Cytotoxicity and exhaustion markers of chimeric antigen receptor T cells targeting BCMA in multiple myeloma cell lines between patients and healthy donors.

OBJECTIVES: Multiple myeloma (MM) accounts for 10% of hematologic malignancies. However, most of the patients suffered from relapsed/refractory disease. We would like to expand CAR T cell therapy to treat MM using our current platform. METHODS: BCMA CAR T lymphocytes were generated for volunteers or MM patients. The transduction efficiency was detected by the ddPCR technique. Immunophenotyping and exhaustion markers were monitored by flow cytometry. The efficacy of BCMA CAR T cells was tested using coculturing with BCMA CAR or mock, and the positive and negative targets, K562/hBCMA-ECTM and K562, respectively. RESULTS: BCMA CAR T cells were generated from consented volunteers or MM patients and could be detected CAR BCMA expression at a mean of 4.07 ± 1.95 or 4.65 ± 1.21 copies/cell, respectively. Those modified T cells were primarily effector memory T cells. Our BCMA CAR T cells could explicitly eradicate the K562/hBCMA-ECTM cell line while the K562 cell line survived. Interestingly, the BCMA CAR, mock T cells, and peripheral blood mononuclear cells from MM patients expressed similar levels of the exhaustion makers, TIM-3, LAG-3, and PD1. CONCLUSIONS: Our BCMA CAR T cells, mainly effector/effector memory, could eliminate BCMA-expressing cells in vitro and had similar levels of exhaustion markers among different populations.

[Challenges and strategies for improving efficacy in CAR-T therapy].

CAR-T cell therapy targeting CD19 and BCMA for relapsed or refractory hematopoietic tumors has been adopted in routine practice and has shown dramatic results. However, half of patients who achieve remission with CAR-T therapy eventually relapse, and thus efforts to improve the efficacy of CAR-T therapy are gaining momentum. Notably, studies have described innovative technologies that enable control of cell kinetics after infusion, which is not possible with conventional CAR-T therapies. In this article, we review the challenges of CAR-T cell therapy and the development of new technologies.

Novel anti-tumor mechanism of galanin receptor type 2 in head and neck squamous cell carcinoma cells.

Galanin and its receptors, GALR1 and GALR2, are known tumor suppressors and potential therapeutic targets in head and neck squamous cell carcinoma (HNSCC). Previously, we demonstrated that, in GALR1-expressing HNSCC cells, the addition of galanin suppressed tumor proliferation via upregulation of ERK1/2 and cyclin-dependent kinase inhibitors, whereas, in GALR2-expressing cells, the addition of galanin not only suppressed proliferation, but also induced apoptosis. In this study, we first transduced HEp-2 and KB cell lines using a recombinant adeno-associated virus (rAAV)-green fluorescent protein (GFP) vector and confirmed a high GFP expression rate (>90%) in both cell lines at the standard vector dose. Next, we demonstrated that GALR2 expression in the presence of galanin suppressed cell viability to 40-60% after 72 h in both cell lines. Additionally, the annexin V-positive rate and sub-G0/G1 phase population were significantly elevated in HEp-2 cells (mock vs GALR2: 12.3 vs 25.0% (P < 0.01) and 9.1 vs 32.0% (P < 0.05), respectively) after 48 h. These changes were also observed in KB cells, although to a lesser extent. Furthermore, in HEp-2 cells, GALR2-mediated apoptosis was caspase-independent, involving downregulation of ERK1/2, followed by induction of the pro-apoptotic Bcl-2 protein, Bim. These results illustrate that transient GALR2 expression in the presence of galanin induces apoptosis via diverse pathways and serves as a platform for suicide gene therapy against HNSCC.

Adeno-associated Virus-mediated Ezh2 Knockdown Reduced the Increment of Newborn Neurons Induced by Forebrain Ischemia in Gerbil Dentate Gyrus.

It is established that neurogenesis of dentate gyrus is increased after ischemic insult, although the regulatory mechanisms have not yet been elucidated. In this study, we focused on Ezh2 which suppresses gene expression through catalyzing trimethylation of lysine 27 of histone 3. Male gerbils were injected with adeno-associated virus (AAV) carrying shRNA targeting to Ezh2 into right dentate gyrus 2 weeks prior to forebrain ischemia. One week after ischemia, animals were injected with thymidine analogue to label proliferating cells. Three weeks after ischemia, animals were killed for histological analysis. AAV-mediated knockdown of Ezh2 significantly decreased the ischemia-induced increment of proliferating cells, and the proliferated cells after ischemia showed significantly longer migration from subgranular zone (SGZ), compared to the control group. Furthermore, the number of neural stem cells in SGZ significantly decreased after ischemia with Ezh2 knockdown group. Of note, Ezh2 knockdown did not affect the number of proliferating cells or the migration from SGZ in the non-ischemic condition. Our data showed that, specifically after ischemia, Ezh2 knockdown shifted the balance between self-renewal and differentiation toward differentiation in adult dentate gyrus.

CD19 target-engineered T-cells accumulate at tumor lesions in human B-cell lymphoma xenograft mouse models.

Adoptive T-cell therapy with CD19-specific chimeric antigen receptors (CARs) is promising for treatment of advanced B-cell malignancies. Tumor targeting of CAR-modified T-cells is likely to contribute therapeutic potency; therefore we examined the relationship between the ability of CD19-specific CAR (CD19-CAR)-transduced T-cells to accumulate at CD19(+) tumor lesions, and their ability to provide anti-tumor effects in xenograft mouse models. Normal human peripheral blood lymphocytes, activated with immobilized RetroNectin and anti-CD3 antibodies, were transduced with retroviral vectors that encode CD19-CAR. Expanded CD19-CAR T-cells with a high transgene expression level of about 75% produced IL-2 and IFN-γ in response to CD19, and lysed both Raji and Daudi CD19(+) human B-cell lymphoma cell lines. Furthermore, these cells efficiently accumulated at Raji tumor lesions where they suppressed tumor progression and prolonged survival in tumor-bearing Rag2(-/-)γc(-/-) immunodeficient mice compared to control cohorts. These results show that the ability of CD19-CAR T-cells to home in on tumor lesions is pivotal for their anti-tumor effects in our xenograft models, and therefore may enhance the efficacy of adoptive T-cell therapy for refractory B-cell lymphoma.

Novel immunotherapies in multiple myeloma.

For a substantial period, options for the treatment of multiple myeloma (MM) were limited; however, the advent of novel therapies into clinical practice in the 1990s resulted in dramatic changes in the prognosis of the disease. Subsequently, new proteasome inhibitors and immunomodulators with innovations in efficacy and toxicity were introduced; yet there remains a spectrum of patients with poor outcomes with current treatment strategies. One of the causes of disease progression in MM is the loss of the ability of the dysfunctional immune environment to control virulent cell clones. In recent years, therapies to overcome the immunosuppressive tumor microenvironment and activate the host immune system have shown promise in MM, especially in relapsed and refractory disease. Clinical use of this approach has been approved for several immunotherapies, and a number of studies are currently underway in clinical trials. This review outlines three of the newest and most promising approaches being investigated to enhance the immune system against MM: (1) overcoming immunosuppression with checkpoint inhibitors, (2) boosting immunity against tumors with vaccines, and (3) enhancing immune effectors with adoptive cell therapy. Information on the latest clinical trials in each class will be provided, and further developments will be discussed.

Higher Transduction Efficiency of AAV5 to Neural Stem Cells and Immature Neurons in Gerbil Dentate Gyrus Compared to AAV2 and rh10.

The safety and high efficiency of adeno-associated virus (AAV) vectors has facilitated their wide-scale use to deliver therapeutic genes for experimental and clinical purposes in diseases affecting the central nervous system (CNS). AAV1, 2, 5, 8, 9, and rh10 are the most commonly used serotypes for CNS applications. Most AAVs are known to transduce genes predominantly into neurons. However, the precise tropism of AAVs in the dentate gyrus (DG), the region where persistent neurogenesis occurs in the adult brain, is not fully understood. We stereotaxically injected 1.5 × 1010 viral genomes of AAV2, 5, or rh10 carrying green fluorescent protein (GFP) into the right side of gerbil hippocampus, and performed immunofluorescent analysis using differentiation stage-specific markers 1 week after injection. We found that AAV5 showed a significantly larger number of double-positive cells for GFP and Sox2 in the DG, compared with the AAV2 and rh10 groups. On the contrary, AAVrh10 presented a substantially larger number of double-positive cells for GFP and NeuN in the DG, compared with AAV2 and AAV5. Our findings indicated that AAV5 showed high transduction efficiency to neural stem cells and precursor cells, whereas AAVrh10 showed much higher efficiency to mature neurons in the DG.

Establishment and characterization of transplantable, luminescence labeled rat renal cell carcinoma cell lines.

PURPOSE: Since renal cell carcinoma is considered an immunogenic tumor, testing therapeutic strategies has been impeded by the lack of relevant tumor models in immunocompetent animals. Recent advances in bioluminescence imaging permit sensitive in vivo detection and quantification of cells emitting light. Thus, we established bioluminescent rat renal cell carcinoma cell lines for immunocompetent rats. MATERIALS AND METHODS: The rat renal cell carcinoma cell line ACI-RCC stemming from chemically induced renal cell carcinoma in syngeneic ACI rats was stably transfected with a recombinant retroviral vector encoding luciferase genes derived from fireflies (ACI-RCC-ffLuc) or click beetles (ACI-RCC-cbLuc). Cell line growth patterns were characterized by bioluminescence imaging. RESULTS: Linear correlations noted observed between cell number and photon counts in each cell type. ACI-RCC-cbLuc emitted light about 500-fold higher than ACI-RCC-ffLuc. When transplanted subcutaneously, only ACI-RCC-ffLuc grew, possibly because of less antigenicity. ACI-RCC-ffLuc photon emission correlated significantly with subcutaneous tumor size. Orthotopic tumor growth and subsequent metastatic spread were monitored with time by increased photon intensity on bioluminescence imaging. Based on ACI-RCC-cbLuc bioluminescent intensity the in vitro screening test allowed the identification of several anticancer agents, including molecules related to human renal cell carcinoma progression. CONCLUSIONS: The new in vivo rat renal cell carcinoma model with luciferase labeled tumor cells allowed us to monitor tumor growth noninvasively and semiquantitatively by bioluminescence imaging. This model system coupled with in vitro screening permits precise evaluation of tumor behavior in intact animals and determination of the therapeutic efficacy of anticancer agents for renal cell carcinoma.

The NOTCH-FOXM1 Axis Plays a Key Role in Mitochondrial Biogenesis in the Induction of Human Stem Cell Memory-like CAR-T Cells.

Recent studies have shown that stem cell memory T (TSCM) cell-like properties are important for successful adoptive immunotherapy by the chimeric antigen receptor-engineered-T (CAR-T) cells. We previously reported that both human and murine-activated T cells are converted into stem cell memory-like T (iTSCM) cells by coculture with stromal OP9 cells expressing the NOTCH ligand. However, the mechanism of NOTCH-mediated iTSCM reprogramming remains to be elucidated. Here, we report that the NOTCH/OP9 system efficiently converted conventional human CAR-T cells into TSCM-like CAR-T, "CAR-iTSCM" cells, and that mitochondrial metabolic reprogramming played a key role in this conversion. NOTCH signaling promoted mitochondrial biogenesis and fatty acid synthesis during iTSCM formation, which are essential for the properties of iTSCM cells. Forkhead box M1 (FOXM1) was identified as a downstream target of NOTCH, which was responsible for these metabolic changes and the subsequent iTSCM differentiation. Like NOTCH-induced CAR-iTSCM cells, FOXM1-induced CAR-iTSCM cells possessed superior antitumor potential compared with conventional CAR-T cells. We propose that NOTCH- or FOXM1-driven CAR-iTSCM formation is an effective strategy for improving cancer immunotherapy. SIGNIFICANCE: Manipulation of signaling and metabolic pathways important for directing production of stem cell memory-like T cells may enable development of improved CAR-T cells.

Retroviral vector-producing mesenchymal stem cells for targeted suicide cancer gene therapy.

BACKGROUND: Mesenchymal stem cells (MSCs) are a promising vehicle for targeted cancer gene therapy because of their potential of tumor tropism. For efficient therapeutic application, we developed retroviral vector-producing MSCs that enhance tumor transduction via progeny vector production. METHODS: Rat bone marrow-derived MSCs were nucleofected with the proviral plasmids (vesicular stomatitis virus-G protein-pseudotyped retroviral vector components) (VP-MSCs) or pLTR plasmid alone (non-VP-MSCs). The luciferase-based in vivo imaging system was used to assess gene expression periodically. To evaluate the anticancer effects, we administered MSCs expressing herpes simplex virus-thymidine kinase (HSV-tk) into the left ventricular cavity of nude mice engrafted with 9L glioma cells subcutaneously. RESULTS: In vivo imaging revealed that administration of luciferase-expressing non-VP-MSCs enhanced the bioluminescence signal at the inoculation sites of 9L cells, whereas no accumulation was observed in mice at the site of the control Rat-1 fibroblasts. Compared to non-VP-MSCs, the administration of VP-MSCs resulted in significant augmentation of the signal with an increase in transgene copy number. Immunohistochemical analysis showed marked luciferase expression at the tumor periphery in mice injected with VP-MSCs, whereas little expression was detected in those injected with non-VP-MSCs. Under the continuous infusion of ganciclovir, systemic administration of VP-MSCs expressing HSV-tk suppressed tumor growth more effectively than non-VP-MSC administration, whereas no anticancer effect was observed without ganciclovir treatment. Furthermore, VP-MSC administration caused no transgene transduction in the normal tissues and organs. CONCLUSIONS: VP-MSCs accumulated at the site of tumors after intravascular injection in tumor-bearing mice, followed by in situ gene transfer to tumors without transduction of normal organs. When applied to the HSV-tk/ganciclovir suicide gene therapy, more efficient tumor growth suppression was observed using VP-MSCs compared to non-VP-MSCs. This VP-MSC-based system has great potential for improved cancer gene therapy.

Interleukin-10 expression mediated by an adeno-associated virus vector prevents monocrotaline-induced pulmonary arterial hypertension in rats.

Pulmonary arterial hypertension (PAH) is a fatal disease associated with inflammation and pathological remodeling of the pulmonary artery (PA). Interleukin (IL)-10 is a pleiotropic antiinflammatory cytokine with vasculoprotective properties. Here, we report the preventive effects of IL-10 on monocrotaline-induced PAH. Three-week-old Wistar rats were intramuscularly injected with an adeno-associated virus serotype 1 vector expressing IL-10, followed by monocrotaline injection at 7 weeks old. IL-10 transduction significantly improved survival rates of the PAH rats 8 weeks after monocrotaline administration compared with control gene transduction (75% versus 0%, P<0.01). IL-10 also significantly reduced mean PA pressure (22.8+/-1.5 versus 29.7+/-2.8 mm Hg, P<0.05), a weight ratio of right ventricle to left ventricle plus septum (0.35+/-0.04 versus 0.42+/-0.05, P<0.05), and percent medial thickness of the PA (12.9+/-0.3% versus 21.4+/-0.4%, P<0.01) compared with controls. IL-10 significantly reduced macrophage infiltration and vascular cell proliferation in the remodeled PA in vivo. It also significantly decreased the lung levels of transforming growth factor-beta1 and IL-6, which are indicative of PA remodeling. In addition, IL-10 increased the lung level of heme oxygenase-1, which strongly prevents PA remodeling. In vitro analysis revealed that IL-10 significantly inhibited excessive proliferation of cultured human PA smooth muscle cells treated with transforming growth factor-beta1 or the heme oxygenase inhibitor tin protoporphyrin IX. Thus, IL-10 prevented the development of monocrotaline-induced PAH, and these results provide new insights into the molecular mechanisms of human PAH.

CRISPR/Cas9-mediated cervical cancer treatment targeting human papillomavirus E6.

High-risk human papillomavirus (HPV) is a common cause of cervical cancer. HPV E6 oncoprotein promotes the degradation of host tumor suppressor gene p53, leading to the development of tumors. Therapeutic strategies that specifically target E6, which is constitutively expressed in tumors and is not present in normal tissues, may be highly effective and safe. CRISPR-CRISPR associated protein 9 (Cas9) is one of the genome editing technologies that has recently garnered attention, and is used to knockout target gene expression. By combining cervical cancer cell lines engineered to constitutively express Cas9 and an adeno-associated virus (AAV) vector carrying a single guide (sg) RNA targeting E6 (AAV-sgE6), the present study sought to investigate the effects of this novel therapeutic approach on cervical cancer. The Cas9 gene was transfected into three high-risk HPV-positive cervical cancer cell lines (HeLa, HCS-2, and SKG-I) to establish cell lines that constitutively expressed Cas9. Using these cell lines, genetic mutations and their frequencies, as well as the levels of protein expression, apoptosis and cell proliferation were examined in vitro. In addition, the effects of AAV-sgE6 were examined in a mouse model of cervical cancer in vivo by a single administration of AAV-sgE6 directly into subcutaneous tumors. The results demonstrated that multiple mutations occurred frequently in the targeted E6 genomic sequence in cervical cancer cells transduced with AAV-sgE6. In addition, these AAV-sgE6-transduced cells had reduced expression of E6, increased expression of p53, increased apoptosis and their growth was suppressed in a concentration-dependent manner. Furthermore, subcutaneous tumor growth was significantly suppressed in vivo following intratumoral administration of AAV-sgE6, and adverse events due to AAV-sgE6 administration were not observed. Collectively, the present results indicated that targeting E6 expression in high-risk HPV by CRISPR-Cas9 is a highly specific and effective strategy that may be effective in treating patients with cervical cancer.

Functional Analysis of an Inducible Promoter Driven by Activation Signals from a Chimeric Antigen Receptor.

Adoptive transfer of T cells expressing a chimeric antigen receptor (CAR) is a promising cell-based anticancer therapy. Although clinical studies of this approach show therapeutic efficacy, additional genetic modification is necessary to enhance the efficacy and safety of CAR-T cells. For example, production of an antitumor cytokine from CAR-T cells can potentially enhance their tumor-killing activity, but there are concerns that constitutive expression of anticancer molecules will cause systemic side effects. Therefore, it is important that exogenous gene expression is confined to the tumor locality. Here, we aimed to develop an inducible promoter driven by activation signals from a CAR. Transgene expression in T cells transduced with the CD19-targeted CAR and an inducible promoter, including inducible reporter genes (CAR-T/iReporter), was only induced strongly by co-culture with CD19-positive target cells. CAR-T/iReporter cells also showed redirected cytolysis toward CD19-positive, but not CD19-negative, tumor cells. Overall, our study indicated that the inducible promoter was selectively driven by activation signals from the CAR, and transduction with the inducible promoter did not affect original effector activities including interleukin-2 and interferon-γ production and the antitumor activity of CAR-redirected cytotoxic T lymphocytes. Moreover, this inducible promoter permits visualization and quantification of the activation status in CAR-T cells.

AAV6-Mediated IL-10 Expression in the Lung Ameliorates Bleomycin-Induced Pulmonary Fibrosis in Mice.

Idiopathic pulmonary fibrosis (IPF) is a fibroproliferative disorder with limited therapeutic options. An aberrant wound healing process in response to repetitive lung injury has been suggested for its pathogenesis, and a number of cytokines including transforming growth factor ß1 play pivotal roles in the induction and progression of fibrosis. Thus, the regulation of these pro-inflammatory conditions may reduce the progression of IPF and ameliorate its symptoms in patients. Interleukin-10 (IL-10), a pleiotropic cytokine, exerts anti-inflammatory and anti-fibrotic effects in numerous biological settings. In the present study, we investigated the preventive effects of IL-10 on bleomycin-induced pulmonary fibrosis in mice with the continuous expression of this cytokine via an adeno-associated virus serotype 6 vector. Mice were administered the adeno-associated virus serotype 6 vector encoding mouse IL-10 by intratracheal injection, and osmotic minipumps containing bleomycin were subcutaneously implanted seven days later. Lung histology and the expression levels of pro-inflammatory cytokines and fibrogenic cytokines were then analyzed. In mice exhibiting persistent IL-10 expression on day 35, the number of infiltrated inflammatory cells and the development of fibrosis in lung tissues were significantly reduced. Increases in transforming growth factor ß1 and decreases in IFN-γ were also suppressed in treated animals, with changes in these cytokines playing important roles in the pathogenesis of pulmonary fibrosis. Furthermore, IL-10 significantly improved survival in bleomycin-induced mice. Our results provide insights into the potential benefit of the anti-fibrotic effects of IL-10 as a novel therapeutic approach for IPF.

Efficient transduction of adeno-associated virus vectors into gerbil hippocampus with an appropriate combination of viral capsids and promoters.

Adeno-associated virus (AAV) is an ideal vector for gene transduction into the central nervous system because of its safety and efficiency. While it is currently widely used for clinical trials and is expected to become more widespread, the appropriate combination of viral serotypes and promoters have not been fully investigated. In this study, we compared the transduced gene expression of AAVrh10 to AAV5 in gerbil hippocampus using three different promoters, including cytomegalovirus (CMV), chicken ß-actin promoter with the CMV immediate-early enhancer (CAG), and the Synapsin 1 (Syn1) promoter. Four-week-old male gerbils underwent stereotaxic injection with 1 × 1010 viral genome of AAV carrying green fluorescent protein (GFP). Quantification of the GFP-positive areas 3 weeks after injection showed that AAVrh10-CMV and AAVrh10-CAG were the most efficient (p < 0.001, compared with the control) and AAVrh10-Syn1 and AAV5-CMV were the next most efficient (p < 0.05, compared with the control). On the other hand, AAV5-Syn1 showed little expression, which was only observed at the injected site. In conclusion, we should note that some combinations of viral capsids and promoters can result in failure of gene delivery, while most of them will work appropriately in the transgene expression in the brain.

Eradication of cervical cancer in vivo by an AAV vector that encodes shRNA targeting human papillomavirus type 16 E6/E7.

The major causative agent of cervical cancer is human papilloma virus (HPV); the viral proteins E6 and E7 induce carcinogenesis through the inactivation of the host tumor-suppressor gene. Therefore, the stable expression of specific inhibitors of E6 and E7 in cancer cells is expected to provide effective treatment for cervical cancer without affecting normal tissue. In this study, we propose a novel therapeutic approach using an adeno-associated virus (AAV) vector encoding short hairpin RNA (shRNA) against the oncoproteins E6 and E7 (shE6E7) of HPV type 16 (HPV­16), termed AAV­shE6E7. Three different HPV­16-positive cervical cancer cell lines (BOKU, SiHa and SKG-IIIa cells) were tested for gene transfer efficiency using serotypes of AAV vectors. For in vitro analysis, the cells were transduced AAV­shE6E7; alternatively, in vivo studies were performed via the administration of a direct injection of AAV­shE6E7 into cervical cancer cell-derived tumors in mice. The high gene transfer efficiency was observed using AAV2 in all three cervical cancer cell lines. Following transduction, we observed apoptosis, G1 phase arrest and cell growth inhibition. Additionally, in the transduced cells, the E6, E7 and p16 expression levels decreased, whereas the expression levels of p53, p21 and pRb levels were enhanced. The growth of subcutaneously transplanted tumors was markedly inhibited by the single administration of AAV2­shE6E7, and the tumors were almost completely eradicated without any adverse effects. These results provided evidence of the utility of AAV2­shE6E7 as a novel treatment approach for cervical cancer.

The activated conformation of integrin ß7 is a novel multiple myeloma-specific target for CAR T cell therapy.

Cancer-specific cell-surface antigens are ideal targets for monoclonal antibody (mAb)-based immunotherapy but are likely to have previously been identified in transcriptome or proteome analyses. Here, we show that the active conformer of an integrin can serve as a specific therapeutic target for multiple myeloma (MM). We screened >10,000 anti-MM mAb clones and identified MMG49 as an MM-specific mAb specifically recognizing a subset of integrin ß7 molecules. The MMG49 epitope, in the N-terminal region of the ß7 chain, is predicted to be inaccessible in the resting integrin conformer but exposed in the active conformation. Elevated expression and constitutive activation of integrin ß7 conferred high MMG49 reactivity on MM cells, whereas MMG49 binding was scarcely detectable in other cell types including normal integrin ß7+ lymphocytes. T cells transduced with MMG49-derived chimeric antigen receptor (CAR) exerted anti-MM effects without damaging normal hematopoietic cells. Thus, MMG49 CAR T cell therapy is promising for MM, and a receptor protein with a rare but physiologically relevant conformation can serve as a cancer immunotherapy target.

Large-scale production of recombinant viruses by use of a large culture vessel with active gassing.

Adenovirus and adeno-associated virus (AAV) vectors are increasingly used for gene transduction experiments. However, to produce a sufficient amount of these vectors for in vivo experiments requires large-capacity tissue culture facilities, which may not be practical in limited laboratory space. We describe here a large-scale method to produce adenovirus and AAV vectors with an active gassing system that uses large culture vessels to process labor- and cost-effective infection or transfection in a closed system. Development of this system was based on the infection or transfection of 293 cells on a large scale, using a large culture vessel with a surface area of 6320 cm2. A minipump was connected to the gas inlet of the large vessel, which was placed inside the incubator, so that the incubator atmosphere was circulated through the vessel. When active gassing was employed, the productivity of the adenovirus and AAV vectors significantly increased. This vector production system was achieved by improved CO2 and air exchange and maintenance of pH in the culture medium. Viral production with active gassing is particularly promising, as it can be used with existing incubators and the large culture vessel can readily be converted for use with the active gassing system.

Cancer gene therapy using mesenchymal stem cells.

Cellular and gene therapies represent promising treatment strategies at the frontier of medicine. Hematopoietic stem cells, lymphocytes, and mesenchymal stem cells (MSCs) can all serve as sources of cells for use in such therapies. Strategies for gene therapy are often based on those of cell therapy, and it is anticipated that some examples will be put to practical use in the near future. Given their ability to support hematopoiesis, MSCs may be useful for the enhancement of stem cell engraftment, and the acceleration of hematopoietic reconstitution. Furthermore, MSCs may advance the treatment of severe graft-versus-host disease, based on their immunosuppressive ability. This application is also based on the homing behavior of MSCs to sites of injury and inflammation. Interestingly, MSCs possess tumor-homing ability, opening up the possibility of applications in the targeted delivery of anti-cancer genes to tumors. Many reports have indicated that MSCs can be utilized to target tumors and to deliver anti-cancer molecules locally, as tumors are recognized as non-healing wounds with inflammatory tissue. Here, we review both the potential of MSCs as cellular vehicles for targeted cancer therapy and the molecular mechanisms underlying MSC accumulation at tumor sites.