A novel MVMp-based vector system specifically designed to reduce the risk of replication-competent virus generation by homologous recombination

Recent work highlights the potential usefulness of MVM-based vectors as selective vehicles for cancer gene therapy (Dupont et al, Gene Therapy, 2000; 7: 790-796). To implement this strategy, however, it is necessary to develop optimized methods for producing high-titer, helper-free parvovirus stocks. Recombinants of MVMp (rMVMp) are currently generated by transiently co-transfecting permissive cell lines with a plasmid carrying the vector genome and a helper plasmid expressing the capsid genes (replaced with a foreign gene in the vector genome). The resulting stocks, however, are always heavily contaminated with replication-competent viruses (RCV), which precludes their use in vivo and particularly in gene therapy. In the present work we have developed a second-generation MVMp-based vector system specifically designed to reduce the probability of RCV generation by homologous recombination. We have constructed a new MVMp-based vector and a new helper genome with minimal sequence overlap and have used the degeneracy of the genetic code to further decrease vector-helper homology. In this system, the left homologous region was almost completely eliminated and the right sequence overlap was reduced to 74 nt with only 61% homology. We were thus able to substantially reduce (∼ 200 ×), but not completely eliminate, generation of contaminating viruses in medium-scale rMVMp preparations. Since the remaining sequence homology between the new vector and helper genomes is weak, our results suggest that contaminating viruses in this system are generated by nonhomologous recombination. It is important to note, unlike the autonomously replicating helper viruses produced from the first-generation vector/helper genomes, the contaminating viruses arising from the new packaging system cannot initiate secondary infection rounds (so they are not 'replication-competent viruses'). Our findings have important implications for the design of new MVMp-based vectors and for the construction of trans-complementing packaging cell lines.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Gene therapy clinical trials in Belgium

The present paper briefly describes the missing information about gene therapy clinical trials authorized in Belgium in relation with the regulatory framework. It also proposes a basic database format, complying with legal confidentiality rules. We then discuss transparency in the gene therapy field within the European Union.</p

Efficacy and safety of the bisphosphonate Tiludronate for the treatment of tumor-associated hypercalcemia

Tiludronate is a new bisphosphonate whose efficacy has already been reported for the prevention of postmenopausal bone loss. We have evaluated its efficacy and tolerance by a dose-finding study in 19 hypercalcemic cancer patients after adequate intravenous (iv) rehydration. Treatment consisted of 3 days of iv tiludronate given at doses of 3.0 mg/kg/day (n = 3), 4.5 mg/kg/day (n = 3), or 6.0 mg/kg/day (n = 13); this iv therapy was followed by 17 days of oral tiludronate, 400 mg (n = 13) or 800 mg (n = 6)daily. Treatment had to be discontinued in 9 patients, including 3 because of evident treatment failure and 1 because of severe toxicity. After iv tiludronate, 13/18 patients had a normal Ca level, including 10/12 who had received 6.0 mg/kg/day, but Ca2+ levels were fully normalized in only 4/18 and 3/12 patients, respectively. After 6.0 mg/kg/day, Ca levels had fallen from 12.1 ± 0.3 to 10.0 ± 0.4 mg/dl (P < 0.0005), whereas fasting urinary calcium excretion went from 0.639 ± 0.099 to 0.272 ± 0.054 mg Ca/mg creatinine on d4 (P < 0.001). On the other hand, oral tiludronate was unable to normalize Ca in patients who were still hypercalcemic after the iv course, although the daily administration of 800 mg appeared to be more efficient than the 400 mg daily dosage. The administration of tiludronate caused an increase in serum phosphate levels, from 2.9 ± 0.2 to 3.7 ± 0.2 mg/dl after the iv course, probably through an increase in the TmP/GFR index, which went from 2.3 ± 0.2 to 3.6 ± 0.4 mg/dl (P < 0.05). Three patients had an increase in serum creatinine levels after the iv course, one obese patient developing an acute renal insufficiency; during oral tiludronate therapy, 5 other patients also presented an increase in serum creatinine levels. Oral tiludronate administration was also associated with occasional nausea and vomiting. In summary, compared with aminobisphosphonates, tiludronate is not indicated for the treatment of tumor-associated hypercalcemia because of the need for high iv doses which are potentially nephrotoxic.SCOPUS: ar.jinfo:eu-repo/semantics/publishe