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

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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. For exhibited by the wild-type gene in both activated and the hGM-CSF AUGUA mutant, the expression level nonactivated human PBLs. The AUGUA modification to increased by about 50-fold over that of the wild type. The mGM-CSF gene had a minimal effect over wild-type murine (m) GM-CSF AUGUA mutant, however, did not gene expression levels. exhibit such a pronounced increase in gene expression In parallel with the normal human lymphocytes, with levels up from the wild type by about five-fold. In human T-cell lymphoma cells (HUT-78) were also tested. contrast to the results obtained from human and murine B16 cells were included as a positive control. We found GM-CSF-modified genes, the effect of modifications to that the AUGUA mutation conferred no significant human and murine IL-2 transgene expression appears to enhancement of expression levels as compared to wild- be negligible. Likewise, results obtained from 30UTR- type GM-CSF, in either activated or nonactivated deleted IFN-g revealed a modest 10-fold increase in primary lymphocytes, or HUT-78 cell cultures (Figure expression over wild type, but no substantial difference 1a–c), although a five-fold increase was readily repeated to transgene expression with regard to the AUGUA for the control (B16) cells (Figure 1d). Greatly enhanced mutant. expression over the wild-type gene and AUGUA mutant

Gene Therapy Augment cytokine transgene expression N-S Yang et al 102 Table 1 Gene-gun-mediated transfer and expression levels of ﬁve cytokine transgenes in transfected cell cultures*

Cytokine cDNA Luciferase Wild type Truncated AUGUA mutant

(A) B16 cells murine melanoma (ng/106 cells/24 h) hGM-CSF 0.0770.04c 0.2470.15c 71.31721.52a 40.73712.97b hIL-2 0.07570.008c 0.5370.095b 1.8470.372a NT mIFN-g 0.0070.00c 0.6470.244b 5.6171.725a 0.3970.11b mGM-CSF 0.3470.01d 1.9870.66c 148.68732.85a 12.7572.21b mIL-2 2.2370.086c 13.4773.53b 37.4673.01a 31.6975.43a

(B) MCF-7 cells human mammary carinoma (ng/106 cells/24 h) hGM-CSF 0.0170.01b 0.0770.02b 15.2975.43a 11.1373.00a hIL-2 0.0870.006c 0.4170.134b 4.1771.95a NT mIFN-g 0.0070.00c 0.2270.06b 2.9570.56a 0.2770.07b mGM-CSF 0.6970.15c 1.3470.32c 108.62734.88a 4.2270.98b mIL-2 2.2670.00c 10.4575.29b 30.7075.19a 32.1674.72a

Duncan’s multiple-range test was used for statistical analyses. Values within the same row bearing unlike letters (eg, a, b, c) differ signiﬁcantly in statistics (Po0.05); those with the same letters are not statistically different. *Material and methods: An Accellshelium gas-driven gene gun was used for all DNA transfections.26 Plasmid DNA was precipitated onto 27 1–3 mm gold particles using a spermidine/CaCl2-coating method. The DNA/gold particle preparation was then loaded into 0.5 in DNA cartridge, each delivering 0.5 mg of gold particles and 0.5 mg of plasmid DNA. B16 and MCF-7 cells were suspended in medium at a density of 5 Â 107 cells/ml, and PBLs at 25 Â 107 cells/ml, from which 20 ml of cell suspension was spread evenly onto a 1.5 cm diameter smear area in a 35 mm dish, and immediately gene gun transfected. Particle-mediated DNA delivery was performed with a 220 pounds per square inch (psi) helium pulse. A volume of 2 ml of culture medium was immediately added to gene gun-treated cell samples, and cultures were

incubated at 371C in a humidified 5% CO2 atmosphere. Aliquots were collected 24 h later, frozen at À201C and later assayed for specific transgenic cytokine levels by ELISA. The activated PBL cell cultures were incubated for 3 days prior to transfection in standard growth medium supplemented with concanavalin A (ConA) and phytohemagglutinin (PHA)-M (Sigma) at 5 mg/ml each, plus natural rat IL-2 protein at 55 U/ml (Collaborative Biomedical Products, Bedford, MA, USA). Cells were grown and maintained in a standard culture medium containing RPMI 1640 basal medium supplemented with 10% heat- inactivated fetal bovine serum, 2 mml-glutamine, 1 mm sodium pyruvate, 1% minimal Eagle’s medium nonessential amino acids and 12 mg/ ml ciprofloxacin. Human wild-type GM-CSF cDNA was obtained from Dr James Malter (Pathology Department, University of Wisconsin-Madison, WI, USA) and murine GM-CSF cDNA was provided by Dr Nicholas Gough (Walter and Elisa Hall Institute, Melbourne, Australia). Human IL-2 and murine IL-2 cDNAs were obtained from American Type Culture Collection (ATCC, Rockville, MA, USA). Murine IFN-g and all IL-12 cDNA were cloned from cDNA libraries using published cDNA sequences. Luciferase cDNA was purchased from Promega (Madison, WI, USA). All the constructs for cytokine expression were driven by the same CMV promoter containing only the basic promoter element (without any intron supplement), followed by an SD/SA site, the test cytokine cDNA and an SV40 polyA signal (pNAss vector). Truncated and mutated variations of specific cytokine genes were subcloned from correspondent wild-type cDNA coding sequences, via PCR cloning to remove the entire 30UTR and 50UTR sequences, with the exception of the last seven base pairs of the 50UTR prior to the translation start codon. These seven nucleotides were kept to retain the ‘start codon context’.28 The truncated IFN-g cDNA was produced via restriction digestion of the wild-type cDNA at the HindIII and DraI restriction sites. Mutated versions of the tested cytokine genes retained their 3’UTR, but the AU-rich regions containing ATTTA pentamers were replaced by four tandem repeats of AUGUA via an overlap extension PCR cloning as described by Rajagopalan and Malter.19 PCR products were selected from amplification reactions performed in 12 different buffers from a PCR optimization kit (Stratagene). PCR-amplified DNA fragments were purified via gel electrophoresis, phosphorylated with T4 polynucleotide kinase (NEB, Beverly, MA, USA), and if necessary, treated with Klenow DNA polymerase (NEB). The PCR fragments were then ligated with a linearized and dephosphorylated expression vector using 2000 U T4 DNA ligase at a 3:1 molar ratio of the PCR insert to plasmid DNA. The resulting ligation mixture was used to transform DH5-a competent cells, then discrete colonies selected, screened via PCR and restriction digested to confirm the correct orientation of PCR fragments. Transformed bacteria were grown in 500 ml LB/kanamycin broth, and the plasmid DNA isolated and purified on an ion exchange column, Qiagen (Chatsworth, CA, USA). ELISA assays for various cytokines: hGM-CSF and mGM-CSF in supernatants of transfected cell cultures were analyzed with ELISA kits obtained from Pharmingen (San Diego, CA, USA); murine IFNg and human IL-2 levels in culture media were assayed using kits from BioSource (Camarilla, CA, USA); murine GM-CSF and IFN-g in skin tissue extracts analyzed with kits from Endogen (Cambridge, MA, USA) and Genzyme (Cambridge, MA, USA), respectively. NT: not tested.

was obtained with the truncated 30UTR vector in all three increases in the expression of transgenic mGM-CSF types of lymphocyte cultures (Figure 1a–c). proteins in serum were obtained for the AUGUA- modified and 30UTR-deleted genes, respectively. For the In mouse skin tissue and serum sample. It was 30UTR-deleted and AUGUA-mutant vectors for human important to distinguish whether the results obtained GM-CSF, however, it was D220 and D135-fold higher from in vitro experiments, regarding 30UTR modification than the wild-type counterparts. The in vivo data in Table of cytokine genes and their relationship to transgenic 2 show that transgenic mGM-CSF levels in serum for the expression, could be reflected in in vivo gene transfer 30 UTR-deleted vector is Dsix-fold higher than the systems. Human and murine GM-CSF and murine IFN-g AUGUA mutants. On the other hand, both the hGM- cDNA gene constructs together with 30UTR modifica- CSF and mIFN-g levels were of only D1.6-fold difference tions described above were gene gun transfected into for these two vector systems, again showing expression mouse skin tissue and the activity was determined in diversity for different cytokines. This trend was detected skin tissue and serum of mice. As shown in Table 2, in the skin at the site of gene transfer, as well as in the when compared to the wild-type cDNA, 8.5- and 50-fold serum of test mice. A similar result was observed for the

Gene Therapy Augment cytokine transgene expression N-S Yang et al 103

Figure 1 Expression of mGM-CSF plasmid constructs in freshly isolated (nonactivated) or the activated, normal human lymphocyte primary cultures: (a) freshly isolated and nonactivated lymphocyte cells; (b) lymphocytes activated for 3 days in primary culture; (c) HUT-78 immortalized, human T-cell lymphoma cells and (d) B16 mouse melanoma cells. Normal primary blood lymphocytes (PBLs) were obtained from whole human blood samples that were removed from a volunteer donor via venal-puncture using sodium heparin (20 U/ml) as an anticoagulant, and diluted 1:1 with phosphate-buffered saline (PBS). The diluted blood was layered over one-half volume of 1.077 speciﬁc gravity Ficoll (Accurate Chemical, Westbury, NY, USA) and centrifuged to separate peripheral blood mononuclear cells (PBMC) from polymorphonuclear leukocytes and red blood cells. The mononuclear cell layer was collected and washed with PBS to remove platelets and residual Ficoll. The cell culture condition is the same as that described in Table 1.

Table 2 Cytokine transgenic expression levels in transfected mouse skin tissue and serum samples*

Cytokine cDNA Luciferase Wild type Truncated AUGUA mutant

Serum levels (pg/ml) hGM-CSF 1.7970.96b 1.6270.52b 352.817104.00a 219.00713.70a mGM-CSF 0.0170.01c 1.1171.30c 55.14723.66a 9.4372.23b mIFN-g 100.90713.80c 236.0076.10b 512.70781.90a 323.00793.30b

Skin level (ng/transfection site) hGM-CSF 0.01570.006b 0.01270.013b 4.82471.032a 3.18971.20a mGM-CSF NT 0.19170.075c 31.55972.097a 7.54771.787b mIFN-g 1.4670.001c 2.43870.899c 17.16973.023a 6.63672.438b

Statistical analyses of the data as shown by attached letters are the same as those described in Table 1. *Material and methods: In vivo gene transfer was performed on female 6–8-week-old female BALB/cByJ mice (purchased from the Animal Care Center and housed at the Animal Care Facility, Harlan–Sprague Dawley, Indianapolis, IN, USA). Abdominal hair was removed with surgical clippers, and the exposed epidermis was topically transfected directly with an Accellsgene gun. Each mouse received four transfection treatments at widely separated abdominal sites, at a discharge pressure of 330 psi. Skin samples from the transfection sites and serum samples were collected at 8 and/or 24 h postgene transfer. Transfected tissues were homogenized, centrifuged and the supernatant was quantitatively assayed for specific cytokines using corresponding ELISA kit assays as previously described.3,5,23,29 For others, see footnote in Table 1. modified murine IFN-g and human GM-CSF. These methods. For in vivo gene transfer into skin tissue, single in vivo results (Table 2) in general correlate well with needle injection or electroporation of naked DNA into the in vitro data shown in Table 1, reaffirming our initial intradermal tissue has not been demonstrated, to our findings. It would be important if the current in vivo knowledge, as a routine, reliable and efficient means for results could also be evaluated using other gene transfer gene transfer. Future studies using multiple needle/skin

Gene Therapy Augment cytokine transgene expression N-S Yang et al 104 microseeding, intramuscular injection or hydrodynamic generates the highest expression levels of transgenic gene delivery methods may provide additional relevant cytokine proteins in all tested systems. These cytokines information on the behavior and use of various cytokine were repeatedly shown to exhibit functional biological transgene constructs, as a follow-up of our current study. activity, as many researchers have previously re- Using ex vivo and in vivo gene therapy protocols for ported.22,29,30 A comparison of the results from our immunotherapy studies in mouse tumor models, we had current study versus those reported by Rajagopalan previously demonstrated that all the 30UTR-deleted and Malter22 suggests that 30cDNA structural features cytokine transgenes and their recombinant products may influence transgenic protein expression via addi- were biochemically and physiologically active. This tional mechanisms besides mRNA stability and poly- was demonstrated by their capacity to elicit CTL or some-run-on efficiency. It is currently unclear to us as to histological inflammatory activity and potent antitumor which mechanism(s) (eg, RNA stability) is playing the activities effectively.12,13,29,30–32 Together with the findings key role for enhanced cytokine gene expression, and of the relative capacities of different cDNA constructs in additional experiments are needed to demonstrate the the current study, we believe that these results can be possible post-transcriptional, translational or other pro- usefully employed by researchers for the proper and cessing regulations of these transgenic RNA or DNA specified use of 30UTR deletion for cytokine expression vector systems. The results presented here and the report vectors in a clinically relevant study, that is, different as from Rajagopalan and Malter22 also suggest that the gene well as the same cytokine species can differ drastically in gun-mediated delivery technique for intracellular trans- transgene expression under different in vivo and ex vitro fer of mRNA or of corresponding cDNA expression conditions, and hence specific cytokine expression vectors can be effectively employed to analyze and vectors do need to be carefully tested in target or related compare nucleic acid structural features and their effects cells/tissues before clinical studies. on post-transcriptional, translational or post-transla- In previous studies on cytokine gene expression, it has tional events required for transgene expression. been documented that mutating the AU-rich sequences of mRNA in the 30UTR can effectively enhance mRNA stability, resulting in an augmented transgenic cytokine Effects of CMV promoter/intron A and EF-1a protein expression18–20,22,30 These results were accom- promoter/intron 1 on cytokine gene expression plished via an overlapping PCR technique, in which the In this experiment, cytokine genes were constructed to AUUUA regions of the 30UTR were replaced with contain a CMV immediate/early promoter sequence4 AUGUA pentamer repeats.20,22 There were also sugges- with either a CMV-derived intron A sequence or a short, tions that a group of cytokine gene constructs, with the SV40-derived splice donor/splice acceptor (SD/SA) 30UTR totally removed by cDNA cloning, expressed very sequence. Alternatively, cytokine transgenes were con- well in transgenic studies.29,31 Unfortunately, these structed under the control of a human EF-1a promoter findings were sporadic, and direct, systematic compar- linked with an SV40 intron-1 enhancer. As shown in isons of wild-type and modified or 30UTR-truncated Figure 1a–c, substantial increases in mGM-CSF expres- cytokine cDNAs for transgene expression had not been sion were demonstrated by using intron A as an reported. As a result, the molecular strategy for the use enhancer as compared to the CMV SD/SA. An interest- of cytokine transgenes remains uncertain to some gene ing result was obtained with the human EF-1a promo- therapy researchers. This report directly addresses these ter/intron 1 enhancer, which enhanced expression in B16 issues with comparative experiments in which both and HUT-78 cells, but conferred absolutely no enhancing in vitro and in vivo gene transfer systems are tested. We activity in either fresh/nonactivated or activated lym- show here that when using the DNA plasmid construct phocyte cultures. (as now commonly used in gene therapy clinical trials), Figure 2 reveals that use of the CMV intron A 30UTR deletion mutants are in fact more effective than promoter sequence to drive expression significantly the AUGUA mutants. This study hence provides a firm (Po0.05) increased the expression levels of three foundation for the use of deletion mutants for clinical different cytokine cDNA constructs in B16 cells. When studies, not the AUGUA mutants. compared to the SV40 SD/SA (mini-intron), CMV intron Rajagopalan and Malter22 reported that when cells A improved expression levels 4.4-, 1.8- and 4.7-fold for were transfected using the gene gun with in vitro the cytokines hGM-CSF, mGM-CSF and hIL-12, respec- transcribed mRNA of the wild type, the AUGUA- tively. mutated or the truncated GM-CSF gene variants, the Using a plasmid vector containing the EF-1a promo- truncated RNA constructs exhibited the longest mRNA ter/intron-1 sequence driving mGM-CSF cDNA, we half-lives. However, when transgenic cytokine protein compared the transient expression patterns over 7 days levels were measured, the AUGUA mutant mRNA in B16 and primary lymphocyte cultures, and both conferred the highest levels, suggesting differences in CMV/intron A and CMV/SD/SA were studied in translational efficiency of these various mRNAs on parallel. Figure 3 shows that the EF-1a promoter/in- polysomes.19,22 It was therefore unclear whether the tron-1 directed the highest transgene expression. ‘The AUGUA mutant or the 30UTR-deleted version of expression of CMV/intronA or EF-1a/intron1 mGM-CSF cytokine cDNA genes could better serve in current or expression was greatly reduced by days 4–5 to approxi- future cytokine gene therapy trials. With this observation mately 13–19% of the day 2 peak levels’. In comparison, in mind, we have clearly shown in this study that when the CMV/SD/SA combination appeared to confer a the plasmid vector DNA, not mRNA, is gene gun faster decline in transgenic activity (Figure 3a). We delivered into freshly isolated lymphocytes, activated observed a very similar effect in primary canine tumor lymphocytes, murine and human cell lines or mouse skin cell cultures (data not shown). Figure 3b shows the tissues, the reverse is true; the 30UTR-deleted cDNA cumulative totals of GM-CSF levels over the 7-day

Gene Therapy Augment cytokine transgene expression N-S Yang et al 105

Figure 3 Time course (a) and cumulative production (b) of transgenic mGM-CSF protein in B16 cells as directed by three different promoter/ regulatory elements. Three sets of transcriptional control units, EF-1a/ intron-1, CMV/SD/SA and CMV/intron A, were employed to direct mGM-CSF cDNA expression in transfected B16 melanoma cell cultures. (*Po0.05).

over the CMV/SD/SA and the CMV/intron A construct, respectively (Figure 3b).

Optimization of concomitant/coordinated expression of two subunit genes of IL-12 Given that some therapeutic proteins are composed of heterosubunits encoded by multiple genes, we decided to evaluate different arrangements of cDNA gene constructs using IL-12 as a model protein. IL-12 consists of two subunit proteins, p35 and p40. Transgenic murine IL-12 protein expression was measured in B16 tumor cells transfected in vitro, or in mouse skin tissue transfected in vivo with cDNA constructs. The plasmids containing coding sequences for the p35 and p40 subunits of murine IL-12 were both driven by their individual CMV promoters and linked tandemly in the Figure 2 Effect of intron A or SV40 SD/SA element on cytokine transgene same direction (Figure 4a). The afore-mentioned plas- expression in mouse B16 cells. Cytokine transgene expression vector mids gave more than three-fold higher levels of the IL-12 containing a CMV promoter followed by (a) intron A or (b) SV40 SD/SA protein heterodimer than bipartite cotransfections with a sequence. ( P 0.05). * o mixture of two separate plasmids, one encoding p35 and the other encoding p40 (compare’direct tandem’ and culture period (sum of the seven single-day values) for ‘bipartite’ in Figure 5, Po0.05). When the two subunit the three promoter/intron combinations. The expression genes were linked in a head-to-head manner, each still levels for the EF-1a promoter/intron-1 translate over the driven by its own promoter (Figure 4b; ‘retrograde 7-day experimental period to a 514 and 157% increase tandem’ in Figure 5), the expression level was only

Gene Therapy Augment cytokine transgene expression N-S Yang et al 106

Figure 4 Human IL-12 subunit genes, p35 and p40 cDNAs, were subcloned into four configurations of plasmid expression vectors: (a) tandem arrangement in the same direction (head to tail); (b) tandem units in the retrograde direction (head to head); (c) IRES spacer-linked arrangement and (d) two separate plasmids, each encoding one of the p35 and p40 subunit proteins. The primary plasmid vector, pNAss (Clontech, Palo Alta, CA, USA), has a pUC19 backbone with an ampicillin-resistance gene, a CMV promoter, a SV40 splicing donor/splicing acceptor (SD/SA) sequence and an SV40 polyadenylation signal sequence. Two other plasmid expression vectors that were also evaluated were: the pWRG3147 vector contains an elongation factor (EF)-1a promoter,33 the SV40 intron 1 sequence, and an SV40 polyadenylation sequence. The pWRG 7007 vector contains a CMV promoter followed by the CMV intron A enhancer sequence, a bovine growth hormone (bGH) polyadenylation signal sequence and a kanamycin-selection gene. The CMV promoter was purchased from Clontech, the CMV/intron A element was obtained from Drs J Arthoson and JI Mullins, University of Washington Health Sciences Center, Seattle, Washington4 and the EF-1a promoter subcloned from an EF containing plasmid was provided by Dr Shigekazu Nagata, Osaka Bioscience Institute, Osaka, Japan.33 Human IL-12 subunit p35 and p40 cDNA fragment genes were cloned from mRNA of a human lymphoblastoid cell line using a commercial RT-PCR kit (Gibco/BRL). Murine IL-12 subunits were cloned from mouse cDNA libraries as previously described.32 The four murine IL-12 expression vectors were constructed as follows: the first and second contain the p35 and p40 coding sequence arranged in tandem either in the same orientation or head-to-head orientation, with each subunit gene driven by its own CMV promoter (a, b). The third vector contains a single operon drivenby a CMV promoter, with p35 and p40 coding sequences linked by an IRES sequence, obtained from Novagen, Madison, WI, USA (c). The fourth vector system employed two separate plasmids, each encoding one of the IL-12 subunits (d). The quantity of transgenic murine IL-12 protein heterodimer was determined by a cell proliferation bioassay using ConA-activated splenocytes, as previously described.30 Human IL-12 levels were determined via a p35/p40 heterodimer-specific ELISA kit (R&D Systems, Minneapolis, MN, USA).

D60% of that obtained with the direct tandem config- uration. The direct tandem mIL-12 vector was about two- fold more efficient than a single operon construct linked with an internal ribosome entry site (IRES) sequence (Figure 4c; ‘IRES’ in Figure 5). Thus a single plasmid, engineered with a unidirectional orientation of two separate, tandem expression units for the two IL-12 subunits, was the most efficient of the vector strategies tested. For DNA vaccines and cancer gene therapies to be practical for human applications, it is critically important that candidate therapeutic genes be efficiently expressed at the protein level. It is highly desirable that an initial or pretherapeutic determination of the level of transgene expression be systematically evaluated under various standardized or relevant in vivo and/or in vitro conditions.5,22,26,34,35 Different cell types, tissues/tumors can express the same transgenic CMV promoter with highly variable levels; and different subclones of CMV promoters perform very differently. This study thus may serve as an example for such a broad level and high diversity on transgene gene expression in various target tissue systems, and these may serve as useful information for future clinical applications. The current study systematically readdressed the Figure 5 Comparative analysis of in vivo hIL-12 transgene expression in following factors contributing to the low levels of B16 cells transfected using various subunit gene arrangements in therapeutic cytokine transgene expression often found expression vector construction. Bars from left to right represent pCMV- in humans/large mammal systems: low efficiencies of Luc, and the four hIL-12 vectors depicted in Figure 4, in the order a, b, d gene transfer, the specific capacity of different cell or and c. Statistical analysis was performed using the paired t-test: ‘a’ denotes Po0.05 when Retrograde tandem set was tested against the labeled tissue types in transgene expression and the lack of treatment; ‘b’ denotes Po0.05 when the Bipartite set was tested against generic method available for effectively pre-evaluating the labeled treatment; a/b means Po0.05 for both tests. the possible levels and patterns in targeted tissues

Gene Therapy Augment cytokine transgene expression N-S Yang et al 107 of individual patients. Collectively, results from the 8 Morse MA. Technology evaluation: gene therapy (IL-2), Valentis present comparative study provide more clear verifica- Inc. Curr Opin Mol Ther 2000; 2: 448–452. tion and strong support for utilizing a combination of 9 Lim M, Simons JW. Emerging concepts in GM-CSF gene- specific molecular modifications on cytokine the cDNA transduced tumor vaccines for human prostate cancer. Curr sequence in order to upgrade cytokine transgene Opin Mol Ther 1999; 1: 64–71. expression in in vivo and ex vitro transgenic systems. 10 Barouch DH et al. Control of viremia and prevention of clinical Based on results from this study, we estimate that an AIDS in rhesus monkeys by cytokine-augmented DNA increase of at least seven to 30-fold in various cytokine vaccination. Science 2000; 290: 486–492. transgene expression levels could be readily obtained by 11 Rosenberg SA. 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