Pharmacogenetic heterogeneity of transgene expression in muscle and tumours

BACKGROUND: Recombinant adenoviruses are employed to deliver a therapeutic transgene in the liver, muscle or tumour tissue. However, to rationalise this delivery approach, the factors of variation between individuals need to be identified. It is assumed that differences between inbred strains of laboratory animals are considered to reflect differences between patients. Previously we showed that transgene expression in the liver of different rat strains was dependent on the transcription efficiency of the transgene. In the present paper we investigated if transfection of muscle and tumour tissue were also subject to such variations. METHODS: Variation, in transgene expression, after intramuscular gene delivery was determined in different rodent strains and gene expression in tumours was investigated in different human and rodent cell lines as well as in subcutaneously implanted rodent tumours. The molecular mechanisms involved in transgene expression were dissected using an adenovirus encoding luciferase. The luciferase activity, the viral DNA copies and the luciferase transcripts were assessed in cultured cells as well as in the tissues. RESULTS: Large differences of luciferase activity, up to 2 logs, were observed between different rodent strains after intramuscular injection of Ad Luciferase. This inter-strain variation of transgene expression was due to a difference in transcription efficiency. The transgene expression level in tumour cell lines of different tissue origin could be explained largely by the difference of infectibility to the adenovirus. In contrast, the main step responsible for luciferase activity variation, between six human breast cancer cell lines with similar phenotype, was at the transcriptional level. CONCLUSION: Difference in transcriptional efficiency in muscles as observed between different inbred strains and between human breast cancer cell lines may be expected to occur between individual patients. This might have important consequences for clinical gene therapy. The variation between tumour types and tissues within a species are mainly at the levels of infectivity

A comparison of efficacy and toxicity between electroporation and adenoviral gene transfer

BACKGROUND: Electroporation of skeletal muscle after injection of naked DNA was shown by others to increase transgene expression. Information regarding tissue damage caused by electroporation is conflicting. It is also not well known how plasmid electroporation compares with transfection by adenoviral vectors. To investigate these questions the most used protocol for muscle electroporation was used, i.e. 8 pulses of 200 V/cm and 20 ms at a frequency of 1 Hz. RESULTS: Intra-muscular DNA transfer of pLuciferase was increased by 2 logs after electroporation, confirming data described by others. However, the blood levels of the encoded protein were still lower than those obtained after injection of first generation adenoviral vectors. Also, the electroporation procedure, on its own, caused severe muscle damage consisting of rhabdomyolysis and infiltration, whereas the adenoviral vectors caused only a slight infiltration. As damage of targeted tissue may be an advantage in the case of tumour transfection, we also compared the two transfection methods in tumour tissue. In case of poorly permissive tumours, adenoviral vectors cannot transfect more than 2% of the tumour tissue without inducing significant liver damage. In contrast, the electroporation seems to offer a wider therapeutic window since it does not cause any systemic toxicity and still induce's significant transfection. CONCLUSIONS: Plasmid electroporation of the muscle induce severe local damage and is of no advantage over adenoviral vectors for obtaining high blood levels of a vector encoded protein. In contrast, electroporation of tumours might be safer than adenoviral gene transfer

Genetic heterogeneity in response to adenovirus gene therapy

BACKGROUND: After intravenous delivery of the adenoviral vector into rats or mice, 95–99% of the encoded protein is produced in the hepatocytes. We observed, as have others, that the early expression levels of the vector encoded protein vary, greatly, within a species, from one animal strain to another. This study was initiated to determine the molecular mechanism causing the difference: hepatic transfection, transcription or translation. For this purpose different doses of Ad5 luciferase and Ad5 LacZ were intravenously injected into Brown Norway rats and Wag/Rij rats, two strains that differ by a factor of 10 in encoded protein levels. The proportion of LacZ positive hepatocytes, the adenoviral DNA, specific transgenic RNA and luciferase protein were compared in the two strains. RESULTS: The number of transduced hepatocytes and the amounts of Ad5 DNA in the livers was similar in both strains, whereas the Brown Norway rats produced 8 to 10 times more of both vector encoded proteins and of transgene mRNA than the Wag/Rij rats. CONCLUSIONS: It is concluded that the difference between strains in vector encoded protein expression is due to different transcriptional events. No evidence was obtained to suggest that the differences are related to liver damage influenced by vector toxicity or immune reactions

Life-threatening hypokalaemia and quadriparesis in a patient with ureterosigmoidostomy

We report quadriparesis as a result of severe hypokalaemia and acidosis in a 50-year-old man who had undergone ureterosigmoidostomy for bladder extrophy 48 years earlier. Aggressive suppletion with intravenous potassium and bicarbonate combined with potassium-sparing diuretics and ACE inhibitors resulted in complete restoration of the serum potassium and resolution of the neurological symptoms. The underlying mechanism as well as the treatment of hypokalaemia and hyperchloraemic metabolic acidosis after ureterosigmoidostomy are briefly discussed

Adenoviral gene transfer of angiostatic ATF-BPTI inhibits tumour growth

BACKGROUND: The outgrowth of new vessels – angiogenesis – in the tumour mass is considered to be a limiting factor of tumour growth. To inhibit the matrix lysis that is part of the tumour angiogenesis, we employed the chimeric protein mhATF-BPTI, composed of the receptor binding part of the urokinase (ATF) linked to an inhibitor of plasmin (BPTI). METHODS: For delivery, recombinant adenovirus encoding the transgene of interest was injected intravenously or locally into the tumour. The anti tumour effect of this compound was compared to that of human endostatin and of mhATF alone in two different rat bronchial carcinomas growing either as subcutaneous implants or as metastases. RESULTS: Significant inhibition of the tumour growth and decrease of the number of lung metastasis was achieved when the concentration of mhATF-BPTI at the tumour site was above 400 of ng / g tissue. This concentration could be achieved via production by the liver, only if permissive to the recombinant adenovirus. When the tumour cells could be transduced, local delivery of the vector was enough to obtain a response. In the case of metastasis, the capacity of the lung tissue to concentrate the encoded protein was essential to reach the required therapeutic levels. Further, endostatin or mhATF could not reproduce the effects of mhATF-BPTI, at similar concentrations (mhATF) and even at 10-fold higher concentration (endostatin). CONCLUSION: The ATF-BPTI was shown to inhibit tumour growth of different rat lung tumours when critical concentration was reached. In these tumour models, endostatin or ATF induce almost no tumour response

Exploitation of Herpesvirus Immune Evasion Strategies to Modify the Immunogenicity of Human Mesenchymal Stem Cell Transplants

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent cells residing in the connective tissue of many organs and holding great potential for tissue repair. In culture, human MSCs (hMSCs) are capable of extensive proliferation without showing chromosomal aberrations. Large numbers of hMSCs can thus be acquired from small samples of easily obtainable tissues like fat and bone marrow. MSCs can contribute to regeneration indirectly by secretion of cytokines or directly by differentiation into specialized cell types. The latter mechanism requires their long-term acceptance by the recipient. Although MSCs do not elicit immune responses in vitro, animal studies have revealed that allogeneic and xenogeneic MSCs are rejected. METHODOLOGY/PRINCIPAL FINDINGS: We aim to overcome MSC immune rejection through permanent down-regulation of major histocompatibility complex (MHC) class I proteins on the surface of these MHC class II-negative cells through the use of viral immune evasion proteins. Transduction of hMSCs with a retroviral vector encoding the human cytomegalovirus US11 protein resulted in strong inhibition of MHC class I surface expression. When transplanted into immunocompetent mice, persistence of the US11-expressing and HLA-ABC-negative hMSCs at levels resembling those found in immunodeficient (i.e., NOD/SCID) mice could be attained provided that recipients' natural killer (NK) cells were depleted prior to cell transplantation. CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate the potential utility of herpesviral immunoevasins to prevent rejection of xenogeneic MSCs. The observation that down-regulation of MHC class I surface expression renders hMSCs vulnerable to NK cell recognition and cytolysis implies that multiple viral immune evasion proteins are likely required to make hMSCs non-immunogenic and thereby universally transplantable