Construction of a fusion plasmid containing the PSCA gene and cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and its anti-tumor effect in an animal model of prostate cancer

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is a negative regulator of T cell activation, which competes with CD28 for B7.1/B7.2 binding, and which has a greater affinity. Fusion of specific antigens to extracellular domain of CTLA4 represents a promising approach to increase the immunogenicity of DNA vaccines. In this study, we evaluated this interesting approach for CTLA4 enhancement on prostate stem cell antigen (PSCA)-specific immune responses and its anti-tumor effects in a prostate cancer mouse model. Consequently, we constructed a DNA vaccine containing the PSCA and the CTLA-4 gene. Vaccination with the CTLA4-fused DNA not only induced a much higher level of anti-PSCA antibody, but also increased PSCA-specific T cell response in mice. To evaluate the anti-tumor efficacy of the plasmids, murine models with PSCA-expressing tumors were generated. After injection of the tumor-bearing mouse model, the plasmid carrying the CTLA4 and PSCA fusion gene showed stronger inhibition of tumor growth than the plasmid expressing PSCA alone. These observations emphasize the potential of the CTLA4-fused DNA vaccine, which could represent a promising approach for tumor immunotherapy.

Death switch for gene therapy: application to erythropoietin transgene expression

The effectiveness of the caspase-9-based artificial "death switch" as a safety measure for gene therapy based on the erythropoietin (Epo) hormone was tested in vitro and in vivo using the chemical inducer of dimerization, AP20187. Plasmids encoding the dimeric murine Epo, the tetracycline-controlled transactivator and inducible caspase 9 (ptet-mEpoD, ptet-tTAk and pSH1/Sn-E-Fv’-Fvls-casp9-E, respectively) were used in this study. AP20187 induced apoptosis of iCasp9-modified C2C12 myoblasts. In vivo, two groups of male C57BI/6 mice, 8-12 weeks old, were injected intramuscularly with 5 µg/50 g ptet-mEpoD and 0.5 µg/50 g ptet-tTAk. There were 20 animals in group 1 and 36 animals in group 2. Animals from group 2 were also injected with the 6 µg/50 g iCasp9 plasmid. Seventy percent of the animals showed an increase in hematocrit of more than 65 percent for more than 15 weeks. AP20187 administration significantly reduced hematocrit and plasma Epo levels in 30 percent of the animals belonging to group 2. TUNEL-positive cells were detected in the muscle of at least 50 percent of the animals treated with AP20187. Doxycycline administration was efficient in controlling Epo secretion in both groups. We conclude that inducible caspase 9 did not interfere with gene transfer, gene expression or tetracycline control and may be used as a safety mechanism for gene therapy. However, more studies are necessary to improve the efficacy of this technique, for example, the use of lentivirus vector.

Terapia gênica: o que é, o que não é e o que será / Gene therapy: what is, what is not and what will be

Terapia gênica é o tratamento baseado na introdução de genes sadios com uso de técnicas de DNA recombinante. O primeiro teste clínico bem-sucedido dessa técnica foi divulgado em 1990. Em que pese a ocorrência, em certos estudos clínicos, de efeitos adversos, alguns dos quais graves, laboratórios de pesquisa e empresas vêm continuamente desenvolvendo novos materiais e procedimentos mais seguros e eficazes. Embora ainda em estágio experimental, progressos recentes indicam oportunidades crescentes de investimento pela indústria, bem como justificam a expectativa de que, em alguns casos, essa tecnologia poderá chegar à prática clínica dentro de poucos anos.

Gene therapy is the therapeutic procedure based on the introduction of healthy genes using recombinant DNA techniques. The first successful clinical trial of this technique was published in 1990. Despite the occurrence, in certain clinical trials, of adverse effects, some of which serious, both laboratories and companies are continuously developing novel materials and establishing both safer and more effective procedures. Although still in experimental stages, recent progress both points to growing opportunities for investment by industry, as well as justify the expectation that, in some cases, this technology may reach clinical practice within a few years.

Effects of therapeutic angiogenesis with plasmid VEGF165 on ventricular function in a canine model of chronic myocardial infarction / Efeito da angiogênese terapêutica com VEGF165 sobre a função ventricular em infarto do miocárdio crônico

OBJECTIVE: Therapeutic angiogenesis is currently under investigation in ischemic heart disease. We examined the effect on left ventricular function induced by therapeutic angiogenesis by intramyocardial injection of plasmid VEGF165, in a canine model of chronic myocardial infarction. METHODS: Left thoracotomy was performed in 10 mongrel dogs, and myocardial infarction induced by ligation of the major diagonal coronary artery. At 7 postoperative (p.o.) day (pre-treatment), left ventricular ejection fraction was assessed by echocardiogram, and a second procedure was done: saline or plasmid VEGF165 at 200 mg/mL was injected over 10 points of the ischemic areas of control or treated groups, respectively. Fourteen days later (post-treatment, day 21) a control echocardiogram was performed, the animals were sacrificed and histological examination was performed. RESULTS: Ejection fraction was maintained in the treated group: 52.45 + 15.1 percent on day 7 and 48.53 + 11.74 percent on day 21 (P=0.59), and tended to decrease in the control group, from 59.3 + 4 percent to 39.37 + 19.43 percent (P=0.04), although absolute values did not differ significantly between groups. Histological examination revealed a non significant increase in capillary vessels number in all areas in treated group. Paradoxically, arterioles were significantly less in number in all areas of treated dogs. CONCLUSION: Intramyocardial injection of plasmid VEGF165, in this canine model of chronic myocardial infarction, resulted in preservation of left ventricular ejection fraction, contrary to the control group where left ventricular ejection fraction showed continuous decline during the experiment. Histological examination, however, was unable to explain completely these results.

OBJETIVO: Angiogênese por terapia gênica é alternativa ainda experimental para revascularização miocárdica. Este estudo objetivou verificar a indução de angiogênese e melhora funcional miocárdica pela injeção transmural de plasmídeo contendo VEGF165 em áreas de infarto crônico do miocárdio. MÉTODOS: Em 10 cães anestesiados, por toracotomia lateral esquerda, foi induzido infarto agudo do miocárdio (IAM) por meio da ligadura do ramo diagonal principal da artéria coronária descendente anterior. Após 7 dias, realizado ecocardiograma para avaliação da fração de ejeção (FE) ventricular esquerda. Os animais foram divididos em dois grupos: Tratado (GT) e Controle (GC) e submetidos a segundo procedimento, para injeção intramiocárdica de solução contendo plasmídeo VEGF165 na concentração de 200mg/ml (GT) ou solução salina (GC), distribuída em 10 pontos da área infartada. Após 14 dias, novo ecocardiograma, sacrifício dos animais e retirada do coração para estudo histológico. RESULTADOS: Houve tendência à manutenção da FE no GT e de queda da FE no GC, conforme os valores: FE ao ecocardiograma pós-IAM inicial: GC 59,33 + 4,6 por cento e GT 52,45 + 15,1 por cento (P=0,359). FE após 14 dias: GC 39,37 + 19,43 por cento e GT 48,53 + 11,74 por cento (P=0,394). Comparação intra-grupo: Variação negativa da FEVE GC: 59,37 + 4 por cento para 39,37 + 19,43 por cento (P=0,04) e manutenção GT: 52,45 + 15,1 por cento para 48,53 + 11,74 por cento (P=0,59). No GT observou-se aumento do número de vasos capilares, mais intenso nas regiões injetadas, porém presente em todo miocárdio. Paradoxalmente, no GT houve redução do número de arteríolas. CONCLUSÃO: A injeção transmural de plasmídeo VEGF165 resultou em tendência para atenuar a perda de contratilidade consequente ao dano miocárdico, na fase crônica do IAM. O exame histológico da rede vascular, entretanto, não explica completamente os eventos funcionais.

Efecto diferencial de la 4'-epi-doxorubicina sobre plásmido y cromosoma en aislados de enterobacterias con resistencia a antibióticos ß-lactámicos / Diferential effect of the 4'-epi-doxorubicin on plasmid and cromosome in aislated of enterobacteries with ß-lactamase antibiotics resistance

La resistencia bacteriana a los antibióticos puede residir en el cromosoma y/o plásmidos; sin embargo una bacteria puede modificar la resistencia por el uso de agentes tales como los intercaladores del ADN, entre los cuales están los antibióticos antraciclínicos. En este trabajo se evaluó el efecto de la 4'-epidoxorubicina (4EPI) sobre los plásmidos de Enterobacterias nosocomiales con resistencia a la ampicilina (Amp). Tanto la Amp como la (4EPI) mostraron un efecto variado sobre las cepas en estudio. Los resultados muestran que la metodología debe ser adaptada a cada microorganismo en estudio y el resultado más relevante fue el efecto negativo sobre el plásmido a las concentraciones 2mg/mL y 0,00002 mg/ml de 4EPI para Klebsiella pneumoniae PIN147