Efficacy Of Gene Transduction Research Articles

B-Type Natriuretic Peptide Inhibits the Expression and Function of SERCA2a in Heart Failure.

B-type natriuretic peptide (BNP) possesses protective cardiovascular properties; however, there has not been sufficient serious consideration of the side effects of BNP. As for sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA2a), it was once considered a new target for the treatment of heart failure (HF). Nevertheless, clinical trials of SERCA2a gene therapy in HF have finally become unsuccessful. Research has found that elevated BNP levels and decreased SERCA2a expression are two important HF characteristics, which are always negatively correlated. We hypothesize that BNP inhibits SERCA2a expression and, therefore, exerts negative effects on SERCA2a expression and function.The effects of BNP on endogenous SERCA2a expression and function were tested in mice with HF induced by transverse aortic constriction and neonatal rat cardiomyocytes (NRCM). Furthermore, to verify the effects of BNP on exogenous SERCA2a gene transduction efficacy, BNP was added to the myocardium and cardiomyocytes infected with an adenovirus overexpressing SERCA2a.In vivo, BNP levels were increased, SERCA2a expression was reduced in both the BNP intervention and HF groups, and BNP reduced the overexpressed exogenous SERCA2a protein in the myocardium. Our in vitro data showed that BNP dose-dependently inhibited the total and exogenous SERCA2a expression in NRCM by activating the cGMP-dependent protein kinase G. BNP also inhibited the effects of SERCA2a overexpression on Ca2+ transience in NRCM.The expression and function of endogenous and exogenous SERCA2a are inhibited by BNP. The opposite relationship between BNP and SERCA2a should be given serious attention in the treatment of HF via BNP or SERCA2a gene therapy.

Cardiac vein retroinjections provide an efficient approach for global left ventricular gene transfer with adenovirus and adeno-associated virus

Heart failure (HF) is a major burden worldwide, and new therapies are urgently needed. Gene therapy is a promising new approach to treat myocardial diseases. However, current cardiac gene delivery methods for producing global myocardial effects have been inefficient. The aim of this study was to develop an endovascular, reproducible, and clinically applicable gene transfer method for global left ventricular (LV) transduction. Domestic pigs (n = 52) were used for the experiments. Global LV myocardium coverage was achieved by three retrograde injections into the three main LV vein branches. The distribution outcome was significantly improved by simultaneous transient occlusions of the corresponding coronary arteries and the main anastomotic veins of the retroinjected veins. The achieved cardiac distribution was visualized first by administering Indian Ink solution. Secondly, AdLacZ (2 × 1012vp) and AAV2-GFP (2 × 1013vg) gene transfers were performed to study gene transduction efficacy of the method. By retrograde injections with simultaneous coronary arterial occlusions, both adenovirus (Ad) and adeno-associated virus (AAV) vectors were shown to deliver an efficient transduction of the LV. We conclude that retrograde injections into the three main LV veins is a potential new approach for a global LV gene transfer.

Peptide Amphiphiles as Biodegradable Adjuvants for Efficient Retroviral Gene Delivery.

Retroviral gene delivery is the key technique for in vitro and ex vivo gene therapy. However, inefficient virion-cell attachment resulting in low gene transduction efficacy remains a major challenge in clinical applications. Adjuvants for ex vivo therapy settings need to increase transduction efficiency while being easily removed or degraded post-transduction to prevent the risk of venous embolism after infusing the transduced cells back to the bloodstream of patients, yet no such peptide system have been reported thus far. In this study, peptide amphiphiles (PAs) with a hydrophobic fatty acid and a hydrophilic peptide moiety that reveal enhanced viral transduction efficiency are introduced. The PAs form β-sheet-rich fibrils that assemble into positively charged aggregates, promoting virus adhesion to the cell membrane. The block-type amphiphilic sequence arrangement in the PAs ensures efficient cell-virus interaction and biodegradability. Good biodegradability is observed for fibrils forming small aggregates and it isshown that via molecular dynamics simulations, the fibril-fibril interactions of PAs are governed by fibril surface hydrophobicity. These findings establish PAs as additives in retroviral gene transfer, rivalling commercially available transduction enhancers in efficiency and degradability with promising translational options in clinical gene therapy applications.

Abstract 12241: Low-Efficiency Gene Editing is Capable of Suppressing Arrhythmogenesis in Long QT Syndrome Type II: Computer Simulation Study

Background: Long QT Syndrome Type II (LQT2) is a congenital cause of arrhythmia and sudden cardiac death due to mutations in ion channel hERG responsible for the rapid inward rectifying potassium current (I Kr ). Progress in gene therapy may result in a viable treatment for LQT2. However, the gene transduction efficacy required to prevent cardiac arrhythmias in LQT2 is unknown. We used computer simulation to examine the minimum gene transduction efficacy necessary to suppress early afterdepolarizations (EADs). Hypothesis: Low transduction efficacy of hERG is sufficient to suppress EADs through electrotonic coupling between transduced I Kr (+) and non-transduced I Kr (-) cardiomyocytes (Fig 1A). Methods: Computational models of rabbit and human cardiac tissue were implemented in 2D (100 x 100 cells) and 3D (13 x 13 x 13 cells) with a multi-state Markov model for I Kr and a dynamic gap junction resistance model. Gene transduction efficacy was modeled by varying the percentage of I Kr (+) cardiomyocytes from 0-100%. Spatial heterogeneity in gene delivery was modeled by varying the cluster size of I Kr (+) cells from 1-400 cells. I Kr (+) cell clusters were distributed randomly in the tissue. Results: In 2D, EADs were suppressed with 14% I Kr (+) cells (rabbit model) and 12% I Kr (+) cells (human model) (Fig 1B). In 3D, the minimum transduction efficacy was further reduced to 10% (rabbit model) which likely reflects the impact of increased cell-cell coupling. Clustering I Kr (+) cells required greater transduction efficacy (Fig 1C). Detailed ionic current trajectory analysis suggests that the sufficiently large gap junction current from I Kr (+) cells during phase 2 of the action potential can prevent I Kr (-) cells from undergoing the Andronov-Hopf bifurcation that leads to EADs. Conclusions: Computational models suggest low-efficiency gene editing is a viable approach for suppressing arrhythmogenesis in LQT2 patients.

Spacer Length Modification Facilitates Discrimination between Normal and Neoplastic Cells and Provides Clinically Relevant CD37 CAR T Cells.

Despite the remarkable initial efficacy of CD19 chimeric Ag receptor T (CAR-T) cell therapy, a high incidence of relapse has been observed. To further increase treatment efficacy and reduce the rate of escape of Ag-negative cells, we need to develop CAR-T cells that target other Ags. Given its restricted expression pattern, CD37 was considered a preferred novel target for immunotherapy in hematopoietic malignancies. Therefore, we designed a CD37-targeting CAR-T (CD37CAR-T) using the single-chain variable fragment of a humanized anti-CD37 Ab, transmembrane and intracellular domains of CD28, and CD3ζ signaling domains. High levels of CD37 expression were confirmed in B cells from human peripheral blood and bone marrow B cell precursors at late developmental stages; by contrast, more limited expression of CD37 was observed in early precursor B cells. Furthermore, we found that human CD37CAR-T cells with longer spacer lengths exhibited high gene transduction efficacy but reduced capacity to proliferate; this may be due to overactivation and fratricide. Spacer length optimization resulted in a modest transduction efficiency together with robust capacity to proliferate. CD37CAR-T cells with optimized spacer length efficiently targeted various CD37+ human tumor cell lines but had no impact on normal leukocytes both in vitro and in vivo. CD37CAR-T cells effectively eradicated Raji cells in xenograft model. Collectively, these results suggested that spacer-optimized CD37CAR-T cells could target CD37-high neoplastic B cells both in vitro and in vivo, with only limited interactions with their normal leukocyte lineages, thereby providing an additional promising therapeutic intervention for patients with B cell malignancies.

Improving Gene Therapy Efficacy Using Impella

Gene therapy is a promising option for heart failure patients, but there remains a lack of effective delivery mechanisms. The Impella device is a heart pump that can increase coronary flow and pressure, important factors that determines efficacy of gene transduction when using intracoronary delivery. Because it also lends hemodynamic support, the Impella can be coupled with balloon coronary occlusion to increase cardiac vector dwell time. We hypothesized that use of the Impella offers safe and efficient delivery of genes using intracoronary administration of adeno-associated viral (AAV)-6. AAV6 encoding luciferase gene (5.0 × 10E13) was injected into the coronary artery one week after MI induction in Yorkshire pigs. A total of twelve pigs were allocated to one of the following groups: slow antegrade delivery (n=3), slow antegrade delivery with Impella (n=3), delivery during coronary artery occlusion with Impella hemodynamic support (n=3), delivery during coronary artery and sinus occlusions with Impella hemodynamic support (CA + CS, n=3), Four weeks later, tissues were harvested from various heart regions and other organs to assess luciferase expression and viral uptake. Impella support offered safe vector delivery during 1-2 minutes of CA and CS occlusion. Impella + coronary artery block enhanced luciferase expression globally in the heart but not in non-cardiac tissues, such as liver, as compared to injection without coronary artery block. Impella with CA + CS block even further increased luciferase expression by increasing viral uptake, as evidenced by increased vector genome in the tissues detected by PCR. At the infarct border zone, an important area for cardiac gene therapy, CA + CS occlusion led to greater than 300-fold increase in luciferase (p=0.005) compared to viral injection alone. Impella unloading with CA + CS occlusion dramatically improves AAV gene expression in the heart without increasing off-target expression; it is thus a promising method for clinical application of cardiac gene therapy.

Acoustic waves improves retroviral transduction in human retinal stem cells

BackgroundsThe plasticity of retinal stem cells (RSCs), a type of cells that can differentiate into neuron cells and photoreceptor cells, endows them with potential therapeutic properties that can be applied to regenerative medicine. Gene modification of these stem cells before trans-differentiation and transplantation enhances their survival and increases their therapeutic function. The different ways to effectively deliver gene into RSCs are still discussed. This study aimed to use the acoustic waves to improve the efficacy of gene delivery for RSCs. MethodsRSCs were obtained from non-fetal human ocular pigmented ciliary margin tissues. The enhanced green fluorescent protein-encoded murine stem cell retroviruses (MSCV) were prepared and used to infect RSCs. Glass chambers containing RSCs, retroviruses, and various concentrations of polybrene (0, 0.8, 2, 4 and 8 μg/mL) were exposed under 20 or 25 Vp-p ultrasonic standing wave fields (USWF) for 5 min. The percentage of green fluorescent protein positive cells in each sample was calculated and compared to test the efficacy of gene transduction. ResultsOur results showed that the efficiency of gene transduction by MSCV infection was enhanced following the concentration of polybrene and the energy of USWF. The percentage of green fluorescent protein positive cells was significantly higher in chambers that contained 8 μg/mL of polybrene and was exposed to 20Vp-p of USWF for 5 min. In addition, the percentage increased in chambers contained 2, 4 and 8 μg/mL of polybrene when they were exposed to 25Vp-p of USWF. Comparing to those did not treated with ultrasound, the efficiency of retroviral transduction to RSCs increased 4-fold after exposed to USWF for 5 min. ConclusionWe demonstrated the ability of ultrasound standing waves to improve retroviral transduction into RSCs. We believe that this may be applied to the experimental designs of future studies and may have possible therapeutic uses.

Wnt11 plays an important role in the osteogenesis of human mesenchymal stem cells in a PHA/FN/ALG composite scaffold: possible treatment for infected bone defect.

BackgroundInfected bone defect poses a great challenge for orthopedists because it is difficult to cure. Tissue-engineered bone based on the human mesenchymal stem cells (hMSCs), has currently taken a promising treatment protocol in clinical practice. In a previous study, a porous hydroxyapatite/fibronectin/alginate (PHA/FN/ALG) composite scaffold displayed favorable biological properties as a novel scaffold, which was considered better than single-material scaffolds. In addition, Wnt11 has been demonstrated to play an important role in the development of osteoblasts, but until recently, its role in the osteogenic differentiation of hMSCs in infectious environment remained unclear.MethodsIn this study, we constructed a PHA/FN/ALG composite scaffold with layer-by-layer technology. Furthermore, we also constructed Wnt11-silenced (RNAi) and -overexpressing hMSCs by lentiviral transduction. The gene transduction efficacy was confirmed by quantitative PCR assay and Western blot analysis. Tissue-engineered bone was constructed with hMSCs and PHA/FN/ALG composite scaffolds, and then was implanted into an infected bone defect model for evaluating the osteogenic capacity by quantitative PCR, gross observation, micro-CT and histology analysis.ResultsAll those cells showed similar adhesion abilities and proliferation capacities in scaffolds. After tissue-engineered bone implantation, there were high levels of systemic inflammatory factors in vivo, which significantly declined three days after antibiotic therapy. One or two months after implantation, the results of osteogenic-related gene analyses, gross observation, micro-CT and histology consistently showed that the Wnt11 over-expression hMSC group displayed the strongest osteogenesis capacity, whereas the Wnt11-RNAi hMSC group displayed inferior osteogenesis capacity, when compared with the other cell-containing groups. However, the blank control group and the only composite scaffold without cell implantation group both showed extremely weak osteogenesis capacity.ConclusionOur results revealed that the Wnt11 gene plays an important role in hMSCs for enhancing the osteogenesis in an infectious environment.

Study of recombinant adeno-associated virus 2-mediated cotransfer of hTGF-β1 and hTGF-β3 encoding genes to the rabbit degenerative nucleus pulposus cells

Objective 1) To verify the the potential of the adenoassociated viral vector as a strategy for intradiscal gene transfer in degenerative rabbit intervertebral discs. 2) To investigate the gene transduction efficacy and to quantify the biologic effects on the matrix synthesis after single gene transfer and combined gene transfer. Methods Rabbit models of disc degeneration were established by injecting the N-terminal 30×103 fibronectin fragment (Fn-f), 4 weeks later, saline with or without virus was injected directly into 144 lumbar discs of 36 skeletally mature New Zealand white rabbits. Group A (n =8) received the rAAV2-hTGFβ1; Group B (n=6) received rAAV2-hTGFβ3;Group C (n=6) recived rAAV2-hTGFβ1 and rAAV2-hTGFβ3; Group D (n=8) recived rAAV2-EGFP as the experimental control. Group E (n=8) recived PBS as the blank control. Two rabbits of the group A and group E were sacrefied 1 week after injection, immunohistochemical staining for hTGF-β1 was performed on the slices of nucleus pulposus (NP) tissues. On 4,8 and 12 weeks after gene transferring, NP tissues were cultured or decomposed to quantify the biochemical changes of the matrix using 35S-sulfate incorporation assay and western blot detection. The expression of EGFP was observed 12 weeks after injection. Results Discs in group A exhibited extensive and intense positive immunostaining for hTGF-β1 than the control discs in group E 1 week after gene transferring. The nucleus pulposus tissues in group A, B and C exhibited a 1.28-2.06 fold increase in proteoglycan synthesis and a 1.25-1.73 fold increase in collagen type Ⅱ production over those in group E (P＜0.05 or P＜0.01).Combination of two gene transfer in group C makes a significantly increased level of proteoglycan (1.195-1.290 fold)and collagen type Ⅱ (1.152-1.219 fold) than single gene transfer in group A and B(P＜0.05 or P＜0.01).No statistic differences shows between A group and B group. The difference of the matrix synthesis between group D and group E was also not statistically significant (P＞0.05). Extensive and intensive green fluorescence was observed on the slice of nucleus pulposus tissues received rAAV2-EGFP 12 weeks after gene delivery. The expression of EGFP kept for more than 12 weeks. Conclusion Findings showed that the disc tissue injected with rAAV2 mediated genes highly expressed the therapeutic proteins from 1 week to more than 12 weeks after delivery. It is suggested that adenoassociated virus be an valid vector for the transfer of the exogenous genes in the degenerative disc. The therapeutic factors hTGF-β1 and hTGF-β3 could efficiently increase the synthesis of proteoglycan and collagen type Ⅱ in the degenerative NP cells and combined transfer of two genes was more effective than single gene transfer. The two factors have an positive synergistic effects. Key words: Intervertebral disk; Transforming growth factor beta; Transfection; Gene therapy

Successful cross‐breeding of cloned pigs expressing endo‐β‐galactosidase C and human decay accelerating factor

For successful organ xenotransplantation, genetically engineered pigs have been actively produced. Our attention has focused on (i) reduction of alphaGal expression by its digestion enzyme, endo-beta-galactosidase C (EndoGalC), and (ii) inhibition of complement activation by human decay accelerating factor (hDAF). Cell sorting and nuclear transfer enabled the effective production of cloned pigs expressing transgene at high levels. We report the successful cross-breeding of pigs expressing EndoGalC and hDAF. After hDAF and EndoGalC genes were transfected into pig fibroblasts from the fetus of Landrace x Yorkshire and Meishan, respectively, transfected cells expressing transgenes effectively were collected using a cell sorter. Cloned pigs were produced using the technology of somatic cell nuclear transfer. After cross-breeding of cloned pigs, kidneys expressing both EndoGalC and hDAF were transplanted into baboons to examine the efficacy of gene transduction. Well-designed cloned pigs were produced by cross-breeding. alphaGal expression levels in cloned pigs were reduced up to 2 to 14%, compared to that in wild-type pigs. hDAF expression reached about 10- to 70-fold, compared to that in human umbilical vein endothelial cells. No congenital deformity was observed. There was no problem of increased stillbirth rate or growth retardation. Hyperacute rejection could be avoided in such a cloned pig to baboon kidney transplantation without any treatment for anti-pig antibody removal. However, grafts suffered from fibrin deposition as early as 1 h after transplantation, and were rejected after 1 week. Using a cell sorting system for effective collection of transfected cells, two types of cloned pigs were produced with a very high level of hDAF expression and a low level of alphaGal expression. Such genetic modification was effective in preventing hyperacute rejection, but there was an immediate lapse into procoagulation after transplantation, resulting in acute vascular rejection. Effective suppression of antibody binding to the graft would be necessary, even if a high level of hDAF is expressed.

Efficient delivery of angiostatin K1-5 into tumors following insertion of an NGR peptide into adenovirus capsid

Adenovirus (Ad)-mediated delivery of anti-angiogenic molecules into tumors constitutes an appealing approach for growth inhibition. However, lack of expression on tumors of Ad receptors leads to weak tumor transduction. Therefore, to provide Ad with a new entry pathway into tumors, an NGR peptide was inserted into either fiber (AdFNGR) or hexon (AdHNGR) capsid proteins. This strategy provided Ad with a very efficient entry pathway in both endothelial cells and tumor cells, with the highest efficacy observed for AdHNGR. Using pharmacological, biochemical and genetic approaches, AdHNGR and AdFNGR were shown to bind not only to CD13 receptor, but also to alphavbeta3 integrins. Both vectors were efficient tools to deliver angiostatin K1-5 cDNA into endothelial cells, thus leading to a dramatic inhibition of their proliferation and increased cell death. Although AdHNGR and Adwt were found to display similar gene transduction efficacy in Lewis lung carcinoma (LLC), pseudotyping AdHNGR with an Ad3-fiber unmasked the ability of NGR-peptide to target these tumors. As a result, delivery of angiostatin K1-5 cDNA into highly aggressive tumors translated into a stronger inhibition of their growth. Altogether, our results suggest that NGR-bearing Ad are valuable tools to realize the potential of this anti-angiogenic approach to anti-tumor therapy.

669. Light-Induced Adenovirus Gene Transfer

The development of methods for efficient and specific delivery of therapeutic genes into target tissues is an important issue for further development of in vivo gene therapy. In the present study the physical targeting technique, photochemical internalization (PCI), has been used in combination with adenovirus. PCI is based on the use of photosensitizing compounds that localize in endocytic membranes of cells. Upon light exposure the photosensitizer induce damage to the membrane, facilitating release of internalized particles from the endocytic vesicles into the cytosol. The combination of PCI and adenoviral transduction has previously been shown to be favorable compared to adenovirus alone, and enables up to 20 times increase in gene transfer in a variety of cell lines. The observed enhancement in gene transduction efficacy may prove important in the development of a viable gene therapeutic strategy, which is dependent on an efficient, targeted and safe methodology for gene delivery. The aim of this study was to increase the understanding of the molecular mechanism behind photochemical enhancement of adenoviral transduction. K44A Hela cells, mutated in dynamin, were treated with photosensitizer before infection with adenovirus and exposure to light. Cells expressing normal dynamin showed an enhanced gene expression after light treatment, while exposure to light did not have a pronounced effect in cells expressing negative mutated dynamin. Dynamin is involved in both clathrin and caveolar mediated endocytosis. To investigate if both pathways were involved during photochemical transduction, cells were treated with chlorpromazine and filipinIII before infection and light treatment. Chlorpromazine inhibits clathrin-mediated endocytosis, while filipinIII inhibits the caveolar mediated uptake. Treating the cells with chlorpromazine induced a dose dependent reduction of gene transfer in photochemically treated cells. Furthermore, the cellular localization of both viral particles and photosensitizer, after treatment with inhibitor agent, were investigated using fluorescence microscope.

Improvement of retroviral vectors by coating with poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL).

Although some cationic reagents, such as polybrene, improve gene transduction in vitro, their use in vivo is prohibited due to their toxicity to the exposed cells. This paper demonstrates that a new cationic reagent, poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL), improves gene transduction with retroviral vectors without increasing cell toxicity. A retroviral vector derived from the Moloney leukemia virus, containing the lacZ gene, was modified with PEG-PLL prior to transduction into NIH3T3, Lewis lung carcinoma, and primary cultured mouse brain cells. LacZ transduction efficacy was evaluated by counting the number of X-Gal-positive cells. We have demonstrated that PEG-PLL is able to stably modify the viral particle surface due to the affinity of the PEG moiety to the biomembrane, and neutralizes negative charges by the cationic nature of the poly-lysine residue. Thus, PEG-PLL increased the gene transduction efficiency and minimized cell toxicity because free PEG-PLL was removable by centrifugation. We have shown that PEG-PLL increased the viral gene transduction efficiency 3- to 7-fold with NIH3T3 or Lewis lung carcinoma cell lines without increasing cytotoxicity. It improved retroviral gene transduction efficacy even against labile cells, such as primary cultured brain cells. PEG-PLL is a novel reagent that improves retroviral gene transduction efficacy without increasing cytotoxicity.

Gene therapy of cancer with interleukin-12.

IL-12 has demonstrated remarkable antitumor activity when used directly as a recombinant protein or when different viral or non-viral vectors transfer its genes. At enhancing tumor immunity, IL-12 acts as a bridge between innate and adaptive immune responses due to its ability to induce proliferation and activation of NK, NKT, and T cells. In addition, IL-12 inhibits tumor angiogenesis mainly through IFN gamma-dependent production of the chemokine IP10. As a result, IL-12 can eliminate several types of tumors developed in rodents. Pre-clinical experience forecasted a quick and successful clinical translation, but the encouraging results observed in animals were not reproduced in patients. Moreover, unacceptable toxicity resulting from IFN gamma overproduction was observed in 2 renal carcinoma patients included in a phase II clinical trial that consisted in systemic administration of rIL-12. As a consequence, development of IL-12 as an antitumor agent was temporarily halted while the high expectations raised among clinicians faded away. Gene transfer methods are designed to confine IL-12 production in the tumor environment preventing systemic toxicity. Tumor cells, dendritic cells, or autologous fibroblasts have been transfected with recombinant adenoviruses or retroviruses to secrete IL-12 locally, showing good efficacy and safety profiles. IL-12 combination with other immunotherapy approaches synergizes to achieve even better results. Encouraging pilot clinical results have been recently obtained from the first phase I trial studying adenovirus mediated in vivo gene transfer of IL-12 into lesions of advanced cancer patients. Further improvements will follow from: i) increases in the efficacy of gene transduction; ii) development of tumor specific promoters; iii) development of regulatable and long-term expression vectors and iv) combination with other immunological and non-immunological anticancer therapies.

Improvement of transduction efficiency of recombinant adenovirus vector conjugated with cationic liposome for human oral squamous cell carcinoma cell lines

Adenovirus (Ad) vectors are commonly used in gene therapy trials because of their efficiency in gene transfer. However, their use is limited by immune responses that reduce transgene expression and decrease the efficiency of repeated vector administration. In this study, the efficacy of gene transduction and the tumor-cell killing effect on four human oral (SAS, HSC-2, HSC-3, HSC-4) and one murine squamous cell carcinoma cell (SCC-7, a kind gift of Dr. M. Hiraoka, Kyoto University) lines in vitro with Ad vector conjugated with catioic liposome (Ad/SUV) was evaluated. Ad/SUV resulted in two to five-fold over higher transduction efficiency in four human and one murine cell lines in vitro than Ad vector alone. The optimal Ad-SUV ratio was determined as 10 6 pfu of Ad vector with 1 μmol SUV. Ad/SUV showed more tumor-cell killing effect than Ad vector alone. Furthermore, the shielding effects of Ad vector with Ad/SUV from neutralizing antibody were evaluated. We also found that Ad/SUV is less susceptible to inactivation by neutralizing antibodies in vitro. The efficacy of gene transduction with Ad vector was blocked more than 70% with neutralizing serum, while Ad/SUV retained approximately 50% of the control activity in vitro. On the basis of these results, the anti-tumor effect with suicide gene therapy using Ad/SUV in vivo was evaluated. Three injections of Ad/SUV showed the inhibition of tumor growth compared with control in vivo. Our results suggested that an enhanced anti-tumor effect on human oral squamous cell carcinoma would be obtained with repeated administrations of Ad/SUV.

Tumor necrosis factor alpha-gene therapy for an established murine melanoma using RGD (Arg-Gly-Asp) fiber-mutant adenovirus vectors.

Although adenovirus vectors (Ad) provide high‐level transduction efficacy to many cell types, extremely high doses of Ad are required for sufficient gene transduction into several tumors, including melanoma. Here, we demonstrated that the expression of coxsackie‐adenovirus receptor, a primitive Ad‐receptor, was very low in murine and human melanoma cells. We also found that fiber‐mutant Ad containing the Arg‐Gly‐Asp (RGD) sequence in the fiber knob remarkably augmented gene transduction efficacy in melanoma cells by targeting αv‐integrins. In addition, intratumoral injection of RGD fiber‐mutant Ad containing the tumor necrosis factor α gene (AdRGD‐TNFα) revealed dramatic anti‐tumor efficacy through hemolytic necrosis in an established murine B16 BL6 melanoma model. Ad‐RGD‐TNFα required one‐tenth the dosage of Ad‐TNFα to induce an equal therapeutic effect. These results suggest that αv‐integrin‐targeted Ad will be a very powerful tool for the advancement of melanoma gene therapy.

Fiber-mutant technique can augment gene transduction efficacy and anti-tumor effects against established murine melanoma by cytokine-gene therapy using adenovirus vectors

Melanoma cells are relatively resistant to adenovirus vector (Ad)-mediated gene transfer due to the low expression of Coxsackie-adenovirus receptor (CAR), which acts as a primitive Ad-receptor. Therefore, extremely high doses of Ad are required for effective gene therapy against melanoma. In the present study, we investigated whether fiber-mutant Ad containing the Arg-Gly-Asp (RGD) sequence in the fiber knob could promote gene delivery and anti-tumor effects in the murine B16 BL6 tumor model. B16 BL6 cells (in vitro) and tumors (in vivo) infected with RGD fiber-mutant Ad containing a tumor necrosis factor α gene (Ad-RGD-TNFα) produced more TNFα than those infected with conventional Ad-TNFα. In addition, Ad-RGD-TNFα required about one-tenth the dosage of Ad-TNFα for induction of equal therapeutic effects upon intratumoral injection into established B16 BL6 tumors. Furthermore, the combination of both TNFα- and interleukin 12-expressing RGD fiber-mutant Ads exhibited more effective tumor regression than the Ad expressing each alone. These results suggested that the fiber-mutant for altering Ad-tropism is a very potent technology for advancing gene therapy for melanoma.

A retroviral vector system 'STITCH' in combination with an optimized single chain antibody chimeric receptor gene structure allows efficient gene transduction and expression in human T lymphocytes.

Genetic engineering of T lymphocytes for adoptive clinical immunotherapy calls for efficient gene transduction methods. Therefore, a transient retroviral gene transduction system 'STITCH' was developed comprising pSTITCH retroviral vector encoding the transgene, plasmids encoding Moloney murine leukemia virus gag/pol and gibbon ape leukemia virus envelope, and the human kidney cell line 293T as a packaging line. Cotransfection of retroviral vector and packaging plasmids in 293T cells results in the production of GALV env pseudotyped viral particles with a titer of 10(7) infectious units per milliliter. The 'STITCH' gene transduction system efficiently transduces genes into activated human T lymphocytes derived from healthy donors and cancer patients. The efficacy of gene transduction is donor-independent. A direct application of the 'STITCH' gene transduction system is the genetic engineering of activated human T lymphocytes to induce expression of antibody based chimeric receptors in their membrane. Introduction of these chimeric receptors into activated human T lymphocytes graft these cells with specificity for, for example, renal cell carcinoma. In order to study the effect of the chimeric receptor gene structure on the processes ultimately leading to functional membrane expression, we designed a number of different chimeric receptor gene structures and subsequently compared their membrane expression on 293T cells and activated human T lymphocytes. Distinct membrane expression densities were observed on 293T cells and human T lymphocytes for the different chimeric receptor gene constructs. Gene transduction of activated human T lymphocytes with four out of five chimeric receptor gene constructs resulted in functional expression of chimeric receptor as demonstrated by specific recognition and cytolysis of renal cell carcinoma.

In vivo adenovirus-mediated prodrug gene therapy for carcinoembryonic antigen-producing pancreatic cancer.

In gene therapy for malignancy, the herpes simplex virus thymidine kinase (HSVtk)‐ganciclovir (GCV) system has been widely used. For pancreatic cancer targeting, we estimated the therapeutic efficacy of gene transduction by an adenovirus‐carrying HSVtk gene under the control of a carci‐noembryonic antigen (CEA) promoter (AdCEAtk) followed by systemic administration of GCV. Four cell lines, CEA‐producing Su.86.86, BxPC‐3 (pancreatic cancer cells), MKN45 (gastric cancer cells) and CEA‐nonproducing HeLa, were used for analysis of GCV sensitivity induced by adeno‐viral gene transduction. To evaluate the therapeutic efficacy of AdCEAtk and GCV administration in human CEA‐positive pancreatic cancer in vivo, a subcutaneously implanted tumor‐bearing nude mouse model was used. When the HSVtk gene was transduced with a ubiquitous promoter into these cells, increase of the GCV sensitivity was independent of CEA‐production. In contrast, when the cells were transduced with a CEA promoter, the cell‐killing effect of GCV was increased in only CEA‐producing cells. For in vivo analysis, AdCEAtk was delivered into subcutaneously established tumors of Su.86.86 cells. Immunohistochemical staining of the tumor showed that HSVtk protein was expressed only in tumor cells, and tumor growth was markedly suppressed by administration of GCV. These results suggest that the adenovirus‐mediated transfer of HSVtk gene with CEA promoter specifically increases the GCV sensitivity of CEA‐producing pancreatic cancer cells in vitro and in vivo. This strategy may provide a useful tool for treating pancreatic cancer, especially CEA‐producing tumor cells.

皮膚悪性腫ようのＢＲＭ療法のその後の展開 遺伝子治療 悪性黒色腫に対する臨床応用

In this study, I review the newest and attractive technique for the treatment of metastatic melanoma, that is, gene therapy. First, the contents of guide line for gene therapy in Japan was described. Second, the background of gene therapy, for exam ple, the way of gene transfer and advantages and disadvantages of viral vectors and/or nonviral vectors were described. Third, the current status of melanoma gene therapy in USA was described; that is, retroviralmediated gene transduction and/or direct gene transfer with liposome is safe, and feasible gene transductions and treatment with IL-2/TNF and/or HLA B7 gene into TIL and/or melanoma cells are being carried out in USA. Further improvement of efficacy of gene transduction is needed and longterm follow up of patients should be done for safety.