Adeno-associated virus vector system controlling capsid expression improves viral quantity and quality.

Adeno-associated virus (AAV) vectors are promising tools for gene therapy. The current AAV vector system produces an abundance of empty capsids that are eliminated before clinical use, leading to increased costs for gene therapy. In the present study, we established an AAV production system that regulates the timing of capsid expression using a tetracycline-dependent promoter. Tetracycline-regulating capsid expression increased viral yield and reduced empty capsids in various serotypes without altering AAV vector infectivity in vitro and in vivo. The replicase expression pattern change observed in the developed AAV vector system improved viral quantity and quality, whereas timing control of capsid expression reduced empty capsids. These findings provide a new perspective on the development of AAV vector production systems in gene therapy.

Highly efficient genome editing for single-base substitutions using optimized ssODNs with Cas9-RNPs.

Target-specific genome editing using engineered nucleases has become widespread in various fields. Long gene knock-in and single-base substitutions can be performed by homologous recombination (HR), but the efficiency is usually very low. To improve the efficiency of knock-in with single-stranded oligo DNA nucleotides (ssODNs), we have investigated optimal design of ssODNs in terms of the blocking mutation, orientation, size, and length of homology arms to explore the optimal parameters of ssODN design using reporter systems for the detection of single-base substitutions. We have also investigated the difference in knock-in efficiency among the delivery forms and methods of Cas9 and sgRNA. The knock-in efficiencies for optimized ssODNs were much higher than those for ssODNs with no blocking mutation. We have also demonstrated that Cas9 protein/sgRNA ribonucleoprotein complexes (Cas9-RNPs) can dramatically reduce the re-cutting of the edited sites.

Phase I clinical trial of adoptive transfer of expanded natural killer cells in combination with IgG1 antibody in patients with gastric or colorectal cancer.

Natural killer (NK) cells exhibit strong cytotoxic activity against tumor cells without prior sensitization, and have the potential to exert antibody-dependent cellular cytotoxicity (ADCC). In this clinical trial, we examined the safety and efficacy of the use of NK cells, generated using a novel expansion system, in combination with IgG1 antibodies for the treatment of advanced gastric or colorectal cancers. Treatment consisted of trastuzumab- or cetuximab-based chemotherapy, plus adoptive NK cell therapy. For administration of expanded NK cells, dose escalation with a sequential 3 + 3 design was performed in three steps, at doses of 0.5 × 109 , 1.0 × 109 , and 2.0 × 109 cells/injection (N = 9). After 3 days of IgG1 antibody administration, patients were infused with expanded NK cells three times at triweekly intervals. NK cell populations expanded with our system were confirmed as being enriched in NK cells (median 92.9%) with high expression of NKG2D (97.6%) and CD16 (69.6%). The combination therapy was very well tolerated with no severe adverse events. Among six evaluable patients, four presented stable disease (SD) and two presented progressive disease. Of the four SD patients, three showed an overall decrease in tumor size after combination therapy. Immune monitoring suggested that combination therapy enhanced whole blood IFN-γ production and reduced peripheral regulatory T cells (Tregs). In conclusion, this phase I trial provides evidence of good tolerability, induction of Th1 immune responses, and preliminary anti-tumor activity for this combination therapy, in patients with advanced gastric and colorectal cancer that have received previous therapy.

Phase I clinical trial of autologous NK cell therapy using novel expansion method in patients with advanced digestive cancer.

BACKGROUND: NK cells can destroy tumor cells without prior sensitization or immunization. Tumors often lose expression of MHC molecules and/or antigens. However, NK cells can lyse tumor cells in a non-MHC-restricted manner and independent of the expression of tumor-associated antigens. NK cells are therefore considered ideal for adoptive cancer immunotherapy; however the difficulty of obtaining large numbers of fully functional NK cells that are safe to administer deters its clinical use. This phase I clinical trial seeks to address this obstacle by first developing a novel system that expands large numbers of highly activated clinical grade NK cells, and second, determining if these cells are safe in a mono-treatment so they can be combined with other reagents in the next round of clinical trials. METHODS: Patients with unresectable, locally advanced and/or metastatic digestive cancer who did not succeed with standard therapy were enrolled. NK cells were expanded ex vivo by stimulating PBMCs with OK432, IL-2, and modified FN-CH296 induced T cells. Patients were administered autologous natural killer cell three times weekly via intravenous infusions in a dose-escalating manner (dose 0.5 × 10(9), 1.0 × 10(9), 2.0 × 10(9) cells/injection, three patients/one cohort). RESULTS: Total cell population had a median expansion of 586-fold (range 95-1102), with a significantly pure (90.96 %) NK cell population. Consequently, NK cells were expanded to approximately 4720-fold (range 1372-14,116) with cells being highly lytic in vitro and strongly expressing functional markers such as NKG2D and CD16. This NK cell therapy was very well tolerated with no severe adverse events. Although no clinical responses were observed, cytotoxicity of peripheral blood was elevated approximately twofolds up to 4 weeks post the last transfer. CONCLUSION: We successfully generated large numbers of activated NK cells from small quantities of blood without prior purification of the cells. We also determined that the expanded cells were safe to administer in a monotherapy and are suitable for the next round of clinical trials where their efficacy will be tested combined with other reagents. TRIAL REGISTRATION: UMIN UMIN000007527.

Stimulation through very late antigen-4 and -5 improves the multifunctionality and memory formation of CD8⁺ T cells.

T cells express multiple integrin molecules. The significance of signaling through these molecules on acquisition of T-cell effector functions and memory formation capacity remains largely unknown. Moreover, the impact of stimulation through these signals on the generation of T cells for adoptive immunotherapy has not been elucidated. In this study, using a recombinant fragment of fibronectin, CH-296, we demonstrated that stimulation via very late Ag (VLA)-4 and VLA-5 in human and BALB/c mouse CD8(+) T cells, in combination with TCR stimulation, enhances effector multifunctionality and in vivo memory formation. Using TCR-transgenic mouse-derived CD8(+) T cells expressing TCR specific for the syngeneic CMS5 fibrosarcoma-derived tumor Ag, we showed that stimulation by CH-296 improved the ability of tumor-specific CD8(+) T cells to inhibit CMS5 tumor growth when adoptively transferred into hosts with progressing tumors. Improved antitumor effects were associated with decreased infiltration of Foxp3(+) CD4(+) Treg cells in tumors. These results suggest that stimulation via VLA-4 and VLA-5 modulates the qualities of effector T cells and could potentially increase the efficacy of adoptive therapy against cancer.

Phase I clinical trial of fibronectin CH296-stimulated T cell therapy in patients with advanced cancer.

BACKGROUND: Previous studies have demonstrated that less-differentiated T cells are ideal for adoptive T cell transfer therapy (ACT) and that fibronectin CH296 (FN-CH296) together with anti-CD3 resulted in cultured cells that contain higher amounts of less-differentiated T cells. In this phase I clinical trial, we build on these prior results by assessing the safety and efficacy of FN-CH296 stimulated T cell therapy in patients with advanced cancer. METHODS: Patients underwent fibronectin CH296-stimulated T cell therapy up to six times every two weeks and the safety and antitumor activity of the ACT were assessed. In order to determine immune function, whole blood cytokine levels and the number of peripheral regulatory T cells were analyzed prior to ACT and during the follow up. RESULTS: Transferred cells contained numerous less-differentiated T cells greatly represented by CD27+CD45RA+ or CD28+CD45RA+ cell, which accounted for approximately 65% and 70% of the total, respectively. No ACT related severe or unexpected toxicities were observed. The response rate among patients was 22.2% and the disease control rate was 66.7%. CONCLUSIONS: The results obtained in this phase I trial, indicate that FN-CH296 stimulated T cell therapy was very well tolerated with a level of efficacy that is quite promising. We also surmise that expanding T cell using CH296 is a method that can be applied to other T- cell-based therapies. TRIAL REGISTRATION: UMIN UMIN000001835.

Drawing a high-resolution functional map of adeno-associated virus capsid by massively parallel sequencing.

Adeno-associated virus (AAV) capsid engineering is an emerging approach to advance gene therapy. However, a systematic analysis on how each capsid amino acid contributes to multiple functions remains challenging. Here we show proof-of-principle and successful application of a novel approach, termed AAV Barcode-Seq, that allows us to characterize phenotypes of hundreds of different AAV strains in a high-throughput manner and therefore overcomes technical difficulties in the systematic analysis. In this approach, we generate DNA barcode-tagged AAV libraries and determine a spectrum of phenotypes of each AAV strain by Illumina barcode sequencing. By applying this method to AAV capsid mutant libraries tagged with DNA barcodes, we can draw a high-resolution map of AAV capsid amino acids important for the structural integrity and functions including receptor binding, tropism, neutralization and blood clearance. Thus, Barcode-Seq provides a new tool to generate a valuable resource for virus and gene therapy research.

Anti-tumor-promoting activities of agaro-oligosaccharides on two-stage mouse skin carcinogenesis.

We have previously reported that agaro-oligosaccharides (AGOs) suppressed the elevated levels of nitric oxide (NO), prostaglandin E2(PGE2), and pro-inflammatory cytokines in activated monocytes/macrophages, via heme oxygenase-1 induction. In this report, we initially demonstrated that AGOs intake inhibited NO production in activated peritoneal macrophages. Then, we tested for the ability of AGOs to prevent tumor promotion on the two-stage mouse skin carcinogenesis model. As a result, AGOs feeding led to delayed tumor appearance and decreased tumor number. It is known that PGE2 is one of key players in carcinogenesis. Thus, we confirmed that PGE2 production was suppressed by AGOs intake in TPA-induced ear edema model. We also demonstrated that cyclooxygenase-2 and microsomal PGE synthase-1, rate-limiting enzymes in PGE2 production, were down-regulated by AGOs in human monocytes. Consequently, AGOs are expected to prevent tumor promotion by inhibiting PGE2 elevation in chronic inflammation site.

Angelica keiskei extract improves insulin resistance and hypertriglyceridemia in rats fed a high-fructose drink.

Angelica keiskei is a traditional herb peculiar to Japan and abundantly contains vitamins, dietary fiber and such polyphenols as chalcone. We investigated in the present study the effect of A. keiskei on insulin resistance and hypertriglyceridemia in fructose-drinking rats as a model for the metabolic syndrome. Male Wistar rats were given a 15% fructose solution as drinking water for 11 weeks. Fructose significantly increased the levels of serum insulin and triglyceride (TG) compared with the control level. Treatment with an ethanol extract of A. keiskei (AE) significantly reduced the levels of blood glucose (-16.5%), serum insulin (-47.3%), HOMA-R (-56.4%) and TG (-24.2%). A hepatic gene analysis showed that fructose reduced the expression of the genes related to fatty acid ß-oxidation and high-density lipoprotein (HDL) production. Treatment with AE enhanced the expression of the acyl-CoA oxidase 1 (ACO1), medium-chain acyl-CoA dehydrogenase (MCAD), ATP-binding membrane cassette transporter A1 (ABCA1) and apolipoprotein A1 (Apo-A1) genes. These results suggest that AE improved the insulin resistance and hypertriglyceridemia of the fructose-drinking rats.

Six new chalcones from Angelica keiskei inducing adiponectin production in 3T3-L1 adipocytes.

Angelica keiskei (Ashitaba in Japanese), a traditional herb in Japan, contains abundant prenylated chalcones. It has been reported that the chalcones from A. keiskei showed such bioactivities as anti-bacterial, anti-cancer and anti-diabetic effects. Xanthoangelol, 4-hydroxyderricin and six new chalcones were isolated in this study from an ethanol extract of A. keiskei by octadecyl silyl (ODS) and silica gel chromatography, and identified by 1D- and 2D-nuclear magnetic resonance (NMR) and high-resolution mass spectrometric analyses. The chalcones from A. keiskei markedly increased the expression of the adiponectin gene and the production of adiponectin in 3T3-L1 adipocytes. These results suggest that the chalcones from A. keiskei might be useful for preventing the metabolic syndrome.

Synergistic effect of CH-296 and interferon gamma on cytokine-induced killer cells expansion for patients with advanced-stage malignant solid tumors.

BACKGROUND: Cytokine-induced killer cells (CIKs) are heterogenous antitumor effectors including interferon gamma (IFN-γ)-amplified CD3(+)CD56(+) cells. CH-296 has been shown to stimulate T-cell proliferation in the presence of T cell receptor (TCR)-stimulating signals. The purpose of this study was to investigate the synergistic effect of CH-296 and IFN-γ on expansion of CIKs for treating patients with advanced-stage malignant solid tumors. METHODS: CIKs were cultured with immobilized CH-296 in the presence (retronectin [RN]-CIKs) or absence of IFN-γ (RN-CIKs/del) for 14 days. Proliferation, apoptosis, phenotype, and cytotoxicity were detected. Twenty (20) patients (18 patients with stage IV solid tumors) received three cycles of RN-CIKs treatment. The clinical responses were evaluated using Karnofsky Performance Status scoring and computed-tomography scanning. RESULTS: CH-296 promoted CIKs expansion in a time-dependent manner by inhibiting apoptosis and increasing proliferation. Costimulation of CH-296 and IFN-γ amplified more antitumor effectors of CIKs with activated T-cell phenotype, which displayed potent cytotoxicity and increased cytokines secretion upon antigen priming. Sixteen (16) patients receiving RN-CIKs experienced relief of clinical symptoms. The overall clinical response rate was 65% (13/20) and the mean overall survival was 16.95±6.10 months. No severe adverse events were observed in the clinical trial. CONCLUSIONS: CH-296 and IFN-γ synergistically promote antitumor efficiency of CIKs by increasing proliferation, inhibiting apoptosis, and enhancing cytotoxicity.

Oligosaccharides from agar inhibit pro-inflammatory mediator release by inducing heme oxygenase 1.

We investigated whether agaro-oligosaccharides have any immunological effects on RAW264.7 mouse macrophages and human monocytes in vitro. We demonstrate that agaro-oligosaccharides suppressed the elevated levels of nitric oxide, prostaglandin E(2), and such pro-inflammatory cytokines as tumor necrosis factor-alpha, interleukin-1beta and interleukin-6 in lipopolysaccharide-stimulated monocytes and macrophages. We also demonstrate that those effects of agaro-oligosaccharides on activated monocytes and macrophages may have been caused by heme oxygenase-1 induction. It is therefore proposed that agaro-oligosaccharides might be a good candidate for a functional food to prevent inflammatory diseases.

Highly efficient isothermal DNA amplification system using three elements of 5'-DNA-RNA-3' chimeric primers, RNaseH and strand-displacing DNA polymerase.

We developed an efficient method of isothermally amplifying DNA termed ICAN, Isothermal and Chimeric primer-initiated Amplification of Nucleic acids. This method allows the amplification of target DNA under isothermal conditions at around 55 degrees C using only a pair of 5'-DNA-RNA-3' chimeric primers, a thermostable RNaseH and a DNA polymerase with strong strand-displacing activity. ICAN is capable of amplifying DNA at least several times greater than the amount produced with PCR by increasing primer concentration. This method would be applicable for on-site DNA detection including gene diagnosis, and would also be suitable for 'real time' detection when combined with a cycling probe.

Antidiabetic activities of chalcones isolated from a Japanese Herb, Angelica keiskei.

Diabetes mellitus is a chronic disease that is characterized by hyperglycemia caused by insufficient insulin action. We have explored the edible ingredients from folk medicines in Japan that contain substances complementing insulin action, such as the induction of adipocyte differentiation and the enhancement of glucose uptake. We eventually found that the ethanol extract from a Japanese herb "Ashitaba", Angelica keiskei, contained two major chalcones of 4-hydroxyderricin (4-HD) and xanthoangelol that showed strong insulin-like activities via a pathway independent of the peroxisome proliferator-activated receptor-gamma activation. The 4-HD especially showed the preventive effects on the progression of diabetes in genetically diabetic KK-Ay mice.