Efficient Gene Transduction Research Articles

Establishment of a cell culture platform for human liver organoids and its application for lipid metabolism research.

Human liver organoids (HLOs) are reliable tools to represent physiological human liver biology. However, their use is limited especially in basic sciences. One of the reasons for this would be the insufficient systematic methodology to handle HLOs, including culture system, functional assessment, and gene transduction. Here, we generated and characterized mouse L cells stably and simultaneously overexpressing R-spondin1, hepatocyte growth factor, fibroblast growth factor (FGF) 7, and FGF10 via lentiviral transduction. The conditioned medium of the cells contributed to HLO growth as a replacement of commercially available recombinant proteins, which leads to a significant reduction of their culture cost. Proliferative and maturation phases of the cells were controlled by switching the medium to facilitate the evaluation of hepatocyte function, including insulin responsiveness and intracellular lipid accumulation. Gene expression analysis revealed that HLOs highly expressed genes involved in lipid metabolism. Importantly, HLOs secreted physiologically matured very low-density lipoprotein, which is rarely observed in mice and in established cell lines. Efficient gene transduction into HLOs was achieved via a transient 2-dimensional culture during viral infection. This study provides an invaluable platform for utilizing HLOs in various research fields, such as molecular biology, pharmacology, toxicology, and regenerative medicine.

PB0183 Efficient Gene Transduction in Pigs and Macaques with the Engineered AAV Vector AAV.GT5 for Hemophilia B Gene Therapy

PB0183 Efficient Gene Transduction in Pigs and Macaques with the Engineered AAV Vector AAV.GT5 for Hemophilia B Gene Therapy

Efficient gene transduction in pigs and macaques with the engineered AAV vector AAV.GT5 for hemophilia B gene therapy

Gene therapy using adeno-associated virus (AAV)-based vectors has become a realistic therapeutic option for hemophilia. We examined the potential of a novel engineered liver-tropic AAV3B-based vector, AAV.GT5, for hemophilia B gene therapy. Invitro transduction with AAV.GT5 in human hepatocytes was more than 100 times higher than with AAV-Spark100, another bioengineered vector used in a clinical trial. However, liver transduction following intravenous injection of these vectors was similar in mice with a humanized liver and in macaques. This discrepancy was due to the low recovery and short half-life of AAV.GT5 in blood, depending on the positive charge of the heparin-binding site in the capsid. Bypassing systemic clearance with the intra-hepatic vascular administration of AAV.GT5, but not AAV-Spark100, enhanced liver transduction in pigs and macaques. AAV.GT5 did not develop neutralizing antibodies (NAbs) in two of four animals, while AAV-Spark100 induced serotype-specific NAbs in all macaques tested (4 of 4). The NAbs produced after AAV-Spark100 administration were relatively serotype specific, and challenge with AAV.GT5 through the hepatic artery successfully boosted liver transduction in one animal previously administered AAV-Spark100. In summary, AAV.GT5 showed different vector kinetics and NAb induction compared with AAV-Spark100, and intra-hepatic vascular administration may minimize the vector dose required and vector dissemination.

Extracellular Vesicle-Encapsulated AAVs for Therapeutic Gene Delivery to the Heart.

Adeno-associated virus (AAV) has emerged as one of the best tools for cardiac gene delivery due to its cardiotropism, long-term expression, and safety. However, a significant challenge to its successful clinical use is preexisting neutralizing antibodies (NAbs), which bind to free AAVs, prevent efficient gene transduction, and reduce or negate therapeutic effects. Here we describe extracellular vesicle-encapsulated AAVs (EV-AAVs), secreted naturally by AAV-producing cells, as a superior cardiac gene delivery vector that delivers more genes and offers higher NAb resistance. We developed a 2-step density-gradient ultracentrifugation method to isolate highly purified EV-AAVs. We compared the gene delivery and therapeutic efficacy of EV-AAVs with an equal titer of free AAVs in the presence of NAbs, both in vitro and in vivo. In addition, we investigated the mechanism of EV-AAV uptake in human left ventricular and human induced pluripotent stem cell cardiomyocytes in vitro and mouse models in vivo using a combination of biochemical techniques, flow cytometry, and immunofluorescence imaging. Using cardiotropic AAV serotypes 6 and 9 and several reporter constructs, we demonstrated that EV-AAVs deliver significantly higher quantities of genes than AAVs in the presence of NAbs, both to human left ventricular and human induced pluripotent stem cell cardiomyocytes in vitro and to mouse hearts in vivo. Intramyocardial delivery of EV-AAV9- sarcoplasmic reticulum calcium ATPase 2a to infarcted hearts in preimmunized mice significantly improved ejection fraction and fractional shortening compared with AAV9-sarcoplasmic reticulum calcium ATPase 2a delivery. These data validated NAb evasion by and therapeutic efficacy of EV-AAV9 vectors. Trafficking studies using human induced pluripotent stem cell-derived cells in vitro and mouse hearts in vivo showed significantly higher expression of EV-AAV6/9-delivered genes in cardiomyocytes compared with noncardiomyocytes, even with comparable cellular uptake. Using cellular subfraction analyses and pH-sensitive dyes, we discovered that EV-AAVs were internalized into acidic endosomal compartments of cardiomyocytes for releasing and acidifying AAVs for their nuclear uptake. Together, using 5 different in vitro and in vivo model systems, we demonstrate significantly higher potency and therapeutic efficacy of EV-AAV vectors compared with free AAVs in the presence of NAbs. These results establish the potential of EV-AAV vectors as a gene delivery tool to treat heart failure.

The Magic Staff: A Comprehensive Overview of Baculovirus-Based Technologies Applied to Human and Animal Health.

Baculoviruses are enveloped, insect-specific viruses with large double-stranded DNA genomes. Among all the baculovirus species, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most studied. Due to its characteristics regarding biosafety, narrow host range and the availability of different platforms for modifying its genome, AcMNPV has become a powerful biotechnological tool. In this review, we will address the most widespread technological applications of baculoviruses. We will begin by summarizing their natural cycle both in larvae and in cell culture and how it can be exploited. Secondly, we will explore the different baculovirus-based protein expression systems (BEVS) and their multiple applications in the pharmaceutical and biotechnological industry. We will focus particularly on the production of vaccines, many of which are either currently commercialized or in advanced stages of development (e.g., Novavax, COVID-19 vaccine). In addition, recombinant baculoviruses can be used as efficient gene transduction and protein expression vectors in vertebrate cells (e.g., BacMam). Finally, we will extensively describe various gene therapy strategies based on baculoviruses applied to the treatment of different diseases. The main objective of this work is to provide an extensive up-to-date summary of the different biotechnological applications of baculoviruses, emphasizing the genetic modification strategies used in each field.

Abstract 12178: Extracellular Vesicle-Encapsulated AAVs for Therapeutic Gene Delivery to the Heart

Background: Adeno-associated virus (AAV) has emerged as one of the best tools for cardiac gene delivery due to its cardiotropism, long-term expression, and safety. However, a significant challenge to its successful clinical use are pre-existing neutralizing antibodies (NAbs), which bind to free AAVs, prevent efficient gene transduction and reduce or negate therapeutic effects. Here we describe extracellular vesicle-encapsulated AAVs (EV-AAVs), secreted naturally by AAV-producing cells, as a superior cardiac gene delivery vector that delivers more genes and offers higher NAb resistance. Method: We developed a two-step density-gradient ultracentrifugation method to isolate highly purified EV-AAVs. Results: Using cardiotropic AAV serotypes 6 and 9 and several reporter constructs, we demonstrated that EV-AAVs deliver significantly higher quantities of genes than AAVs in the presence of NAb, both to human left ventricular and hiPSC cardiomyocytes in vitro, and to mouse hearts in vivo. Intramyocardial delivery of EV-AAV9-SERCA2a to infarcted hearts in pre-immunized mice significantly improved ejection fraction and fractional shortening compared to AAV9-SERCA2a delivery. These data validated NAb evasion by and therapeutic efficacy of EV-AAV9 vectors. Interestingly, in vivo trafficking studies in mouse hearts showed significantly higher expression of EV-AAV9-delivered genes in cardiomyocytes compared to non-cardiomyocytes, even with comparable cellular uptake. Using cellular sub-fraction analyses and pH-sensitive dyes, we discovered that EV-AAVs were internalized into acidic endosomal compartments of cardiomyocytes for releasing and acidifying AAVs for their nuclear uptake. Conclusion: Together, using six different in vitro and in vivo model systems, we demonstrate significantly higher potency and therapeutic efficacy of EV-AAV vectors compared to free AAVs. These results establish the potential of EV-AAV vectors as a gene delivery tool to treat heart failure. 1

Ecotropic HIV-1 vectors pseudotyped with R-peptide-deleted envelope protein variants reveal improved gene transfer efficiencies

Viral vectors derived from human immunodeficiency virus type 1 (HIV-1) mediate efficient stable gene transduction. Consequently, these vectors are utilized in gene therapeutic approaches. We here aimed for improving HIV-1 pseudotype vector formation using envelope proteins (Env) of ecotropic murine leukemia virus (MLV) suffering deletions of the R-peptide and further amino acid substitutions in their cytoplasmatic domains. All examined Env variants revealed cell-surface expression and showed elevated fusogenicity as compared to wildtype (eMLV-wt) Env but failed to efficiently pseudotype MLV particles. However, two variants generated ecotropic HIV-1 pseudotype vectors with superior infectivity. Most importantly, pseudotyping with the variant eMLV-GaLVΔR encompassing the R-peptide-deleted cytoplasmic domain of the gibbon ape leukemia virus Env yielded titers three-fold higher than HIV(eMLV-wt) vectors. We anticipate that superior ecotropic HIV(eMLV-GaLVΔR) pseudotype vectors will be of utility in preclinical gene therapy studies aiming at the genetic modification of primary murine cells.

Intravenous immunoglobulin prevents peripheral liver transduction of intrathecally delivered AAV vectors

Gene therapy using neurotropic adeno-associated virus vectors represents an emerging solution for genetic disorders affecting the central nervous system. The first approved central nervous system-targeting adeno-associated virus gene therapy, Zolgensma®, for treating spinal muscular atrophy is administered intravenously at high doses that cause liver-associated adverse events in 20%-30% of patients. Intrathecal routes of vector administration, such as the intra-cisterna magna route, provide efficient gene transduction to central nervous system cells while reducing off-target liver transduction. However, significant levels of liver transduction often occur upon intra-cisterna magna vector delivery in preclinical studies. Using vectors expressing monoclonal antibody transgenes, we examined whether passive transfer of adeno-associated virus-neutralizing antibodies as intravenous immunoglobulin before intrathecal adeno-associated virus delivery improved the safety of viral gene therapy targeting the central nervous system in mice and nonhuman primates. We used intracerebroventricular and intra-cisterna magna routes for vector administration to mice and nonhuman primates, respectively, and evaluated transgene expression and vector genome distribution. Our data indicate that pretreatment with intravenous immunoglobulin significantly reduced gene transduction to the liver and other peripheral organs but not to the central nervous system in both species. With further refinement, this method may improve the safety of adeno-associated virus-based, central nervous system-targeting gene therapies in clinical settings.

Transduction catalysis: Doxorubicin amplifies rAAV-mediated gene expression in the cortex of higher-order vertebrates

SummaryRapid and efficient gene transduction via recombinant adeno-associated viruses (rAAVs) is highly desirable across many basic and clinical research domains. Here, we report that vector co-infusion with doxorubicin, a clinical anti-cancer drug, markedly enhanced rAAV-mediated transgene expression in the cerebral cortex across mammalian species (cat, mouse, and macaque), acting throughout the time period examined and detectable at just three days after transfection. This enhancement showed serotype generality, being common to all rAAV serotypes tested (2, 8, 9, and PHP.eB) and was observed both locally and at remote locations consistent with doxorubicin undergoing retrograde axonal transport. All these effects were observed at doses matching human blood plasma levels in clinical therapy and lacked detectable cytotoxicity as assessed by cell morphology, activity, apoptosis, and behavioral testing. Altogether, this study identifies an effective means to improve the capability and scope of in vivo rAAV applications, amplifying cell transduction at doxorubicin concentrations paralleling medical practice.

Transduction Catalysis: Doxorubicin Accelerates and Enhances rAAV-Mediated Gene Expression in the Cortex of Mouse, Cat and Monkey

Rapid and efficient gene transduction via recombinant adeno-associated viruses (rAAVs) is highly desirable across many basic and clinical research domains. Here we report vector co-infusion with doxorubicin, a clinical anti-cancer drug, markedly enhanced rAAV-mediated gene expression in the cerebral cortex across mammalian species (cat, mouse, and macaque), acting throughout the time-period examined and detectable at just three days post-transfection. This enhancement showed serotype generality, being common to rAAV serotypes 2, 8, 9 and PHP.eB tested, and was observed both locally, and at remote locations consistent with doxorubicin undergoing retrograde axonal transport. All these effects were observed at doses matching human blood plasma levels in clinical therapy, and lacked detectable cytotoxicity as assessed by cell morphology, activity, apoptosis and behavioral testing. Altogether, this study identifies an effective means to improve the capability and scope of in vivo rAAV applications, accelerating and augmenting gene transduction at doxorubicin concentrations paralleling medical practice.

F-Deletion Non-Integrating Measles Virus Vector: A Promising Tool for T-Cell Engineering and Naive iPSCs Generation

Although great successes of chimeric antigen receptor T-cell (CAR-T) therapy highlighted the importance of anti-cancer immunity for cancer treatment, there are still some problems remained, i.e., long preparation time, extremely high cost and potential risk by insertional mutagenesis. To developmore rapid and safer T-cell engineering systems than current procedures using retrovirus or lentiviral vectors, we have long been focusing on measles virus as a new vector because of its high infectivity to T cells including resting state and rapid gene expression without chromosome integration. Presently, Sendai virus vectors (SVs), which is also a Paramyxoviridaevirus-based vector, are widely used for gene delivery and induced pluripotent stem cells (iPSCs), but the transduction to undifferentiated T cells (UTs) is a big challenge for SVs. We, therefore, compared the gene transduction efficiency between our measles vector (MVs) and SVs. We also compared iPSCs generation efficiencies between MVs and SVs. We engineered our non-replicating and non-integrating measles virus vectors (MV)with F deletion to eliminate cell membrane fusion-associated cytotoxicity.Based on the original property of measles virus,our recombinant MVsallowed more efficient gene transduction to various hematopoietic cells including UTs and B cells than SVs. Importantly, MVs induced less apoptosis compared withSVs due to their slower amplification of viral RNA in transduced cells. Moreover, we could establish iPSCs from UTs with MVs harboring reprogramming genes 50 times more efficiently than SVs harboring the same reprogramming genes. MV-induced iPSCs derived from CD3+T cells (MV-TiPSCs) were similar to regular human pluripotent stem cells (hPSCs: embryonic stem cells and iPSCs), which are in primed state, in morphology, the expressions of pluripotent markers and the ability to differentiate into three germ layers. On the other hand, without using naive induction culture condition, MV-induced iPSCs derived from CD34+hematopoietic progenitor cells (MV-HPC-iPSCs) presented a dome shape and showed a transcriptome profile close to naive iPSCs. To further confirm naive-like properties of MV-HPC-iPSCs, we evaluated gene expression patterns of these cells for 22 common genes most differently expressed in naive and primed hPSCs reported in previous reports (Fig.1).As expected, MV-HPC-iPSCs were clustered in naive hPSCs group while MV-HPC-iPSCs after culturing in primed induction condition showed primed-like features (Fig.2). Moreover, whole genome bisulfite sequencing analysis showed that MV-HPC-iPSCs had lower methylation than primed MV-HPC-iPSCs. These results strongly suggested that MV could induce naive-like iPSCs directly, and primed induction culture changed the cells to primed state with increasing genomic methylation. Considering the very safe history of MV vaccine, the capabilities of simultaneous expressions of multiple genes and the high transduction efficiency for hematopoietic cells including UTs, our MVs will be useful to directly induce naive state iPSCs, and be a promising tool for developing new T-cell immunotherapies. Disclosures Liao: TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding. Soda:TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding. Miura:Neoprecision therapeutics: Research Funding. Tahara:TAKARA BIO, INC.: Research Funding. Miyamoto:neopharma Japan Co. Ltd: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Takeda:TAKARA BIO, INC.: Research Funding. Tani:Oncolys BioPharma Inc.: Equity Ownership; SymBio Pharmaceuticals Limited: Equity Ownership; TAKARA BIO, INC.: Research Funding; Neoprecision therapeutics: Equity Ownership, Research Funding; neopharma Japan Co. Ltd: Research Funding; Shinnihonseiyaku Co., Ltd: Equity Ownership.

Non-viral Gene Therapy for Stargardt Disease with ECO/pRHO-ABCA4 Self-Assembled Nanoparticles.

Stargardt disease (STGD) is an autosomal recessive retinal disorder caused by a monogenic ABCA4 mutation. Currently, there is no effective therapy to cure Stargardt disease. The replacement of mutated ABCA4 with a functional gene remains an attractive strategy. In this study, we have developed a non-viral gene therapy using nanoparticles self-assembled by a multifunctional pH-sensitive amino lipid ECO and a therapeutic ABCA4 plasmid. The nanoparticles mediated efficient intracellular gene transduction in wild-type (WT) and Abca4-/- mice. Specific ABCA4 expression in the outer segment of photoreceptors was achieved by incorporating a rhodopsin promoter into the plasmids. The ECO/pRHO-ABCA4 nanoparticles induced substantial and specific ABCA4 expression for at least 8months, 35% reduction in A2E accumulation on average, and a delayed Stargardt disease progression for at least 6months in Abca4-/- mice. ECO/plasmid nanoparticles constitute a promising non-viral gene therapy platform for Stargardt disease and other visual dystrophies.

Three-dimensional microenvironmental priming of human mesenchymal stem cells in hydrogels facilitates efficient and rapid retroviral gene transduction via accelerated cell cycle synchronization

There are numerous approaches to improve the low transduction efficiency of retroviral vectors in two-dimensional (2D) cell culture substrates. However, the effect of a three-dimensional (3D) microenvironment, which better mimics in vivo conditions, is unknown. Cytocompatible hyaluronic acid (HA) hydrogels are a good candidate to study this issue. Here, photocrosslinkable HA hydrogels with an elastic modulus of 1.0–2.7 kPa were successfully prepared by varying the degree of methacrylation in the HA backbone. Culturing human adipose-derived stem cells (hASCs) in a 3D microenvironment significantly reduces the amount of time required for retroviral gene transduction compared with the conventional 2D method and maintains a high transduction efficiency. This acceleration of retroviral gene transduction correlates with the rate of cell-cycle synchronization. hASCs cultured in a 3D microenvironment have a shorter G1 phase and total cell-cycle length than hASCs cultured using the conventional 2D method. This cell-cycle regulation is dependent on expression of cyclin D1. In summary, prior culturing of hASCs in a 3D microenvironment accelerates retroviral gene transduction by regulating cyclin D1 expression and accelerating cell-cycle synchronization. We conclude that priming via culturing in a 3D microenvironment facilitates efficient and rapid retroviral gene transduction of hASCs without inducing apoptosis.

Efficient Gene Transduction and Reprogramming of Hematopoietic Cells Including T-Cells By Using a Non-Integrating Measles Virus Vector

By the ectopic expression of reprogramming genes OCT, KLF4, SOX2 and MYC (OKSM), somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs). Human iPSCs are considered a promising cell source to provide an import tool for the basic investigation and the advanced medicine including gene therapy and regenerative medicine. To establish iPSCs, integration-free Sendai virus (SV) vectors have been most widely used do far, but transduction and reprogramming of T cells without stimulation is still very challenging. On the other hand, a great success of chimeric antigen receptor T cell (CAR-T) therapies highlighted the importance of anti-cancer immunity for the cancer treatment. Particularly, many refractory patients with acute lymphoblastic leukemia and B-cell lymphoma were successfully treated with CD19-CAR-T therapies, however, some patients died before receiving the treatment due to long preparation time of CAR-Ts. Therefore, rapid production systems of CAR-Ts are desired, and for this purpose, efficient and safe gene transduction systems to T cells should be developed.In this study, we developed a new non-integrating measles virus (MV) vector-based delivery system with F deletion to eliminate cell membrane fusion-associated cytotoxicity. MV vectors transduced genes through MV receptors including CD46 and signaling lymphocyte activation molecule (CD150/SLAM). First, we examined transduction efficiencies of MV vectors and SV vectors in hematopoietic cells by using GFP expression vectors (MV-Gs and SV-Gs). Compared to SV-Gs, our MV-Gs allowed more efficient gene transfer into most hematopoietic cell type including T (3-fold) and B cells (7-fold) (Fig. 1). Furthermore, at the same multiplicity of infection (MOI) of viral transduction, MV-Gs induced less apoptosis in T cell subset compared to SV-Gs (Fig. 2) due to the slower kinetics of viral RNA amplification in the transduced cells 24 h ,48 h and 72 h post transduction.Those results encouraged us to examined if MV vectors are more potent than SV vectors in iPSC generation from unstimulated T cells. To address this question, we developed MV vectors harboring four reprogramming genes (MV-OKSMGs) and compared with SV vectors harboring these genes (SV-OKSMGs). As expected, with the MV-OKSMGs, we could generate high-quality iPSCs with the similar morphology, pluripotency markers, karyotype and differentiation capacity as human embryonic stem cells. Upon the less cytotoxicity, iPSC generation efficiency of MV-OKSMGs was much higher than that of SV-OKSMGs for unstimulated T cells (0.47 ± 0.25% vs 0.008 ± 0.009%).Considering the safe history of MV vaccine, carrying capabilities of multiple genes, more flexible receptors and higher transduction efficiency for resting T cells, our exclusive MV vector would be a potential gene transfer system for iPSC generation and lymphocyte-based-immunotherapies such as CAR-T therapies. [Display omitted] DisclosuresLiao:neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding. Soda:Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Sugawara:neopharma Japan Co. Ltd: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Miura:neopharma Japan Co. Ltd: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Tahara:TAKARA BIO, INC.: Research Funding. Takishima:neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding. Hirose:TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding. Hijikata:Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Miyamoto:Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding; neopharma Japan Co. Ltd: Research Funding. Takeda:TAKARA BIO, INC.: Research Funding. Tani:neopharma Japan Co. Ltd: Research Funding; Oncolys BioPharma Inc.: Equity Ownership; SymBio Pharmaceuticals Limited: Equity Ownership; TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding.

Progress of lentiviral vector in cancer gene therapy

Cancer gene therapy replies on efficient, safe and stable gene transduction. Lentiviral vector (LVs) stands out due to its good performance, which brings new hope to cancer treatment. In recent years, exquisite design, improved production process and advanced safty endow LVs broad prospects for application. The progresses of LVs in cancer vaccine production, chimeric antigen receptor-engineered T cells, suicide gene therapy, RNA-mediated interference, inhibition of tumor angiogenesis, help not only the development of cancer gene therapy, but also the practice of precision medicine. Key words: Lentiviral vector; Gene therapy; Tumor

Rapid and efficient gene delivery into the adult mouse brain via focal electroporation.

In vivo gene delivery is required for studying the cellular and molecular mechanisms of various biological events. Virus-mediated gene transfer or generation of transgenic animals is widely used; however, these methods are time-consuming and expensive. Here we show an improved electroporation technique for acute gene delivery into the adult mouse brain. Using a syringe-based microelectrode, local DNA injection and the application of electric current can be performed simultaneously; this allows rapid and efficient gene transduction of adult non-neuronal cells. Combining this technique with various expression vectors that carry specific promoters resulted in targeted gene expression in astrocytic cells. Our results constitute a powerful strategy for the genetic manipulation of adult brains in a spatio-temporally controlled manner.

Lentivirus-Based Stable Gene Delivery into Intestinal Organoids.

Lentivirus-based gene delivery works efficiently for the majority of mammalian cells cultured under standard two-dimensional conditions. By contrast, intestinal epithelial organoids embedded into three-dimensional extracellular matrix appear to be resistant to lentiviral transduction. We observed that Matrigel, a matrix that reconstitutes a basement membrane and is indispensable for cell survival and proliferation, prevents lentiviruses from binding to intestinal cells. In this chapter, we describe a simple method of a highly efficient gene transduction into intestinal organoids. This method involves organoid dispersion into single intestinal epithelial cells, mixing these individual cells with lentiviral particles, plating on Matrigel, and subsequent re-embedding into Matrigel. Under these conditions, the majority of the cells are exposed to the virus in the absence of the matrix barrier while remaining attached to the matrix. Using a GFP-labeled lentivirus, we demonstrate that this method allows for highly efficient infection of intestinal organoids after overnight incubation of Matrigel-attached cells with lentiviral particles.

Efficient Gene Transduction of Dispersed Islet Cells in Culture Using Fiber-Modified Adenoviral Vectors.

To establish novel islet-based therapies, our group has recently developed technologies for creating functional neo-islet tissues in the subcutaneous space by transplanting monolithic sheets of dispersed islet cells (islet cell sheets). Improving cellular function and viability are the next important challenges for enhancing the therapeutic effects. This article describes the adenoviral vector-mediated gene transduction of dispersed islet cells under culture conditions. Purified pancreatic islets were obtained from Lewis rats and dissociated into single islet cells. Cells were plated onto laminin-5-coated temperature-responsive polymer poly(N-isopropylacrylamide)-immobilized plastic dishes. At 0 h, islet cells were infected for 1 h with either conventional type 5 adenoviral vector (Ad-CA-GFP) or fiber-modified adenoviral vector (AdK7-CA-GFP) harboring a polylysine (K7) peptide in the C terminus of the fiber knob. We investigated gene transduction efficiency at 48 h after infection and found that AdK7-CA-GFP yielded higher transduction efficiencies than Ad-CA-GFP at a multiplicity of infection (MOI) of 5 and 10. For AdK7-CA-GFP at MOI = 10, 84.4 ± 1.5% of islet cells were found to be genetically transduced without marked vector infection-related cellular damage as determined by viable cell number and lactate dehydrogenase (LDH) release assay. After AdK7-CA-GFP infection at MOI = 10, cells remained attached and expanded to nearly full confluency, showing that this adenoviral infection protocol is a feasible approach for creating islet cell sheets. We have shown that dispersed and cultured islet cells can be genetically modified efficiently using fiber-modified adenoviral vectors. Therefore, this gene therapy technique could be used for cellular modification or biological assessment of dispersed islet cells.

Abstract 4401: Tumor-associated antigen gene-loading polyplex micelle is a promising platform for anti-cancer DNA vaccine

Abstract Backgrounds: Polyplex micelle attracts an increasing attention as a gene carrier, because of efficient gene transduction and biocompatibility in vivo. In this study, we examined the potential of polyplex micelle carrying genes encoding tumor-associated antigen (TAA): squamous cell carcinoma antigen recognized by T cells-3, CD40L and GM-CSF as a DNA vaccine platform in mouse tumor models with different types of major histocompatibility antigen complex (MHC). Methods and Results: Intraperitoneal administration of polyplex micelles were predominantly distributed and expressed in the lymph nodes, spleen and liver. The triple DNA vaccine significantly prolonged the survival of mice harboring peritoneal dissemination of CT26 colorectal cancer compared with mock controls, of which long-survivor mice induced complete rejection of re-challenged CT26 tumors. The DNA vaccine also inhibited the growth and metastasis of subcutaneous CT26 and Lewis lung cancers in BALB/c and C57BL/6 mice, respectively, which represent different MHC haplotypes. The DNA vaccine highly stimulated both cytotoxic T lymphocyte and natural killer cell activities, and increased the infiltration of CD11c+-DC, CD4+ and CD8a+ T cells into tumors. Depletion of CD4+ or CD8a+ cells using their neutralizing antibodies deteriorated the anti-tumor efficacy of the DNA vaccine. Conclusions: TAA/CD40L+GM-CSF gene-loaded polyplex micelle is a novel vaccine platform to elicit tumor rejection immunity regardless of recipient's MHC-haplotype. Note: This abstract was not presented at the meeting. Citation Format: Lin Cui, Kouichi Furugaki, Kensuke Osada, Kazunori Kataoka, Kenji Nakano. Tumor-associated antigen gene-loading polyplex micelle is a promising platform for anti-cancer DNA vaccine. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4401. doi:10.1158/1538-7445.AM2015-4401

Creating a Future of Transplantation.

On behalf of the Japan Society for Organ Preservation and Medical Biology (JSOPMB), I express my sincere appreciation to Dr. David J. Eve and Professor Cesar V. Borlongan, Coeditors of Cell Medicine, for providing us with such an excellent opportunity to publish the data that were presented at the annual meeting of the JSOPMB. I also thank Dr. David J. Eve for the editing of our articles in detail. I am very sure that the relationship between Cell Medicine and JSOPMB has enhanced the motivation of JSOPMB members as well as board members and will continue to do so in the future, while also encouraging young Japanese researchers to join this organization. One of the extremely important missions of the annual meeting of the JSOPMB is to exchange new research outcomes and create new therapeutic concepts. JSOPMB always encourages and motivates young investigators. JSOPMB was started in 1974 for the study of organ preservation and developed widely in the 1990s with the participation of researchers in various fields of medicine, pharmacology, engineering, veterinary medicine, and basic science. Currently, JSOPMB has more than 700 members and is run under the direction of Professor Takashi Kondo, the president of the JSOPMB. Excellent presentations conducted at the 41th annual meeting of the JSOPMB held on November 28–29, 2014, in Osaka, Japan, under the supervision of Dr. Yoshiki Sawa (Professor, Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Osaka, Japan), were selected and given an opportunity to be published in this special issue of Cell Medicine. Seven of these presentations are herein published in this special JSOPMB issue. Stem cell research was a major topic of interest. There were two articles regarding stem cells. Miyagi-Shiohira et al. reviewed cryopreservation of mesenchymal stem cells, especially adipose-derived stem cells. Dimethyl sulfoxide (DMSO) is commonly used as a cryopreservation medium as it diffuses into the cell through the plasma membrane and protects the cells from the damage caused by freezing. As substitutes for DMSO or animal-derived serum, cell banker series, polyvinylpyrrolidone (PVP), sericin and maltose, and methyl cellulose (MC) have been investigated for their clinical applications. Saitoh et al. evaluated feeder cells for establishing induced pluripotent stem cells (iPSCs) from human deciduous tooth dental pulp cells (HDDPCs). Their data showed that mouse embryonic fibroblasts (MEFs) were better feeder cells than an immortalized line established from Santos inbred mouse (SIM) embryonic fibroblasts resistant to 6-thioguanine and ouabain known as STO cells for establishing iPSCs. There were three articles regarding pancreatic islets. Noguchi et al. reviewed islet culture/preservation before islet transplantation. In the current clinical culture conditions, it seems that islets cultured at 37°C cannot receive enough oxygen because central necrosis of islets during culture was frequently observed. Low-temperature storage may be better than culture, although a future study should carefully evaluate the effects of islet equivalent and tissue volume in relation to the antigenicity of 4°C-preserved islets. Hanayama et al. showed efficient gene transduction of dispersed islet cells in culture using fiber-modified adenoviral vectors harboring a polylysine (K7) peptide in the C-terminus of the fiber knob. The fiber-modified adenoviral vector yielded higher transduction efficiencies than conventional adenoviral vector at a multiplicity of infection (MOI) of 5 and 10. Yamashita et al. evaluated human laminin (HL) isotype coating for creating islet cell sheet. HL-332 was an optimal human-derived extracellular matrix (ECM) for coating temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm) surfaces. Miyamoto et al. showed a Tapered Stencil for Cluster Culture (TASCL) device developed to create liver spheroids in vitro. The TASCL device will be useful for application as a toxicity evaluation system for drug testing. Pillai et al. have achieved the fluorescence off state of quantum dots (QDs) by the conjugation of black hole quencher (BHQ) molecules intermediated with a peptide using streptavidin-QDs585 and biotin-pep-BHQ-1. The technology is expected to be useful as molecular imaging with on–off control of QD fluorescence. The theme of this JSOPMB issue is “Creating a Future of Transplantation.” The board members and I are looking forward to seeing further progress in JSOPMB in conjunction with Cell Medicine.