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Molecular cloning, genomic characterization and over-expression of a novel gene, XRRA1, identified from human colorectal cancer cell HCT116Clone2_XRR and macaque testis

Background As part of our investigation into the genetic basis of tumor cell radioresponse, we have isolated several clones with a wide range of responses to X-radiation (XR) from an unirradiated human colorectal tumor cell line, HCT116. Using human cDNA microarrays, we recently identified a novel gene that was down-regulated by two-fold in an XR-resistant cell clone, HCT116Clone2_XRR. We have named this gene as X-ray radiation resistance associated 1 (XRRA1) (GenBank BK000541). Here, we present the first report on the molecular cloning, genomic characterization and over-expression of the XRRA1 gene. Results We found that XRRA1 was expressed predominantly in testis of both human and macaque. cDNA microarray analysis showed three-fold higher expression of XRRA1 in macaque testis relative to other tissues. We further cloned the macaque XRRA1 cDNA (GenBank AB072776) and a human XRRA1 splice variant from HCT116Clone2_XRR (GenBank AY163836). In silico analysis revealed the full-length human XRRA1, mouse, rat and bovine Xrra1 cDNAs. The XRRA1 gene comprises 11 exons and spans 64 kb on chromosome 11q13.3. Human and macaque cDNAs share 96% homology. Human XRRA1 cDNA is 1987 nt long and encodes a protein of 559 aa. XRRA1 protein is highly conserved in human, macaque, mouse, rat, pig, and bovine. GFP-XRRA1 fusion protein was detected in both the nucleus and cytoplasm of HCT116 clones and COS-7 cells. Interestingly, we found evidence that COS-7 cells which over-expressed XRRA1 lacked Ku86 (Ku80, XRCC5), a non-homologous end joining (NHEJ) DNA repair molecule, in the nucleus. RT-PCR analysis showed differential expression of XRRA1 after XR in HCT116 clones manifesting significantly different XR responses. Further, we found that XRRA1 was expressed in most tumor cell types. Surprisingly, mouse Xrra1 was detected in mouse embryonic stem cells R1. Conclusions Both XRRA1 cDNA and protein are highly conserved among mammals, suggesting that XRRA1 may have similar functions. Our results also suggest that the genetic modulation of XRRA1 may affect the XR responses of HCT116 clones and that XRRA1 may have a role in the response of human tumor and normal cells to XR. XRRA1 might be correlated with cancer development and might also be an early expressed gene.

Background
Treatment with X-radiation (XR) remains a major modality for eradicating cancer and it has been estimated that half of all cancer patients undergo radiotherapy at some point during their treatment [1]. At least partly because of the relevance of radiotherapy to cancer treatment, the genetic control of the expression of resistance, or conversely, sensitivity to XR has been investigated for several decades. It is now generally accepted that there are several components to the cellular response to XR stress: sensing of the DNA damage, cell signaling pathways, and repair of the DNA damage [2]. However, the molecular mechanisms of how cells detect, communicate, and cope with XR-induced DNA damage are not well characterized. There is strong evidence that ataxia telangiectasia mutated (ATM) protein act as damage sensors and that their activation initiates mobilization and/or activation of repair complexes [3]. In addition, the ATM kinase activity mediates the prompt induction of various signaling pathways with some of these events leading to either cellular apoptosis or cell cycle arrest [4]. There is also evidence that non-homologous end joining involving Ku86/Ku70 is likely the major DNA double strand breaks rejoining pathway following XR with a smaller role for homologous recombination in post replicated DNA [5].
Many of the studies on the genetic basis of cellular response to XR have benefited either from mutants that have increased ionizing radiation (IR) sensitivity compared to the parental cell lines or from the study of human genetic syndromes that are associated with increased IR sensitivity (i.e. Fanconi's anemia, Bloom's, and Werner's syndromes) [6,7]. By contrast, mutants or human genetic syndromes associated with increased IR resistance (as exemplified by Li Fraumeni) are rare [8]. Furthermore, Bennett et al [9] recently identified 107 new loci that are associated with response to γ-radiation in yeast. While over 50% of these newly identified putative yeast genes shared homology with human genes, it remains to be established that the human counterparts of these genes are actually associated with similar responses to γ-radiation.
In order to further investigate the genetic basis of tumor cell radioresponse, we have isolated several clones with a wide range of responses to X-radiation from an unirradiated human colorectal tumor cell line, HCT116. Interestingly, we found a cell clone, HCT116 Clone2_XRR that was resistant to both fractionated and single dose X-radiation when compared to the parental cells. In addition, we also isolated two other clones: (a) HCT116 CloneK_XRS that was more sensitive than the parental cells, and (b) HCT116 Clone10 that possessed similar X-radiation responses as the parental cells, to both of these types of Xradiation (Qutob, S.S., Ng, C.E., to be published else-where).
Global gene analysis between cells HCT116 Clone2_XRR and HCT116 Clone10 using cDNA microarray identified an unknown GenBank expression sequence tag (EST) accession no. R40588, as one of the spots on the 19,200 human cDNA microarray (University Health Network, Ontario Cancer Institute, Toronto), that was down regulated by approximately two-fold in HCT116 Clone2_XRR in comparison with HCT116 Clone10 cells (Qutob, S.S., Ng, C.E., to be published elsewhere).
Here we describe the in silico analysis of R40588 that resulted in the identification of a novel gene, XRRA1. We cloned the XRRA1 full-length cDNA from macaque testis and an XRRA1 splice variant from HCT116 Clone2_XRR cells. We then extended our analysis to identify XRRA1 in several mammals (i.e. mouse, rat, pig and bovine). This paper also reports the experiments to evaluate XRRA1 expression in human and macaque normal tissue/organs and in various human cancer and normal cell lines, as well as over-expression of GFP-XRRA1 fusion protein in HCT116 clones and COS-7 cells. We did immunostaining of Ku86 in the presence of over-expressed GFP-XRRA1 24 hours after XR treatment at 4 and 10 Gy of COS-7 cells. This is the first report on the molecular cloning, genomic characterization and over-expression of the novel XRRA1 gene.

Genomic approaches revealed a novel XRRA1 gene
Using the EST sequence of R40588 as template, we performed ESTs "walking" either towards the 5' or 3' direction on both the sense and anti-sense strands that we refer to as ESTs-based ORF assembling (EBOA). By EBOA, we acquired a cDNA candidate that putatively contained start-, stop-codon and poly-A signal. ESTs that overlapped each other sequentially were as follows: GenBank accession nos R40588, BQ648278, BU187847, BE782795, BI819448, BQ925455, and BM563944. Out of this ORF candidate, we found 31 ESTs as primary sequences that could be constructed into one potential novel gene in silico (Table 1, Fig. 1). We have named this gene as X-ray radiation resistance associated 1 (XRRA1) (GenBank accession no. BK000541). BLAST [10] analysis demonstrated that it was a novel gene.
We used human genomic clones RP11-147I3 and CMB9-8M21 (GenBank accession nos AP001992 and AP000560) of chromosome 11q13.3 as template for GrailEXP [11] analysis. The analysis predicted 11 exons including a putative promoter region for the human (Homo sapiens, Hs) XRRA1 gene (Fig. 2, Fig. 3A). These 11 exons spliced perfectly to create the mRNA for Hs XRRA1. The exon-intron junctions, comprising the 5' splice donor and 3' splice acceptor, follow Kozak consensus (Table 3) [12]. Cloning of the 5' region of XRRA1 gene that comprised the first four exons from HCT116 Clone2_XRR and HCT116 Clone10 revealed that Hs XRRA1 had those exons. However, in addition to the one from HCT116 Clone2_XRR , we obtained one splice variant that lacked exon three and ended on exon four (GenBank accession no. AY163836) (Fig. 3A).
To test whether these exon-intron borders were actually unique to XRRA1, we searched for possible XRRA1 homologues from other organisms. Thus far, we could only retrieve Xrra1 containing genomic clones from mouse (Mus musculus, Mm) (GenBank accession no. NW_000328) and rat (Rattus norvegicus, Rn) (GenBank accession no. AC127923). Mm Xrra1 gene is located on chromosome 7. GrailEXP analyses showed that Mm Xrra1 gene also contained 11 exons. Alignment of mouse and rat genomic sequences resulted in information for rat exons, which also consist of 11 exons. Interestingly, all the exon-intron borders of Hs XRRA1, Mm and Rn Xrra1 genes are well conserved (Table 3). We again employed Human XRRA1 cDNA sequences and its translation Figure 1 Human XRRA1 cDNA sequences and its translation. The gene comprises 11 exons, and produces 559 aa protein. XRRA1 protein consists several motifs i.e. leucine rich repeats, PEST sequence, nuclear localization signal, and two sites of tyrosine phosphorylation (*). Poly A-signal, AATAA is underlined.
EBOA to look for all possible ESTs of mouse and other organisms ( We used cDNA libraries from macaque (Macaca fascicularis, Mf) brain and testis to obtain Mf XRRA1. Since we have created and sequenced these libraries, we were able to look for the Mf XRRA1. We found only three testis clones (QtsA-12093, 10089 and 20433) from 10,400 testis and 53,000 brain cDNAs that were homologous to Hs XRRA1. One of the clones, QtsA-20433 (GenBank Promoter region of human XRRA1 gene as predicted by GrailExp [11]

Figure 2
Promoter region of human XRRA1 gene as predicted by GrailExp [11]. Two putative sites of TATA box were found at -506 and -400 from the start codon. Predicted transcription factor binding sites revealed several members of bZIP family such as C/ EBPa, c-Jun, c-Fos, AP-1 and CREB.
Representation of the XRRA1 genomic structure accession no. AB072776) had the full-coding sequence and showed strong identity to Hs XRRA1, sharing 96 and 95% identities in cDNA and protein sequences, respectively. SIM4 [13] and BLAST analysis showed that Mf XRRA1 sequence dispersed properly into 11 exons on human chromosomal band 11q13 (Fig. 3A).

XRRA1 is conserved among human, macaque, mouse, rat, pig and bovine
Multiple alignments of both XRRA1 cDNA and protein sequence from human, macaque, mouse, rat, pig, and bovine Xrra1s showed a conservative molecule. Percent identities at both the nucleotide and protein level among these mammals resulting from two by two alignments are listed in Table 4. As expected, phylogenetically, human XRRA1 is closer to macaque XRRA1 and both of them are closer to bovine and pig Xrra1s than murine and rat Xrra1s. We failed to find possible XRRA1 homologues in other organisms ranging from microorganisms, plants, and animals. The latter included organisms whose genomes have been sequenced completely such as yeast, plasmodium, fruit fly, zebra fish and fugu.
The Hs XRRA1 gene produces a novel 559 aa protein (Fig.  3B). The XRRA1 protein contains two possible sites of leucine-rich repeats (aa 20-41 and aa 138-164), a PEST sequence at aa 247-264, two tyrosine phosphorylation sites at aa 81 and aa 142, and bipartite nuclear targeting sequences are found at aa 357-373 and aa 485-501. Besides human, Mf XRRA1, Mm, Rn, and Bt Xrra1s retain the above-mentioned motifs.

XRRA1 responded differentially to XR in clones of varying radiation responses
The RT-PCR results confirmed earlier observation from our cDNA microarray analysis that unirradiated HCT116 Clone2_XRR cells had lower basal expression of XRRA1 than unirradiated HCT116 Clone10 cells (Fig. 4, Table  5). However, XRRA1 expression in HCT116 Clone2_XRR cells increased almost immediately (~10 minutes) after XR and decreased thereafter. By  The intron sizes of Rn Xrra1 gene could not be determined since the sequencing of rat genomic clone CH230-188L13 (GenBank AC127923) containing the gene was in progress. Nevertheless, their splicing sites can be identified, thus the exon sizes are also known. contrast, HCT116 Clone10 cells down regulated XRRA1 expression approximately ten minutes following XR. Interestingly, XRRA1 expression in HCT116 CloneK_XRS cells was initially similar to HCT116 Clone10 cells but regained the basal levels by 24 hours following XR.

XRRA1 was expressed predominantly in the testis in human and macaque
We carried out DNA microarray where PCR-amplified QtsA-20433 (Mf XRRA1) was included. Because we found that Mf XRRA1 was more commonly present in testis than in brain cDNA libraries, we further investigated whether Mf XRRA1 was predominantly expressed in the testis. The ratio of Mf XRRA1 expression in testis to that in mixed tissues was 3.02. We also compared the expression of XRRA1 between human and macaque testis. The ratio of XRRA1 expression in human testis to macaque testis was 1.04. In addition, we examined Hs XRRA1 expression from various normal human tissues/organs. We found that Hs XRRA1 was expressed predominantly in testis followed by prostate and ovary (Fig. 4, Table 5). Other tissues/organs such as peripheral blood leukocyte, spleen, thymus, small intestine and colon demonstrated low expression of Hs XRRA1.

XRRA1 was expressed ubiquitously in various types of cancer cells
We evaluated the expression of Hs XRRA1 in various cancer cell lines (neuroblastoma, glioma, breast, lung, leukemia, renal, ovarian, prostate, another colorectal). XRRA1 was present in all of those cells, although the expression level was variable (Fig. 4, Table 5). We also detected XRRA1 expression in immortalized normal fibroblast AG1522 and COS-7 cells, as well as in a dermal microvascular endothelial cell line (HDMEC). Surprisingly, besides mouse fibrosarcoma cells and tumor biopsy from a mouse model of fibrosarcoma, we were also able to detect Mm Xrra1 expression in pluripotent cells such as murine embryonic stem cells R1 (Fig. 4, Table 5).

Over-expression of GFP-XRRA1 fusion protein was only achieved transiently and showed nucleo-cytoplasmic protein distribution
To understand further the possible function of the XRRA1 gene, we fused the XRRA1 cDNA immediately downstream of a green fluorescent protein (GFP) cDNA. We over-expressed the GFP-XRRA1 in HCT116 clones, but after 48 hours post-transfection the fluorescence clearly decreased both in the number of cells and intensity of fluorescence. We obtained similar results when we overexpressed GFP-XRRA1 in COS-7 cells. After three weeks selection in 500 µg/ml G418, very few COS-7 cells remained fluorescent (Fig. 5). Only transient expression of the GFP-XRRA1 was observed in both types of cells. The fluorescence of the GFP-XRRA1 did not show an exclusive nuclear localization (although the predicted XRRA1 protein contains NLS motif). Instead, the fluorescence appeared in most cells to be nucleo-cytoplasmic (Fig. 5).

Over-expressed COS-7 cells with GFP-XRRA1 might modulate Ku86 expression
Ku86 was exclusively localized in nuclei with or without XR (Fig 6). The over-expression of GFP-XRRA1 abolished nuclear immunostaining of Ku86 in COS-7 (Fig 6A).
Immunostaining of α-Tubulin, as additional control for the experiment, did not change in the presence of GFP-XRRA1 (Fig 6B). The secondary antibody conjugated with Cyanine-3 (Cy3) was used for both Ku86 and α-Tubulin immunostaining.

Discussion
EBOA and in silico analysis helped us to identify a novel gene XRRA1 from a previously unknown EST R40588 not only in human, but also in mouse, bovine, and rat.
Recently, several clones that contained Hs XRRA1 spliced variants have been deposited to GenBank database by others. Hs XRRA1 clone from testis (GenBank accession no. BC037294) lacked exon 4; the one from spleen (Gen-Bank accession no. AK074152) lacked exons 3, 4 and a possible partial deletion of exon 5, and the one from NT2 cells (GenBank accession no. AK056364) lacked exons 2, 3, 4, and 10 (Fig. 3A). The testis clone with GenBank no. BC037294 did not appear to have a proper open reading frame. EBOA and GrailExp methods failed to produce a start site further upstream of Hs XRRA1 (GenBank no. BK000541).
Surprisingly, when we searched from approximately 63,400 macaque brain and testis cDNAs, we found only three testis clones that were homologous to Hs XRRA1, suggesting that the Mf XRRA1 gene is expressed only at a very low level in brain. The Mf XRRA1 is highly homologous with Hs XRRA1. Because the Hs XRRA1 gene consists of 11 exons, it is likely that the Mf XRRA1 gene also contains 11 exons (see Fig. 3A). In general, the XRRA1 gene seems to be highly conserved among mammals (i.e. human, macaque, mouse, rat, pig, and bovine), suggesting that it has similar function(s). Moreover, we were unable to find any convincing evidence of XRRA1 gene from fully sequenced genomic DNA of fish, fly, worm, yeast and microbes. This suggests that XRRA1 might be specific for, or evolutionary-enriched in, mammals. A specific role(s) for XRRA1 in mammals was further supported by the observation that the predicted protein sequences for motifs such as LRR, PEST, and NLS, as well as Tyr phosphorylation sites, are preserved among the abovementioned mammals. Additionally, these motifs suggested that the XRRA1 protein may interact dynamically with other proteins, or might be subjected to rapid degra-dation via the proteasomal pathway, or might suggest a possible role for XRRA1 in cellular signaling.
Although the protein contains a NLS motif, it appears that XRRA1 is not exclusively located in the nucleus, as shown by the over-expression of the GFP-XRRA1 fusion protein.
HCT116 clones and COS-7 cells containing the fusion protein (either with full length or truncated XRRA1 protein) had lost the auto-fluorescence. This suggested that the protein concentration above the basal expression levels of XRRA1 might be lethal to the cells.
It is noteworthy that the expression of XRRA1 in HCT116 clones was variably affected by 4 Gy XR. The results sug-RT-PCR of XRRA1 and a house-keeping gene G3PDH from HCT116 clones treated with X-radiation, various cancer and normal cells, and normal tissues/organs Figure 4 RT-PCR of XRRA1 and a house-keeping gene G3PDH from HCT116 clones treated with X-radiation, various cancer and normal cells, and normal tissues/organs. The same set of primers was used to amplify corresponding mRNA from mouse embryonic stem cells and fibrosarcoma cells/tumor. GFP-XRRA1 fusion protein in COS-7 and HCT116 clones gested that the XRRA1 gene might be involved in the immediate response following XR and might further be linked to XR resistance and/or sensitivity. Thus the return of XRRA1 to almost pre-irradiated basal levels by 24 hours post-irradiation in radiosensitive HCT116 CloneK_XRS cells (but not in the other two clones) might be necessary for the subsequent manifestation of enhanced sensitivity to XR by this clone. One possibility is that the XRRA1 gene may play a role either in the expression of cell death and/ or signaling of DNA damage following XR stress. Therefore, genetic modulation of the expression of XRRA1 in these clones may, specifically lead to either enhanced or decreased XR-induced lethality, and, generally, may be relevant for determining cellular response to XR in both tumor and normal cells.
In mammalian cells, DNA double strand breaks caused by XR are preferentially repaired by non-homologous end joining (NHEJ) mechanism. The major molecules involved in the NHEJ pathway are heterodimeric DNA end-binding complexes Ku86/Ku70 [14]. Therefore, we used Ku86 as the first candidate to test whether XRRA1 is involved in the modulation of the NHEJ pathway. The immunocytochemistry study on both HCT116 Clone2_XRR and COS-7 cells showed that there was no significant modulation of Ku86 with XR. However, COS-7 cells overexpressing GFP-XRRA1 were devoid of nuclear Ku86. This phenomenon suggested that over-expression of XRRA1 might have resulted in the down-regulation of Ku-86. Further studies are required to determine whether there is a negative correlation between XRRA1 and Ku86 expressions during cellular response to XR.
The varying expression of XRRA1 following XR described above could be related to the role of transcription factors. In fact, we were able to detect a possible region between nt -506 and -400 from the start codon for transcription factor binding sites that regulate the expression of XRRA1.
These factors were C/EBPα, HSF-1, c-Jun, c-Fos, AP-1, and CREB. Interestingly, most of the previously mentioned transcription factors are members of the bZIP superfamily. The latter is associated with various functions, including mediating G1 arrests [15,16], regulating responses against environmental stresses including IR and UV [17], acting as an immediate-early response factor [18], controlling wide range plasticity processes [19], and serving as a promoter element that mediates transcriptional activation in response to increased levels of intracellular essential secondary messenger cAMP [20]. Thus, putative binding sites for these transcription factors further strengthen the suggestion that the expression of XRRA1 was rapidly affected after XR treatment.
XRRA1 was expressed predominantly in the testis in both human and macaque. It may be relevant to note that the testis is generally regarded as one of the most sensitive organs to XR [21]. Thus upregulation of XRRA1 in an XRsensitive organ would appear to be generally consistent with our observation that it is downregulated in the XRresistant HCT116 Clone2_XRR cells. Because there was comparable expression level of XRRA1 in both human and macaque, it suggests that there is a functional necessity for XRRA1 in mammalian testis (i.e. XRRA1 might serve as a testis-specific molecule). By contrast, the low expression of XRRA1 in healthy colon and relatively much stronger expression of XRRA1 in HCT116 cells, may suggest an upregulation of this gene in cancer development. The ubiquitous expression of XRRA1 in numerous cancer cells and in immortalized normal cells might be indicative of a role of this gene in tumor development.
Interestingly, from the SAGEmap, we identified three tags that specifically represent XRRA1 anchored with restriction enzyme NlaIII. The tags were TATTCAGGGG, ACCT-GGTGCC, and GAATCAAGTG. SAGE libraries where XRRA1 was less than 20 tags per million were excluded. We found that the XRRA1 was present in mammary gland epithelium ductal in situ carcinoma, normal cerebellum, normal gastric body epithelium, well differentiated oligodendroglioma, pancreatic epithelium ductal adenocarcinoma, metastasis mammary gland carcinoma, ovarian clear cell poorly differentiated carcinoma, brain glioblastoma multiform cell line, ovarian serous adenocarcinoma, mammary gland ductal in situ high grade carcinoma, prostate carcinoma, brain juvenile ependymoma, and medulloblastoma cerebellum. The foregoing data further suggested that XRRA1 might be correlated with carcinomas in breast, brain, prostate, pancreas and ovary. Finally, the expression of Mm Xrra1 expression in mouse embryonic stem (ES) cells (R1) as well as in mouse fibrosarcoma cells and in fibrosarcoma biopsy suggest that XRRA1 might function as an early expressed gene.

Conclusions
XRRA1 is a novel molecule that is expressed selectively in normal healthy tissues/organs. It is expressed at relatively higher levels in primary sex organs such as testis (of both human and macaque), prostate and ovary. The expression of XRRA1 in normal proliferative cells, embryonic stem cells, and various tumor cells is also generally high and comparable to testicular expression of XRRA1. These findings suggest that XRRA1 expression may be important for cell proliferation, development, and differentiation as well as carcinogenesis. In regard to XR, we found that XRRA1 expression was rapidly modulated after treatment, suggesting the potential involvement of this gene/protein in the manifestation/expression of radioresistance/sensitivity by tumor cells such as human colorectal cancer cells.
Over-expression of GFP-XRRA1 fusion protein was located in both nucleus and cytoplasm of HCT116 clones or COS-7 cells. A possible correlation with NHEJ pathway for repairing DNA double strand break after XR is shown by the lack of Ku86 immunostaining in COS-7 cells that over-expressed the GFP-XRRA1. Further experiments to determine the function of XRRA1 in DNA damage sensor, repair and apoptosis following radio-and or chemotherapy are ongoing. Also the function of XRRA1 in carcinogenesis as well as embryogenesis and reproduction biology needs to be elucidated.

Construction of macaque cDNA libraries and DNA sequencing of their cDNA inserts
Oligo-capped cDNA libraries were made from macaque brain and testis. cDNAs were isolated according to the method described previously [22,23]. The 5'-end sequences of the clones were sequenced using ABI 3700 sequencer (Applied Biosystems, Tokyo, Japan) and categorized using DYNACLUST (Dynacom Co., Chiba, Japan) based on a BLAST search against the GenBank database. The entire sequences of clones were determined by the primer walking method with an ABI PRISM BigDye Terminator Sequencing kit (Applied Biosystems) according to the manufacturer' s instructions.

Construction of plasmid expressing Mf XRRA1 fused to GFP
Macaque cDNA clone of QtsA-20344 was found from the cDNA libraries to contain Mf XRRA1 transcript. A 1709-bp XhoI fragment of Mf XRRA1 was removed from pME18S-FL3 and sub-cloned into XhoI sites of pEGFP-C1 (BD Biosciences, Mississauga, ON, Canada), immediately downstream of the GFP sequence (pFM1709). To create a truncated Mf GFP-XRRA1 (pFM584), 1.1-kb EcoRI fragment was removed from pFM1709 leaving a 584-bp of the 5' XRRA1 cDNA fused with GFP.
Transfection of GFP-XRRA1 into HCT116 Clone2_XRR , HCT116 Clone10 , HCT116 CloneK_XRS , and COS-7 cells HCT116 clones and COS-7 cell were transfected with a mixture of pFM584, pFM1709 or pEGFP-C1 and lipid complexes FuGENE 6 (Roche Diagnostics, Laval, QC, Canada) transiently or stably. Briefly, HCT116 clones and COS-7 were grown on cover slips in a 6-well plate and reached 60-70% confluences before transfection. The ratio of FuGENE 6 and pure plasmid was 6:1 in serum free medium. G418 (Sigma-Aldrich, Oakville Ltd., ON, Canada) at a concentration of 400 µg/ml was used for selection. Cells were fixed with 3.7% formaldehyde in PBS for 10 min, and then washed three times with 1x PBS. Coverslips were mounted upside down onto slides with Dako Fluorescent Mounting Medium (DAKO, Carpinteria, CA, USA). Visualization was performed using a fluorescence microscope Axioskop 2 MOT (Carl Zeiss MicroImaging Inc., Thornwood, NY, USA). Images of cells were captured with a CCD camera fitted with a FITC filter.

Immunocytochemistry
Transfected COS-7 and HCT116 Clone2_XRR cells with pFM1709 or pEGFP-C1 were grown on cover slips until they reached 70-80% confluency before being treated with 4 or 10 Gy XR. Cells were fixed in 2% formaldehyde in 1x PBS for 15 min 24 h after XR treatment. Cells were then permeabilized with 0.2% Triton X-100 for 10 min. Ku86 antibody was obtained from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA. The secondary antibody was conjugated with Cy3 (Amersham Biosciences Corp, Baie d' Urfe, QC, Canada). Controls for the immunostaining were the secondary antibody alone, and control for the experiment was α-Tubulin antibody that was obtained from Calbiochem, San Diego, CA, USA.

RT-PCR
Total RNAs of normal/cancer cells were extracted using RNAeasy kit (Qiagen Inc.) according to the manufacturer' s instructions. TRIZOL Reagent (Invitrogen Inc., Burlington, ON, Canada) was used to isolate RNA of mouse fibrosarcoma tumor biopsy. RNAs of human peripheral blood leukocyte, spleen, thymus, small intestine, colon, ovary, testis and prostate were purchased from BD Biosciences. First Strand cDNA Synthesis kit (MBI Fermentas) was used to create first strand cDNA from RNA samples. A specific primer pair that amplified ~1 kb house keeping gene glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as the control. The primers were 5'-TGAAGGTCGGTGTGAACGGATTTGGC-3' and 5'-CATG-TAGGCCA TGAGGTCCACCAC-3'. Conditions of PCR were as follows: 94°C for 30 s, 61°C for 15 s, and 72°C for 40-60 s for 35 cycles. The PCR products were then separated on 1.5% agarose gels, stained with ethidium bromide, and visualized with the gel documentation imager Epi Chemi II (UVP Inc., Upland, CA, USA). Densitometry of bands were analyzed using Labworks™ software (UVP). Relative expression of XRRA1 was calculated as percentages of the mean raw densities of grayscale level of XRRA1 to G3PDH.


extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Medicine

text:
Inhibition of established collagen-induced arthritis with a tumour necrosis factor-α inhibitor expressed from a self-contained doxycycline regulated plasmid

Tumor necrosis factor (TNF)-α is produced by cells of the immune system and is a key mediator in immune and inflammatory reactions. Through interaction with widely expressed receptors (TNF receptor 1 and TNF receptor 2), TNF-α is able to orchestrate the expression of a range of downstream proinflammatory molecules. Over the past decade novel biologics that inhibit TNF-α have been developed as extremely effective treatments for rheumatoid arthritis. Structurally, these biologics are antibodies, or TNF receptors on an antibody backbone that bind TNF-α directly and are delivered to patients by repeated injection. Gene therapy offers an improved approach to delivering biologics as a single administration of their encoding genetic material. In the present study we demonstrate the therapeutic effect of a small molecular weight dimeric TNF receptor 2 (dTNFR) constitutively expressed from plasmid DNA, delivered intramuscularly with electroporation, after disease onset in a collagen-induced arthritis model. Regulated promoters that enable the production of a transgene to be controlled are more suited to the application of gene therapy in the clinic. Regulated expression of dTNFR from the plasmid pGTRTT was also therapeutic in the mouse collagen-induced arthritis model when the inducer doxycycline was also administered, whereas no therapeutic effect was observed in the absence of doxycycline. The therapeutic effect of dTNFR expressed from a constitutive or regulated plasmid was dependent on the degree of disease activity at the time of DNA injection. The observations of this study are considered with regard to the disease model, the magnitude of gene regulation, and the path to clinical application.

We previously constructed dTNFR, which is a smaller inhibitor of TNF-α than existing biologics and consists of two extracellular subunits of the human TNF receptor TNFR2 connected by a short flexible serine glycine linker [3]. This molecule inhibits TNF-α in vitro [3] and has been shown to inhibit disease when delivered before onset in arthritis models [4] and was also therapeutic in a model of multiple sclerosis [5].
Gene therapy is potentially an improvement on protein therapy. Following the appropriate delivery of genetic material, the body's own cells are able to produce a protein therapeutic. Delivery of genetic material can be achieved with viruses such as adenoviruses, which give transient high-level expression of transgenes and have been widely applied in gene therapy in experimental RA models. Alternatively, retroviruses that integrate transgenes into the genome of dividing cells have proved effective tools in experimental ex vivo strategies in arthritis models [6]. The initial enthusiasm for viral vectors as candidate vehicles for gene delivery was tempered by safety concerns in clinical trials: first the death of a patient following administration of adenovirus [7]; and second the development of leukaemia in severe combined immunodeficient patients who received retrovirally transduced haematopoietic stem cells [8].
Plasmid DNA isolated from bacteria has no innate mechanism of cell entry or propagation, and does not encode accessory proteins or integrate into the genome, but it has the potential to be an efficient vehicle for gene delivery in vivo when administered by intramuscular injection. First reported by Wolff and coworkers in 1990 [9], injection of plasmid DNA into mouse skeletal muscle results in longterm (at least 2 months) transgene expression. More recently it was shown that when muscle was electroporated after DNA injection the efficiency of transfection was further enhanced by a factor of 100-fold, with transgene expression persistent up to 1 year [10]. Plasmid DNA also has the advantage of being very stable and both easy and cheap to produce in large quantities.
Ideally, gene therapy for a chronic disease such as RA will permit long-term production of a therapeutic molecule, so reducing the need for repeat administration. In combination with regulated promoter systems, therapeutic levels of a molecule are produced, so avoiding excess (toxicity) and inadequate (no effect) production, and this also provides a means by which to terminate expression in the event of adverse effects. Principal among the transcriptionally regulated systems that have been developed are the tetracycline 'off' [11], 'on' [12] and 'repressor' [13] systems, and the ecdysone [14], rapamycin [15] and streptogramin [16] systems, which are all two-or three-component systems that utilize a small molecule to regulate the activity of a responsive promoter (for review [17]). The tetracycline regulated systems have the inherent advantage that tetracycline and several derivatives have well defined pharmacokinetics and pharmacodynamics [18]. We and others previously combined the components of the tetracycline 'on' system into a self-contained autoregulated plasmid vector [19].
In the present study we constructed plasmids encoding dTNFR from constitutive and regulated promoters, from which the expression of dTNFR was characterized in vitro. Function of the vectors was demonstrated in vivo after intramuscular delivery, and we examined the therapeutic effect of dTNFR expressed constitutively or in a regulated manner from plasmid DNA administered after the onset of disease in the mouse collagen-induced arthritis (CIA) model.

Materials and method DNA and cloning
The vectors pGT, pGTL, pGTE, pGTRTL, and pGCMV were previously reported [19]. The construct pGTTRD, which encodes dTNFR from a Ptet, was constructed by removing the dTNFR sequence from the construct pTRIP [3] by restriction with NcoI filled in with Klenow, and cut with XbaI, and inserting it into pGT restricted with EcoRV and XbaI. The self-contained regulated plasmid pGTRTT encoding dTNFR was then contructed by removing the Ptet-dTNFR cassette from pGTTRD by restriction with Nhe1and PflMI and ligating it into pGTRTL restricted with the same enzymes. The plasmid pcdTNFR, in which dTNFR is located downstream of a cytomegalovirus promoter, was constructed by removing the dTNFR gene from pGTTRD by restriction with HindIII and XbaI and inserting it into pcDNA3 (Invitrogen, Leek, The Netherlands) restricted with the same enzymes.
A control construct pGTRTEmpty was also prepared, which retained all the elements of pGTRTT except the dTNFR gene. pGTRTE is an autoregulatory plasmid from which enhanced green fluorescent protein (EGFP) expression is regulated, and it was constructed by removing the Ptet-EGFP cassette from pGTE by restriction with XhoI and PflMI and inserting it into pGTRTL restricted with the same enzymes. The EGFP gene was removed from pGTRTE by restriction with ClaI and re-ligation of the plasmid formed pGTRTEmpty.
The plasmid pcLuc+, which encodes luciferase from a cytomegalovirus promoter, was constructed by removing the improved luciferase gene (Luc+) from pGL3 Basic (Promega Corp., Madison, WI, USA) with the restriction enzymes XhoI and XbaI and inserting the gene into pcDNA3 restricted with the same enzymes.
Plasmids were expanded in Escherichia coli DH5α except autoregulatory vectors, which were propagated in E. coli R105 DH 21. Plasmid DNA was purified using the Plasmid Mega Kit (Qiagen Ltd, Crawley, UK), or when required for injection into mice the EndoFree™ Plasmid Mega Kit (Qiagen Ltd) was used. All plasmids that were used in expression studies are depicted schematically in Fig. 1.

Transfections
Transfections were performed using the calcium phosphate precipitation method used previously [19]. Cos-7 cells were plated on 12-well plates at a density of 0.4 × 10 6 /well and were transfected the next day with 2 µg DNA. Cells were subjected to an osmotic shock on the second day, after which fresh media was added with or without doxycycline at a concentration of 1 µg/ml. Supernatants were collected 24 or 48 hours later and levels of dTNFR were determined by ELISA.

In vivo electroporation
Mice were treated according to approved UK Home Office and institutional guidelines. Naïve or arthritic DBA/1 mice were injected intraperitoneally with the muscle relaxant Hypnorm™ (Janssen Animal Health, Janssen Pharmaceuticals, Beerse, Belgium) and were anaesthetized with halothane (Concord Pharmaceuticals Ltd, Dunmow, UK) using Boyle's apparatus (British Oxygen Company, London, UK). The fur covering the right quadracep was shaved and the exposed skin sprayed with disinfectant. Endotoxin-free plasmid for injection was prepared in a solution of 0.9% NaCl at a concentration of 250 µg/ml for reporter gene studies or 833 µg/ml for therapeutic studies. DNA (20 µl) was injected intramuscularly at three sites, and Camcare ECG gel (Camcare Gels, Mepal, UK) was then applied to the surface of the skin. Caliper electrodes 384L (BTX Instrument Division, Harvard Apparatus Inc., Holliston, MA, USA) were applied transversely across the quadriceps and the muscle was electroporated with four pulses at 200 V/cm and 20 ms duration at a frequency of 2 Hz using a BTX Electro Square Porator ECM 830 (Harvard Apparatus Inc.). The polarity of the electrodes was then reversed and the procedure repeated.

Regulated expression of luciferase in vivo
Regulated expression of luciferase from pGTRTL was assessed in DBA/1 mice following intramuscular injection and electroporation. Expression of luciferase was compared with control plasmids pGmCMV, pGTL and pGCMV. Plasmid DNA was injected intramuscularly into 10-to 12-week-old naïve DBA/1 mice along with electroporation. Luciferase expression in control groups was assessed 4 weeks after DNA injection, when animals were killed and muscle tissue was snap frozen until further processing.
The plasmid pGTRTL was injected into 30 mice, which were divided into six groups of five animals. These mice received normal drinking water for 2 weeks, and then two groups were given distilled water containing 10% sucrose, three groups drank doxycycline 200 µg/ml and one group was given doxycycline 2 mg/ml; the doxycycline solutions were prepared in distilled water containing 10% sucrose. All drinking bottles were wrapped in aluminium foil and were renewed every 2-3 days. After a further 2 weeks a group of animals from each treatment group was killed and the quadriceps muscles dissected and snap frozen. In order to analyze the reversal of gene induc-Available online http://arthritis-research.com/content/6/2/R103

Figure 1
Expression plasmids used in this study. Numbers in parenthesis represent the length of each vector in base pairs. ᭢, SV40 early/late poly A signal; , β-globin poly A signal; , downstream SV40 untranslated region. CMV, immediate-early enhancer/promoter; dTNFR, dimeric human tumour necrosis factor receptor 2; Luc, luciferase gene; Luc+, improved luciferase gene; Ptet, tetracycline responsive promoter; rtTA, reverse tetracycline transactivator. tion from pGTRTL following the removal of doxycycline, a group of animals that had received doxycycline 200 µg/ml were switched to the sucrose drink. Three days later the remaining mice were killed and muscles collected as for the others.

Luciferase assay
Luciferase activity in transfected muscle was determined using the luciferase assay system (Promega Corp.). Quadriceps muscles dissected from treated mice were snap frozen and stored at -70°C. Frozen muscle was processed by a method similar to that described by Hartikka and coworkers [20]. Briefly, they were first crushed in a freezer mill (model 6750; Spex Centriprep Inc., Metuchen, NJ, USA) and the powder was transferred to an Eppendorf tube containing 0.5 ml reporter lysis butter. The suspension was then subjected to three rounds of freeze thawing, followed by 15 min of vortexing at 4°C. Samples were centrifuged (13 000 rpm for 5 min) and the lysate collected; pelleted material was resuspended in a second aliquot of lysis buffer and was subjected to the 15 min of vortexing followed by centrifugation. The second lysate was combined with the first and luciferase activity was determined in a 20 µl aliquot of the sample, which was automatically mixed with 100 µl of luciferase assay substrate, and light emission measured using a MLX Microtiter ® Plate Luminometer (Dynex Technologies Inc., Chantilly, VA, USA). Protein concentrations of muscle lysate were determined using the Bradford protein assay (Bio-Rad Laboratories Inc., Hercules, CA, USA) and values for luciferase activity were expressed as relative light units per microgram of protein.

Collagen-induced arthritis
DBA/1 mice aged between 10 and 12 weeks were administered Hypnorm™ (0.1 ml, intraperitoneally) and were shaved at the base of the tail. Bovine collagen type II (CII) was emulsified with complete Freund's adjuvant at a final concentration of 2 mg/ml, and a total of 0.1 ml was injected intradermally at three sites at the base of the tail. Twenty-one days later, a booster (0.1 ml) consisting of CII emusified with incomplete Freund's adjuvant (2 mg/ml) was injected intradermally across three sites at the tail base. A further 3 days later animals were injected with lipopolysaccharide (40 µg in 0.1 ml phosphate-buffered saline [PBS]; E. coli serotype 055:B5; Sigma-Aldrich Co. Ltd, Poole, UK) intraperitoneally to synchronize disease [21]. The development and progression of arthritis was monitored every 2-3 days and was assigned a clinical score based on visual signs of arthritis (0.25 = swelling in a single digit; 0.5 = swelling in more than one digit; 1 = swelling and erythema of the paw; 2 = swelling of the paw and ankle; 3 = complete inflammation of the paw; the maximum score for each mouse was therefore 12) and the thickness of hind paws was measured using POCO 2T calipers (Kroeplin Längenmesstechnik, Schlüchtern, Germany). Mice were monitored until 40 days after immunization, when they were killed and blood was collected for serum and draining lymph nodes were collected for assessment of stimulated cytokine secretion.
Three days after injection of lipopolysaccharide (day 27), animals were assessed for development of arthritis. Animals with a clinical score of 0.5 or above were used in gene therapy experiments and were administered 50 µg of DNA intramuscularly in 60 µl at three sites and were electroporated utilizing conditions described above.

Cytokine expression from draining lymph node cells
Inguinal draining lymph nodes were removed from mice on day 40 after immunization. Incisions were made in the lymph nodes and cells were dispersed using a nylon cell strainer (70 µm; Becton Dickinson Labware, Franklin Lakes, NJ, USA). Cell suspensions were centrifuged and resuspended at 3 × 10 6 /ml in Dulbecco's modified Eagle's medium supplemented with 10% foetal calf serum, glutamine (2 mmol/l), penicillin (100 U/ml) and streptomycin (100 µg/ml). Cells (6 × 10 5 cells in 200 µl) were aliquoted into wells in a 96-well microtitre plate and were stimulated with either CII (50 µg/ml) or ConA (2.5 µg/ml; Sigma-Aldrich Co. Ltd) for 48 hours, after which supernatants were collected and stored at -80°C until measurements of IFN-γ or IL-4 were performed by ELISA (see below).

Detection of dTNFR and antihuman TNFR2 by ELISA
To measure levels of dTNFR, a microtitre plate was coated with 50 µl of a mouse monoclonal antihuman TNFR2 (R&D Systems, Minneapolis, MN, USA) at 4 µg/ml overnight at 4°C. Plates were washed with PBS and then blocked with 200 µl of 2% casein solution in PBS for 1 hour at room temperature. Plates were washed with PBS containing 0.05% Tween 20 (PBS/Tween) before incubation of standards (50 µl of human TNFR2 [R&D Systems] 1 pg/ml to 1 µg/ml) and samples (50 µl of serum or culture medium) for 3 hours at room temperature. Plates were washed extensively with PBS/Tween before incubation with 50 µl of biotinylated goat antihuman TNFR2 (R&D Systems) at a concentration of 100 ng/ml for 1 hour at room temperature. Signal was detected using the TMB microwell substrate system (Kirkegaard and Perry Laboratories Inc., Gaithersburg, MD, USA) and the reaction stopped by addition of 4 mol/l sulphuric acid (100 µl) and absorbance measurements were performed at 450 nm using an EL 312e microplate biokinetics reader (Bio-Tek Instruments Inc., Winooski, VT, USA). The detection limit of this ELISA was 10 pg/ml.
In order to determine the immunogenicity of the dTNFR, an ELISA was also performed to measure antihuman TNFR2 in the sera from experimental mice. Microtitre plates were coated overnight at 4°C with 50 µl of human TNFR2 (2 µg/ml). Plates were washed with PBS and then Arthritis Research & Therapy Vol 6 No 2 Gould et al. blocked with 2% casein solution in PBS for 2 hours at room temperature. Plates were then washed with PBS/Tween and then incubated with serum (diluted 1 : 500) or dilutions of control mouse monoclonal antibody (R&D Systems: 1 µg/ml to 0.1 pg/ml) for 3 hours at room temperature. Plates were again washed with PBS/Tween and bound antibody was detected using peroxidase conjugated sheep antimouse IgG (The Binding Site, Birmingham, UK), and after 1 hour the signal was developed and measured as described above. The control mouse monoclonal antibody was detected down to a concentration of 0.1 pg/ml.

ELISA for detection of anti-CII IgG
Microtitre plates (96-well) were coated with 50 µl of 2 µg/ml CII dissolved in Tris-buffered saline overnight at 4°C. After blocking for 2 hours with 2% casein (200 µl), wells were repeatedly washed with PBS/Tween and then 50 µl of 10-fold dilutions of mouse sera from 1/10 to 1/100 000 000 were applied to the wells and incubated overnight at 4°C. Total anti-CII IgG and IgG isotypes were quantitated using 50 µl of peroxidase conjugated sheep antimouse IgG, IgG 1 and IgG 2a (The Binding Site) at a dilution of 1/5000 with PBS/Tween. Signal was detected as described above. Each plate included a standard curve of a positive serum obtained from untreated CIA mice at day 40, which was used to define arbitrary units of total IgG, IgG 1 and IgG 2a anti-CII antibodies.

Measurement of IL-4 and IFN-γ γ by ELISA
Microtitre plates were coated with 50 µl of capture antibody for IL-4 (rat antimouse IL-4 used at 2 µg/ml; Pharmingen, San Diego, CA, USA) or IFN-γ (rat antimouse IFN-γ used at 5 µg/ml; Pharmingen), both antibodies were diluted with 0.5 mol/l carbonate/bicarbonate buffer (pH 9.6), and plates were incubated overnight at 4°C. Plates were washed with PBS and then blocked with 200 µl of 2% casein solution in PBS for 1 hour at room temperature. Plates were washed with PBS/Tween before incubation of 50 µl of samples and standards (mIL-4 [10 ng/ml to 3 pg/ml] or IFN-γ [300 ng/ml to 100 pg/ml]) for 3 hours at room temperature. Plates were washed extensively with PBS/Tween before incubation with 50 µl biotinylated mouse monoclonal antibody to IL-4 (rat antimouse IL-4 used at 0.5 µg/ml; Pharmingen) or IFN-γ (rat antimouse IFN-γ used at 1 µg/ml; Pharmingen). Plates were again washed and then incubated with streptavidin biotinylated horseradish peroxidase complex (diluted 1 : 1000; Amersham Biosciences UK Ltd, Little Chalfont, Bucks, UK). Signal was detected as described above and the limits of detection for the IL-4 and IFN-γ ELISAs were 3 pg/ml and 100 pg/ml, respectively.

Statistical analysis
Descriptive statistics and significant differences between groups were calculated using Student's t-tests for two samples data of unequal variance (Microsoft ® Excel 98 software).

Expression of dTNFR in vitro
Transient transfection of Cos-7 cells was used to demonstrate expression of dTNFR from the construct pcdTNFR (Fig. 2). A basal level of TNFR2 was also detected in the control transfection of Cos-7 with pcDNA3, which indicates that the ELISA cross-reacts with the monkey TNFR2 produced spontaneously by this cell line. We previously found that this ELISA reacts with monkey TNFR2 (unpublished data).
Expression of dTNFR from pGTRTT was determined in transiently transfected Cos-7 cells. Induction of transfected Cos-7 cells with doxycycline (1 µg/ml) for 48 hours achieved expression levels of dTNFR from pGTRTT that were equivalent to the levels produced from cells transfected with pcdTNFR (Fig. 2). As previously demonstrated for pGTRTL, there was some basal activity of the Ptet in pGTRTT, with dTNFR expression detected in the absence of doxycycline induction. As expected, the control vector pGTRTEmpty expressed no dTNFR above background in either the absence or presence of doxycycline.

Expression of luciferase following in vivo plasmid injection and electroporation
Electroporation has been reported to enhance the transfection efficiency of plasmid DNA delivered in vivo by intramuscular injection [10]. In order to confirm this effect in naïve DBA/1 mice, the constitutively expressing plasmid pcLuc+ was injected intramuscularly and electroporated. Measurement of luciferase expression 3 days later indicated that electroporation enhanced transfection efficiency, with expression levels 245-fold greater than achieved with DNA injection alone (Fig. 3a).
The self-contained autoregulatory plasmid pGTRTL, which was previously shown to function efficiently in vitro, was assessed for function in vivo following delivery to naïve DBA/1 mice. Expression of luciferase from pGTRTL was compared with luciferase expression from the control vectors pGCMV, pGmCMV and pGTL. In each case, after DNA was injected and electroporated the mice were left for 2 weeks; then those that were injected with pGTRTL either were maintained non-induced with a drink of 10% sucrose, or they were induced with doxycycline drinks prepared in 10% sucrose at concentrations of 200 µg/ml or 2 mg/ml. After a further 2 weeks mice were killed and expression levels of luciferase determined. Results in Fig. 3a confirmed that regulated expression of luciferase from pGTRTL is observed in vivo, with 43-fold and 27-fold induction observed in groups that received doxycycline drinks of 200 µg/ml and 2 mg/ml, respectively. Induced levels of luciferase from pGTRTL with 200 µg/ml doxycy-Available online http://arthritis-research.com/content/6/2/R103 cline exceeded the expression levels observed with pGCMV by more than threefold. Interestingly, basal expression of luciferase from pGTRTL was significantly greater than from the control vectors pGmCMV and pGTL.
The downregulation of luciferase expression from pGTRTL in vivo was assessed in mice that were untreated for 2 weeks after DNA injection, then induced with doxycycline (200 µg/ml) for 2 weeks, and then had doxycycline removed for 3 days before terminating the experiment. Fig. 3c illustrates that removal of doxycycline for 3 days resulted in return to basal levels of luciferase expression observed for the non-induced pGTRTL group, whereas a group that continued to receive doxycycline for the extra 3 days maintained induced levels of luciferase expression.

Treatment of collagen-induced arthritis with pcdTNFR after disease onset
Control plasmid pcDNA3 (n = 11) or the expression plasmid pcdTNFR (n = 14) were delivered by intramuscular injection and electroporation to arthritic (clinical score at least 0.5) DBA/1 mice on day 27 after CII immunization. Clinical score and hind paw swelling was monitored and is shown in Fig. 4a and 4b. The results indicate that there was no therapeutic effect of pcTNFR treatment. The clinical score of mice at the time of DNA delivery on day 27 was in the range 0.5-4, and a published study indicates that the therapeutic outcome of TNF-α inhibition is related to the level of disease activity when treatment is initiated in Arthritis Research & Therapy Vol 6 No 2 Gould et al.

Figure 3
Constitutive and regulated luciferase expression in vivo. (a) Plasmid pcLuc+ (15 µg) was injected intramuscularly into the right quadriceps of six naïve DBA/1 mice. The muscle of three mice was then electroporated (8 pulses, 200 V/cm, 20 ms duration, 2 Hz), and the other three mice were untreated. Three days later the experiment was terminated and muscle processed for measurement of luciferase. Levels of luciferase are the mean of three animals and are normalized for protein concentration of the muscle lysate. Vertical lines represent standard error, and a significant difference (P ≤ 0.01) between the luciferase level in the muscle of electroporated and non-electroporated mice is indicated (*). (b) Plasmid DNA (15 µg) was injected (intramuscularly) and electroporated in naïve DBA/1 mice. After 2 weeks GTRTL injected mice (n = 5 for all groups) were given 10% sucrose (white bar), or doxycycline drinks (black bars) prepared in 10% sucrose at 200 µg/ml and 2 mg/ml for the subsequent 2 weeks. Luciferase expression in muscle was determined in dissected muscle and was compared with that in mice receiving the control plasmids pGCMV, pGmCMV and pGTL. Significant differences between the pGTRTL plus doxycycline groups and the non-induced pGTRTL group (P ≤ 0.05) are indicated (*). (c) Downregulation of luciferase expression from pGTRTL was assessed in a group of mice that had received doxycycline 200 µg/ml for 2 weeks; they were switched to sucrose for 3 days, after which luciferase levels in muscle were compared with those in groups that were non-induced or continuously induced for the duration of the experiment. A significant difference between the pGTRTL plus doxycycline group and the non-induced pGTRTL group (P ≤ 0.05) is indicated (*), and a significant difference between the pGTRTL doxycycline removed group and the pGTRTL doxycycline 200 group (P ≤ 0.05) is indicated ($). RLU, relative light unit.

Figure 2
Expression of dimeric human tumour necrosis factor receptor 2 (dTNFR) from pcdTNFR and pGTRTT transfected Cos-7 cells. Cos-7 cells plated in 12-well plates at 0.4 × 10 6 /well were transiently transfected with 2 µg of the constructs pGTRTT, pcdTNFR, pGTRTEmpty and pcDNA3. Cells transfected with pGTRTT or pGTRTEmpty were either cultured in normal media (lighter bars) or media supplemented with doxycycline (1 µg/ml; darker bars). Levels of human TNFR2 were measured in culture supernatants collected after 48 hours and are expressed as the mean of triplicate values, with vertical lines representing the standard error. mouse CIA [22]. Animals were therefore subdivided into those that had a clinical score of 2 or less and those with a higher clinical score at the time of DNA injection. In terms of clinical score, a significant therapeutic effect of pcdTNFR was observed in mice (n = 6) with lower disease activity at the time of DNA injection, as compared with those injected with pcDNA3 (n = 9), and no therapeutic effect was observed in mice with a clinical score greater than 2 when treatment (pcdTNFR, n = 8; pcDNA3, n = 2) was initiated (Fig. 4c and 4e). Data for paw swelling also illustrate a significant therapeutic effect of pcdTNFR in mice with lower disease activity than in those that had more established disease at the initiation of treatment ( Fig. 4d and 4f).

Inhibition of collagen-induced arthritis with dTNFR expressed from pGTRTT delivered after disease onset
Animals injected with the plasmid pGTRTT on day 27 and administered doxycycline (n = 17) developed significantly reduced arthritis, as measured by paw thickness and clinical score, when compared with control animals that received pGTRTT but no doxycycline (n = 16) and animals that received the control vector pGTRTEmpty and doxycycline (n = 16; Fig. 5a and 5b). When therapeutic effect was assessed on the basis of disease activity at the time of DNA delivery, it was clear that animals with a clinical score under 2 responded to pGTRTT plus doxycycline treatment in terms both of clinical score and of hind paw swelling ( Fig. 5c and 5d). Where disease had progressed to give a clinical score greater than 2 at the time of DNA delivery, no benefit of pGTRTT plus doxycycline was observed ( Fig. 5e and 5f).
Levels of dTNFR in the serum of pGTRTT treated mice was below the detection level of the ELISA (data not shown), and no antihuman TNFR2 was detected in these sera (data not shown), indicating that the dTNFR was not immunogenic during the time frame of the experiment.
The average daily fluid intakes per mouse for the groups in which the treated animals were housed were determined at 3.6 ml, 3.1 ml and 3.4 ml for the pGTRTT plus doxycycline, pGTRTT without doxycycline, and pGTRTEmpty plus doxycycline treatment groups, respectively. The average amount of doxycycline administered was therefore 720 µg in the pGTRTT treated group, which equates to approximately 30 mg/kg doxycycline/day.

Immunological status of collagen-induced arthritis mice was not altered by dTNFR treatment
The anti-CII antibody profile of mice treated with pGTRTT with or without doxycycline and pGTRTEmpty was similar at the end of the experiment for all groups (Table 1). In addition, cytokine release from draining lymph node cells in response to CII or ConA stimulation was also unaffected by any treatment (Table 2).

Discussion
In the present study we demonstrated a therapeutic effect of dTNFR in CIA when expressed from plasmid DNA under the control of constitutive and regulated promoters. Importantly, the therapeutic effect was achieved when DNA was delivered after disease onset, and all of the components for regulated expression were combined in a single vector.
We confirmed in DBA/1 mice that electroporation increases transfection efficiency in vivo following intramuscular injection of plasmid DNA, with transgene expression detected beyond 4 weeks from pGTRTL. In Available online http://arthritis-research.com/content/6/2/R103

Figure 4
Effect of pcdTNFR on progression of collagen-induced arthritis. Development of arthritis was monitored by (a, c and e) clinical score and (b, d and f) hind paw swelling in DBA/1 mice. Treatment was administered after onset of clinical arthritis. All mice injected with pcdTNFR (᭺; n = 16) and pcDNA3 (ٗ; n = 11) are depicted in panels a and b; those with a clinical score less than 2 at the time of DNA injection are presented in panels c and d; and those with a clinical score above 2 when treatment was initiated are plotted in panels e and f. Significant differences between the pcdTNFR and pcDNA3 group (P ≤ 0.05) are indicated (*). agreement with other studies using mice, these observations indicate that there is long-term persistence of plasmid DNA [10], and expression of the transgene luciferase [10] and rtTA [23] encoded by the autoregulatory plasmid. Regulation from pGTRTL in vivo is comparable to in vitro function [19], as demonstrated by the 43-fold increase in luciferase expression induced by 2 weeks of doxycycline induction, and return to basal expression 3 days after removal of doxycycline. Interestingly, the basal expression from pGTRTL was elevated compared with pGTL in vivo, whereas the basal expression from the two vectors was similar in cultured fibroblasts [19], which may indicate an effect that is related to expression in skeletal muscle. Improvements in the tetracycline system have led to the development of tetR targeted repressors such as tetR-KRAB [24] and tTS [25], which have been shown to reduce efficiently basal expression from the Ptet regulated promoters in vitro and when codelivered to skeletal muscle [26,27]. Incorporating the tetR-KRAB gene into the self-contained plasmid would reduce basal expression and increase the magnitude of regulation, as was recently achieved with an adenoviral vector [28].
According to data obtained in clinical trials, transfection of human skeletal muscle with injected plasmid DNA does not appear to be as efficient as in mice because only short-term transgene expression has been demonstrated [29]. Enhanced transfection efficiency by electroporation has yet to be conducted in humans, but observations with primates indicate a beneficial effect [10]. The tetracycline gene regulatory system has also been demonstrated to function in primates, but there is evidence of immunogenicity in this species [30]. Although the components of the system may also be immunogenic in humans, further research will be needed to determine whether regulated expression of an immunomodulatory cytokine enables transfected cells to evade detection by the immune system.
Constitutive expression of therapeutic genes has been utilized extensively and successfully in experimental gene therapy. However, for clinical application regulated promoter systems are more flexible because they enable the level of expression to be controlled and provide a means by which to terminate gene expression. These features are likely requirements for gene therapy application in chronic relapsing conditions such as RA. Although the present study demonstrates the effectiveness of the pGTRTT autoregulated vector, the full utility was not harnessed because dTNFR was continuously induced. More stringent examination requires the use of chronic arthritis models such as CIA in Vβ12 T-cell receptor transgenic mice [31] and use of therapeutic molecules that actually reverse established disease, for example IL-4 [32] and IL-1 inhibitors [22,33].
In the present study the therapeutic effect of dTNFR was clearly demonstrated in CIA when expressed from plasmids with constitutive or regulated promoters, but in both cases the therapeutic effect was related to the disease severity at the time of DNA injection. The first reports of anti-TNF-α treatment by protein therapy in CIA indicated that therapeutic effect was observed when anti-TNF-α treatment was initiated before disease onset [34,35] or immediately after onset [35]. The therapeutic effect of anti-Arthritis Research & Therapy Vol 6 No 2 Gould et al.

Figure 5
Gene therapy treatment of collagen-induced arthritis (CIA) with regulated expression of dimeric human tumour necrosis factor receptor 2 (dTNFR). Progression of CIA was monitored by clinical score (a, c and e) and hind paw swelling (b, d and f) in groups that received pGTRTEmpty plus doxycycline (ٗ; n = 16), pGTRTT without doxycycline (᭺; n = 16) and pGTRTT plus doxycycline (᭹; n = 17). Treatment was administered after onset of clinical arthritis. All treated animals are illustrated in panels a and b; those with a clinical score of 2 or less at the time of DNA injection (day 27) are illustrated in panels c and d; and animals with a clinical score greater than 2 at the start of treatment are depicted in panels e and f. Significant differences between the pGTRTT plus doxycycline and the pGTRTT without doxycycline groups are indicated (*P < 0.05, **P < 0.02 and ***P < 0.01).
TNF-α in CIA was later shown to relate directly to the stage of CIA development at the time treatment was initiated, with maximum effect achieved when treatment was started just after disease onset. The effect was less when administered 2 days after onset and it was ineffective when treatment was started 7 days after onset [22]. Essentially, where disease activity was lower anti-TNF-α was beneficial, but when disease was more advanced TNF-α inhibition was ineffective. This is akin to observations in our gene therapy experiments. By contrast, anti-TNF-α therapy causes a reversal of chronic symptoms in a large proportion of RA patients, which clearly highlights a differential outcome from anti-TNF-α therapy in CIA and human disease.
The tetracycline system has previously been utilized in CIA gene therapy for regulated expression of vIL-10 [27,36]. In one study [36] regulated expression of vIL-10 was achieved by injection of two adeno-associated virus vectors intramuscularly before immunization of DBA/1 mice, and doxycycline administration started 23 days after immunization. The study conducted by Perez and coworkers [27] involved coinjection of a single plasmid from which vIL-10 was regulated, along with a plasmid encoding the transcriptional silencer tTS. Doxycycline and plasmids (intramuscular and electroporated) were both delivered before onset of disease and resulted in a modest delay in onset and reduced incidence of arthritis.
Doxycycline is reported to have direct effects on inflammatory processes, which theoretically could be of direct benefit in the treatment of arthritis. In vitro studies have shown that doxycycline inhibits bone and cartilage breakdown [37], inhibits matrix metalloproteinases (particularly the activity of matrix metalloproteinase-13 and -8 against CII [38]), increases inducible nitric oxide synthase mRNA degradation [39], and induces Fas/Fas ligand mediated apoptosis of activated T cells [40]. However, these effects of doxycycline in general have a 50% inhibitory concentration in excess of 10 µg/ml, whereas maximal activation of the Ptet occurs at a concentration of 1 µg/ml. Indeed, no beneficial effect of doxycycline treatment with the control vector pGTRTEmpty was evident in this study, and a recent clinical trial showed no benefit of doxycycline treatment of RA patients [41]. However, the tetracycline analogue minocycline administered at a similar concentration (800 µg/ml) to that used this study was beneficial in adjuvant arthritis and CIA in rats [42].
When anti-TNF-α treatments were initiated before onset of disease in CIA and in transfer models of arthritis, effects have been observed on disease severity [4,34,35,43,44]. Changes in immune response were also noted, including reduced proinflammatory cytokine production, anti-CII antibody levels in sera [4], and changes in anti-CII immunoglobulin isotype ratio [45,46], indicating a downregulation of the T-helper-1 response.
Available online http://arthritis-research.com/content/6/2/R103 R111 Table 1 Anticollagen II antibodies in mice with collagen-induced arthritis  When gene therapy has been started at the onset of disease, the therapeutic effects of anti-TNF-α have been less consistent. Expression of a dimeric chimeric human TNFR1-IgG fusion protein from adenovirus was effective in a rat CIA model when delivered intravenously after onset (low level) of disease, but it was ineffective when delivered intra-articularly [43]. In mouse CIA, intravenous administration of adenovirus encoding the same dimeric TNFR1 molecule at onset ameliorated disease development for 10 days and was followed by rebound exacerbated disease [47]. Injection of retrovirus encoding TNFR1-IgG peri-articularly at the onset of disease inhibited its progression [48]. Injection of retrovirally transduced syngeneic fibroblasts encoding dTNFR at onset of CIA did not prevent disease development [4]. These previous studies used viral vectors, and in this regard our study is the first to observe a therapeutic effect with a TNF-α inhibitor expressed from plasmid constructs.

Anti-CII antibodies
Few studies have examined the effects of anti-TNF-α on the immune system when treatment commenced at or after the onset of CIA. Expression of dTNFR from fibroblasts injected at CIA onset did not alter anti-CII levels [4], while expression of TNFR1-immunoglobulin following injection (peri-articularly) of encoding retrovirus at onset in arthritic paws has been reported to reduce anti-CII levels, particularly IgG 2a , at 7 days after onset [48]. We were unable to show any effect of dTNFR treatment on anti-CII levels or cytokine expression from stimulated draining lymph node cells. Although we examined immunological markers at a similar time point as did Muhkerjee and coworkers [48], we used different TNF-α inhibitors, vectors and routes of delivery, and we initiated treatment after disease onset.
The observation that expression of dTNFR for 13 days in the CIA experiments was not immunogenic is encouraging. In RA patients there has been no report of immunogenicity against Etanercept ® , which also contains the extracellular domain of the human TNFR2. By contrast, expression of TNFR1-IgG encoded from adenovirus in mouse CIA led to the development of autoantibodies to mouse TNFR1 [47]. There was some evidence that these antibodies had agonistic TNF-α activity in vitro, and the immunogenicity of the TNFR1-IgG was associated with the rebound exacerbation of disease that was observed.
The overall picture that emerges is that before disease onset inhibition of TNF-α can inhibit disease development by targeting the immune response. But after onset of disease the target for TNF inhibition is to block the cascade of inflammatory cell recruitment and for optimal effect the inhibitory molecule should be delivered as soon after disease onset as possible.
This study illustrates the potential for performing gene therapy with a single injection of regulated vector in CIA.
Improvements in plasmid delivery and gene regulation may permit adoption of a similar therapeutic approach in RA patients in the future.

Conclusion
In the present study we inhibited disease progression in established CIA with dTNFR expressed from plasmid DNA in a constitutive or regulated manner. Response to anti-TNF-α therapy occurs in a large proportion of RA patients. However, in CIA inhibition of TNF-α is less potent. In the study we showed that inhibition of disease progression, by a plasmid encoding dTNFR, is mainly observed when the arthritis score is low at the start of treatment. Regulated vectors are powerful tools in gene therapy but will require stringent long-term testing in chronic models of disease in order to demonstrate their full potential. Plasmid delivery of therapeutic molecules such as TNF-α inhibitors represents a feasible and simple gene therapy approach for RA treatment and demands further improvements in gene regulation and methods of delivery.