Phase I Study of a Recombinant Adenovirus-Mediated Gene Transfer in Lung Cancer Patients
Thomas TurszAxel Le CesneP BaldeyrouErwan GautierP. OpolonC. SchatzAndréa PaviraniMichael CourtneyD. LamyThierry RagotPatrick SaulnierAntoine AndremontR. MonierM. PerricaudetT. Le Chevalier
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Abstract:
Despite vigorous efforts at curbing tobacco consumption and aggressive combined-modality treatment programs, both the incidence of and the mortality from lung cancer have remained virtually unchanged in the last 10 years. More effective innovative therapies are clearly needed. The direct transfer into tumor cells of tumor suppressor genes or toxic gene products that specifically promote tumor cell death and spare nonmalignant cells is a potentially novel anticancer treatment approach that should be investigated. Purpose: On the basis of compelling preclinical data, we initiated a phase I study involving six patients with inoperable lung cancer and an endobronchial lesion accessible by bronchoscopy. Our purpose was to evaluate the feasibility, tolerance, and clinical, biologic, and immunologic effects of the intratumoral administration of a recombinant, replication-deficient adenovirus (rAd.RSVβ-gal), using the Rous sarcoma virus promoter to drive transcription of the Escherichia coli lacZ marker gene that encodes for the bacterial enzyme β-galactosidase (β-gal). From June 1994 through April 1995, six patients (five males and one female) were enrolled in the trial. A single dose of recombinant virus suspension containing 10 7 or 10 8 plaque-forming units (PFU) was injected intratumorally into two successive cohorts of three patients. Eligible patients received concomitant chemotherapy. Patients were kept under isolation conditions from 3 days before the injection was given until virus excretion was undetectable. Biopsyspecimens of the tumor and surrounding mucosa were collected on the 8th day and at 1, 2, and 3 months after injection. They were analyzed by cell culture, polymrase chain reaction (PCR), and β-gal expression for the presence of recombinant adenovirus. So that the risk of virus recombination or complementation could be minimized, wild-type adenovirus carriers among the hospital staff (identified by PCR) were excluded from contact with patients who were potentially excreting recombinant virus. β-gal was expressed in tumor biopsy specimens of three patients (one who received the 10 7 PFU dose level and two who received 10 8 ). Bronchoalveolar lavage specimens collected immediately after injection were positive for recombinant adenovirus when analyzed in culture and by PCR. All biologic fluids were negative for recombinant virus as judged by PCR after day 12, with the exception of bronchoalveolar lavage specimens (positive PCR up to 90 days in two of three patients treated with 10 8 PFU). The blood samples obtained from the three patients treated with 10 8 PFU showed positive PCR results immediately after virus injection. Patients were kept in isolation for a median of 17 days. The most common toxic effects were moderate bleeding (occurring in two patients) during bronchoscopy and fever (seen in four patients). Endoscopic and clinically objective antitumor responses were seen in four patients, including one patient who showed a complete response by pathologic evaluation. The median survival for the patients was 12.5 months (range, 3–16+ months). Throughout the study, hospital staff remained negative for recombinant adenovirus infection. This ongoing phase I study has demonstrated that a recombinant adenovirus-mediated marker gene, such as rAd.RSVβ-gal, can be safely introduced into humans and that the gene product is expressed by lung tumor cells of the host. [J Natl cancer Inst 1996;88:1857–63]Keywords:
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To investigate the methods and solve the technical bottlenecks in the preparation of recombinant human protein hZP3 using the baculovirus expression system and pave the technical ground for the production and application of recombinant hZP3.The recombinant vector pFASTBAC HTa-hZP3 was constructed and transferred to competent E. coli cells carrying bacmid to produce recombinant bacmid by homologous recombination. Sf9 cells were transfected with the recombinant bacmid to produce recombinant baculovirus. Full-length recombinant hZP3 (amino acids 1-424) and truncated recombinant hZP3 (amino acids 23-348) were expressed in the sf9 cells by infection with the recombinant baculovirus. The expression time of hZP3 was determined by Western blot and its purification was explored.The recombinant bacmid and baculovirus were successfully constructed for expressing both the full-length and truncated hZP3. The maximal expression of recombinant hZP3 in the sf9 cells was achieved at 72-96 hours after baculovirus infection. Some of the recombinant hZP3 with His-tag could bind affinity matrix and got purified but most of the solubilized hZP3 passed through and the reasons remained unknown. Purified recombinant hZP3 labeled with Dylight Dye488 was able to bind human sperm.It is feasible to express recombinant hZP3 in insect cells using the baculovirus system though the yield of hZP3 needs to be optimized. The methods for efficient enrichment and purification of recombinant hZP3 require further exploration.
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The treatment of rheumatoid arthritis(RA)in the last decade has got enormous advances with using biological technologies.However,there are several serious limitations of these therapies,such as the high expense,side-effects,and the requirement for repeated injections.Gene therapy for the treatment of RA is effective and stably to deliver the specific target.The gene therapy for treating RA has been accomplished in animal model,and it is in the early stage of using in human patients.With the development of molecular technologies,the gene therapy for RA will be widely used in future.
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The first recombinant SARS-CoV-2 variants were identified in 2022, causing public health concerns. The importance of recombinant variants has increased especially since the WHO designated the recombinant variant XBB and its lineages as subvariants that require monitoring on 20 November 2022. In this study, we provide the first insights into the new SARS-CoV-2 variant named XAN, a recombinant composed of Omicron sub-lineages BA.2 and BA.5. To our knowledge, this is the first report on the recombinant SARS-CoV-2 XAN variant identified in Bulgaria.
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Gene therapy provides the best prospect of a fundamental new treatment for cystic fibrosis. The lungs are the most important target, because this organ is the most severely affected by the disease and is also accessible for topical treatment. Advances in this field have been very rapid, and the prospects remain good although a number of problems need to be overcome. The two main approaches to gene transfer, namely adenoviruses and liposomes, are efficient in vitro, but early clinical trials have shown that they work less well in vivo. A number of proof of concept studies have shown that gene transfer is possible, but full functional correction of the cystic fibrosis defect has not yet been achieved. Adenoviruses have provoked an inflammatory response, and new viral vectors are being developed to overcome this. Existing lipids are relatively inefficient, but new liposomes are being developed to enhance gene transfer. Much work needs to be done to improve safety and efficacy of gene transfer before materials are ready for large scale clinical trials. However, progress is very rapid, and there is a real prospect of developing an effective gene therapy for cystic fibrosis within the next decade.
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The expression of rhCu/Zn SOD in recombinant E.coli BL21(DE3) at different induction temperatures was investigated. The growth of E.coli and the expression of the recombinant protein were studied and compared at 37°C or 30°C .The recombinant E.coli was cultivated in 5L fermentor, results of which showed that at 30°C the specific growth rate was reduced while the viability of recombinant E.coli and plasmid stability were greatly increased. The stability and solubility of the recombinant protein were also improved. The maximum enzyme activity of rhCu/Zn SOD expressed at 30°C was increased to 4 757 U/mL,which was 2.7 times as that at 37°C .
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This chapter contains sections titled: Introduction General methods for recombinant protein expression Recombinant antibodies Human recombinant antibodies Human recombinant monoclonal anti-D Recombinant antigens Microbial antigens Recombinant enzymes Recombinant coagulation factors Conclusions Key points Further reading
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Great progress has been made in the last decade in treatment of reumatoid arthritis(RA)by using biological technologies.However,there are several serious limitations of these therapies,such as the high expense,side-effects,and the requirement for repeated injections.Gene therapy for the treatment of RA is effective and stably on the specific target.Gene transfer vector,transfer cytokine gene therapy,tissue repair and gene therapy,therapy gene transfer for apoptosis gene were discussed in this paper.
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