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Molecular Strategies for Improving Cytokine Transgene Expression in Normal and Malignant Tissues

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Molecular Strategies for Improving Cytokine Transgene Expression in Normal and Malignant Tissues Gene Therapy (2004) 11, 100–108 & 2004 Nature Publishing Group All rights reserved 0969-7128/04 $25.00 www.nature.com/gt BRIEF COMMUNICATION Molecular strategies for improving cytokine transgene expression in normal and malignant tissues N-S Yang, J-H Wang and J Turner Institute of BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan, ROC The augmentation and optimization of specific targeted ex vivo transgene expression assay systems. The results transgene expression systems are important strategies for from these comparative experiments demonstrated that a clinical research into gene therapy and DNA vaccination, due number of molecular biology manipulations can be readily to safety considerations. In this study, we introduced 30 adapted to define and significantly enhance the level or/and untranslated regions and transcriptional control modifications duration of transgene expression for a group of clinically and direct tandem or combinational vector design strategies relevant cytokine genes, with very similar effects for both in into a number of specific cytokine cDNA expression vivo and in vitro test systems. This cytokine transgene plasmids. The experiments were performed in parallel using expression system may offer a favorable means for improv- both in vivo and in vitro transgene expression systems. In ing the efficiency of cytokine gene therapy and DNA vaccines vivo studies were carried out using gene gun delivery of test in future clinical studies. vectors into mouse skin tissues. A combination of specific Gene Therapy (2004) 11, 100–108. doi:10.1038/sj.gt.3302137 cell lines and fresh cell explants were used for in vitro and Keywords: cancer vaccine; GM-CSF; IL-2; IL-12; interferon-g; gene gun; promoter usage; 30UTR Introduction concerns, we believe that it is very important to start developing specific transgene expression systems Researchers in the field of human gene therapy have that can be augmented by molecular biology strategies generally employed relatively high-dose transgene de- for upgrading gene expression levels. So rather than livery systems in the hope that this approach would overloading the vector systems, we have revisited result in maximal but tolerable levels of transgene/vec- molecular means for upgrading specific cytokine trans- tor delivery without seriously harming patients. This gene expression systems that are still being actively rationale may have been overly optimistic and caused considered for cancer gene therapy or cancer vaccines. concerns, resulting in the gene therapy research com- We propose that a combination of vector design, refined munity actively evaluating a spectrum of approaches promoter choice and manipulation of 30 untranslated regarding the safety of human gene therapy clinical regions (30UTR) sequences for cytokine transgene ex- trials.1,2 Cytokine gene therapy approaches have been pression can meet the practical needs of and be actively evaluated for a variety of potential applications advantageous for future cancer gene therapy clinical in the fields of cancer immunotherapy, cancer vaccines trials. Potentially, an increase in transgenic expression of and gene-based vaccinations against infectious dis- 20-fold over wild-type/conventional vector expression eases.3–9 For many cytokine gene therapy approaches, a could be generated from factors relating to promoter sharp transient gene expression peak, rather than a high usage (X), vector design (Y) and plasmid selection (Z), and prolonged transgenic cytokine expression is valued when taken together that is (X) Â (Y) Â (Z). This could by investigators.3–11 It has also been suggested that provide highly desirable therapeutic gene expression although using current transgenic experimental systems systems for safe and clinically efficacious studies in the results in low specific transgenic cytokine levels in very near future. regional or localized tissue, a 10–20 fold increase in Through extensive literature search, we observed that expression levels might effectively facilitate certain systematic and comparative studies addressing different physiological activities and an efficacious response, as cytokine genes and different cell/tissue types had not indicated in some tumor vaccine studies.12,13 Taking into been previously documented to address the above issue. consideration the aforementioned advantages of increas- Using the gene gun method, we show in this report that a ing transgene expression and the necessary caution of wide range of transgenic expression levels for four not ‘overdosing’ gene transfer vectors due to safety specific, commonly used cytokines can be observed when various cDNA gene constructs or expression vector systems are tested under different in vitro, ex vivo Correspondence: Dr N-S Yang, Institute of BioAgricultural Sciences, Academia Sinica No. 128, Academia Sinica Rd Sec 2, Nankang District, or in vivo conditions. This information on the capacity Taipei 11529, Taiwan and behavioral diversity of different cell/tissue types to Received 28 August 2002; accepted 07 July 2003 transgene expression vector systems, we believe, could Augment cytokine transgene expression N-S Yang et al 101 be usefully employed in future gene therapy clinical In MCF-7 human mammary carcinoma cells. In order trials for cancer therapy and DNA vaccines. to examine the potential effects of cell type difference on cytokine transgene modification and expression, the same cytokine genes tested previously in B16 cells were 0 Effect of 3 UTR modifications on cytokine transgene transfected in human mammary carcinoma MCF7 cells. expression The results are summarized in Table 1B. The greatest AU-rich elements (AREs) are commonly present in the increase in transgenic expression levels over wild type 30UTR of most cytokine genes, inflammatory genes and were shown by 30UTR-deleted human and murine GM- oncogene mRNAs, which confer instability.14–21 Rajago- CSF, with expression levels increased by about 100-fold palan and Malter22 reported that mRNA containing the in both cases. Also, AUGUA mutation of human and (AUGUA) Â 4 mutant sequence, the 30UTR-truncated mouse GM-CSF had a completely different effect on sequence or the normal/wild-type AUUUA sequence expression levels as compared to the wild-type gene. The revealed dramatic differences in mRNA stability and AUGUA mutant of hGM-CSF resulted in an increase of polyribosome-binding affinity. However, whereas 30- 100-fold over the expression level of wild type. The truncated cDNA expression versions of various cytokine AUGUA mutant of mGM-CSF, however, had a negligible genes have already been used in many studies,22–25 their effect on wild-type expression levels. Murine IL-2- utility as compared to 30UTR-containing or AUGUA- truncated and AUGUA mutants displayed a similar mutated constructs has not undergone systematic study. effect on wild-type transgene expression levels as shown In this study, we investigated modifications of cytokine in B16 cells. Truncated human IL-2, in contrast, displayed cDNA expression vectors, in which either the AUUUA a 10-fold increase in expression over that of wild type, pentamer reiterations were replaced with a mutant form whereas in B16 cells the increase in expression was less (AUGUA) Â 4 or the 30UTR was completely deleted/re- pronounced. The IFN-g-truncated and AUGUA mutants moved to inhibit the binding of mRNA degrading also displayed similar effects on wild-type gene expres- proteins. These two 30UTR modifications were intro- sion levels as revealed in tests with B16 cells. duced into the cDNA constructs of murine IL-2, human IL-2, murine IFN-g, human GM-CSF and murine GM- CSF, after which transgenic expression levels of these In human primary blood lymphocytes and T-cell cDNAs, both in vitro and in vivo, were compared to those lymphoma cells. GM-CSF gene expression is known from the wild-type cDNA containing the wild-type to vary with respect to the use of malignant or normal 30UTR sequences. cells for transfection.17,22 We therefore tested whether the effect of 30UTR cytokine modifications on transgenic expression were influenced by the use of cancerous cells. In B16 mouse melanoma cells. Complete 30UTR Activated lymphocytes were obtained by incubating the deletion and a single base-pair change (AUUUA- primary lymphocyte cultures for 3 days prior to AUGUA) in the 30UTR wild-type sequence of five transfection in a culture medium containing ConA, different cytokine cDNA genes were chosen as the PHA-M and natural rat IL-2. Murine GM-CSF wild system for studying the effect of 30UTR mutation on type and 30UTR modified genes were transfected into cytokine transgene expression. In this and all the primary cell cultures of freshly isolated human following experiments of this study, triplicate cell blood lymphocytes, and the results are summarized in cultures or skin tissue samples were assayed for each Figures 1a and b. In this experiment, a strong promoter/ test sample, and the mean7s.ds. were represented. The enhancer element, namely the CMV immediate/early results are summarized in Table 1A. All gene expression promoter linked with a CMV-derived intronA sequence levels were taken 24 h post-transfection. Generally, it can as an enhancer, was used to drive various cytokine be noted that removal of the 30UTR in all cytokine genes cDNA gene constructs. Among the tested cytokines, tested resulted in the greatest level of transgene expres- the mGM-CSF gene exhibited the three most dramati- sion, as compared to wild-type and mutated 30UTR- cally distinguishable levels of transgene expression in modified genes. The greatest increases in transgenic relation to the genotypes under test (the wild type, expression levels were shown by 30UTR-deleted human 30UTR-truncated and AUGUA mutants). Upon inspec- and murine GM-CSF with expression levels improved by tion of Figures 1a and b, one can distinguish that the about 100-fold in both cases. The AUGUA 30UTR 30UTR deletion of the mGM-CSF gene resulted in a mutations of human and murine GM-CSF had, however, transgenic expression level 10-fold greater than that different effects on the transgenic expression levels.
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