Cómo finalizar un ensayo clínico Fase III en Terapia Celular sin morir en el intento

Esta conferencia tiene como base el primer ensayo clínico con células madre mesenquimales con éxito, que será publicado pronto en la prestigiosa revista "The Lancet".Desde el año 2004 los tratamientos celulares se rigen por la Ley de Medicamentos, lo cual implica que las células son un fármaco que deben someterse a la misma rigidez de producción que cualquier fórmula magistral química o biológica, pero con una diferencia esencial, son un fármaco vivo. Otro aspecto a considerar es el desconocimiento por parte de la comunidad científica, así como la comunidad reguladora, de los mecanismos de acción y de la farmacopea de las células. Ambos aspectos hacen que el desarrollo de los ensayos clínicos sea más complicado y aumente su dificultad hasta llegar a ser un fármaco disponible para las farmacias hospitalarias. Nuestra andadura empezó en el año 2001; en aquel momento no existía legislación asociada al producto celular. No obstante decidimos seguir el protocolo de cualquier otro fármaco; dicha decisión nos ha permitido obtener el primer fármaco de terapia celular a nivel mundial: células troncales mesenquimales alogénicas derivadas del tejido adiposo para el tratamiento de la patología fistulosa perianal. Si intentamos resumir este proceso en un párrafo debemos destacar los buenos resultados de seguridad del uso autólogo de las células y la dificultad de “educar” a los cirujanos a la hora de aplicar su tratamiento; este hecho, junto con un diseño/seguimiento de aplicación no ajustado a un medicamento vivo, nos hizo fracasar en nuestro primer ensayo clínico en fase III. Afortunadamente, tanto la empresa que “apostó” por nuestra idea, como nosotros, aprendimos enormemente de dicho “fracaso”, aunque costó una enorme cantidad de dinero y puestos de trabajo. Una vez repasados los errores, empezamos de nuevo el ciclo del ensayo clínico,:Fase IIa (seguridad), Fase II (seguridad y primeros datos de eficacia) y Fase III (eficiencia frente al tratamiento convencional), pero en en esta ocasión, y por cuestiones económico-operativas, se decidió utilizar las células de forma alogénica, aprovechando una propiedad de las células mesenquimales que las hace inmunoprivilegiadas. Como conclusión, me gustaría destacar las enormes dificultades legislativas, económicas y farmacológicas que implica el proceso de un ensayo clínico. Pero paralelamente me gustaría animar a todo el mundo a insistir en las ideas y seguir los protocolos de las buenas prácticas de investigación, tanto de laboratorio como clínicas, para alcanzar el éxito. Y por último, exponer que, para avanzar en este camino, son tan importantes los errores/fracasos como los éxitos; sin las enseñanzas de los errores es difícil progresar hasta obtener resultados óptimos. Me gustaría acabar con una frase de paulo Coelho: “una búsqueda comienza siempre con la suerte del principiante y termina con la prueba del conquistador”.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Avances en Terapia Celular

Todos los tejidos de nuestro organismo se van desgastando con el paso del tiempo, y de forma natural son capaces de limitar este desgaste desarrollando una interesante capacidad de regeneración y reparación, lo cual nos permite alcanzar a la mayoría de los seres humanos la madurez y una esperanza de vida que se va elevando continuamente. Estos mecanismos de regeneración intrínsecos son muy limitados, posiblemente asociado a que durante el desarrollo y evolución de la especie hemos preferido aumentar nuestro sistema defensivo ante agresiones externas, mediante un entramado inmunológico muy eficaz, en lugar de seleccionar la regeneración de nuestros tejidos como ocurre en otras especies animales como el ocelote, la estrella de mar... a expensas de una menor capacidad defensiva ante agresiones externas; lo cual impide a nuestro organismo reparar o renovar grandes cantidades de tejido dañado de forma aguda por medios naturales. Actualmente, para paliar estar grandes agresiones a tejidos específicos, la medicina y en especial la cirugía ha desarrollado un sistema de trasplantes de órganos que, aún siendo en nuestro país uno de los más eficaces del mundo, no está exento de complicaciones como la escasez de donantes o el rechazo inmunológico

Clonación y caracterización de un gen (APA1) implicado en el control de la expresión de la H+-ATPasa de la membrana plasmática de Saccharomyces cerevisiae

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Bioquímica. Fecha de lectura: 4-7-199

Preliminary study on non-viral transfection of F9 (factor IX) gene by nucleofection in human adipose-derived mesenchymal stem cells

Background. Hemophilia is a rare recessive X-linked disease characterized by a deficiency of coagulation factor VIII or factor IX. Its current treatment is merely palliative. Advanced therapies are likely to become the treatment of choice for the disease as they could provide a curative treatment. Methods. The present study looks into the use of a safe non-viral transfection method based on nucleofection to express and secrete human clotting factor IX (hFIX) where human adipose tissue derived mesenchymal stem cells were used as target cells in vitro studies and NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice were used to analyze factor IX expression in vivo studies. Previously, acute liver injury was induced by an injected intraperitoneal dose of 500 mg/kg body weight of acetaminophen. Results. Nucleofection showed a percentage of positive cells ranging between 30.7% and 41.9% and a cell viability rate of 29.8%, and cells were shown to secrete amounts of hFIX between 36.8 and 71.9 ng/mL. hFIX levels in the blood of NSG mice injected with ASCs transfected with this vector, were 2.7 ng/mL 48 h after injection. Expression and secretion of hFIX were achieved both in vitro cell culture media and in vivo in the plasma of mice treated with the transfected ASCs. Such cells are capable of eventually migrating to a previously damaged target tissue (the liver) where they secrete hFIX, releasing it to the bloodstream over a period of at least five days from administration. Conclusions. The results obtained in the present study may form a preliminary basis for the establishment of a future ex vivo non-viral gene/cellular safe therapy protocol that may eventually contribute to advancing the treatment of hemophilia

Células progenitoras multipotentes obtenidas del tejido adiposo y su aplicación clínica

La primera vez que se describió la existencia de células progenitoras en el tejido adiposo subcutáneo fue en el año 2001. Se comunicó la exis- tencia de un grupo de células homogéneas con aspecto fibroblástico, obtenidas a partir de la fracción vásculo-estromal de tejido adiposo ex- traido por liposucción, que tenían la capacidad de diferenciarse a osteo- citos, condriocitos y adipocitos. En el año 2004 fueron definidas como ASC (Adipose-derived Stem Cells) y se establecieron los criterios de identificación: 1. Células con morfología fusiforme que se aíslan median- te digestión enzimática y adhesión a los plásticos. 2. Con capacidad de autorrenovación por largos periodos de tiempo. 3. Multipotenciales (se di- ferencian a adipocitos, condrocitos y osteocitos) y 4. Que tienen el si- guiente perfil de expresión: marcaje positivo para CD29, CD44, CD 90 y CD105; y un marcaje negativo para CD34, CD45 y CD133. Se han descrito más de doscientos ciclos de replicación ex vivo de las ASC sin alteraciones genéticas o fenotípicas. En los últimos años se ha observado la expresión de marcadores de células madre embrionarias en la superficie de las ASC (Oct-4, Rex-1 y Sox-2). También se han descrito algunas vías de señalización, que regulan la proliferación y/o la diferen- ciación celular (Wnt, Esfingosilfosforilcolina y FGF-2). También se ha comunicado diferenciación a músculo esquelético y cardiaco, tejido neu- ronal, endotelio vascular, epitelio, tejido hepático y tejido pancreático. Lo que propone a las ASCs como una de las células más prometedoras para su uso en terapia celular

Recurrent anal fistulae: Limited surgery supported by stem cells

AIM: To study the results of stem-cell therapy under a Compassionate-use Program for patients with recurrent anal fistulae. METHODS: Under controlled circumstances, and approved by European and Spanish laws, a Compassionate-use Program allows the use of stem-cell therapy for patients with very complex anal fistulae. Candidates had previously undergone multiple surgical interventions that had failed to resolve the fistulae, and presented symptomatic recurrence. The intervention consisted of limited surgery (with closure of the internal opening), followed by local implant of stem cells in the fistula- tract wall. Autologous expanded adipose-derived stem cells were the main cell type selected for implant. The first evaluation was performed on the 8th postoperative week; outcome was classified as response or partial response. Evaluation one year after the intervention confirmed if complete healing of the fistula was achieved. RESULTS: Ten patients (8 male) with highly recurrent and complex fistulae were treated (mean age: 49 years, range: 28-76 years). Seven cases were non- Crohn’s fistulae, and three were Crohn’s-associated fistulae. Previous surgical attempts ranged from 3 to 12. Two patients presented with preoperative incontinence (Wexner scores of 12 and 13 points). After the intervention, six patients showed clinical response on the 8th postoperative week, with a complete cessation of suppuration from the fistula. Three patients presented a partial response, with an evident decrease in suppuration. A year later, six patients (60%) remained healed, with complete reepithelization of the external opening. Postoperative Wexner Scores were 0 in six cases. The two patients with previous incontinence improved their scores from 12 to 8 points and from 13 to 5 points. No adverse reactions or complications related to stem-cell therapy were reported during the study period. CONCLUSION: Stem cells are safe and useful for treating anal fistulae. Healing can be achieved in severe cases, sparing fecal incontinence risk, and improving previous scorin

Liquid biopsy by NGS: Differential presence of exons (DPE) is related to metastatic potential in a colon-cancer model in the rat

Differential presence of exons (DPE) is a method of interpretation of exome sequencing, which has been proposed to design a predictive algorithm with clinical value in patients with colorectal cancer (CRC). The goal of the present study was to examine the reproducibility in a rat model of metastatic colon cancer. DHD/K12-TRb cells were injected in syngenic immunocompetent BD-IX rats. Cells were from two stocks with low and normal metastatic potential, and injected into two separate groups of rats. Five to ten weeks after injection, blood samples were taken prior euthanasia and whole exome sequencing performed. Through DPE analysis, we identified a set of exons whose differential presence in plasma allowed us to compare both groups of tumor-bearing animals. A verification test was performed to confirm that the algorithm was able to classify extracted samples into their corresponding groups of origin. The highest mean probability was 0.8954. In conclusion, the DPE analysis in tumor-bearing animals was able to discriminate between different disease status, which fully supports previous results in CRC patients.This studywas funded by two grants from"Instituto de Salud Carlos III", Spain (FIS; refs. PS09/01815 and PI13/01924

The role of mucin cell-free DNA detection as a new marker for the study of acellular pseudomyxoma peritonei of appendicular origin by liquid biopsy

Background: Acellular pseudomyxoma peritonei (aPMP) is a rare peritoneal malignancy characterized by the accumulation of large amounts of mucin (lacking tumor cells) in the peritoneum. Many cases account for several kilograms of mucin to be screened by the pathologist. This is a comprehensive study of three patients with aPMP, whose tumors showed KRAS mutation, allowing for the tracking of this marker by liquid biopsy. Methods: Pre and post-surgery plasma, and mucin removed during cytoreductive surgery were collected from the patients. KRAS mutations were analyzed using droplet digital polymerase chain reaction (ddPCR). Mucin was injected in mice. KRAS and cytokine levels were measured in plasma of the mice using ddPCR and a magnetic bead-based assay. Mucin microbiome was analyzed by 16S rRNA sequencing. Results: KRAS mutations were detected in mucin cell-free DNA (cfDNA) from the three patients but not in the pre or post-surgery plasma. Electron microscopy detected microparticles (diameter <0.4 µm) in mucin. Mucin from one patient grew up inside the peritoneal cavity of mice and human KRAS was identified in mucin cfDNA, but not in plasma. All mucins showed the same bacterial profile. Cytokine levels were slightly altered in mice. Conclusions: The three aPMP patients included in this study shared some common aspects: the absence of tumor cells in mucin, the presence of KRAS mutated cfDNA in mucin, and the absence of this tumor-derived mutation in the bloodstream, providing additional information to the routine pathological examinations and suggesting that mucin cfDNA could potentially play a role in aPMP recurrence and prognosis.The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was funded by a grant from the ‘FIS-ISCIII-FEDER’ [Fondo de Investigaciones Sanitarias-Instituto de Salud Carlos III-Fondo Europeo de Desarrollo Regional (European Regional Development Fund)], Ministry of Health, Spain (grant number PI17/01233)

Toxicity study in a pig model of intraperitoneal collagenase as an “enzymatic scalpel” directed to break stroma in order to generate a new perspective for peritoneal carcinomatosis approach: an experimental research

Background: This study aimed to measure the toxicity resulting from collagenase administration to the peritoneal cavity in a pig model as a preliminary step to break down the stroma surrounding tumors. Methods: Eight pigs were treated with 2 different collagenase concentrations previously tested in rats by our group. Time and temperature were controlled using a peritoneal lavage system (PRS System, Combat Medical Ltd.) identical to that used in human surgeries through hyperthermic intraperitoneal chemotherapy (HIPEC); 2 additional pigs were treated with peritoneal lavage only. Samples of blood and peritoneal fluid were collected pre-treatment, immediately after treatment, and 24 h postoperatively. In addition, histological studies and blood collagenase levels were measured. Results: No complications were observed during the surgeries. Intraoperative images evidenced the release of peritoneal tissue during collagenase treatment. After surgery, the animals showed no signs of pain. Diet and mobility were normal at 4 h postoperatively, and there were no significant differences in hematologic or biochemical parameters. Quantification of MMP1 and MMP2 in all samples as measured by absorbance showed no differences in blood collagenase levels between pre-treatment, post-treatment, and 24 h postoperatively. None of the animals treated with collagenase showed peritoneal adhesions during the second surgery. Histologically, peritoneal organs and serous structures did not show any microscopic alterations associated with collagenase treatment in any group. Conclusion: Lavage of the peritoneal cavity with doses of up to 100,000 collagen digestion units/animal for 30 min is safe and removes connective tissue from the peritoneal cavity

Main histological parameters to be evaluated in an experimental model of myocardial infarct treated by stem cells on pigs

Myocardial infarction has been carefully studied in numerous experimental models. Most of these models are based on electrophysiological and functional data, and pay less attention to histological discoveries. During the last decade, treatment using advanced therapies, mainly cell therapy, has prevailed from among all the options to be studied for treating myocardial infarction. In our study we wanted to show the fundamental histological parameters to be evaluated during the development of an infarction on an experimental model as well as treatment with mesenchymal stem cells derived from adipose tissue applied intra-lesionally. The fundamental parameters to study in infarcted tissue at the histological level are the cells involved in the inflammatory process (lymphocytes, macrophages and M2, neutrophils, mast cells and plasma cells), neovascularization processes (capillaries and arterioles) and cardiac cells (cardiomyocytes and Purkinje fibers). In our study, we used intramyocardial injection of mesenchymal stem cells into the myocardial infarction area 1 hour after arterial occlusion and allowed 1 month of evolution before analyzing the modifications on the normal tissue inflammatory infiltrate. Acute inflammation was shortened, leading to chronic inflammation with abundant plasma cells and mast cells and complete disappearance of neutrophils. Another benefit was an increase in the number of vessels formed. Cardiomyocytes and Purkinje fibers were better conserved, both from a structural and metabolic point of view, possibly leading to reduced morbidity in the long term. With this study we present the main histological aspects to be evaluated in future assays, complementing or explaining the electrophysiological and functional findingsThis study has been funded by Instituto de Salud Carlos III (ISCIII) through the Spanish Net of Cell Therapy (TerCel), RETICS subprogram of the I+D+I 2013-2016 [RD16/0011/0013 funded by ISCIII and co-founded by European Regional Development Fund (ERDF)