SPARC (secreted protein acidic and rich in cysteine) knockdown protects mice from acute liver injury by reducing vascular endothelial cell damage.

Secreted protein, acidic and rich in cysteine (SPARC) is involved in many biological process including liver fibrogenesis, but its role in acute liver damage is unknown. To examine the role of SPARC in acute liver injury, we used SPARC knock-out (SPARC(-/-)) mice. Two models of acute liver damage were used: concanavalin A (Con A) and the agonistic anti-CD95 antibody Jo2. SPARC expression levels were analyzed in liver samples from patients with acute-on-chronic alcoholic hepatitis (AH). SPARC expression is increased on acute-on-chronic AH patients. Knockdown of SPARC decreased hepatic damage in the two models of liver injury. SPARC(-/-) mice showed a marked reduction in Con A-induced necroinflammation. Infiltration by CD4+ T cells, expression of tumor necrosis factor-α and interleukin-6 and apoptosis were attenuated in SPARC(-/-) mice. Sinusoidal endothelial cell monolayer was preserved and was less activated in Con A-treated SPARC(-/-) mice. SPARC knockdown reduced Con A-induced autophagy of cultured human microvascular endothelial cells (HMEC-1). Hepatic transcriptome analysis revealed several gene networks that may have a role in the attenuated liver damaged found in Con A-treated SPARC(-/-) mice. SPARC has a significant role in the development of Con A-induced severe liver injury. These results suggest that SPARC could represent a therapeutic target in acute liver injury.

A phase I clinical trial of thymidine kinase-based gene therapy in advanced hepatocellular carcinoma.

The aim of this phase I clinical trial was to assess the feasibility and safety of intratumoral administration of a first-generation adenoviral vector encoding herpes simplex virus thymidine kinase (HSV-TK) gene (Ad.TK) followed by systemic ganciclovir to patients with advanced hepatocellular carcinoma (HCC). Secondarily, we have analyzed its antitumor effect. Ten patients were enrolled in five dose-level cohorts that received from 10¹° to 2 × 10¹² viral particles (vp). Ad.TK was injected intratumorally and patients received up to three doses at 30-day intervals. Positron emission tomography was used to monitor TK gene expression. Ad.TK injection was feasible in 100% of cases. Treatment was well tolerated and dose-limiting toxicity was not achieved. Cumulative toxicity was not observed. Hepatic toxicity was absent even in cirrhotic patients. Fever, flu-like syndrome, pain at the injection site and pancytopenia were the most common side effects. No partial responses were observed and 60% of patients showed tumor stabilization of the injected lesion. Importantly, two patients who received the highest dose showed signs of intratumoral necrosis by imaging procedures. One of them achieved a sustained stabilization and survived for 26 months. In conclusion, Ad.TK can be safely administered by intratumoral injection to patients with HCC up to 2 × 10¹² vp per patient.

Mesenchymal stem cells as therapeutic tools and gene carriers in liver fibrosis and hepatocellular carcinoma.

Mesenchymal stem (stromal) cells (MSCs) are a source of circulating progenitors that are able to generate cells of all mesenchymal lineages and to cover cellular demands of injured tissues. The extent of their transdifferentiation plasticity remains controversial. Cells with MSC properties have been obtained from diverse tissues after purification and expansion in vitro. These cellular populations are heterogeneous and under certain conditions show pluripotent-like properties. MSCs present immunosuppressive and anti-inflammatory features and high migratory capacity toward inflamed or remodeling tissues. In this study we review available data regarding factors and signaling axes involved in the chemoattraction and engraftment of MSCs to an injured tissue or to a tissue undergoing active remodeling. Moreover, experimental evidence in support of uses of MSCs as vehicles of therapeutic genes is discussed. Because of its regenerative capacity and its particular immune properties, the liver is a good model to analyze the potential of MSC-based therapies. Finally, the potential application of MSCs and genetically modified MSCs in liver fibrosis and hepatocellular carcinoma (HCC) is proposed in view of available evidence.

Critical appraisal of the current status of dendritic cell based therapy

Una serie de circunstancias históricas han otorgado a las células dendríticas un lugar importante en la inmunoterapia antitumoral. El descubrimiento de su papel fundamental en la presentación antigénica ha propiciado su uso como adyuvantes en protocolos de vacunación en modelos tumorales de ratón. En estos modelos se llegaron a obtener remisiones tumorales. A finales de los años 90 la coincidencia de (i) estos prometedores resultados preclínicos, (ii) la disponibilidad en hospitales de áreas de terapiacelular (desarrolladas sobre todo para transplantes de médula ósea y (iii) protocolos de cultivo sencillos para diferenciar células dendríticas a partir de monocitos o precursores mieloides han impulsado la realización de múltiples ensayos clínicos. Los primeros datos clínicos no cumplieron las expectativas, pero se están extrayendo conclusiones sobre la actividad biológica e incluso se han obtenido algunos casos de respuestas clínicas en pacientes con cánceres avanzados. En cualquier caso, algunos de los ensayos que mejores resultados estaban obteniendo se encuentran bajo sospecha de fraude científico y otros buenos resultados obtenidos en los grupos piloto no se han reproducido cuando se han utilizado los mismos protocolos en grupos más amplios de pacientes.La situación actual del campo puede ser vista como una botella medio llena o medio vacía. En cualquier caso, se necesita mejorarla potencia terapéutica a nivel preclínico incluso con el riesgo de efectos adversos (AU)

A set of historical circumstances has centred the field of cancer immunotherapy on dendritic cells (DC). The discovery of their central role in antigen presentation for immunization gave rise to their use as adjuvants for cancer vaccination in transplantable tumour models in mice. Tumour rejections of experimental transplantable tumour models were achieved. During the late 90s concurrence of (i) this encouraging preclinical data, (ii) the availability of hospital cell therapy facilities that have been mainly set up for bone marrow transplantation and (iii) simple culture means to differentiate DC from monocytes or myeloid precursors spurred many clinical trials worldwide. Early clinical experience has not met the great expectations raised, but many lessons are being learned in terms of biological activity and cases of clinical response in advanced cases are routinely reported. However, some of the trials that reported the most encouraging clinical responses have confronted controversy due to scientific misconduct orto accumulation of successful cases in the pilot groups of patients that were not so reproducible when more patients were studied under the same protocols. The current status of the field could be seen as a half empty or a half full bottle. In either case, improvements are needed from the lab bench to increase therapeutic potency, even at the risk of side effects (AU)

Posibilidades de la terapia génica en el sistema musculoesquelético / Possibilities of gene therapy in the musculoskeletal system

La terapia génica consiste en la introducción de material genético en el interior de un órgano o tejido con la intención de producir un efecto biológico que permita prevenir o tratar una enfermedad. Por tanto, abre nuevas posibilidades al tratamiento de las patologías del sistema musculoesquelético, como son la reparación de fracturas óseas, la reparación de lesiones del cartílago articular, la artrodesis de la columna vertebral, la reparación de lesiones tendinosas o ligamentarias y los tumores musculoesqueléticos. Sus procedimientos son muy variados, lo que obliga a una intensa investigación de las posibilidades de cada procedimiento para una enfermedad determinada. Además, se pretende incrementar la eficacia de los vectores en la transducción celular, desarrollar promotores específicos de tejido que permitan reducir los efectos tóxicos asociados a la expresión génica en tejidos no deseados, aplicar sistemas que permitan que la expresión del gen terapéutico pueda ser regulable, e identificar el gen o la combinación de genes más apropiada para permitir la reparación tisular

Gene therapy consist in the introduction of genetic material in an organ or tissue to produce a biological effect capable of preventing or treating a disease. Therefore opens new possibilities for treatment of musculoskeletal disease, fusion of the spinal column, repair of tendon or ligament injuries, and musculoskeletal tumors. Procedures vary widely, meaning that the possibilities of each procedure for a given disease should be investigated. In addition, it is proposed that the efficacy of vectors in cell transduction be increased, specific tissue promoters for reducing toxic effects associated with gene expression in nontarget tissues be developed, systems allowing the expression of the therapeutic gene be regulated, and the most important gene or combination of genes for tissue repair be identified

Critical appraisal of the current status of dendritic cell based therapy

Una serie de circunstancias históricas han otorgado a las célulasdendríticas un lugar importante en la inmunoterapia antitumoral.El descubrimiento de su papel fundamental en la presentaciónantigénica ha propiciado su uso como adyuvantes en protocolosde vacunación en modelos tumorales de ratón. En estosmodelos se llegaron a obtener remisiones tumorales. A finales delos años 90 la coincidencia de (i) estos prometedores resultadospreclínicos, (ii) la disponibilidad en hospitales de áreas de terapiacelular (desarrolladas sobre todo para transplantes de médulaósea y (iii) protocolos de cultivo sencillos para diferenciar célulasdendríticas a partir de monocitos o precursores mieloides hanimpulsado la realización de múltiples ensayos clínicos. Los primerosdatos clínicos no cumplieron las expectativas, pero se estánextrayendo conclusiones sobre la actividad biológica e incluso sehan obtenido algunos casos de respuestas clínicas en pacientescon cánceres avanzados. En cualquier caso, algunos de los ensayosque mejores resultados estaban obteniendo se encuentran bajosospecha de fraude científico y otros buenos resultados obtenidosen los grupos piloto no se han reproducido cuando se hanutilizado los mismos protocolos en grupos más amplios de pacientes.La situación actual del campo puede ser vista como una botellamedio llena o medio vacía. En cualquier caso, se necesita mejorarla potencia terapéutica a nivel preclínico incluso con el riesgode efectos adversos

A set of historical circumstances has centred the field of cancerimmunotherapy on dendritic cells (DC). The discovery of theircentral role in antigen presentation for immunization gave rise totheir use as adjuvants for cancer vaccination in transplantabletumour models in mice. Tumour rejections of experimental transplantabletumour models were achieved. During the late 90s concurrenceof (i) this encouraging preclinical data, (ii) the availabilityof hospital cell therapy facilities that have been mainly set upfor bone marrow transplantation and (iii) simple culture meansto differentiate DC from monocytes or myeloid precursors spurredmany clinical trials worldwide. Early clinical experience hasnot met the great expectations raised, but many lessons are beinglearned in terms of biological activity and cases of clinical responsein advanced cases are routinely reported. However, someof the trials that reported the most encouraging clinical responseshave confronted controversy due to scientific misconduct orto accumulation of successful cases in the pilot groups of patientsthat were not so reproducible when more patients were studiedunder the same protocols. The current status of the field could beseen as a half empty or a half full bottle. In either case, improvementsare needed from the lab bench to increase therapeuticpotency, even at the risk of side effects

Pancreatic cancer escape variants that evade immunogene therapy through loss of sensitivity to IFNgamma-induced apoptosis.

Combined injections into experimental tumor nodules of adenovirus encoding IL-12 and certain chemokines are capable to induce immune-mediated complete regressions. In this study, we found that the combination of two adenoviruses, one encoding IL-12 and other MIP3alpha (AdCMVIL-12+AdCMVMIP3alpha) was very successful in treating CT-26-derived colon carcinomas. However, in experimental tumors generated from the pancreatic carcinoma cell line Panc02 such combined treatment induces 50% of macroscopic complete regressions, although local relapses within 1 week are almost constant. We derived cell lines from such relapsing tumors and found that experimental malignancies derived from their inoculum were not amenable to treatment in any case with AdCMVIL-12+AdCMVMIP-3alpha. Importantly, relapsing cell lines were insensitive to in vitro induction of apoptosis by IFNgamma, in clear contrast with the original Panc02 cells. Comparative analyses by cDNA arrays of relapsing cell lines versus wild-type Panc02 were performed revealing an important number of genes (383) whose expression levels were modified more than two-fold. These changes grouped in certain gene ontology categories should harbor the mechanistic explanations of the acquired selective resistance to IFNgamma.

Vectores adenovirales de primera generación, el vector por excelencia en inmunoterapia génica del cáncer / First generation adenoviral vectors, the vector par excellence for immuno-gene therapy of cancer

La terapia génica constituye una novedosa alternativa terapéutica para muchas enfermedades cuando las terapias habituales no tienen efecto. Básicamente la terapia génica consiste en la introducción de material genético en el interior de una célula diana con el objeto de producir en ella un cambio funcional que se traduzca en un efecto terapéutico. El intenso desarrollo de esta área de la biomedicina ha llevado a la puesta en marcha de numerosos protocolos clínicos de terapia génica para el tratamiento experimental de enfermedades de diverso origen: tumoral, infeccioso, autoinmune, degenerativo, genético. En la mayoría de los casos, es necesario recurrir al empleo de un vehículo, denominado vector, para introducir el material genético en las células. Éstos pueden provenir de virus modificados genéticamente (vectores virales) o pueden ser formulaciones fisicoquímicas (vectores no virales). Actualmente existe un amplio abanico de vectores para transferencia génica, sin embargo no se dispone del vector ideal que pueda ser tan versátil como para adaptarse a las numerosas situaciones experimentales o clínicas. Debido a las propiedades de los vectores adenovirales, tales como su alta eficacia de transducción, amplio tropismo, fácil construcción y producción, éstos han sido ampliamente utilizados y se cuenta con una amplia experiencia en campos como la terapia génica del cáncer. En este sentido, y a pesar de la corta duración de expresión del transgén debido a su alta inmunogenicidad, los adenovirus de primera generación han demostrado su eficacia tanto en estrategias de inmunoterapia en modelos tumorales experimentales como también en ensayos clínicos de fase I. En este trabajo se revisan críticamente los distintos vectores virales empleados en terapia génica profundizando en las características biológicas de los adenovirus, los distintos tipos de vectores adenovirales, los diferentes sistemas de construcción de adenovirus de primera generación así como su empleo en distintas aproximaciones de inmunoterapia génica del cáncer (AU)

The promise of gene therapy in gastrointestinal and liver diseases.

Gene therapy consists of the transfer of genetic material to cells to achieve a therapeutic goal. In the field of gastroenterology and hepatology gene therapy has produced considerable expectation as a potential tool in the management of conditions that lack effective therapy including non-resectable neoplasms of the liver, pancreas and gastrointestinal tract, chronic viral hepatitis unresponsive to interferon therapy, liver cirrhosis, and inflammatory bowel disease.

Cytokine gene transfer into dendritic cells for cancer treatment.

Bone marrow-derived dendritic cells have been used to treat established experimental tumors by unleashing a cellular immune response against tumor antigens. Such antigens are artificially loaded onto dendritic cells' antigen-presenting molecules by different techniques including incubation with synthetic antigenic determinants, tumor lysates or nucleic acids encoding for those relevant antigens. Ex vivo gene transfer with viral and non-viral vectors is frequently used to obtain expression of the tumor antigens and thereby to formulate the therapeutic vaccines. Efficacy of the approaches is greatly enhanced if dendritic cells are transfected with a number of genes which encode immunostimulating factors. In some cases, such as with IL-12, IL-7 and CD40L genes, injection inside experimental malignancies of thus transfected dendritic cells induces complete tumor regression in several models. In this case tumor antigens are captured by dendritic cells by still unclear mechanisms and transported to lymphoid organs where productive antigen presentation to T-cells takes place. Many clinical trials testing dendritic cell-based vaccines against cancer are in progress and partial clinical efficacy has been already proved. Transfection of genes further strengthening the immunogenicity of such strategies will join the clinical club soon.

Gene therapy for liver diseases: recent strategies for treatment of viral hepatitis and liver malignancies.

Gene therapy has emerged as a powerful and very plastic tool to regulate biological functions in diseased tissues with application in virtually all medical fields. An increasing number of experimental and clinical studies underline the importance of genes as curative agents in the future. However, intense research is needed to evaluate the potential of gene therapy to improve efficacy and minimise the toxicity of the procedure.

Estrategias de terapia génica aplicadas al tratamiento de los tumores hepáticos / Gene therapy strategy applied to the treatment of liver tumors

La terapia génica representa una prometedora alternativa terapéutica para los tumores hepáticos avanzados en los que los tratamientos habituales como la cirugía, radioterapia y quimioterapia no resultan eficaces. Básicamente la terapia génica consiste en la introducción de material genético en el interior de las células de un tejido para producir en ellas un efecto terapéutico beneficioso. Los sistemas diseñados para introducir los "genes terapéuticos" se denominan vectores, que pueden ser de tipo vira¡ o no vira¡, siendo los primeros los más eficaces. Existen diversas modalidades en la terapia génica contra el cáncer como el empleo de genes suicidas, reemplazo génico, terapia antisentido, inhibición de la angiogénesis e inmunoterapia génica. La aplicación de estos tratamientos en modelos tumorales experimentales ha permitido obtener respuestas antitumorales muy significativas. Fruto de estos datos pre-clínicos se han iniciado varios protocolos clínicos de terapia génica para el tratamiento de tumores hepáticos avanzados. El desarrollo, tanto de promotores específicos de tumor como de sistemas de transferencia génica dirigidos y más eficaces son algunos de los retos que debe superar aún la terapia génica (AU)

Thrombopenic purpura induced by a monoclonal antibody directed to a 35-kilodalton surface protein (p35) expressed on murine platelets and endothelial cells.

OBJECTIVE: With the aim of obtaining monoclonal antibodies (mAbs) against mouse endothelial surface antigens, immunization of rats with a mouse-derived endothelial cell line (PY4.1) and subsequent hybridoma production were performed. MATERIALS AND METHODS: One of the mAbs produced by hybridoma EOL5F5 was selected for its surface binding to endothelial cell lines, and identification of the mAb-recognized antigen was performed by immunoprecipitation. Experiments were performed to analyze the effects of EOL5F5 on systemic administration to mice. RESULTS: EOL5F5-recognized antigen was a single band of 35 kDa under reducing and nonreducing conditions, features that do not match other known differentiation antigens with comparable tissue distribution. In vivo administration of purified EOL5F5 mAb to mice (n = 20) induced intense cutaneous purpura as well as severe but transient thrombocytopenia. Expression of EOL5F5-recognized antigen was detected on platelets from which it immunoprecipitated a moiety of identical electrophoretic pattern in SDS-PAGE, as the one recognized on endothelial cells. Immunohistochemically, EOL5F5-recognized antigen (p35) also was expressed on dermal capillaries, suggesting that, in addition to thrombocytopenia, damaging effects of the antibody on endothelial cells also might cause the observed purpura. CONCLUSIONS: Our results show induction of thrombocytopenic purpura in mice with an mAb against a single antigenic determinant expressed on both platelets and endothelium. EOL5F5 mAb injection sets the stage for useful experimental models that resemble immune thrombocytopenic purpura.

Alpha(v)beta(3) integrin-mediated adenoviral transfer of interleukin-12 at the periphery of hepatic colon cancer metastases induces VCAM-1 expression and T-cell recruitment.

We previously reported that systemic injection of recombinant adenovirus resulted in a rim of gene transduction around experimental liver tumor nodules. This zone of higher infection is dependent on the alpha(v)beta(3) integrin, acting as an adenovirus internalization receptor, which is overexpressed in tissues surrounding liver metastases. When a recombinant adenovirus encoding interleukin-12 (AdCMVIL-12) is given into a subcutaneous tumor nodule in mice also bearing concomitant liver tumors, a fraction of AdCMVIL-12 reaches the systemic circulation and infects liver tissue, especially at the malignant/healthy tissue interface. As a result of the expression at this location of the interleukin-12 transgenes, VCAM-1 is induced on vessel cells and mediates the recruitment of adoptively transferred anti-tumor cytolytic T-lymphocytes. These studies provide mechanistic explanations for the potent therapeutic synergy observed between interleukin-12 gene transfer and adoptive T-cell therapy.

IL-12 gene therapy for cancer: in synergy with other immunotherapies.

In preclinical models of cancer, gene therapy with interleukin 12 (IL-12) has reached unprecedented levels of success when combined with immunotherapy approaches such as gene transfer of other cytokines and/or chemokines, costimulatory molecules or adoptive cell therapy. These combinations have been found to produce synergistic rather than additive effects. Meanwhile, IL-12 gene therapy is beginning clinical testing as a single agent, but combination strategies are at hand.

Genetic heterogeneity in the toxicity to systemic adenoviral gene transfer of interleukin-12.

Despite the efficacy of IL-12 in cancer experimental models, clinical trials with systemic recombinant IL-12 showed unacceptable toxicity related to endogenous IFNgamma production. We report that systemic administration of a recombinant adenovirus encoding IL-12 (AdCMVmIL-12) has a dramatically different survival outcome in a number of mouse pure strains over a wide range of doses. For instance at 2.5 x 10(9) p.f.u., systemic AdCMVmIL-12 killed all C57BL/6 mice but spared all BALB/c mice. Much higher IFNgamma concentrations in serum samples of C57BL/6 than in those from identically treated BALB/c were found. Causes for heterogeneous toxicity can be traced to differences among murine strains in the levels of gene transduction achieved in the liver, as assessed with adenovirus coding for reporter genes. In accordance, IL-12 serum concentrations are higher in susceptible mice. In addition, sera from C57BL/6 mice treated with AdCMVmIL-12 showed higher levels of IL-18, a well-known IFNgamma inducer. Interestingly, lethal toxicity in C57BL/6 mice was abolished by administration of blocking anti-IFNgamma mAbs and also by simultaneous depletion of T cells, NK cells, and macrophages. These observations together with the great dispersion of IFNgamma produced by human PBMCs upon in vitro stimulation with IL-12, or infection with recombinant adenovirus encoding IL-12, suggest that patients might also show heterogeneous degrees of toxicity in response to IL-12 gene transfer.

Gene therapy of orthotopic hepatocellular carcinoma in rats using adenovirus coding for interleukin 12.

The use of gene therapy to enhance antitumor immunity has emerged as a promising procedure to fight cancer. In this study we have tested the ability of an adenovirus carrying interleukin 12 (IL-12) gene (AdCMVIL-12) to eliminate tumoral lesions in 3 animal models of orthotopic hepatocellular carcinoma (HCC). Intratumoral injection of AdCMVIL-12 in animals with a single big tumor nodule implanted in the liver resulted in significant inhibition of tumor growth in a dose-dependent manner. Fifty percent of animals that received a dose of 5 x 10(9) plaque-forming units, showed complete regression of the tumor 2 weeks after treatment. In animals with 2 independent tumor nodules in the left liver lobe, injection in only one of them of 5 x 10(9) pfu AdCMVIL-12 induced, 15 days after therapy, complete regression of 50% of treated tumors and also of 50% of untreated lesions, with 60% long-term survival. Rats that were tumor free after therapy with AdCMVIL-12 showed protection against tumor rechallenge. A group of rats received the carcinogen diethylnitrosamine and developed multiple hepatic dysplasic nodules of 1 to 5 mm in diameter. These animals were treated by intrahepatic artery injection of either AdCMVIL-12 (5 x 10(9) pfu) or control vector. In this model AdCMVIL-12 induced complete tumor regression in 20% of treated rats and inhibited tumor growth in 60% of cases with an increase in rat survival. Activation of natural killer (NK) cells and inhibition of angiogenesis were found to be antitumor mechanisms set in motion by AdCMVIL-12. Our data indicate that experimental HCC can be efficiently treated by intratumoral or intravascular injection of adenovirus expressing IL-12.

Gene therapy of hepatocellular carcinoma.

The extraordinary versatility of gene therapy opens new possibilities for the treatment of incurable diseases, including hepatocellular carcinoma. Gene therapy strategies against tumors include prodrug activation therapy by the transfer of suicide genes, immunogene therapy, tumoral cell phenotype correction by the inhibition of oncogenes or the transfer of tumor suppressor genes, antiangiogenesis and transfer of oncolytic viruses. The experience accumulated during the last decade of clinical gene therapy indicates that genes can be expressed inside the tumor tissue, but the overall results of the studies conducted so far are still disappointing, mainly due to the poor performance of the currently available gene therapy vectors. This review covers the general aspects of gene therapy vectors, preclinical data available in animal models of hepatocellular carcinoma, and finally a brief summary of the gene therapy clinical trials aimed at the treatment of liver cancer.