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Only two decades after discovering miRNAs, our understanding of the functional effects of deregulated miRNAs in the development of diseases, particularly cancer, has been rapidly evolving. These observations and functional studies provide the basis for developing miRNA-based diagnostic markers or new therapeutic strategies. Adenoviral (Ad) vectors belong to the most frequently used vector types in gene therapy and are suitable for strong short-term transgene expression in a variety of cells. Here, we report the set-up and functionality of an Ad-based miRNA vector platform that can be employed to deliver and express a high level of miRNAs efficiently. This vector platform allows fast and efficient vector production to high titers and the expression of pri-miRNA precursors under the control of a polymerase II promoter. In contrast to non-viral miRNA delivery systems, this Ad-based miRNA vector platform allows accurate dosing of the delivered miRNAs. Using a two-vector model, we showed that Ad-driven miRNA expression was sufficient in down-regulating the expression of an overexpressed and highly stable protein. Additional data corroborated the downregulation of multiple endogenous target RNAs using the system presented here. Additionally, we report some unanticipated synergistic effects on the transduction efficiencies in vitro when cells were consecutively transduced with two different Ad-vectors. This effect might be taken into consideration for protocols using two or more different Ad vectors simultaneously.

In this article, we will review the main vaccination strategies currently being implemented by the health authorities and analyze the main vaccines authorized by the EMA. As practical aspects of vaccination, we must make it clear that until collective immunity is reached, the preventive measures being implemented will have to be kept in place. In the words of the WHO Accelerator Project, There is no time to waste in the fight against COVID-19. No one is safe until everyone is safe.

Journal of Current Science and Technology, 12, 1, 151-161

Seminal demonstration of the possibility of stable genetic modification of mammalian cells performed by Wacław and Elisabeth Szybalski opened the doors for gene therapy, the term coined by Wacław Szybalski already in 1962. In the next 60 years, numerous tools for gene delivery have been developed and applied for clinical research, culminating in the registration of several genetic therapies in Europe and the USA. Some of these strategies, aimed to treat severe combined immunodeficiencies, inherited forms of blindness, spinal muscular atrophy, some cancers, and genetic anemias, are the real hope for patients suffering from previously incurable diseases or the ones whose treatment was not effective. On the approaching 60th anniversary of gene therapy, combined with the 100th anniversary of the birth of Professor Wacław Szybalski (September 9th, 1921), who passed away on December 16, 2020, here I present the summary of the most important aspects of clinical applications of genetic therapies.

VITT is a rare, life-threatening syndrome characterized by thrombotic symptoms in combination with thrombocytopenia, which may occur in individuals receiving the first administration of adenoviral non replicating vectors (AVV) anti Covid19 vaccines. Vaccine-induced immune thrombotic thrombocytopenia (VITT) is characterized by high levels of serum IgG that bind PF4/polyanion complexes, thus triggering platelet activation. Therefore, identification of the fine pathophysiological mechanism by which vaccine components trigger platelet activation is mandatory. Herein, we propose a multistep mechanism involving both the AVV and the neo-synthetized Spike protein. The former can: i) spread rapidly into blood stream, ii), promote the early production of high levels of IL-6, iii) interact with erythrocytes, platelets, mast cells and endothelia, iv) favor the presence of extracellular DNA at the site of injection, v) activate platelets and mast cells to release PF4 and heparin. Moreover, AVV infection of mast cells may trigger aberrant inflammatory and immune responses in people affected by the mast cell activation syndrome (MCAS). The pre-existence of natural antibodies binding PF4/heparin complexes may amplify platelet activation and thrombotic events. Finally, neosynthesized Covid 19 Spike protein interacting with its ACE2 receptor on endothelia, platelets and leucocyte may trigger further thrombotic events unleashing the WITT syndrome.

Background There is an urgent need for targeted biological therapies for prostate cancer with greater efficacy and less toxicity, particularly for metastatic disease, where current therapies are not curative. Therapeutic adenoviral vectors or oncolytic adenoviruses offer the possibility of a competent, nontoxic therapeutic alternative for prostate cancer. However, free viral particles must be delivered locally, an approach that does not address metastatic disease, and they display poor tumor penetration. To fully exploit the potential of these vectors, we must develop methods that improve intratumoral dissemination and allow for systemic delivery. This study establishes a proof-of-principle rationale for a novel human mesenchymal stem (stromal) cell-based approach to improving vector delivery to tumors. Methods/results We have generated mesenchymal stem cell-derived packaging cells for adenoviruses (E1-modified mesenchymal stem cells) by modifying human mesenchymal stem cells with the adenovirus (type C) E1A/B genes needed for viral replication. Using cell-based assays, we have demonstrated that two adenoviral vectors, replication-defective adenovirus expressing p14 and p53 or conditionally replicating oncolytic adenovirus, packaged by E1A/B-modified mesenchymal stem cells, suppress the growth of prostate cancer cells in culture. Using subcutaneous xenograft models for human prostate cancer in mice, we have shown that E1A/B-modified mesenchymal stem cells display tumor tropism in tumor-bearing nude mice, that E1A/B-modified mesenchymal stem cells disseminate well within tumors, and that replication-defective adenovirus expressing p14 and p53 or conditionally replicating oncolytic adenovirus-loaded E1-modified mesenchymal stem cells suppresses tumor growth in mice. Conclusion The results show that this approach, if optimized, could circumvent the obstacles to efficient gene delivery encountered with current gene delivery approaches and provide an effective, nontoxic therapeutic alternative for metastatic disease.

Adenoviral vectors (AdVs) have attracted much attention in the fields of vaccine development and treatment for diseases such as genetic disorders and cancer. In this review, we discuss the utility of AdVs in cancer therapies. In recent years, AdVs were modified as oncolytic AdVs (OAs) that possess the characteristics of cancer cell-specific replication and killing. Different carriers such as diverse cells and extracellular vesicles are being explored for delivering OAs into cancer sites after systemic administration. In addition, there are also various strategies to improve cancer-specific replication of OAs, mainly through modifying the early region 1 (E1) of the virus genome. It has been documented that oncolytic viruses (OVs) function through stimulating the immune system, resulting in the inhibition of cancer progression and, in combination with classical immune modulators, the anti-cancer effect of OAs can be even further enforced. To enhance the cancer treatment efficacy, OAs are also combined with other standard treatments, including surgery, chemotherapy and radiotherapy. Adenovirus type 5 (Ad5) has mainly been explored to develop vectors for cancer treatment with different modulations. Only a limited number of the more than 100 identified AdV types were converted into OAs and, therefore, the construction of an adenovirus library for the screening of potential novel OA candidates is essential. Here, we provide a state-of-the-art overview of currently performed and completed clinic trials with OAs and an adenovirus library, providing novel possibilities for developing innovative adenoviral vectors for cancer treatment.

International audience; The last decade have nourished strong doubts on the beneficial prospects of gene therapy for curing fatal diseases. However, this climate of reservations is currently being transcended by the publication of several successful clinical protocols, restoring confidence in the opportunity of therapeutic gene transfer. A strong sign of this present enthusiasm for gene therapy by clinicians and industrials is the market approval of the therapeutic viral vector Glybera, the first commercial product in Europe of this class of drug. This new field of medicine is particularly attractive when considering therapies for a number of neurological disorders, most of which are desperately waiting for a satisfactory treatment. The central nervous system is indeed a very compliant organ where gene transfer can be stable and successful if provided through an appropriate strategy. The purpose of this review is to present the characteristics of the most efficient virus-derived vectors used by researchers and clinicians to genetically-modify particular cell types or whole regions of the brain. In addition, we discuss major issues regarding side effects such as genotoxicity and immune response associated to the use of these vectors; Suite aux récents succès de divers protocoles thérapeutiques de transfert de gène, notamment appliqués aux pathologies de la rétine, et à la mise sur le marché du Glybera, le premier produit commercial en Europe pour cette classe de médicaments, on observe un regain d'intérêts pour la thérapie génique sur les plans clinique et industriel. Ce nouveau domaine de la médecine expérimentale est particulièrement enthousiasmant si l'on considère que la plupart des maladies neurologiques, attendent désespérément l’apparition d’un traitement satisfaisant. Le système nerveux central est en effet un organe où le transfert de gène peut être stable et réussi s’il est administré selon une stratégie appropriée. L’objectif de cette revue est de présenter les qualités des vecteurs viraux les plus efficaces utilisés actuellement par les chercheurs et les cliniciens pour modifier génétiquement des cellules neurales ou des régions entières du cerveau. Nous abordons également des questions concernant les effets secondaires tels que la génotoxicité et la réponse immunitaire associées à l'utilisation de ces vecteurs

A long time has passed since the first clinical trial with adenoviral (Ad) vectors. Despite being very promising, Ad vectors soon revealed their limitations in human clinical trials. The pre-existing immunity, the marked liver tropism and the high toxicity of first generation Ad (FG-Ad) vectors have been the main challenges for the development of new approaches. Significant effort toward the development of genetically and chemically modified adenoviral vectors has enabled researchers to create more sophisticated vectors for gene therapy, with an improved safety profile and a higher transduction ability of different tissues. In this review, we will describe the latest findings in the high-speed, evolving field of genetic and chemical modifications of adenoviral vectors, a field in which different disciplines, such as biomaterial research, virology and immunology, co-operate synergistically to create better gene therapy tools for modern challenges.

Introduction: Drug attrition rates due to hepatotoxicity are an important safety issue considered in drug development. The HepG2 hepatoma cell line is currently being used for drug-induced hepatotoxicity evaluations, but its expression of drug-metabolizing enzymes is poor compared with hepatocytes. Different approaches have been proposed to upgrade HepG2 cells for more reliable drug-induced liver injury predictions. Areas covered: We describe the advantages and limitations of HepG2 cells transduced with adenoviral vectors that encode drug-metabolizing enzymes for safety risk assessments of bioactivable compounds. Adenoviral transduction facilitates efficient and controlled delivery of multiple drug-metabolizing activities to HepG2 cells at comparable levels to primary human hepatocytes by generating an artificial hepatocyte'. Furthermore, adenoviral transduction enables the design of tailored cells expressing particular metabolic capacities. Expert opinion: Upgraded HepG2 cells that recreate known inter-individual variations in hepatic CYP and conjugating activities due to both genetic (e.g., polymorphisms) or environmental (e.g., induction, inhibition) factors seems a suitable model to identify bioactivable drug and conduct hepatotoxicity risk assessments. This strategy should enable the generation of customized cells by reproducing human pheno- and genotypic CYP variability to represent a valuable human hepatic cell model to develop new safer drugs and to improve existing predictive toxicity assays.

Background: A major obstacle for the development of HIV vaccines is the virus' worldwide sequence diversity. Nevertheless, the presence of T cell epitopes within conserved regions of the virus' structural Gag protein and conserved structures in the envelope (env) sequence raises the possibility that cross-reactive responses may be induced by vaccination. In this study, the aim was to investigate the importance of antigenic match on immunodominance and breadth of obtainable T cell responses. Methods: Outbred CD1 mice were immunized with either heterologous (SIVmac239 and HIV-1 clade B consensus) or homologous (SIVmac239) gag sequences using adenovirus (Ad5) and MVA vectors. Env (SIVmac239) was co-encoded in the vectors to study the induction of antibodies, which is a primary target of current HIV vaccine designs. All three vaccines were designed as virus-encoded virus-like particle vaccines. Antibody responses were analysed by ELISA, avidity ELISA, and neutralization assay. T cell responses were determined by intracellular cytokine staining of splenocytes. Results: The homologous Env/Gag prime-boost regimen induced higher Env binding antibodies, and induced stronger and broader Gag specific CD8+ T cell responses than the homologous Env/heterologous Gag prime-boost regimen. Homologous Env/heterologous Gag immunization resulted in selective boosting of Env specific CD8+ T cell responses and consequently a paradoxical decreased recognition of variant sequences including conserved elements of p24 Gag. Conclusions: These results contrast with related studies using Env or Gag as the sole antigen and suggest that prime-boost immunizations based on homologous SIVmac239 Gag inserts is an efficient component of genetic VLP vaccines-both for induction of potent antibody responses and cross-reactive CD8+ T cell responses.

Background/Aims: While recombinant adenoviruses are among the most widely-used gene delivery vectors and usually propagated in HEK-293 cells, generating recombinant adenoviruses remains time-consuming and labor-intense. We sought to develop a rapid adenovirus production and amplification (RAPA) line by assessing human Ad5 genes (E1A, E1B19K/55K, pTP, DBP, and DNA Pol) and OCT1 for their contributions to adenovirus production. Methods: Stable transgene expression in 293T cells was accomplished by using piggyBac system. Transgene expression was determined by qPCR. Adenoviral production was assessed with titering, fluorescent markers and/or luciferase activity. Osteogenic activity was assessed by measuring alkaline phosphatase activity. Results: Overexpression of both E1A and pTP led to a significant increase in adenovirus amplification, whereas other transgene combinations did not significantly affect adenovirus amplification. When E1A and pTP were stably expressed in 293T cells, the resultant RAPA line showed high efficiency in adenovirus amplification and production. The produced AdBMP9 infected mesenchymal stem cells with highest efficiency and induced most effective osteogenic differentiation. Furthermore, adenovirus production efficiency in RAPA cells was dependent on the amount of transfected DNA. Under optimal transfection conditions high-titer adenoviruses were obtained within 5 days of transfection. Conclusion: The RAPA cells are highly efficient for adenovirus production and amplification.

The absence of preexisting immunity against porcine adenovirus (Ad) serotype 3 (PAd3) and bovine Ad serotype 3 (BAd3) in humans makes them attractive alternatives to human Ad serotype 5 (HAd5) vectors. To determine whether there is significant cross-reactivity among HAd5, BAd3, and PAd3 at the level of cell-mediated immune responses, BALB/c mice were inoculated intraperitoneally with wild type (WT) or replication-defective (RD) HAd5, BAd3, or PAd3. Thirty-five days after the first inoculation, cross-reactive CD8+ cytotoxic T cells, as well as CD4+ Th1- and Th2-helper T cells, in the spleen were analyzed by ELISPOT, flow cytometry and cytotoxic T lymphocyte (CTL) assays. Virus neutralization assays were used to evaluate humoral cross-reactivity. CD8+ or CD4+ T cells primed with WT or RD HAd5, PAd3, or BAd3 demonstrated significant (P

Lentiviruses (LVs) are considered one of the most promising tools for gene transfer, however, their potential to induce pro-inflammatory cytokines on delivery into the respiratory tissue remains to be established. Here we tested a third-generation vesicular stomatitis virus (VSV)-G pseudotyped LV vector in the two respiratory epithelial cell lines A549 and CFT1-C2. We observed that the VSV-G LV vector does not induce (a) activation of the nuclear factor (NF)-kappaB, which intervenes in transcription of pro-inflammatory genes; (b) expression of ICAM-1; and (c) transcription of a panel of cytokines, with the exception of a mild and transient (24h) increase of IFN-gamma mRNA. In contrast, an adenovirus-derived vector strongly activated NF-kappaB and different transcripts such as those of ICAM-1, IL-8, RANTES, IP-10, TNF-alpha, IL-6, IL-1 beta. In conclusion, this third-generation VSV-G pseudotyped LV vector does not elicit major pro-inflammatory signals in human airway epithelial cells and appears to be better suited for gene delivery strategies.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a variety of transformed cell lines, but generally spares most normal cells. Transduction by an adenoviral vector expressing human TRAIL cDNA (Ad.TRAIL-GFP) resulted in both direct tumor cell killing as well as a potent bystander effect through presentation of TRAIL by transduced normal cells. Administration of Ad.TRAIL-GFP significantly prolonged survival of mice harboring either intracerebral glioblastomas or breast carcinoma-induced peritoneal carcinomatosis. Additionally, TRAIL induced prolonged transgene expression in normal tissue, presumably as a result of diminished immunemediated destruction of vector-transduced cells. Taken together, these data suggest that vector-mediated transduction of TRAIL may represent an effective strategy for cancer gene therapy.

Spinal muscular atrophy (SMA), the most common genetic cause of infant death, is a neurodegenerative disorder affecting motor neurons. SMA results from a loss in full-length survival of motor neuron (SMN) protein due to deletions/mutations in the SMN1 gene. In this study, we assessed the ability of cell-penetrating peptides (CPP) to deliver recombinant SMN protein to cultured neurons as a prelude for a potential therapeutic to treat SMA. Firstly, we confirmed that E. coli produced recombinant GFP protein fused to TAT (YGRKKRRQRRR; TAT-GFP) transduced rat cortical neurons in a concentration dependent manner. However, due to low yields of recombinant TATSMN protein obtainable from E. coli, we investigated the potential of a modified TAT (TATκ: YARKAARQARA) or R9 (RRRRRRRRR) peptide downstream of the fibronectin (FIB) secretory signal peptide to generate recombinant CPP-fused SMN protein. While U251 cells transduced with an adenoviral vector expressing CMV-FIB-TATκ-SMN secreted recombinant TATκ-SMN protein, we did not detect TATκ-SMN protein transduction of cortical neurons. Further, purified TATκ-SMN was unable to transduce SH-SY5Y cells, nor block apoptosis following LY294002 treatment of these cells. Our findings indicate that TATκ is not a suitable CPP to deliver SMN protein to neurons. Nonetheless, we have developed a novel method to generate full-length recombinant SMN protein using a mammalian expression system, which can be used to explore the application of other CPPs to deliver SMN protein as a treatment for SMA.

Background During human (HIV) and simian (SIV) immunodeficiency virus infection, loss of CD4+ T cells and progression to AIDS are associated with a decline in antibody titers to the viral Gag protein, while antibodies to the Env protein remain high, suggesting a T cell independent antibody response to Env. Results To explore differential regulation of Gag and Env antibody responses, immunocompetent BALB/c and T cell deficient nude mice were immunized with virus like particles (VLP) of simian immunodeficiency virus or adenoviral vectors expressing SIV Gag and Env. High levels of antibodies against Gag and Env could only be induced in immunocompetent mice, but not in the immunodeficient mice. Thus, neither cells expressing Env after adenoviral gene transfer nor VLPs induce a T cell independent primary anti-Env antibody response. However, secondary B cell responses to Env, but not to Gag, were observed in immunodeficient mice after transfer of primed B cells and boosting with VLPs or adenoviral vectors expressing Gag and Env. This T cell independent secondary antibody response to Env was reduced after stimulation with VLPs modified to contain monomeric membrane bound gp130 surface subunit of Env and undetectable after injection of soluble gp130. Conclusions Membrane-bound trimeric Env seems to be responsible for the maintenance of high levels of anti-Env antibodies during progression to AIDS. This T cell independent secondary antibody response may prevent T cell-dependent affinity maturation and thus contribute to viral immune escape by favoring persistence of non-protective antibodies.

Therapies aimed at the protection and/or regeneration of inner ear hair cells are of great interest, given the significant monetary and quality of life impact of balance disorders. Different viral vectors have been shown to transfect various cell types in the inner ear. The past decade has provided tremendous advances in the use of adenoviral vectors to achieve targeted treatment delivery. Several routes of delivery have been identified to introduce vectors into the inner ear while minimizing injury to surrounding structures. Recently, the transcription factor Atoh1 was determined to play a critical role in hair cell differentiation. Adenoviral-mediated overexpression of Atoh1 in culture and in vivo has demonstrated the ability to regenerate vestibular hair cells by causing transdifferentiation of neighbouring epithelial-supporting cells. Functional recovery of the vestibular system has also been documented following adenoviral-induced Atoh1 overexpression. Experiments demonstrating gene transfer in human vestibular epithelial cells reveal that the human inner ear is a suitable target for gene therapy.

Purpose: In preparation for a phase I clinical trial using a combined cytotoxic/immunotherapeutic strategy with adenoviruses (Ad) expressing Flt3L (Ad-Flt3L) and thymidine kinase (Ad-TK) to treat glioblastoma (GBM), we tested the hypothesis that Ad-TK+GCV would be the optimal tumor-killing agent in relation to efficacy and safety when compared with other proapoptotic approaches. Experimental Design: The efficacy and neurotoxicity of Ad-TK+GCV was compared with Ads encoding the proapoptotic cytokines [tumor necrosis factor-α, tumor necrosis factor–related apoptosis-inducing factor (TRAIL), and Fas ligand (FasL)], alone or in combination with Ad-Flt3L. In rats bearing small GBMs (day 4), only Ad-TK+GCV or Ad-FasL improved survival. Results: In rats bearing large GBMs (day 9), the combination of Ad-Flt3L with Ad-FasL did not improve survival over FasL alone, whereas Ad-Flt3L combined with Ad-TK+GCV led to 70% long-term survival. Expression of FasL and TRAIL caused severe neuropathology, which was not encountered when we used Ad-TK+/−Ad-Flt3L. In vitro, all treatments elicited release of high mobility group box 1 protein (HMGB1) from dying tumor cells. In vivo, the highest levels of circulating HMGB1 were observed after treatment with Ad-TK+GCV+Ad-Flt3L; HMGB1 was necessary for the therapeutic efficacy of AdTK+GCV+Ad-Flt3L because its blockade with glycyrrhizin completely blocked tumor regression. We also showed the killing efficacy of Ad-TK+GCV in human GBM cell lines and GBM primary cultures, which also elicited release of HMGB1. Conclusions: Our results indicate that Ad-TK+GCV+Ad-Flt3L exhibit the highest efficacy and safety profile among the several proapoptotic approaches tested. The results reported further support the implementation of this combined approach in a phase I clinical trial for GBM.

Lung cancer is a major public health problem in the western world, and gene therapy strategies to tackle this disease systemically are often impaired by inefficient delivery of the vector to the tumour tissue. Some of the main factors inhibiting systemic delivery are found in the blood stream in the form of red and white blood cells (WBCs) and serum components. Mesenchymal stem cells (MSCs) have been shown to home to tumour sites and could potentially act as a shield and vehicle for a tumouricidal gene therapy vector. Here, we describe the ability of an adenoviral vector expressing TRAIL (Ad.TR) to transduce MSCs and show the apoptosis-inducing activity of these TRAIL-carrying MSCs on A549 lung carcinoma cells. Intriguingly, using MSCs transduced with Ad.enhanced-green-fluorescent-protein (EGFP) we could show transfer of viral DNA to cocultured A549 cells resulting in transgenic protein production in these cells, which was not inhibited by exposure of MSCs to human serum containing high levels of adenovirus neutralizing antibodies. Furthermore, Ad.TR-transduced MSCs were shown not to induce T-cell proliferation, which may have resulted in cytotoxic T-cell-mediated apoptosis induction in the Ad.TR-transduced MSCs. Apoptosis was also induced in A549 cells by Ad.TR-transduced MSCs in the presence of physiological concentrations of WBC, erythrocytes and sera from human donors that inhibit or neutralize adenovirus alone. Moreover, we could show tumour growth reduction with TRAIL-loaded MSCs in an A549 xenograft mouse model. This is the first study that demonstrates the potential therapeutic utility of Ad.TR-transduced MSCs in cancer cells and the stability of this vector in the context of the blood environment.

Regulatable promoter systems allow gene expression to be tightly controlled in vivo. This is highly desirable for the development of safe, efficacious adenoviral vectors that can be used to treat human diseases in the clinic. Ideally, regulatable cassettes should have minimal gene expression in the "OFF" state, and expression should quickly reach therapeutic levels in the "ON" state. In addition, the components of regulatable cassettes should be non-toxic at physiological concentrations and should not be immunogenic, especially when treating chronic illness that requires long-lasting gene expression. In this chapter, we will describe in detail protocols to develop and validate first generation (Ad) and high-capacity adenoviral (HC-Ad) vectors that express therapeutic genes under the control of the TetON regulatable system. Our laboratory has successfully used these protocols to regulate the expression of marker genes, immune stimulatory genes, and toxins for cancer gene therapeutics, i.e., glioma that is a deadly form of brain cancer. We have shown that this third generation TetON regulatable system, incorporating a doxycycline (DOX)-sensitive rtTA(2)S-M2 inducer and tTS(Kid) silencer, is non-toxic, relatively non-immunogenic, and can tightly regulate reporter transgene expression downstream of a TRE promoter from adenoviral vectors in vitro and also in vivo.

OBJECTIVE: Glioblastoma multiforme (GBM) is a devastating brain tumor for which there is no cure. Adenoviral-mediated transfer of conditional cytotoxic (herpes simplex virus [HSV] 1-derived thymidine kinase [TK]) and immunostimulatory (Fms-like tyrosine kinase 3 ligand [Flt3L]) transgenes elicited immune-mediated long-term survival in a syngeneic intracranial GBM model in rodents. However, the lack of a large GBM animal model makes it difficult to predict the outcome of therapies in humans. Dogs develop spontaneous GBM that closely resemble the human disease; therefore, they constitute an excellent large animal model. We assayed the transduction efficiency of adenoviral vectors (Ads) encoding beta-galactosidase (betaGal), TK, and Flt3L in J3T dog GBM cells in vitro and in the dog brain in vivo. METHODS: J3T cells were infected with Ads (30 plaque-forming units/cell; 72 h) encoding betaGal (Ad-betaGal), TK (Ad-TK), or Flt3L (Ad-Flt3L). We determined transgene expression by immunocytochemistry, betaGal activity, Flt3L enzyme-linked immunosorbent assay, and TK-induced cell death. Ads were also injected intracranially into the parietal cortex of healthy dogs. We determined cell-type specific transgene expression and immune cell infiltration. RESULTS: Adenoviral-mediated gene transfer of HSV1-TK, Flt3L, and betaGal was detected in dog glioma cells in vitro (45% transduction efficiency) and in the dog brain in vivo (10-mm area transduced surrounding each injection site). T cells and macrophages/activated microglia infiltrated the injection sites. Importantly, no adverse clinical or neuropathological side effects were observed. CONCLUSION: We demonstrate effective adenoviral-mediated gene transfer into the brain of dogs in vivo and support the use of these vectors to develop an efficacy trial for canine GBM as a prelude to human trials.

To determine whether IL-4 is therapeutic in treating established experimental arthritis, a recombinant adenovirus carrying the gene that encodes murine IL-4 (Ad-mIL-4) was used for periarticular injection into the ankle joints into mice with established collagen-induced arthritis (CIA). Periarticular injection of Ad-mIL-4 resulted in a reduction in the severity of arthritis and joint swelling compared with saline- and adenoviral control groups. Local expression of IL-4 also reduced macroscopic signs of joint inflammation and bone erosion. Moreover, injection of Ad-mIL-4 into the hind ankle joints resulted in a decrease in disease severity in the untreated front paws. Systemic delivery of murine IL-4 by intravenous injection of Ad-mIL-4 resulted in a significant reduction in the severity of early-stage arthritis., Introduction: Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that is characterized by joint inflammation, and progressive cartilage and bone erosion. Recent research has identified certain biologic agents that appear more able than conventional therapies to halt effectively the progression of disease, as well as ameliorate disease symptoms. One potential problem with the use of biologic agents for arthritis therapy is the need for daily or weekly repeat dosing. The transfer of genes directly to the synovial lining can theoretically circumvent the need for repeat dosing and reduce potential systemic side effects [1,2]. However, although many genes have been effective in treating murine CIA if administrated at a time before disease onset, local intra-articular or periarticular gene transfer has not been highly effective in halting the progression of established disease. IL-4, similar to tumor necrosis factor (TNF)-α and IL-1 inhibitors, has been shown be therapeutic for the treatment of murine CIA when administered intravenously as a recombinant protein, either alone or in combination with IL-10. IL-4 can downregulate the production of proinflammatory and T-helper (Th)1-type cytokines by inducing mRNA degradation and upregulating the expression of inhibitors of proinflammatory cytokines such as IL-1 receptor antagonist (IL-1Ra) [3,4]. IL-4 is able to inhibit IL-2 and IFN-γ production by Th1 cells, resulting in suppression of macrophage activation and the production of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNF-α by monocytes and macrophages [4,5,6,7,8,9]. Objective: In order to examine the therapeutic effects of local and systemic IL-4 expression in established CIA, an adenoviral vector carrying the gene for murine IL-4 (Ad-mIL-4) was generated. The ability of Ad-mIL-4 to treat established CIA was evaluated by local periarticular and systemic intravenous injection of Ad-mIL-4 into mice at various times after disease onset. Materials and methods: Male DBA/1 lacJ (H-2q) mice, aged 7-8 weeks, were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The mice were immunized intradermally at the base of tail with 100 μ g bovine type II collagen. On day 21 after priming, mice received a boost injection (intradermally) with 100 μ g type II collagen in incomplete adjuvant. For the synchronous onset of arthritis, 40 μ g lipopolysaccharide (Sigma, St Louis, MO, USA) was injected intraperitoneally on day 28. Ad-mIL-4 was injected periarticularly into the hind ankle joints of mice on day 32 or intravenously by tail vein injection on day 29. Disease severity was monitored every other day using an established macroscopic scoring system ranging from 0 to 4: 0, normal; 1, detectable arthritis with erythma; 2, significant swelling and redness; 3, severe swelling and redness from joint to digit; and 4, maximal swelling with ankylosis. The average of macroscopic score was expressed as a cumulative value for all paws, with a maximum possible score of 16 per mouse. Cytokine production by joint tissue or serum were assessed using enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN, USA). Results: To examine the therapeutic effects of IL-4 gene transfer in a murine model of arthritis, 5×108 particles of Ad-mIL-4 and enhanced green fluorescent protein (Ad-eGFP) were administered by periarticular injection into the ankle joints of mice with established disease 4 days after lipopolysaccharide injection. All mice had established disease at time of injection. As shown in Figure 1, the severity of arthritis (Fig. 1a), paw thickness (Fig. 1b), and the number of arthritic paws (Fig. 1c) were all significantly reduced in the Ad-mIL-4 group, compared with the saline- and Ad-eGFP-treated groups. Analysis of the bones in the ankle joints of control arthritic mice showed evidence of erosion with an associated monocytic infiltrate around the joint space compared with the Ad-mIL-4-treated and nonarthritic control joints. In addition, injection of the ankle joints in the hind legs resulted in a therapeutic effect in the front paws. A similar contralateral effect has been observed with adenoviral-mediated delivery of viral (v)-IL-10. Interestingly, a high level of murine IL-10 also was detected from the joint lysates of Ad-mIL-4-treated naïve and arthritic mice, with the production of endogenous IL-10 correlating with the dose of Ad-mIL-4. The administration of recombinant IL-4 protein systemically has been shown to be therapeutic in murine CIA models if given before disease onset. To examine the effect of systemic IL-4 delivered by gene transfer, 1×109 particles of Ad-mIL-4 were injected via the tail vein of collagen-immunized mice the day after lipopolysaccharide injection. Whereas the immunized control mice, injected with Ad-eGFP, showed disease onset on day 3 after lipopolysaccharide injection, Ad-mIL-4-treated mice showed a delay in disease onset and as a reduction in the total number of arthritic paws. Also, systemic injection of Ad-mIL-4 suppressed the severity of arthritis in CIA mice according to arthritis index. Discussion: Gene therapy represents a novel approach for delivery of therapeutic agents to joints in order to treat the pathologies associated with RA and osteoarthritis, as well as other disorders of the joints. In the present study we examined the ability of local periarticular and systemic gene transfer of IL-4 to treat established and early-stage murine CIA, respectively. We have demonstrated that both local and systemic administration of Ad-mIL-4 resulted in a reduction in the severity of arthritis, as well as in the number of arthritic paws. In addition, the local gene transfer of IL-4 reduced histologic signs of inflammation and of bone erosion. Interestingly, local delivery of Ad-mIL-4 was able to confer a therapeutic effect to the untreated, front paws through a currently unknown mechanism. In addition, both local and systemic expression of IL-4 resulted in an increase in the level of endogenous IL-10, as well as of IL-1Ra (data not shown). Previous experiments have shown that gene transfer of IL-10 and IL-1 and TNF inhibitors at the time of disease initiation (day 28) is therapeutic. However, delivery of these agents after disease onset appeared to have only limited therapeutic effect. In contrast, the present results demonstrate that IL-4, resulting from local periarticular and systemic injection of Ad-mIL-4, was able partially to reverse progression of established and early-stage disease, respectively. These results, as well as those of others, support the potential application of IL-4 gene therapy for the clinical treatment of RA.