Helper virus-free gutless adenovirus (HF-GLAd): a new platform for gene therapy.

Gene therapy is emerging as a treatment option for inherited genetic diseases. The success of this treatment approach greatly depends upon gene delivery vectors. Researchers have attempted to harness the potential of viral vectors for gene therapy applications over many decades. Among the viral vectors available, gutless adenovirus (GLAd) has been recognized as one of the most promising vectors for in vivo gene delivery. GLAd is constructed by deleting all the viral genes from an adenovirus. Owing to this structural feature, the production of GLAd requires a helper that supplies viral proteins in trans. Conventionally, the helper is an adenovirus. Although the helper adenovirus efficiently provides helper functions, it remains as an unavoidable contaminant and also generates replicationcompetent adenovirus (RCA) during the production of GLAd. These two undesirable contaminants have raised safety concerns and hindered the clinical applications of GLAd. Recently, we developed helper virus-free gutless adenovirus (HF-GLAd), a new version of GLAd, which is produced by a helper plasmid instead of a helper adenovirus. Utilization of this helper plasmid eliminated the helper adenovirus and RCA contamination in the production of GLAd. HF-GLAd, devoid of helper adenovirus and RCA contaminants, will facilitate its clinical applications. In this review, we discuss the characteristics of adenoviruses, the evolution and production of adenoviral vectors, and the unique features of HF-GLAd as a new platform for gene therapy. Furthermore, we highlight the potential applications of HF-GLAd as a gene delivery vector for the treatment of various inherited genetic diseases. [BMB Reports 2020; 53(11): 565-575].

No more helper adenovirus: production of gutless adenovirus (GLAd) free of adenovirus and replication-competent adenovirus (RCA) contaminants.

Gene therapy is emerging as an effective treatment option for various inherited genetic diseases. Gutless adenovirus (GLAd), also known as helper-dependent adenovirus (HDAd), has many notable characteristics as a gene delivery vector for this particular type of gene therapy, including broad tropism, high infectivity, a large transgene cargo capacity, and an absence of integration into the host genome. Additionally, GLAd ensures long-term transgene expression in host organisms owing to its minimal immunogenicity, since it was constructed following the deletion of all the genes from an adenovirus. However, the clinical use of GLAd for the treatment of inherited genetic diseases has been hampered by unavoidable contamination of the highly immunogenic adenovirus used as a helper for GLAd production. Here, we report the production of GLAd in the absence of a helper adenovirus, which was achieved with a helper plasmid instead. Utilizing this helper plasmid, we successfully produced large quantities of recombinant GLAd. Importantly, our helper plasmid-based system exclusively produced recombinant GLAd with no generation of helper plasmid-originating adenovirus and replication-competent adenovirus (RCA). The recombinant GLAd that was produced efficiently delivered transgenes regardless of their size and exhibited therapeutic potential for Huntington's disease (HD) and Duchenne muscular dystrophy (DMD). Our data indicate that our helper plasmid-based GLAd production system could become a new platform for GLAd-based gene therapy.

Breathing New Life into TRAIL for Breast Cancer Therapy: Co-Delivery of pTRAIL and Complementary siRNAs Using Lipopolymers.

Preclinical studies showed that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) therapy is safe and effective to combat cancers, but clinical outcomes have been less than optimal due to short half-life of TRAIL protein, insufficient induction of apoptosis, and TRAIL resistance displayed in many tumors. In this study, we explored co-delivery of a TRAIL expressing plasmid (pTRAIL) and complementary small interfering RNAs (siRNAs) (silencing Bcl2-like 12 [BCL2L12] and superoxide dismutase 1 [SOD1]) to improve the response of breast cancer cells against TRAIL therapy. It is desirable to co-deliver the pDNA along with siRNA using a single delivery agent, but this is challenging given different structures of long/flexible pDNA and short/rigid siRNA. Toward this goal, we identified an aliphatic lipid-grafted low-molecular weight polyethylenimine (PEI) that accommodated both pDNA and siRNA in a single complex. The co-delivery of pTRAIL with BCL2L12- or SOD1-specific siRNAs resulted more significant cell death in different breast cancer cells compared with separate delivery without affecting nonmalignant cells viability. Ternary complexes of lipopolymer with pTRAIL and BCL2L12 siRNA significantly retarded the growth of breast cancer xenografts in mice. The enhanced anticancer activity was attributed to increased in situ secretion of TRAIL and sensitization of breast cancer cells against TRAIL by the co-delivered siRNAs. The lipid-grafted PEIs capable of co-delivering multiple types of nucleic acids can serve as powerful carriers for more effective complementary therapeutics. Graphical Abstract [Figure: see text].

Enhancement of TRAIL-induced apoptosis by 5-fluorouracil requires activating Bax and p53 pathways in TRAIL-resistant lung cancers.

Lung cancer, especially lung adenocarcinoma, is one of the main causes of death worldwide. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a primary anticancer agent and a member of the tumor necrosis factor family that selectively induces apoptosis in various tumor cells, but not in normal cells. Combination chemotherapy can be used for treating specific cancer types even at progressive stages. In the present study, we observed that 5-fluorouracil, which exerts anticancer effects by inhibiting tumor cell proliferation, enhanced TRAIL-induced apoptosis of TRAIL-resistant human adenocarcinoma A549 cells. Interestingly, 5-fluorouracil treatment markedly increased Bax and p53 levels and 5-fluorouracil and TRAIL cotreatment increased Ac-cas3 and Ac-cas8 levels compared with those in control cells. Taken together, the present study demonstrated that 5-fluorouracil enhances TRAIL-induced apoptosis in TRAIL-resistant lung adenocarcinoma cells by activating Bax and p53, and also suggest that TRAIL and 5-fluorouracil cotreatment can be used as an adequate therapeutic strategy for TRAIL-resistant human cancers.

DUSP6 is a novel transcriptional target of p53 and regulates p53-mediated apoptosis by modulating expression levels of Bcl-2 family proteins.

p53 regulates various cellular responses through transcriptional regulation of distinct sets of target genes. Dual specificity phosphatase 6 (DUSP6) is a cytosolic phosphatase that inactivates the extracellular-signal-regulated kinase 1/2 (ERK1/2). This study demonstrates that p53 transactivates DUSP6 in human colorectal HCT116 cells to regulate ERK1/2 in p53-mediated cell death. DUSP6 is transactivated by p53 overexpression and genotoxic agents, and chromatin immunoprecipitation revealed two p53-binding sites in the DUSP6 promoter responsible for DUSP6 induction. Expression of shDUSP6 inhibited 5'-FU-induced cell death, whereas overexpression of DUSP6 increased susceptibility to 5'-FU. 5'-FU treatment dephosphorylated ERK in a DUSP6-dependent manner, resulting in destabilization of Bcl-2 and stabilization of Bad. These results provide insights on the modulatory role of p53 in the survival pathway by up-regulating DUSP6.

p53-Independent up-regulation of a TRAIL receptor DR5 by proteasome inhibitors: a mechanism for proteasome inhibitor-enhanced TRAIL-induced apoptosis.

Gliomas are the most common brain tumors in adults and account for more than half of all brain tumors. Despite intensive clinical investigations, average survival for the patients harboring the malignancy has not been significantly improved. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), shown to have potent and cancer-selective killing activity, has drawn considerable attention as a promising anti-cancer therapy. In an attempt to develop TRAIL as an anti-cancer therapy for gliomas, tumor suppressor activity of TRAIL was assessed using human glioma cell lines such as U373MG, U343MG, U87MG and LN18. U343MG, U87MG and LN18 cells were susceptible to TRAIL; however, U373MG cells were completely refractory to TRAIL. Resistance to the applied therapies is a key issue in cancer treatment; thus, various combination treatments were evaluated using U373MG cells to identify a better regimen. Unlike Doxorubicin, Etoposide, Actinomycin D and Wortmannin, a proteasome inhibitor MG132 significantly enhanced TRAIL-induced apoptosis. Similarly, other proteasome inhibitors, including Lactacystin, Proteasome inhibitor I and Velcade (Bortezomib), also enhanced apoptotic activity of TRAIL. Among these proteasome inhibitors, Velcade, the only approved drug, was as effective as MG132 in enhancing TRAIL-induced apoptosis. Both Velcade and MG132 increased the protein levels of DR5, a TRAIL receptor known to be up-regulated by p53, in U373MG cells where p53 is mutated. Our data indicate that proteasome inhibitors up-regulate DR5 in a p53-independent manner and a combination therapy comprising TRAIL and Velcade become a better treatment regimen for gliomas.

Suppression of IRF4 by IRF1, 3, and 7 in Noxa expression is a necessary event for IFN-γ-mediated tumor elimination.

IFN-γ plays a critical role in tumor immunosurveillance by affecting either immune cells or tumor cells; however, IFN-mediated effects on tumor elimination are largely unknown. In this study, we showed that IFN regulatory factors (IRF) modulated by IFNs up- and downregulated Noxa expression, a prodeath BH3 protein, in various cancer cells. Inhibition of Noxa expression using short hairpin RNA in tumor cells leads to resistance against lipopolysaccharide (LPS)-induced tumor elimination, in which IFN-γ is known as a critical effecter in mice. Chromatin immunoprecipitation analysis in both CT26 cells and SP2/0 cells, sensitive and resistant to LPS-induced tumor elimination, respectively, revealed that the responsiveness of IRF1, 3, 4, and 7 in the Noxa promoter region in response to IFN-γ might be crucial in LPS-induced tumor elimination. IRF1, 3, and 7 were upregulated by IFN-γ and activated Noxa expression, leading to the death of Noxa wild-type baby mouse kidney (BMK) cells but not of Noxa-deficient BMK cells. In contrast, IRF4 acts as a repressor for Noxa expression and inhibits cell death induced by IRF1, 3, or 7. Therefore, although IFN-γ alone are not able to induce cell death in tumor cells in vitro, Noxa induction by IFN-γ, which is regulated by the balance between its activators (IRF1, 3, and 7) and its repressor (IRF4), is crucial to increasing the susceptibility of tumor cells to immune cell-mediated cytotoxicity.

Preclinical studies for pharmacokinetics and biodistribution of Ad-stTRAIL, an adenovirus delivering secretable trimeric TRAIL for gene therapy.

Malignant glioma is the most frequent type in brain tumors. The prognosis of this tumor has not been significantly improved for the past decades and the average survival of patients is less than one year. Thus, an effective novel therapy is urgently needed. TNF-related apoptosis inducing ligand (TRAIL), known to have tumor cell-specific killing activity, has been investigated as a novel therapeutic for cancers. We have developed Ad-stTRAIL, an adenovirus delivering secretable trimeric TRAIL for gene therapy and demonstrated the potential to treat malignant gliomas. Currently, this Ad-stTRAIL gene therapy is under phase I clinical trial for malignant gliomas. Here, we report preclinical studies for Ad-stTRAIL carried out using rats. We delivered Ad-stTRAIL intracranially and determined its pharmacokinetics and biodistribution. Most Ad-stTRAIL remained in the delivered site and the relatively low number of viral genomes was detected in the opposite site of brain and cerebrospinal fluid. Similarly, only small portion of the viral particles injected was found in the blood plasma and major organs and tissues, probably due to the brain-blood barrier. Multiple administrations did not lead to accumulation of Ad-stTRAIL at the injection site and organs. Repeated delivery of Ad-stTRAIL did not show any serious side effects. Our data indicate that intracranially delivered Ad-stTRAIL is a safe approach, demonstrating the potential as a novel therapy for treating gliomas.

Effective knockdown of multiple target genes by expressing the single transcript harbouring multi-cistronic shRNAs.

Gene silencing by RNA interference (RNAi) using short interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a valuable tool for evaluating the target gene function. Here, we report an approach for silencing multiple target genes simultaneously by expressing one single transcript encoding different target shRNAs. We first constructed the cytomegalovirus (CMV) promoter-driven expression vectors, each of which expresses microRNA mir-30-mimicked shRNA specifically targeting X-chromosome-linked inhibitor of apoptosis protein (XIAP), Akt, or Bcl-2. Adenovirus harbouring each shRNA expression cassette silenced corresponding target gene expression. Using these mono-cistronic shRNA cassettes, we again constructed the CMV promoter-driven expression vector, into which multi-cistronic shRNAs for XIAP, Akt and Bcl-2 in order were cloned. Adenovirus delivering this multi-cistronic expression cassette silenced each of the target genes as effectively as adenovirus containing individual shRNA did. Our data indicate that single promoter-driven multi-cistronic shRNAs effectively silence multiple target genes. Our approach provides a new smart tool for silencing multiple target genes and will potentially serve as an RNAi-based tailored therapy requiring suppression of target gene expression.

The cell death-inducing activity of the peptide containing Noxa mitochondrial-targeting domain is associated with calcium release.

DNA damage stabilizes the p53 tumor suppressor protein that determines the cell fate by either cell cycle arrest or cell death induction. Noxa, the BH3-only Bcl-2 family protein, was shown to be a key player in p53-induced cell death through the mitochondrial dysfunction; however, the molecular mechanism by which Noxa induces the mitochondrial dysfunction to cause cell death in response to genotoxic agents is largely unknown. Here, we show that the mitochondrial-targeting domain (MTD) of Noxa is a prodeath domain. Peptide containing MTD causes massive necrosis in vitro through cytosolic calcium increase; it is released from the mitochondria by opening the mitochondrial permeability transition pore. MTD peptide-induced cell death can be inhibited by calcium chelator BAPTA-AM. Moreover, MTD peptide shows the potent tumor-killing activities in mice by joining with tumor-homing motifs.

DOBI is cleaved by caspases during TRAIL-induced apoptotic cell death.

Downstream of Bid (DOBI) known as Pus10, has been identified as a modulator of TRAIL-induced cell death using RNAi library screening. The crystal structure of DOBI has revealed that it is a crescent-shaped protein containing the pseudouridine synthase catalytic domain and a THUMP-containing domain. Here, we demonstrated that DOBI is expressed in various tissues such as heart and lung, and is also expressed in various tumor cells such as HeLa and A549. Although ectopic expression of DOBI does not promote TRAIL death signaling in HeLa cells, knock-down of DOBI expression using shRNA inhibited TRAIL death signaling. DOBI is cleaved into a 54 kD cleaved DOBI during cell death, and the recombinant DOBI protein can be directly cleaved by caspases-3, or -8 in vitro. Together, these data suggest that the cleaved DOBI may acquire a new function, possibly by cooperating with tBid in the mitochondrial event of cell death caused by TRAIL.

Homer1 regulates the susceptibility to TRAIL.

TRAIL is an apoptotic cell death-inducing ligand that belongs to a TNF superfamily. To identify the regulators that govern the susceptibility to TRAIL, TRAIL-resistant HeLa (TR) cells were established by repeatedly treating HeLa cells with TRAIL. Here we showed that scaffolding protein Homer1 plays a decisive role in regulating the apoptotic susceptibility to TRAIL. TR cells showing the normal susceptibility to FasL and chemotherapeutic agent etoposide expressed the lower protein levels of Homer1 than parental HeLa cells. They showed the delayed activation of caspases-8, Bid cleavage and Bax translocation to mitochondria in response to TRAIL. Reconstitution of Homer1 expression in TR cells significantly restored the susceptibility to TRAIL. In addition, knock-down of Homer1 using interfering shRNA in parental HeLa cells lost the susceptibility to TRAIL. Together, our data indicate that Homer1 plays a critical role in determining the apoptotic susceptibility to TRAIL.

Hypoxia protects articular chondrocytes from thapsigargin-induced apoptosis.

The present study investigates the effect of low oxygen concentrations on thapsigargin-induced apoptosis and reactive oxygen species (ROS)-related signaling in articular chondrocytes. Chondrocytes were obtained from normal canine knee cartilage and were treated with different concentrations of thapsigargin for 24h under normoxic (21% oxygen tension) or hypoxic (1% oxygen tension) conditions. The cells treated with thapsigargin under normoxic conditions showed a dose-dependent induction of apoptosis. However, the cellular changes and apoptotic events that occurred following thapsigargin treatment, were completely inhibited by hypoxia, including loss of mitochondrial transmembrane potential (MTP), ROS generation and JNK phosphorylation. Moreover, the cells exposed to hypoxic conditions showed increased expression of the anti-apoptotic proteins xIAP-2 and Bcl-2. We demonstrate that hypoxia inhibited thapsigargin-induced apoptosis in chondrocytes by regulating ROS-related signaling and the expression of anti-apoptotic proteins. We propose that maintaining hypoxic conditions in articular cartilage may be required for the prevention of chondrocyte and cartilage diseases such as arthritis.

Possible novel therapy for malignant gliomas with secretable trimeric TRAIL.

Malignant gliomas are the most common primary brain tumors. Despite intensive clinical investigation and many novel therapeutic approaches, average survival for the patients with malignant gliomas is only about 1 year. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown potent and cancer-selective killing activity and drawn considerable attention as a promising therapy for cancers, but concerns over delivery and toxicity have limited progress. We have developed a secretable trimeric TRAIL (stTRAIL) and here evaluated the therapeutic potential of this stTRAIL-based gene therapy in brain tumors. An adenovirus (Ad-stTRAIL) delivering stTRAIL was injected into intra-cranial human glioma tumors established in nude mice and tumor growth monitored using the magnetic resonance imaging (MRI). Ad-stTRAIL gene therapy showed potent tumor suppressor activity with no toxic side effects at therapeutically effective doses. When compared with 1, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a conventional therapy for malignant gliomas, Ad-stTRAIL suppressed tumor growth more potently. The combination of Ad-stTRAIL and BCNU significantly increased survival compared to the control mice or mice receiving Ad-stTRAIL alone. Our data indicate that Ad-stTRAIL, either alone or combined with BCNU, has promise as a novel therapy for malignant gliomas.

Improved purification of recombinant adenoviral vector by metal affinity membrane chromatography.

The increasing importance of adenoviral vectors for gene therapy clinical trials necessitates the development of processes suitable for large-scale and commercial production of adenovirus. Here, we evaluated a novel purification process combining an anion-exchange chromatography and an immobilized metal affinity membrane chromatography for the purification of recombinant adenovirus. Adenovirus was initially purified from clarified infectious lysate by anion-exchange chromatography using Q Sepharose XL resin and further polished using a Sartobind IDA membrane unit charged with Zn(2+) ions as affinity ligands. The metal affinity membrane chromatography efficiently removed residual host cell impurities that co-eluted with adenovirus during the previous anion-exchange chromatography step. The metal affinity membrane chromatography also separated defective adenovirus particles from the infectious adenovirus fraction. Furthermore, the metal affinity membrane chromatography showed an improved yield, when compared with a conventional bead-based metal affinity chromatography. The purity and specific activity of the adenovirus prepared using this two-step chromatography was comparable to those of adenovirus produced by the conventional CsCl density centrifugation. Therefore, our data provide an improved method for the purification of adenoviral vectors for clinical applications.

Up-regulation of IAPs by PI-3K: a cell survival signal-mediated anti-apoptotic mechanism.

Under normal cell physiology, a balance between cell survival and apoptosis is crucial for homeostasis. Many studies have demonstrated that apoptosis is modulated by cell survival stimuli. Active Akt, a common mediator of cell survival signals, has been shown to inhibit apoptosis by attenuating activity of pro-apoptotic factors Bad and caspase-9. However, the anti-apoptotic mechanisms mediated by various cell survival signals are poorly understood. Human prostate cancer LNCaP cells, known to contain constitutively activated Akt as a result of a frame-shift mutation in PTEN, an inhibitor of PI-3K/Akt pathway, were observed to be completely resistant to TRAIL-induced apoptosis. In agreement with the known action of Akt, blockade of the PI-3K/Akt pathway rendered LNCaP cells highly susceptible to TRAIL. Importantly, active PI-3K/Akt prevented processing/activation of caspase-3, a phenomenon associated with the function of inhibitor of apoptosis proteins (IAPs). In fact, inhibition of PI-3K activity using Wortmannin significantly decreased the protein levels of IAPs, concomitantly promoting processing/activation of caspase-3 and TRAIL-induced apoptosis. My data indicate that in addition to blocking Bad and caspase-9 through Akt, PI-3K also inhibits caspase-3 through up-regulating IAPs, thereby attenuates apoptosis.

Blockade of processing/activation of caspase-3 by hypoxia.

Tumor hypoxia, which is caused by the rapid proliferation of tumor cells and aberrant vasculature in tumors, results in inadequate supplies of oxygen and nutrients to tumor cells. Paradoxically, these unfavorable growth conditions benefit tumor cell survival, although the mechanism is poorly understood. We have demonstrated for the first time that hypoxia inhibits TRAIL-induced apoptosis by blocking translocation of Bax from cytosol to the mitochondria in tumor cells. However, it is largely unknown how hypoxia-inhibited Bax translocation attenuates TRAIL-induced apoptosis. Here, we demonstrate that despite its inhibitory activity in TRAIL-induced apoptosis, hypoxia does not affect TRAIL-triggered proximal apoptotic signaling events, including caspase-8 activation and Bid cleavage. Instead, hypoxia inhibited processing of caspase-3, leading to incomplete activation of the caspase. Importantly, hypoxia-blocked translocation of Bax to the mitochondria significantly inhibited releasing the mitochondrial factors, such as cytochrome c and Smac/DIABLO, to the cytosol in response to TRAIL. It is well-known that complete processing/activation of caspase-3 requires Smac/DIABLO released from mitochondria. Therefore, our data indicate that an engagement of the apoptotic mitochondrial events leading to caspase-3 activation is blocked by hypoxia. Our data shed new light on understanding of the apoptotic signal transduction and targets regulated by tumor hypoxia.

Regulation of DR-5 protein and mitochondrial transmembrane potential by gemcitabine, a possible mechanism of gemcitabine-enhanced TRAIL-induced apoptosis.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family and a potent inducer of apoptosis. Gemcitabine, chemotherapeutic agent is well known to be involved in synergistic cytotoxic effect with TRAIL. But the mechanism of this synergistic effect is still unclear. To examine the effects and synergistic mechanism of gemcitabine on TRAIL-induced apoptosis, A549, HCT116 and SNU638 cells were pretreated with gemcitabine and treated with TRAIL protein. Gemcitabine significantly enhanced A549 cell death induced by TRAIL, but it was inhibited by the pan-caspase inhibitor, Z-VAD-fmk. The combined treatment of both induced the activation of caspase-8 and reduced the mitochondrial transmembrane potential (MTP) and also translocated Bax protein and released the cytochrome-c in A549 cells when compared with that of negative control. In addition, the gemcitabine pretreatment up-regulated DR-5 and p53 protein expression in a time-dependent manner, which suggests the possible involvement of the p53 protein as a transcriptional factor for DR-5 up-regulation. Thus, we report our findings that gemcitabine enhanced the TRAIL-induced apoptosis and the apoptotic signals are mediated by DR-5-dependent pathway and mitochondrial pathway. Taken together, gemcitabine enhanced TRAIL-induced apoptosis via DR-5 up-regulation and lowering MTP, and suggest that gemcitabine may be used as a successful chemotherapeutic agent for ligand type tumor therapy combined with TRAIL.

Generation of a novel proform of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein that can be reactivated by matrix metalloproteinases.

TRAIL has been suggested to induce the cell death in various tumor cells but not in normal cells; however, several studies have provided the evidence that TRAIL can induce the cell death in some normal cells including human normal hepatocytes, suggesting that TRAIL may show hepatic toxicity in human. In this study, we designed a pro-form of TRAIL (sTRAIL:IL-18) in that soluble TRAIL (sTRAIL) is fused to IL-18, and a matrix metalloproteinases (MMPs) cleavage site is introduced at the connecting site. We showed that sTRAIL:IL-18 has significantly diminished the killing activity in HeLa cells but regains the activity by releasing the free sTRAIL through MMP-2-mediated cleavage. In addition, the killing activity of sTRAIL:IL-18 was significantly increased in HeLa cells when active MMP-2 was produced by TNF-alpha. Taken together, the data suggested that the sTRAIL:IL-18 can be reactivated at the specialized areas where MMPs are pathologically produced.

Intra-articular injection of recombinant TRAIL induces synovial apoptosis and reduces inflammation in a rabbit knee model of arthritis.

We demonstrated previously that local, intra-articular injection of an adenoviral vector expressing human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a rabbit knee model of inflammatory arthritis stimulated synovial apoptosis and reduced inflammation. To examine whether intra-articular injection of recombinant chimeric human TRAIL protein (rTRAIL) also induces apoptosis of proliferating rabbit synovium and reduces inflammation, we used an experimental rabbit arthritis model of rheumatoid arthritis, induced by intra-articular introduction of allogeneic fibroblasts genetically engineered to secrete human IL-1beta. Analysis of synovium isolated from the rabbits treated with intra-articular injection of rTRAIL, relative to saline control, showed areas of extensive acellular debris and large fibrous regions devoid of intact cells, similar to adenoviral mediated TRAIL gene transfer. Extensive apoptosis of the synovial lining was demonstrated using TUNEL analysis of the sections, corresponding to the microscopic findings in hematoxylin and eosin staining. In addition, leukocyte infiltration into the synovial fluid of the inflamed knee joints following rTRAIL treatment was reduced more than 50% compared with the saline control. Analysis of the glycosaminoglycan synthetic rate by cultured cartilage using radiolabeled sulfur and cartilage histology demonstrated that rTRAIL did not adversely affect cartilage metabolism and structure. Analysis of serum alanine aminotransferase showed that intra-articular injection of rTRAIL did not have adverse effects on hepatic function. These results demonstrate that intra-articular injection of rTRAIL could be therapeutic for treating pathologies associated with rheumatoid arthritis.