Proteasome Inhibition Enhances AAV-Mediated Transgene ...

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doi:10.1016/j.ymthe.2004.10.020

Proteasome Inhibition Enhances AAV-Mediated Transgene Expression in Human Synoviocytes in Vitro and in Vivo Kristi Jennings,1,* Takako Miyamae,2,* Russell Traister,2 Anthony Marinov,2 Shigeki Katakura,1 Dawn Sowders,1 Bruce Trapnell,3 James M. Wilson,4 y Guangping Gao,4 and Raphael Hirsch2, 1

William S. Rowe Division of Rheumatology and 3Division of Pulmonary Biology, Children’s Hospital Medical Center, Cincinnati, OH 45229, USA 2 Division of Rheumatology, Children’s Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA 4 Institute of Human Gene Therapy, University of Pennsylvania Health System, M6.30 Maloney Building, Philadelphia, PA 19104-4283, USA *These authors contributed equally to this work. y

To whom correspondence and reprint requests should be addressed at the Division of Rheumatology, Children’s Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213, USA. Fax: (412) 692 5054. E-mail: [email protected].

Available online 15 December 2004

To explore the potential applicability of recombinant adeno-associated virus (rAAV) vectors in the treatment of rheumatoid arthritis (RA), primary human fibroblast-like synoviocytes (FLS) derived from patients with RA were infected with rAAV encoding mouse IL-10 under the control of the CMV promoter. Addition of the proteasome inhibitor carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (zLLL) to the cultures dramatically enhanced expression of the IL-10 transgene, in a dose-dependent manner. The increased expression was transient, peaking at 3 days and returning to near baseline by 7 days. The enhancement was observed even when zLLL was added 13 days after infection with rAAV. The effect of zLLL was not specific to either the mIL-10 transgene or the CMV promoter, as similar findings were observed using an rAAV construct encoding A1-anti-trypsin under the control of the chick B-actin promoter or GFP, driven by the CMV promoter. Transgene expression could be repeatedly induced by reexposure to zLLL. Transgene mRNA levels increased in parallel with protein levels. Transgene expression could also be repeatedly induced in vivo by administering zLLL to SCID mice previously injected with rAAV-infected FLS. These data demonstrate that proteasome inhibition can dramatically enhance transgene expression in human RA FLS following infection with rAAV and suggest a possible approach to regulating synovial transgene expression in vivo. Key Words: arthritis, AAV, gene therapy, proteasome, synovium

INTRODUCTION Rheumatoid arthritis (RA) and juvenile rheumatoid arthritis are diseases for which current therapies are only partially effective and associated with significant side effects. The autoimmune basis of these diseases is well established; however, the causes are unknown. The inflammation in RA is characterized by recruitment of immune cells, leading to massive thickening of the synovium accompanied by release of inflammatory mediators, ultimately leading to invasion and destruction of articular cartilage and bone [1–3]. Present therapies, while partially effective at controlling symptoms and slowing the disease course, may not

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ultimately prevent disease progression. Thus, there is a great need for innovative therapeutic approaches for arthritis. Local overexpression of anti-inflammatory proteins by synoviocytes represents an attractive approach to treatment. Synoviocytes can be readily accessed by intraarticular injection, making them good targets for gene delivery. These cells have a low mitotic rate [4–6] and therefore are likely to express transduced genes for a considerable length of time, even if the transgene is episomally located. The feasibility of direct in vivo gene transfer to synovium has been well demonstrated with adenoviral vectors in mice, rats, and rabbits [7–16]. However, adenovirusmediated synovial gene transfer results in short-term gene

MOLECULAR THERAPY Vol. 11, No. 4, April 2005 Copyright C The American Society of Gene Therapy 1525-0016/$30.00

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expression persisting only for 1 to 2 weeks, accompanied by an antiviral inflammatory response [7,9,10]. It therefore represents an unlikely candidate vector for a chronic inflammatory process, such as RA, in which long-term expression of therapeutic gene products will be required. Recombinant adeno-associated virus (rAAV) has recently been demonstrated to mediate very long term gene transfer in a number of tissues. Its low immunogenicity makes this vector attractive for the treatment of chronic diseases. Furthermore, its lack of apparent pathogenicity in humans makes its safety profile compelling. rAAV is capable of transducing a variety of tissues, including skeletal muscle [17,18], liver [19], neurons [20–23], and retina [24]. In a recent study, we investigated the use of rAAV for in vivo gene transfer to mouse synovium [25]. Intraarticular injection of mouse knee joints resulted in transgene expression in both periarticular and synovial lining, with the majority of expression in the periarticular areas. The synovial lining consists of type A (macrophage-like) and type B (fibroblast-like) synoviocytes. The ability of rAAV to transduce fibroblasts is species-specific [26,27] and therefore, the poor transducibility of mouse synovial lining does not rule out the potential use of rAAV to target human synovium. Proteasome inhibitors have recently been demonstrated to enhance rAAV-mediated transgene expression in fibroblasts and pulmonary epithelial cells [26,28–30]. Proteasomes modulate the intracellular processing of many foreign antigens and endogenous molecules, including viruses. Several specific, cell-permeable, peptide aldehyde inhibitors of proteasome pathways have been identified that bind to the active sites of proteolytic enzymes within the proteasome core and reversibly block their function [31–33]. These include N-acetyl-l-leucyl-lleucylnorleucine (also called calpain inhibitor I) and carbobenzoxyl-l-leucyl-l-leucyl-l-leucinal (zLLL; also called MG132). In vivo administration of these proteasome inhibitors to mouse lung augmented rAAV2 gene transfer from undetectable levels to a mean of 10% of epithelial cells [29]. Significant in vivo enhancement of transgene expression for over 150 days was observed in mouse lungs without any toxicity, even at doses of proteasome inhibitor 1000-fold higher than necessary for transduction. These proteasome inhibitors, when coadministered with rAAV, also increased rAAV-mediated transgene expression in the liver by 10-fold, but did not affect expression in muscle, suggesting that barriers to rAAV-mediated gene transfer are organ- or tissue-specific. Viral binding and internalization were not adversely affected by the inhibitors. However, a substantial shift in the distribution of virions to the nucleus was observed. Proteasome inhibitors have been reported to increase nuclear accumulation of rAAV particles and the rAAV genome [26,28,29]. The present study investigated the effects of proteasome inhibition on rAAV-mediated transgene expression in human fibroblast-like synoviocytes (FLS).

MOLECULAR THERAPY Vol. 11, No. 4, April 2005 Copyright C The American Society of Gene Therapy

RESULTS Proteasome Inhibition Markedly Enhances rAAV-Mediated Transgene Expression in RA FLS We infected human RA FLS with 1 104 particles/cell of rAAV(mIL-10). During the time of infection, we exposed the cells to the proteasome inhibitor zLLL, which has been reported to increase nuclear accumulation of rAAV particles and the rAAV genome in other cell types [26,28,29]. Incubation with zLLL dramatically enhanced expression of a mouse IL-10 transgene, 3 days following infection with rAAV, in a dose-dependent manner (Fig. 1). Concentrations higher than 40 AM were toxic to the cells. The increased expression was transient, such that by day 7, expression had returned to near baseline. zLLL is a peptide aldehyde and can also inhibit certain lysosomal cysteine proteases and the calpains. Lactacystin, which shows high specificity for the proteasome, showed results similar to those of zLLL (data not shown). To determine whether the increased transgene expression required exposure to zLLL at the time of infection, we added 40 AM zLLL 13 days following infection of FLS with rAAV(mIL-10). We observed enhanced transgene expression (Fig. 2A) similar to that observed when the zLLL was administered at the time of infection. Expression peaked 3 days following exposure to zLLL and returned to baseline over a 7-day period. The effect of zLLL was not specific either to the mIL-10 transgene or to the CMV promoter, as we observed similar findings using an rAAV construct encoding a1anti-trypsin under the control of the chick h-actin promoter (Fig. 2B). Similarly, FLS infected with rAAV encoding GFP, driven by the CMV promoter, showed

FIG. 1. zLLL enhances rAAV-mediated transduction of RA FLS. RA FLS were infected with 1 104 particles/cell of rAAV(mIL-10) in the presence of increasing concentrations of zLLL. Supernatants were assayed by ELISA for mIL-10 on days 3 and 7 after infection. Each time point represents the amount of transgene product secreted into the supernatant over a 24-h period. The experiment was repeated three times, with similar results.

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FIG. 2. Effects of time of exposure, promoter, and transgene on zLLL-induced transgene expression. RA FLS were infected with 1 104 particles/cell of (A) rAAV(mIL-10) or (B) rAAV(a1-anti-trypsin). zLLL (40 AM) was added to some wells 13 days later (arrows) and supernatants were assayed at various time points for mIL-10. Each time point represents the amount of transgene product secreted into the supernatant over a 24-h period. The experiment was repeated three times, with similar results.

marked enhancement of transgene expression following exposure to zLLL (Fig. 3). Proteasome Inhibitor Can Regulate Transgene Expression in Human RA FLS in Vitro We were surprised to observe that the zLLL-induced increase in transgene expression was transient, such that by day 7, expression returned to baseline. This is inconsistent with the proposed mechanism of action of zLLL, that of increasing viral trafficking to the nucleus, as transgene expression should be stable once the viral genome reaches the nucleus and sufficient second-strand synthesis occurs. To explore this finding further, we reexposed synovial cells to zLLL following the loss of transgene expression. Transgene expression could be

FIG. 3. Flow cytometric analysis of eGFP transgene expression in rAAV(eGFP)infected RA FLS treated with zLLL. RA FLS were infected with 1 104 particles/ cell of rAAV(eGFP). Twelve days after infection, some cells were treated with 20 AM zLLL. Three days after the addition of zLLL, the cells were harvested and analyzed in the FITC channel on a FACSCalibur (Becton–Dickinson). The experiment was repeated three times, with similar results.

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repeatedly induced by reexposure to zLLL (Fig. 4A). In each case, expression peaked 2-3 days following exposure to zLLL and then dropped off. We were able to reinduce transgene expression as far out as 32 days after infection. This is in contrast to co-infection with Ad(BglII), a recombinant adenovirus lacking a transgene, which resulted in a stable increase in transgene product (Fig. 4B). Proteasome Inhibitor Can Regulate Transgene Expression in Human RA FLS in Vivo To determine whether proteasome inhibitors would regulate transgene expression in RA FLS in vivo, we infected cells with rAAV(mIL-10) and injected them intraperitoneally into SCID mice. At various times, we administered the mice 1 ml of a 40 AM solution of zLLL in PBS by intraperitoneal (ip) injection and collected sera 3 and 7 days later for measurement of mIL-10. As was observed in vitro, administration of zLLL led to a transient increase in transgene product that could be reinduced with additional treatments (Fig. 5). While the serum levels of IL-10 were lower than observed in vitro, due to a dilutional effect, the results were reproducible. Transgene mRNA Levels Increase in Parallel With Protein Levels in Response to Proteasome Inhibitor Since proteasome inhibitors are known to increase nuclear trafficking of AAV DNA in some cell types, the effects of zLLL on AAV transport were investigated. We isolated cytoplasmic and nuclear fractions from rAAVinfected cells at various time points prior to and after exposure to zLLL and the fractions were analyzed for the presence of AAV DNA (Fig. 6). The majority of the AAV DNA was located in the nucleus within 4 h of infection. However, at 3 days postinfection, we noted increased DNA in the cytoplasm, apparently due to trafficking of AAV DNA between the nucleus and the cytoplasm.


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RNA or protein levels of the transgene product. For instance, transgene DNA levels in the nucleus were similar before and after a second exposure to zLLL (Fig. 6) even though amount of transgene product was markedly different (Fig. 4). To explore further the mechanism by which zLLL increased transgene product, its effects on transgene mRNA were analyzed. We exposed FLS transduced with AAV(mIL-4) to 40 AM zLLL at the time of infection and again at day 13 after infection. Enzyme-linked immunosorbent assay (ELISA) of the supernatants showed an increase in transgene protein levels 2-3 days following each zLLL exposure (Fig. 7). We harvested cells at several time points: 2 days after the first dose of zLLL, before the second addition of zLLL, and 2 days after the second addition of zLLL. We analyzed the mRNA from these samples by quantitative real-time PCR for mIL-4 and GAPDH. zLLL induced a 16-fold increase in mIL-4 mRNA 2 days following the first dose of zLLL (Fig. 6). mIL-4 mRNA was undetectable in the absence of exposure to zLLL. As with the protein levels, the mIL-4 mRNA levels in cells exposed to zLLL had returned to baseline by day 11. Reexposure to zLLL on day 11 led to a second increase in both protein and mRNA levels, albeit smaller than after the first exposure. The observed increase in transgene mRNA following zLLL exposure could be a result of a number of mechanisms, including upregulation of gene transcription or increase in mRNA stability.

DISCUSSION The proteasome represents the major nonlysosomal process for degradation of proteins by ATP/ubiquitin-

FIG. 4. zLLL can regulate rAAV-mediated transduction of RA FLS in vitro. RA FLS were infected with 1 104 particles/cell of rAAV(mIL-10) in the presence or absence of (A) 40 AM zLLL or (B) 100 particles/cell of the recombinant adenovirus Ad(BglII), which lacks a transgene. Arrows indicate exposure to zLLL or adenovirus (Ad). The supernatants were assayed by ELISA for mIL-10 on the days indicated. Each time point represents the amount of transgene product secreted into the supernatant over a 24-h period.

Addition of zLLL led to a transient loss of AAV DNA from the cytoplasm that was maximal at 24 h and was followed by a return of AAV DNA to the cytoplasm. A second exposure to zLLL 7 days later again resulted in a similar transient loss of cytoplasmic AAV DNA. This observation supports the role of proteasome inhibitor in enhancing transport of AAV DNA to the nucleus. However, there was no obvious correlation between nuclear DNA and either


FIG. 5. zLLL can regulate rAAV-mediated transduction of RA FLS in vivo. RA FLS were infected with 1 104 particles/cell of rAAV(mIL-10). After in vitro expansion for 10 days, 2.4 107 cells were injected intraperitoneally into SCID mice. At various time points (arrows), zLLL (40 AM in 1 ml PBS) was administrated ip to one mouse. Sera were collected and titers of mIL-10 were determined by ELISA 3 and 7 days after each zLLL administration. Each line represents an individual mouse and the experiment was repeated three times, with similar results.

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FIG. 6. Effects of zLLL on AAV DNA trafficking. RA FLS were infected with 1 104 particles per cell of rAAV(IL-10). zLLL was added 3 days after infection and again 7 days later. Cytoplasmic and nuclear DNA was isolated at the indicated time points. The first two time points are relative to the time of infection and all subsequent time points are relative to the addition of zLLL. Slot-blot analysis using a transgenespecific biotinylated probe was used to detect the viral DNA in each fraction. The probe did not bind to DNA from uninfected cells (not shown). The studies were repeated to ensure reproducibility, with similar findings.

dependent proteolysis. The bulk of proteins in mammalian cells are hydrolyzed by a distinct pathway that requires ATP and the proteasome particle. In this pathway, which is present in both the nucleus and the cytosol, most substrates are first marked for degradation by covalent linkage to multiple molecules of ubiquitin [34]. This includes certain transcription factors and ratelimiting enzymes. In our studies using human FLS, we saw accumulation of the majority of AAV viral DNA in the nucleus within 4 h after infection. Interestingly, 3 days after

FIG. 7. Transgene protein correlates with mRNA levels in rAAV-infected RA FLS in response to zLLL. RA FLS were infected with 1 104 particles/cell of rAAV(mIL-4) in the presence or absence of 40 AM zLLL. Cells exposed to zLLL were exposed again on day 13. Arrows indicate exposure to zLLL. The supernatants were assayed by ELISA for mIL-10 on the days indicated. mRNA expression levels of mIL-4 were analyzed by real-time reverse transcription PCR and normalized to hGAPDH.

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infection there appeared to be a shift of some of the viral DNA back to the cytoplasm. This is similar to the effect that Xiao et al. saw in AAV-infected HeLa cells in the presence of adenovirus co-infection [35]. Although they dismissed this observation as being due to disruption of nuclei caused by adenovirus cytopathology, our data suggest that this phenomenon may be real. Even more suggestive is the fact that multiple additions of zLLL appear to shift AAV viral DNA from the cytoplasm back to the nucleus soon after addition, with some viral DNA once again returning to the cytoplasm after 48 h. This effect might account for some, but not all, of the increase in transgene seen upon zLLL addition, since 72 h after proteasome inhibition, when maximum transgene protein is observed, the levels of viral DNA in the nucleus are comparable to the levels present prior to zLLL addition. Our data show that zLLL also affects transgene expression at the level of gene transcription and/or mRNA stability. Since nuclear translocation of AAV has previously been shown to be independent of the nuclear pore complex, one possibility is that AAV virions and/ or DNA are stored in a perinuclear compartment that may fuse with the nuclear envelope upon zLLL addition, thereby releasing its contents into the nucleus. In the same fashion, a mechanism for transporting the viral DNA back out of the nucleus could also exist. Further studies on the nuclear trafficking of AAV in human FLS and other cell types are needed to understand these observations fully. The present study demonstrates that the rAAV-mediated transgene expression in human RA FLS can be dramatically enhanced by proteasome inhibition. The most striking observation was that transgene expression could be regulated by repeated exposure to proteasome inhibitors. This was surprising, in light of previous studies suggesting that proteasome inhibitors enhance rAAVmediated transgene expression by increasing nuclear accumulation of rAAV particles and DNA [28,29]. While nuclear transport of rAAV is likely to explain partly the effects of proteasome inhibition, the data presented here suggest that there must be additional mechanisms at


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work. If proteasome inhibitors acted solely by increasing nuclear accumulation of AAV DNA, then once the cells were exposed to the proteasome inhibitor expression is likely to be relatively stable. However, proteasome inhibitors induced a transient and repeatable increase in transgene expression. The effect was observed even when the proteasome inhibitor was added 2 weeks after infection, suggesting a point of action downstream of binding and internalization of the AAV particle. Douar et al. demonstrated a 25- to 50-fold increase in transduction efficiency of HepG2 and HeLa cells in the presence of zLLL [28] and concluded that a significant portion of AAV particles are degraded by the proteasome. It is unclear, however, how the proteasome could degrade intact viral particles, as they are too large to fit into the proteasome structure and would need first to be degraded into proteins that could be ubiquitinated. Proteasome inhibitors may, however, increase the concentration of a protein that enhances nuclear transport or rAAV. Thus, one possibility is that the AAV is harbored in a stable cytoplasmic compartment and that a portion is released and transported to the nucleus upon each exposure to the proteasome inhibitor. However, this would not explain the transient nature of expression for the rAAV that reached the nucleus, unless the rAAV genome is unstable in the nucleus. A more likely explanation is that the AAV genome is transported to the nucleus in an inactive form until acted upon, either directly or indirectly, by the proteasome inhibitor. Proteasomes have been found to degrade numerous transcription factors, which is a likely mechanism to explain the results described here. Potential effects of proteasome inhibitors on protein regulators of gene transcription are of particular interest. For example, such molecules have been shown to influence NF-nB-dependent gene transcription by blocking the degradation of InB [36]. Alterations in tumor suppressor genes, such as p53 [37] and PTEN [38], have also been described. The correlation of enhanced protein expression with increased transgene mRNA suggests an effect on transcription, although enhanced RNA stability is also a possibility. The effect was observed with different transgenes and different promoters, suggesting that this is not idiosyncratic but a general phenomenon. Studies are currently under way to elucidate further the mechanism of action of proteasome inhibitors on rAAVmediated transgene expression in RA FLS. There have been a number of reports on the use of rAAV in models of arthritis [25,39,40]. These studies have provided somewhat conflicting findings; however, mouse synovium appears to be relatively resistant to gene transfer by rAAV. This is consistent with other studies showing that mouse fibroblasts are poorly transduced by rAAV, even though they express rAAV receptors and are permissive for second-strand synthesis [26,27]. The data presented herein demonstrate that the transgene expression in human FLS by rAAV can be dramatically enhanced by proteasome


inhibition. We were unable to duplicate this effect of proteasome inhibition in mouse FLS (data not shown), suggesting that the mouse may not be the most appropriate model for studying rAAV in arthritis. As one of the key safety issues in the application of gene therapy is the ability to control and regulate transgene expression, the finding that proteasome inhibitors can regulate expression in rAAV-transduced RA FLS in vivo suggests a possible approach to the use of rAAV in arthritis. One potential complication to such an approach is that an arthritic joint is characterized by infiltration with inflammatory cells, including neutrophils, lymphocytes, and monocytes. While such cells are generally poorly permissive for AAV, it is possible that administration of rAAV into an inflamed joint might lead to infection of these inflammatory cells, in addition to synoviocytes. It is unclear whether transgene expression in such cells would be influenced in the same way by proteasome inhibitors. The degree to which inflammatory cells in the joint are transduced by rAAV, and the effects of proteasome inhibition on transgene expression in these cells, would need to be determined before such an approach should be tested in the clinical setting. Proteasome inhibitors have been administered to patients as an experimental therapy for cancer without substantial toxicity [41]. Their use in a chronic disease such as RA would require further investigation.

MATERIALS

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METHODS

Mice. Female SCID mice, 5 to 8 weeks of age, were purchased from the National Cancer Institute (Frederick, MD, USA) and housed under Institutional Animal Care and Use Committee-approved conditions in the animal facility at Children’s Hospital of Pittsburgh. Isolation of fibroblast-like synoviocytes. Human RA synovial tissues, obtained during joint replacement surgery, were finely chopped, washed with sterile PBS, resuspended in 4 mg/ml collagenase (Worthington Biochemical Corp., NJ, USA), and incubated in a 378C CO2 humidified incubator overnight. After being washed with PBS, dissociated cells were resuspended in RPMI supplemented with 10% fetal calf serum and adherent cells were passaged in culture for 2–4 weeks. Adherent cell lines were shown to have the phenotype of fibroblast-like synoviocytes (CD90+, CD14) by flow cytometry. Recombinant AAV. The vectors rAAV(mIL-4), encoding the murine IL-4 cDNA; rAAV(mIL-10), encoding the murine IL-10 cDNA; and rAAV(eGFP), encoding the green fluorescent protein (GFP) cDNA, were driven by the CMV promoter, while rAAV(a1AT), encoding the human a1-anti-trypsin cDNA, was driven by the chicken h-actin promoter. All rAAV constructs were derived from AAV2 and were generated by either 293/triple transfection or B50/hybrid method [42]. Adenovirus helper functions were supplemented by pAdDF6, a plasmid construct carrying all essential adenovirus helper genes [42]. Transfection was carried out using the standard calcium phosphate precipitation method. rAAV vector preps were purified by CsCl gradient centrifugation [43]. The genome titers of vector preps were determined by the real-time quantitative PCR method [42], whereas the transducing titers of vector preps were assayed on 84-31 cells as described elsewhere [44]. Virus was stored at 808C in buffer containing 20 mM Tris, pH 7.4, 1 mM MgCl2, 150 mM NaCl, and 10% (v/v) glycerol.

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Recombinant adenovirus. Ad(BglII) is an E1a/E3-deleted replicationdefective adenovirus type 5 backbone vector lacking a transgene and was generously provided by J. A. Bluestone and J. M. Leiden. Recombinant adenovirus was produced and propagated in 293 cells and purified by cesium chloride density centrifugation, as previously described [45,46]. Virus plaques were purified three times before the production of seed stocks, and their identities were confirmed by restriction endonuclease and DNA sequence analysis. Viral titers (particles per milliliter) were calculated by OD260 1012 following lysis of viral stocks in 0.1% SDS, 10 mM Tris-HCl (pH 7.4), and 1 mM EDTA at 568C for 10 min. Virus was stored at 808C in buffer containing 10 mM Tris, pH 7.4, 1 mM MgCl2, 10% (v/v) glycerol. In vitro infection. RA FLS were plated at a density of 5 104 cells per well in 24-well plates and incubated at 378C in a 5% CO2 atmosphere in RPMI medium supplemented with 10% fetal calf serum. Cells were infected with 1 104 particles/cell of rAAV by incubation for 24 h, followed by removal of the virus and replacement with fresh medium. In some cases, zLLL (also called MG-132, purchased from Calbiochem, La Jolla, CA, USA) was added for 24 h either at the time of infection or at later time points. zLLL was prepared as a 35 mM stock solution in DMSO and diluted in RPMI containing 10% fetal calf serum prior to use. Assays for mIL-4, mIL-10, and a1-anti-trypsin. Concentrations of mIL-4 and mIL-10 in cell supernatants were determined by ELISA, as previously described [25]. The concentration of a1-anti-trypsin was also determined by ELISA. Ninety-six-well flat-bottom ELISA plates (Nalge Nunc, Rochester, NY, USA) were coated with 100 Al of 1:500 rabbit anti-a1-anti-trypsin (Sigma, St. Louis, MO, USA) and incubated overnight at 48C. Plates were washed with PBS-Tween, blocked with 1% bovine serum albumin in PBS, and washed again. Supernatant samples were incubated in duplicate wells overnight at 48C. Plates were washed and incubated with peroxidaseconjugated anti-a1-anti-trypsin antibody (EY Laboratories, San Mateo, CA, USA). Plates were washed and developed with ABTS peroxidase substrate (Kirkegaard & Perry) and read at 410 nm with a Kinetic Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). Assays were performed in duplicate wells and the results averaged. In all cases, duplicates differed by b5%. Real-time reverse transcription PCR. Cells were snap frozen in liquid nitrogen and stored at 808C until used. Total RNA was extracted using TRIzol Reagent (Gibco Life Technologies, Rockville, MD, USA) according to the manufacturer’s instructions. RNA concentrations were measured by spectrophotometry. To remove possible genomic DNA contamination, total RNA was treated with amplification-grade DNase I (Gibco Life Technologies). RNA was then subjected to reverse transcription using the SuperScript Preamplification System for First-Strand cDNA Synthesis (Gibco Life Technologies). Serial dilutions of the cDNA template were prepared and PCR was carried out using a LightCycler System (Roche Molecular Biochemicals, Palo Alto, CA, USA). After each elongation phase, the fluorescence of SYBR Green I, which binds double-stranded DNA, was measured. Reactions (10 Al) were performed in microcapillary tubes using 5 Al of diluted cDNA with FastStart DNA SYBR Green I Master Mix (Roche Molecular Biochemicals), upstream and downstream primers, and MgCl2. Sequences of primer pairs were as follows: human glyceraldyhyde-3-phosphate dehydrogenase (hGAPDH), upstream 5V-GCTCQ TCCAGAACATCATCC-3V, downstream 5V-CAGCCCCAGCGTCAAAGG-3V; mouse IL-4 (mIL-4) upstream 5V-GAATGTACCAGGAGCCATATC-3V, downstream 5V-CTCAGTACTACGAGTAATCCA-3V. Reactions containing water, or cDNA synthesized without reverse transcriptase as template, resulted in no PCR products. LightCycler quantification software v3 was used to compare amplification in experimental samples during the loglinear phase to the standard curve from the dilution series of cells at the earliest time point transfected with AAV(mIL-4) and treated with zLLL, which were predicted to have the highest expression of mIL-4. Expression of mIL-4 was normalized to that of hGAPDH at each time point. In vivo RA FLS and zLLL administration. RA FLS were plated at a density of 5 104 cells per well in 24-well plates and incubated at 378C in a 5% CO2 atmosphere in RPMI medium supplemented with 10% fetal calf

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serum. Cells were infected with 1 104 particles/cell of rAAV. After in vitro expansion for 10 days, 2.4 107 cells were injected ip into SCID mice. At various time points, zLLL (40 AM in 1 ml PBS) was administered ip. Sera were collected and titers of IL-10 were determined by ELISA 3 and 7 days after each zLLL administration. Analysis of nuclear and cytoplasmic rAAV DNA. Human fibroblast-like synoviocytes infected with 1 104 particles per cell of rAAV(IL-10) were collected at various time points, washed with PBS, and resuspended in 0.5 ml lysing buffer (1.3 M sucrose, 20 mM MgCl2, 4 mM Tris, 4.2% Triton X100) and 1.5 ml ice-cold water and incubated on ice for 10 min. The nuclei were pelleted by spinning at 1400 rpm for 15 min at 48C. The supernatant containing the cytoplasmic fraction was transferred to a new tube and the nuclear pellet was washed with 2 ml PBS. Both tubes were spun again at 1400 rpm for 15 min. The cytoplasmic fraction was transferred to a new tube and 25 Al of RNase A (200 U/ml) was added. Viral nuclear DNA was isolated using a Qiagen plasmid maxi kit. After isolation, both the cytoplasmic and the nuclear fractions were treated with 50 Al proteinase K (Invitrogen, 10 mg/ml) and incubated at 508C for 1 h. The samples were cooled to room temperature and 10 Al of glycogen (Boehringer Mannheim, 20 mg/ml) was added. The DNA was precipitated with isopropanol, washed with 70% ethanol, air dried, and resuspended in water. Successful isolation of the cytoplasmic and nuclear fractions was confirmed using a cytoplasmic phosphatase assay and Western blotting for histone H3, as previously described [26], and by PCR. Cytoplasmic and nuclear DNA fractions were applied to nitrocellulose using a Bio-Dot SF Microfiltration Apparatus (Bio-Rad). A 500-bp biotinylated probe specific for a region within the mouse IL-10 transgene was prepared using the Random Prime DNA Labeling System with biotin-14-dCTP (Invitrogen). Hybridization, washing, and blocking were performed according to the manufacturer’s instructions. A 1:2000 dilution of SAHRP (Zymed) was applied. Supersignal West Femto Maximum Sensitivity Substrate (Pierce) was added and the blot was exposed to X-OMAT film (Kodak).

ACKNOWLEDGMENT This work was supported, in part, by NIH Grant AI34958. RECEIVED FOR PUBLICATION SEPTEMBER 14, 2004; ACCEPTED OCTOBER 24, 2004.

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