncolytic virotherapy for cancer.

A conditionally replicating adenovirus with strict selectivity in killing cells expressing epidermal growth factor receptor.

Virotherapy of cancer using oncolytic adenoviruses has shown promise in both preclinical and clinical settings. One important challenge to reach the full therapeutic potential of oncolytic adenoviruses is accomplishing efficient infection of cancer cells and avoiding uptake by normal tissue through tropism modification. Towards this goal, we constructed and characterized an oncolytic adenovirus, carrying mutated capsid proteins to abolish the promiscuous adenovirus native tropism and encoding a bispecific adapter molecule to target the virus to the epidermal growth factor receptor (EGFR). The new virus displayed a highly selective targeting profile, with reduced infection of EGFR-negative cells and efficient killing of EGFR-positive cancer cells including primary EGFR-positive osteosarcoma cells that are refractory to infection by conventional adenoviruses. Our method to modify adenovirus tropism might thus be useful to design new oncolytic adenoviruses for more effective treatment of cancer.

Genetic targeting of adenovirus vectors using a reovirus sigma1-based attachment protein.

Targeting adenovirus vectors (AdV's) for selective transduction of specific cell types requires ablation of native adenovirus tropism and introduction of a unique target-binding moiety. To bring these requirements within reach, we developed a novel strategy to target AdV's genetically that relies on replacement of the entire adenovirus fiber protein with a fusion molecule comprising the virion-anchoring domain of fiber and the oligomerization domain of reovirus attachment protein sigma1. The chimeric molecule forms trimers, is transported to the nucleus, and assembles onto the adenovirus capsid. In contrast to previously reported genetically targeted vectors, the AdV presented herein propagates efficiently without a requirement for complementing fiber. Due to ablation of the native adenovirus tropism, the infectivity of this AdV was at least 35-fold reduced on 293 cells. Importantly, a His tag incorporated into the chimeric attachment protein conferred His-tag-dependent tropism to the AdV, which resulted in a 12- to 40-fold greater transduction efficiency on two different cell lines expressing a His-tag-binding receptor. In addition, the infection efficiency was strongly reduced by preincubation with a His-tag-specific Ab. Thus, this sigma1-based chimeric attachment molecule provides a promising new platform for the generation of truly targeted AdV's.

Tissue inhibitor of metalloproteinase-3 expression from an oncolytic adenovirus inhibits matrix metalloproteinase activity in vivo without affecting antitumor efficacy in malignant glioma.

Oncolytic adenoviruses exhibiting tumor-selective replication are promising anticancer agents. Insertion and expression of a transgene encoding tissue inhibitor of metalloproteinase-3 (TIMP-3), which has been reported to inhibit angiogenesis and tumor cell infiltration and induce apoptosis, may improve the antitumor activity of these agents. To assess the effects of TIMP-3 gene transfer to glioma cells, a replication-defective adenovirus encoding TIMP-3 (Ad.TIMP-3) was employed. Ad.TIMP-3 infection of a panel of glioma cell cultures decreased the proliferative capacity of these cells and induced morphologic changes characteristic for apoptosis. Next, a conditionally replicating adenovirus encoding TIMP-3 was constructed by inserting the TIMP-3 expression cassette into the E3 region of the adenoviral backbone containing a 24-bp deletion in E1A. This novel oncolytic adenovirus, AdDelta24TIMP-3, showed enhanced oncolytic activity on a panel of primary cell cultures and two glioma cell lines compared with the control oncolytic virus AdDelta24Luc. In vivo inhibition of matrix metalloproteinase (MMP) activity by AdDelta24TIMP-3 was shown in s.c. glioma xenografts. The functional activity of TIMP-3 was imaged noninvasively using a near-IR fluorescent MMP-2-activated probe. Tumoral MMP-2 activity was significantly reduced by 58% in the AdDelta24TIMP-3-treated tumors 24 hours after infection. A study into the therapeutic effects of combined oncolytic and antiproteolytic therapy was done in both a s.c. and an intracranial model for malignant glioma. Treatment of s.c. (U-87MG) or intracranial (U-87deltaEGFR) tumors with AdDelta24TIMP-3 and AdDelta24Luc both significantly inhibited tumor growth and prolonged survival compared with PBS-treated controls. However, expression of TIMP-3 in the context of AdDelta24 did not significantly affect the antitumor efficacy of this oncolytic agent.

Replication-dependent transgene expression from a conditionally replicating adenovirus via alternative splicing to a heterologous splice-acceptor site.

BACKGROUND: Oncolytic viruses are promising anticancer agents because they selectively kill cancer cells and multiply within a tumor. Their oncolytic potency might be improved by expressing a therapeutic gene from the virus genome. In this regard, proper kinetics and level of transgene expression are important. In addition, expression of cytotoxic transgene products should be confined to cancer cells. Here, we developed oncolytic adenoviruses that provide transgene expression dependent on viral replication. METHODS: We constructed an oncolytic adenovirus that expresses luciferase under regulation of the endogenous major late promoter (MLP) via alternative splicing to an inserted splice-acceptor site analogous to that of the adenovirus serotype 40 long fiber gene. Splicing of the luciferase transcript was studied by RT-PCR analysis. Expression was measured in the presence and absence of the flavonoid apigenin, an inhibitor of viral replication. RESULTS: The inserted splice-acceptor site was properly recognized by the adenoviral splicing machinery. Luciferase expression levels were markedly higher than levels obtained with the cytomegalovirus (CMV) promoter, especially at late stages of infection. Inhibiting adenovirus replication reduced luciferase expression levels dramatically by 4 to 5 logs, whereas expression levels with the CMV-luciferase adenovirus were only moderately affected (2 logs). CONCLUSIONS: Transgene delivery using the endogenous late gene expression machinery resulted in an expression pattern distinct from expression driven by the conventional CMV promoter. The high expression levels and strict coupling of expression to viral replication should be useful for adequate monitoring of replication and might provide a platform for the design of armed conditionally replicating adenoviruses (CRAds) with enhanced oncolytic potency.

Conditionally replicating adenoviruses expressing short hairpin RNAs silence the expression of a target gene in cancer cells.

RNA interference (RNAi) is a posttranscriptional silencing mechanism triggered by double-stranded RNA that was recently shown to function in mammalian cells. Expression of cancer-associated genes was knocked down by expressing short hairpin RNAs (shRNAs) in cancer cells. By virtue of its excellent target specificity, RNAi may be used as a new therapeutic modality for cancer. The success of this approach will largely depend on efficient delivery of shRNAs to tumor cells. Tumor-selective replication competent viruses are especially suited to efficiently deliver anticancer genes to tumors. In addition, their intrinsic capacity to kill cancer cells makes these viruses promising anticancer agents per se. In this study, conditionally replicating adenoviruses were constructed encoding shRNAs targeted against firefly luciferase. These replicating viruses were shown to specifically silence the expression of the target gene in human cancer cells down to 30% relative to control virus. This finding offers the promise of using RNAi in the context of cancer gene therapy with oncolytic viruses.

Immune responses against adenoviral vectors and their transgene products: a review of strategies for evasion.

Human adenoviruses have been adopted as attractive vectors for in vivo gene therapy since they have a well-characterized genomic organization, can be grown to high titres and efficiently transduce a wide spectrum of dividing and non-dividing cells. However, the first-generation of adenoviral (Ad) vectors yielded only transient expression of the transgene in most immunocompetent mice. This constituted a major limitation of this early vector type. In contrast, persistent transgene expression can be established in immunodeficient mice. This suggests that the immunogenicity of adenoviral vectors limits the effective period of adenovirus-based gene therapy. Much effort has been put in devising strategies to circumvent the limitations imposed onto gene therapy by the immune system. Improvements in vector design have significantly improved the performance of the adenovirus vectors. Based on these results it is reasonable to anticipate that new modifications of the vectors will overcome some of the immunological barriers and will further expand the applicability of adenovirus-derived vectors.