[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.

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.

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.

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.

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 (T) 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 (iT) cells by coculture with stromal OP9 cells expressing the NOTCH ligand. However, the mechanism of NOTCH-mediated iT reprogramming remains to be elucidated.

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 , 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.

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.

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 β-actin promoter with the CMV immediate-early enhancer (CAG), and the Synapsin 1 (Syn1) promoter.

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 β1 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.

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 (HPV‑16), termed AAV‑shE6E7.

The activated conformation of integrin β 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 β molecules.

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.

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.

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.

NF-κB activity regulates mesenchymal stem cell accumulation at tumor sites.

Mesenchymal stem cells (MSC) accumulate at tumor sites when injected into tumor-bearing mice, perhaps offering cellular vectors for cancer-targeted gene therapy. However, the molecular mechanisms involved in MSC targeting the tumors are presently little understood. We focused on MSC-endothelial cell (EC) adhesion following TNF-α stimulation in an attempt to elucidate these mechanisms.

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.

Scalable purification of adeno-associated virus serotype 1 (AAV1) and AAV8 vectors, using dual ion-exchange adsorptive membranes.

In vivo gene transduction with adeno-associated virus (AAV)-based vectors depends on laborious procedures for the production of high-titer vector stocks. Purification steps for efficient clearance of impurities such as host cell proteins and empty vector particles are required to meet end-product specifications. Therefore, the development of alternative, realistic methods to facilitate a scalable virus recovery procedure is critical to promote in vivo investigations.

Cell and gene therapy using mesenchymal stem cells (MSCs).

Mesenchymal stem cells (MSCs) are considered to be a promising platform for cell and gene therapy for a variety of diseases. First, in the field of hematopoietic stem cell transplantation, there are two applications of MSCs: 1) the improvement of stem cell engrafting and the acceleration of hematopoietic reconstitution based on the hematopoiesis-supporting ability; and 2) the treatment of severe graft-versus-host disease (GVHD) based on the immunomodulatory ability. Regarding the immunosuppressive ability, we found that nitric oxide (NO) is involved in the MSC-mediated suppression of T cell proliferation.

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.

Adenoassociated virus-mediated prostacyclin synthase expression prevents pulmonary arterial hypertension in rats.

Prostacyclin synthase (PGIS) is the final committed enzyme in the metabolic pathway of prostacyclin production. The therapeutic option of intravenous prostacyclin infusion in patients with pulmonary arterial hypertension is limited by the short half-life of the drug and life-threatening catheter-related complications. To develop a better delivery system for prostacyclin, we examined the feasibility of intramuscular injection of an adenoassociated virus (AAV) vector expressing PGIS for preventing monocrotaline-induced pulmonary arterial hypertension in rats.

Characterization of a novel oncogenic K-ras mutation in colon cancer.

Activating mutations of RAS are frequently observed in subsets of human cancers, indicating that RAS activation is involved in tumorigenesis. Here, we identified and characterized a novel G to T transversion mutation of the K-ras gene at the third position of codon 19 (TTG) which substituted phenylalanine for leucine in 3 primary colon carcinomas. Biological and biochemical activity was examined using transformed NIH3T3 cells expressing mutant or wild-type K-ras.

A histone deacetylase inhibitor enhances recombinant adeno-associated virus-mediated gene expression in tumor cells.

The transduction of cancer cells using recombinant adeno-associated virus (rAAV) occurs with low efficiency, which limits its utility in cancer gene therapy. We have previously sought to enhance rAAV-mediated transduction of cancer cells by applying DNA-damaging stresses. In this study, we examined the effects of the histone deacetylase inhibitor FR901228 on tumor transduction mediated by rAAV types 2 and 5.