TIME TO MOVE TERAPIA CELULAR EN LA LESION MEDULAR

Nuevas estrategias de tratamiento en la LM

“Investigamos para avanzar”

A la búsqueda de soluciones “Investigamos para avanzar”

Contenido

A la búsqueda de soluciones Descripción del proyecto, objetivos y metodología del proceso Fundación Hospital Universitari Vall d’Hebron - Institut de Recerca (HUVH– VHIR) Centro de Investigación Príncipe Felipe (CIPF) Fundació Step by Step (SbS) “Investigamos para avanzar” Impacto e interés social Colaboración y contraprestaciones

Colaboración en el proyecto de investigación TIME TO MOVE: Terapia celular en lesión medular

“Nuevas estrategias de tratamiento de la LM”

Accidentes tráfico

37%

Caidas casuales

34%

Accidentes laborales

10%

Accidentes deportivos

11%

Autolisis8%

Descripción del proyecto

Sólo en España, más de 1.000 personas sufren cada año una lesión medular traumática ( 2,5 por 100.000 habitantes), habiendo en la actualidad cerca de 40.000 personas afectadas. Se calcula que en Europa hay 330.000 personas con lesión medular y se producen alrededor de 11.000 casos nuevos cada año. Hasta el año 2003, la causa más frecuente en España eran los accidentes de tráfico que representaban más del 50% de los casos, reduciéndose a partir de esa fecha hasta el 36,6%. Las caídas casuales representan el 33,6%, los accidentes laborales el 10%, los deportivos el 11,3% y los intentos de autolisis el 8,5%.

A la búsqueda de soluciones: TIME TO MOVE

Origen de las causas de la Lesión Medular

La lesión medular (LM) es un proceso de alta complejidad técnico asistencial que conlleva un elevado consumo de recursos sanitarios. Significa una agresión terrible en la vida de cualquier persona: comporta una ruptura con la vida anterior, un grado severo de discapacidad y una importante pérdida de funcionalidad y de calidad de vida. No existe en la actualidad ningún tratamiento efectivo, y con la suficiente evidencia científica, que mejore el nivel neurológico y la funcionalidad de las personas afectas de

lesión medular. El proyecto TIME TO MOVE(Terapia Celular en Lesión Medular) tiene por objeto:

A. Conocer los mecanismos que intervienen en la inflamación, en la destrucción de tejidos y la muerte celular

B. Determinar la eficacia neuroprotectora y neuroregeneradora de ciertos fármacos y sustancias.

C. Verificar la efectividad de la aplicación de terapias celulares, a partir de los mecanismos de

reparación espontánea e inducida. La finalidad de TIME TO MOVE, coordinado entre grupos de investigación, es pues diseñar, validar y aplicar nuevas estrategias para el tratamiento de las lesiones medulares, agudas y crónicas. Los trabajos de investigación se llevarán a cabo de forma coordinada por los siguientes grupos de investigación en: Unidad de Lesionados Medulares del Hospital Universitario Vall d’Hebron (HUVH); Señalización Celular y Apoptosis del Vall d’Hebron Institut de Recerca (VHIR); Laboratorio de Regeneración Neuronal del Centro de Investigación Príncipe Felipe (CIPF) y Laboratorio Polímeros Terapéuticos del Centro de Investigación Príncipe Felipe (CIPF). La lesión medular es un episodio multifásico, donde convergen múltiples eventos:

1. Pérdida masiva de unidades funcionales (neuronas y células gliales).

2. Invasión de otras células, astrocitos, microglia y células inflamatorias, que generan proteínas de matriz extracelular (condroitin sulfato entre otras), citoquinas y quimocinas, agentes oxidantes,…

3. Productos de la degeneración celular (glutamato, iones, peroxidación lipídica, proteínas derivadas de las vainas de mielina,..).

4. Isquemia y pobre vascularización con pérdida de la barrera hematoencefálica.

La multiplicidad de procesos que se desencadenan indican posibles vías de actuación, tanto en fase aguda como crónica. En la aguda, se trata de lograr detener laexcitotoxicidad (daño y destrucción de las neuronas), de controlar la inflamación y de promover la regeneración, reduciendo el impacto de la lesión primaria y secundaria, y actuando mediante la neuroprotección de los elementos neuronales medulares. En la fase crónica, se busca favorecer la regeneración de los elementos neuronales lesionados.

La finalidad de TIME TO MOVE es aplicar

nuevas estrategias para el tratamiento de las

lesiones medulares, agudas y crónicas.

El trasplante celular puede sustituir a las células perdidas, y a la vez generar un ambiente propiciopara la supervivencia y buen funcionamiento de losaxones supervivientes y lesionados, aliviando elproceso de degeneración, favoreciendo laregeneración endógena, espontánea, en la fasecrónica de la lesión.

La eliminación de la cicatriz glial y la supresión delos inhibidores del crecimiento axonal puede ser unmétodo eficaz para favorecer la reparación de lamédula, en la fase crónica.

El uso de las plataformas biotecnológicas,herramientas innovadoras y útiles para liberarcompuestos de forma controlada y local, permitiríausar fármacos y otras sustancias capaces de repararla médula y regenerar los axones, creando de estemodo un puente sobre el que pueda generarse elcrecimiento axonal, en la fase crónica.

La utilización de polímeros terapéuticos(nanoconstrucciones híbridas) puede ser un sistemade transporte intracelular y extracelular específico,para conseguir terapias de aplicación clínica, en fasecrónica y aguda.

La liberación y actuación de fármacos dirigidosmediante nanotecnología sobre el lugar de la lesión,permitiría limitar y reducir la inflamación. En faseaguda, podría ser una terapia eficaz, evitando losefectos nocivos de la lesión secundaria, y en fasecrónica, crear un ambiente propicio a laregeneración

Hipótesis de trabajo

Determinar los biomarcadores, tanto a nivel clínicocomo experimental, necesarios para conocer elestado y grado de la lesión, y poder predecir suevolución en el tiempo.

Antecedentes

La médula espinal es un cordón nervioso que seextiende desde la base del cerebro hasta la regiónlumbar. Está protegida por la columna, por lasvértebras, y funciona como una línea eléctrica dedoble recorrido, de ida y vuelta, que transmite lasórdenes del cerebro al resto del cuerpo y viceversa.Cuando esta conducción nerviosa se interrumpe poruna lesión –en este caso traumática, sus consecuenciasson más o menos graves según la localización de lalesión. A nivel cervical (tetraplejia) provoca pérdida odisminución de la sensibilidad, de la movilidad deextremidades superiores e inferiores y de todo eltronco, y de la funcionalidad de las vísceras torácicasy abdominales. A nivel torácico y lumbar (paraplejia)provoca pérdida de la sensibilidad, de la movilidad delas extremidades inferiores y de parte del tronco, y dela funcionalidad de las vísceras abdominales. A nivellumbar bajo (cola de caballo) provoca parálisis de lasextremidades inferiores. La mayoría de LM traumáticas no presentan una sección completa de la médula, sino que la lesiónse produce por compresión, laceración o contusión. Además del daño inicial en el tejido medular –mueren neuronas motoras y sensoriales, se produce una degeneración post-traumática de lamédula, que en gran medida es consecuencia de un proceso secundario a la lesión, en el queintervienen múltiples factores: estrés oxidativo por síntesis excesiva de óxido nítrico; activaciónde la microglía; inflamación local; microcirculación alterada; disfunción de la barrerahematoencefálica; y, según se ha descubierto recientemente, un mecanismo de muerte celulardiferida. Todo ello sucede durante los primeros minutos, horas, días o incluso meses después deltrauma inicial. Al mismo tiempo se produce una reparación espontánea por diversos mecanismos,que ha sido observada en modelos animales: generación de nuevos circuitos neuronales a partir delos ya existentes o a partir de una población de células madre multipotente que existen en lamédula espinal (células ependimarias).

Las consecuencias de este proceso secundario sondesastrosas: muerte accidental (necrosis) ymuerte programada (apoptosis) de las neuronas;formación de cicatrices y quistes; pérdida de lamielina (aislante y protector de los axones);alteración de la morfología y funcionalidad de losconductos nerviosos. Los mecanismos por los quese producen son además complejos y pococonocidos. Existe asimismo poco conocimiento yescasa evidencia acerca de los mecanismos,espontáneos o inducidos, de regeneración y/orecuperación de la plasticidad de las neuronas. Hasta el momento actual se han llevado a cabomultitud de aproximaciones para tratar la LMtraumática y paliar sus efectos en la fase aguda.Desde la cirugía precoz de la fractura vertebral,para estabilizar la misma y evitar la progresión

de la lesión, tratamientos farmacológicos para reducir el edema y losradicales libres y para inhibir la toxicidad de los glutamatos, tratamientosantiinflamatorios, antiapoptóticos y antidesmielinizantes, pasando por sustancias para promover la regeneración axonal y estrategias de reparacióncelular y con biomateriales, hasta diversas y numerosas técnicas de terapiasfísicas. Sin embargo, ninguno de los estudios anteriores ha abordado de formaconjunta un proyecto realizado en animales de experimentación, con undesarrollo en laboratorio de investigación básica y de desarrollo de sistemasde biomateriales híbridos biodegradables para la liberación de sustancias ycon potencial translacional inmediato a la práctica clínica.

Metodología El proyecto se estructura en tres etapas, de 12 meses de duración cada una, en las que los cincogrupos de investigación trabajarán simultáneamente y de forma coordinada en los objetivossiguientes:

Abordar el trasplante celular por método quirúrgico de inyección o mediante punción en elespacio subaracnoideo.

Estudiar biomateriales biodegradables para crear nichos que puedan alojar trasplantesautólogos (células madre propias).

Aplicar in vivo, en modelo animal, los nanoconjugados preparados para la liberación de

tratamientos farmacológicos que sirvan para reducir la inflamación y/o para favorecer laregeneración celular axonal

Mostrar la capacidad terapéutica, in vitro e in vivo en modelo animal (roedores), de las

células ependimarias (células madre residentes en la médula espinal que se activan tras lalesión).

Estudiar el perfil de expresión génica (celular) y el tiempo de respuesta en fase aguda, sub-

aguda y crónica.

Estudiar los biomarcadores tras la terapia celular in vivo, a partir de muestras de LCR(líquido cefalorraquídeo) extraídas de los animales tratados.

Contrastar los resultados de biomarcadores obtenidos de las muestras clínicas de los

pacientes y de las de animales de experimentación.

Obtener información de los cambios espacio-temporales, en relación con la inflamación ymuerte celular tras la lesión.

Aplicar y comparar efectos de factores inmunosupresores endógenos y generales.

Analizar el efecto de fármacos (i.e. simvastatina, condroitinasas) que modulan las balsas de

lípidos, en relación a la inflamación y muerte celular.

Analizar los efectos de fármacos neuroprotectores y de terapias celulares en relación a lasmotoneuronas y los oligodendrocitos, en la lesión crónica.

Estudiar el efecto combinado de terapias farmacológicas y celulares en fase crónica.

Analizar biomarcadores en líquidos biológicos humanos (LCR, sangre y orina) comoherramienta de pronóstico de gravedad y como predictores de cambios, en fase aguda ycrónica, y comparar sus posibles diferencias.

Desarrollar y aplicar biomateriales (microcápsulas, microesferas y/o liposomas) para la

liberación controlada de fármacos.

Generar una familia de nanomedicinas con capacidad de paliar o aminorar los dañoscelulares asociados a una lesión medular, en fase aguda y crónica.

 

Los hallazgos y resultados obtenidos, in vitro (en el laboratorio)e in vivo (con animales de experimentación), serán trasladadosa los pacientes con lesión medular. La experimentación clínicase llevará a cabo tanto en fase aguda como en fase crónica deLM.

Fase lesión medular aguda

  Usar biomarcadores de inflamación, a partir de muestras de LCR,

como herramienta pronostica de la profundidad de la lesiónmedular.

Utilizar fármacos neuroprotectores mediante nanotecnología, enrelación a las motoneuronas y los oligodendrocitos.

Aplicar familias de nanomedicinas con capacidad para paliar oaminorar los daños celulares asociados a la lesión medular.

Utilizar terapias de regeneración celular, en relación a lasmotoneuronas y los oligodendrocitos. 

Fase lesión medular crónica

  Conocer el remanente de motoneuronas disponibles en el área

de la lesión medular. Trasplante celular por método quirúrgico de inyección directa. Trasplante celular mediante infiltración intratecal (dentro del

canal medular) dirigida a través de sistema de nanotecnología. Colocación de materiales biodegradables para crear nichos que

puedan alojar trasplantes celulares autólogos (células madrepropias).

Estudiar el efecto combinado de terapias farmacológicas y celulares en fase crónica. 

Usar biomarcadores de inflamación, a partir de muestras de LCR, como herramienta pronostica de la degeneración de la lesiónmedular. 

Experiencia del Equipo Investigador y Medios Disponibles

El proyecto, innovador, se ubica en el Hospital Universitari Valld’Hebron (HUVH), centro terciario destacado en España. Ademásde la Unidad de Lesión Medular de dicho centro, participancinco grupos de investigación y uno de rehabilitación:

de Vall d’Hebron Institut de Recerca (1) de la Universidad de Girona (2) del Centro de investigación Príncipe Felipe de Valencia (2). de la Fundación Step by Step (1)

La selección de pacientes candidatos al estudio en fase clínica se realizará en la Unidad de LesiónMedular del HUVH, así como entre los usuarios de las instalaciones de rehabilitación especializadade la Fundación Step by Step.

Vall d’Hebron Institut de Recerca (VHIR) 

7El VHIR tiene por objeto la investigación biomédica en el ámbito del diagnóstico, la terapéutica yel  tratamiento  de  los  problemas  relacionados  con  la  salud  humana.  En  los  últimos  años  haevolucionado hasta ocupar  las mejores posiciones en Catalunya y España. Sin duda ello ha sidoposible  gracias  a  la  estrecha  relación  entre  el Hospital Universitari Vall  d’Hebron  (HUVH)  y  losinvestigadores, entre la clínica y el laboratorio y viceversa (bedtobenchtobed). La vinculación de laactividad  investigadora en el HUVH es una oportunidad singular e  inmejorable, para potenciar  latraslación  y  la  innovación  centradas  en  la  salud  de  lapoblación.  El  VHIR  tiene  en  la  actualidad  1.200  personas dedicadas  a  la  investigación,  10  áreas  con más  de300  líneas de  investigación abiertas y cerca de 700ensayos  clínicos  activos.  Potenciamos  lainvestigación  clínica  y  la  innovación  en  todos  losámbitos  de  las  ciencias  de  la  salud.  Al  mismotiempo, promovemos activamente la participación ycolaboración  con  otros  centros,  entidades  yempresas.  Durante  el  año  2010,  el  VHIR  es  la  fundación  deinvestigación asociada a hospitales que ha recibido más subvenciones del Instituto de Salud CarlosIII (ISCIII) de todo el Estado. El área con más proyectos financiados (once) es la de neurociencias.En 2011, destacar el inicio del proyecto de investigación “Relevancia fisiológica y patológica de losantagonistas de los receptores de muerte (FAIM, Lifeguard y FLIP) en el sistema nervioso” del Dr.Joan  Comella,  financiado  con  484.000€  por  el  antiguo  Ministerio  de  Ciencia  e  Innovación(MICINN). 

Violencia2% Autolisis

5%Caidas

19%

Tráfico47%

Laboral16%

Deportivo11%

Grupo de Señalización

Celular y Apoptosis - VHIR

El grupo, dirigido por el Dr. Joan X. ComellaCarnicé,posee una amplia experiencia en el estudio de losmecanismos apoptóticos en modelos neuronales. Elprincipal objetivo es caracterizar los mecanismos deacción de los antagonistas de receptores de muerte(FAIM-L, Lifeguard y FLIP) en el sistema nervioso, locual podría permitir desarrollar nuevas estrategiasterapéuticas para enfermedades del sistema nerviosomuy diversas, tales como enfermedadesneurodegenerativas, procesos tumorales, otraumatismos, en los que la apoptosis juegue un papelprimordial.

Unidad de

Lesión Medular Hospital

Universitario Vall d’Hebron

La Unidad de Lesionados Medulares (ULM) del Hospital Universitario Vall d’Hebron fue creada en1976. Desde entonces presta servicio ininterrumpidamente a un promedio de 100 pacientes conlesión medular aguda por año, siendo centro de referencia en el tratamiento de lesiones agudaspara Catalunya, Baleares, Andorra y en ocasiones zonas del sur de Francia.

En el período 1997-2010 se han atendido 1.147 pacientes, de los cuales 917 eran hombres y230 mujeres, con una estancia media de 67 días yalto grado de complejidad de lesión. Al igual que ha Las causas agregadas durante ese mismo

período se indican en el gráfico. No obstante, el perfil ha variado en los últimos años: disminución del porcentaje de accidentes de tráfico, aunque con lesiones más severas por politraumatismos, sin variación en los accidentes deportivos, aumento de los intentos de autolisis (suicidio) en las mujeres, e incremento de caídas fortuitas en personas de edad avanzada.

La Unidad está dirigida por el Dr. Miguel A. GonzálezViejo, quien posee una amplia experiencia en MedicinaFísica y Rehabilitación de las lesiones medulares. Elequipo lo componen la Dra. Lucrecia RamírezGarcerán, adscrita a la ULM desde hace más de 18 añosy experta en la atención al lesionado medular agudo, yla Dra. Lluïsa Montesinos Magraner, que se incorporó enel año 2010 a la Unidad después de su estancia en elTexas InstituteforRehabilitation de Houston (EE.UU.). Cuenta con un equipo multidisciplinarformado porpersonal médico especialista en rehabilitación delesión medular, cirugía ortopédica, medicina intensiva,psiquiatría, psicología, así como personal sanitarioespecializado en cuidados de enfermería, fisioterapia,

terapia ocupacional, trabajo social y tecnología ortopédica y del soporte de losdiferentesespecialistas del Hospital Universitari Vall d’Hebron, necesarios en momentos puntualesen la atención de estos pacientes. Dispone del equipamiento específico y de los habitáculosespecialesnecesarios para la asistencia integral durante el período agudo y subagudo.   

Su finalidad es  investigar soluciones para  la mejora dela  salud  humana,  aplicando  las  tecnologías  másavanzadas  para  desarrollar  nuevas  terapias  y/ométodos  de  diagnóstico,  e  impulsar  la  ciencia  básicabiomédica con el fin de avanzar en  la comprensión delas  bases  moleculares  de  patologías  humanas,  querequieran  nuevos  procedimientos  diagnósticos  yclínicos  para  su  identificación  y  tratamiento.  Y  enparticular, el desarrollo de  terapias avanzadas para eltratamiento de patologías humanas. 

En el campo de las células madre, la misión del CIPF esser  un  centro  europeo  de  referencia  en  terapiascelulares, potenciando la investigación interdisciplinar 

en células madre adultas y embrionarias, con el objeto de regenerar órganos dañados, ycompartir el conocimiento con investigadores y clínicos. Asimismo, ser el integrador de datos"ómicos" de referencia en Europa y computación para el modelado cuantitativo de sistemasbiológicos, potenciando las áreas de Genómica estructural, Regulación genómica y Genómicafuncional.

El CIPF desarrolla métodos (bio) químicos y genéticos para descifrar redes de señalizacióncomplejas, así como agentes terapéuticos potenciales mediante la investigación (cienciabásica). En el campo de desarrollo de nuevas terapias, el CIPF aspira a convertirse en uncentro de referencia internacional, mediante el desarrollo de terapias bio y nano-tecnológicas(nanomedicina) que permitan avanzar significativamente en los aspectos clínicos de patologíashumanas graves.

Centro de Investigación Príncipe Felipe (CIPF) 

El principal objetivo del grupo, dirigido por la Dra. Victoria Moreno es aumentar las posibilidades de éxito en el rescate de la función neuronal tras un proceso traumático,centrándose en el potencial regenerador endógeno del sistema nervioso adulto, con laparticipación de células troncales comrecientemente que el trasplante en fase aguda decélulas ependimarias con capacidad multipotente, procedente de individuos adultos que hansufrido una lesión, en un modelo de contusión en la médula espinal, restaura la funciónneurológica y la actividad locomotora perdida de forma significativa. Mejorar elconocimiento de los procesos moleculares y celulares que ocurren en una lesión del sistemanervioso contribuirá a la búsqueda de herramientas farmacológicas, que favorezcan elescenario más propicio para la regeneración in vivo, con efectos sinérgicos sobre la función neuronal perdida.

Laboratorio de Regeneración Neuronal

De manera específica, el grupo se centra en:

1. La caracterización de células troncales residentes del sistema nervioso central adulto, con especial interés en el estudio del comportamiento de células ependimarias/subependimarias, in vitro y en modelos in vivo.

2. La influencia del microambiente creado tras una lesión medular. 3. La reprogramación del potencial regenerador endógeno por manipulación genética

y/o farmacológica in vivo y ex-vivo. 4. El desarrollo de modelos experimentales de trasplante celular tras lesiones

traumáticas del sistema nervioso central, buscando además efectos sinérgicos con tratamientos farmacológicos.

Los Polímeros Terapéuticos son considerados las primeras nanomedicinas poliméricas. Su valor terapéutico en clínica ya ha sido demostrado sobre todo en cáncer, sin embargo todavía existen retos y oportunidades para mejorar esta plataforma tecnológica. Se considera que las áreas que facilitarán un mayor desarrollo son: (i) el transporte de anticancerígenos dirigidos a nuevas dianas moleculares y su combinación, (ii) el desarrollo de nuevos y complejos materiales poliméricos con estructura definida y (iii) el tratamiento de patologías diferentes al cáncer. Estas líneas de investigación son las directrices actuales del laboratorio, dirigido por la Dra. Ma. Jesús Vicent. El grupo se centra en el desarrollo de conjugados poliméricos de segunda generación, nuevas nanomedicinas con aplicación tanto en terapia anticancerígena como en medicina regenerativa.

Algunas de las líneas de investigación específicas son:

1. Portadores poliméricos con estructura definida, hidrosolubles, biodegradables y biocompatibles basados en ácido-L-glutámico.

2. Desarrollo de sistemas de transporte específico de fármacos a través de la barrera hematoencefálica (BHE).

3. Desarrollo de sistemas de transporte citosólico: terapia génica (transporte DNA, siRNA), transporte intracelular de proteínas.

4. Nanoconjugados poliméricos de fármacos de interés farmacéutico, especialmente con aplicaciones, entre otras, en medicina regenerativa de lesión medular.

5. Conjugados poliméricos con aplicación en neuropatías (sistema nervioso central (neuroinflamación, Alzheimer) y sistema nervioso periférico (FAP).

6. Desarrollo de métodos cuantitativos para el estudio del tráfico intracelular de nanofármacos, endocitosis.

Laboratorio de Polímeros Terapéuticos

Fundación Step by Step (SbS)

La  Fundación  Step  by  Step  es  una  organizaciónsin  ánimo  de  lucro  dedicada  al  lesionadomedular.  Su  Centro  de  Rehabilitación  estáespecializado  en  tratamientos  innovadores  derehabilitación  para  lesionados  medulares  y  hasido  certificado  por  elProject  Walk  de  EE.UU.,centro de  reconocimiento mundial en el  campo

del  tratamiento  de  fisioterapia  del  lesionado medular,  como  el  primer  centro  de  Europa  enaplicar este avanzado método de recuperación de lesionados medulares, implantado con éxito ensu centro pionero en esta metodología.  

SbS cuenta con un Patronato Rector, presidido por Frederic Crespo, y formado porprofesionales de la medicina e investigadores de gran prestigio, que otorgan gran credibilidadal proyecto de la fundación:

Dr. Miguel Ángel González Viejo, Jefe de la Unidad de Lesionados Medulares delHospital Universitari Vall d’Hebron, Barcelona.

Dra. MercèAvellanet, Jefe del Servicio de Rehabilitación del HospitalNostraSenyoraMeritxell, Andorra .

Dra. Anna Veiga, Directora Banco de Líneas Celulares del Centro de MedicinaRegenerativa de Barcelona (CMRB).

Dr. Thomas Graf, Coordinador del Centro de Regulación Genómica de Barcelona (CRG)

Dra. Victoria Fumadó, Responsable de la Unidad de Salud Internacional del Servicio de Pediatría del Hospital de Sant Joan de Déu , Barcelona.

Sr. Jaume López, Director de T3 -Think Tank y Asesor de varias empresas multinacionales.

Sr. Xavier Esteva, Directivo y miembro del consejo de administración de empresas del sector metalúrgico y químico.

Sr. Agustí Jausas, Fundador y Presidente Honorífico de la firma de abogados JAUSAS, Barcelona

La Fundación tiene también como uno de sus objetivos prioritarios la investigación en la curación de la lesión medular. Con esta finalidad, SbS cuenta en la actualidad con un Comité Científico liderado por miembros del Patronato de la Fundación y profesionales de altísimo prestigio profesional y con amplia experiencia investigadora, cuya función en el proyecto TIME TO MOVE es el asesoramiento, seguimiento y la validación del mismo en base a los informes y controles realizados por el propio Comité o, si se precisa, por colaboradores externos designados por él. DR. MIGUEL ÁNGEL GONZÁLEZ VIEJO Vicepresidente Licenciado en Medicina por la Universidad de Zaragoza. Doctor en Medicina por la Universidad Autónoma de Barcelona. Especialista en Rehabilitación y Medicina Física. Jefe de la Unidad de Lesionados Medulares del Hospital Universitario Vall d’Hebron de Barcelona. DRA. MERCÈ AVELLANET VILADOMAT Vicepresidenta Licenciada en Medicina y Cirugía por la Universidad de Barcelona. Especialista en Rehabilitación y Medicina Física. (Añadir web) Doctorada en Medicina y Cirugía por la Universidad Autónoma de Barcelona. European Board of Physical Medicine and Rehabilitation. Jefe del Servicio de Rehabilitación del Hospital Nostra Senyora Meritxell, Andorra. Directora Comité Científico de la Sociedad Española de Rehabilitación y Medicina Física (Añadir web) DRA. ANNA VEIGA, PHD Patrono Doctorada en Biología (Cum Laude) por la Universidad Autónoma de Barcelona. Directora Científica del Servicio de Medicina de la Reproducción del Departamento de Obstetricia y Ginecología de l’Institut Universitario Dexeus, Barcelona. Fue la responsables de la primera Fecundación “in vitro” de España. Directora del Banco de Líneas Celulares de Barcelona. Centro de Medicina Regenerativa de Barcelona CMR[B]. Parc de Recerca Biomèdica de Barcelona DR.THOMAS GRAF, PHD Patrono Doctorado en Ciencias Biológicas por la Universidad de Tuebingen (Alemania). Habilitation por la Universidad de Heidelberg (Alemania). E- director del Einstein Institute de Nueva York. ICREA. Investigador Senior y Coordinador del Centre de Regulació Genòmica de Barcelona. DR. FILIP LIM Doctorado en Bioquímica por la Universidad de Adelaida (Australia). Profesor de Microbiología en el Departamento de Biología Molecular, Universidad Autónoma de Madrid. Investigación en reparación neuronal con vectores virales y células neurales humanas. DRA. Mª TERESA MORENO-FLORES Doctora en Ciencias Biológicas por la Universidad Complutense de Madrid. Profesora en la Facultad de Ciencias Biosanitarias de la Universidad Francisco de Vitoria (Pozuelo de Alarcón, Madrid). Estudio del papel la Glía Envolvente Olfativa como mediadora de regeneración nerviosa.

resupuesto proyecto investigaciónTerapia Celular en Lesión Medular (TIME TO MOVE) 

Total financiación solicitada 

Capítulo  Año 1  Año 2 Año 3  Contratación personal investigador y técnico 

274.500,00 € 274.500,00 € 274.500,00 € 823.500,00 €

Material inventariable  102.500,00 €            0,00 €            0,00 € 102.500,00 €Material fungible  201.000,00 € 201.000,00 € 221.000,00 € 623.000,00 € Viajes y dietas        7.500,00 €    9.000,00 €    9.500,00 €   26.000,00 €Otros gastos      10.000,00 €  14.000,00 €  22.000,00 €   46.000,00 €Canon (% costes indirectos)  151.719,00 € 143.584,00 € 150.584,00 € 445.887,00 €Gestión y coordinación  100.000,00 € 100.000,00 € 100.000,00 € 300.000,00 €Subtotal   847.219,00 € 742.084,00 € 777.584,00 €  Total          2.366.887,00 € 

La Fundación SbS ha realizado más de 15.000 horas de tratamiento de Rehabilitación, atendiendo a lesionados medulares procedentes de toda España y también de otros países, como Inglaterra, Francia, Japón o Estados Unidos. En 2013 cabe destacar la organización del 2nd International SpinalCordRepair Meeting, que contó con la presencia de más de 100 asistentes especialistas y ponentes de alto prestigio internacional en el campo de la lesión medular, así como la firma de convenios de colaboración con centros de investigación acreditados. Durante la realización del Meeting fue presentado el proyecto, con gran aceptación e interés por parte de los asistentes.

Presupuesto global, principales partidas

La financiación solicitada se destinará, según el desglose siguiente, a:

“Investigamos para avanzar”

Impacto e interés social El estudio realizado en la Unidad de Lesión Medular del HUVH en 2010, calcula un coste total de 5.396.313€ para los 71 pacientes ingresados durante el año 2010. Si bien la atención en la fase aguda-subaguda ha sido de 76.004€ por paciente, aunque sólo se factura el 38% del coste real de la asistencia sanitaria.

Severity of injury First Year Each Subsequent Year

High Tetraplegia C1-C4 $ 710,275 $ 127,227

Low Tetraplegia C5- C8 $ 458,666 $ 52,114

Paraplegia $ 259,531 $ 26,410

Incomplete Motor Funcional at any Level $ 209, 324 $ 14,670

No existen datos en España del coste de la lesión medular aguda en otras unidades, ni tampoco datos del coste de la rehabilitación en nuestro país. Los datos que proporciona la NationalSpinalCordInjuryStatistical Center Database de EE.UU. para 2009 ofrecen un marco de costes inclusivo de la fase aguda y rehabilitadora, así como de la discapacidad permanente de la persona.

La discapacidad a largo plazo representa una terrible carga para las personas afectadas, las familias y la sociedad en general, y sus efectos devastadores se traducen también en un elevado coste económico. Las secuelas de la lesión medular son múltiples y deben ser tratadas de forma continuada. Una vez finaliza el proceso de hospitalización y rehabilitación el paciente retorna a su domicilio. Cuando se trata de personas mayores de 65 años resulta particularmente difícil si el cuidador principal es también mayor, o cuando carecen de cobertura en el caso de inmigrantes no regularizados. El proyecto de investigación TIME TO MOVE (Terapia Celular en Lesión Medular) diseña y evalúa un conjunto de acciones y medidas, con el fin de reducir las deficiencias causadas por la lesión medular y la discapacidad que ésta genera en la limitación para la independencia y autonomía de la persona. La demostración de la efectividad y eficacia de TERACELME aportará beneficios tanto para los pacientes, como el sistema sanitario y la sociedad en general. Los resultados que se espera obtener del presente estudio tendrán unos impactos potenciales en diversos ámbitos:

Salud y ámbito social: mejorará la funcionalidad de la persona, mediante una

minimización de la profundidad y nivel de la lesión. Los beneficios esperados son: reducir la morbilidad, mejorar la esperanza de vida, incrementar la calidad de vida, control de los factores de riesgo sobrevenidos a la lesión medular, disminución del número y gravedad de las complicaciones (disreflexia autónoma, úlceras por presión, infecciones urinarias, sepsis, etc), y promover la participación social de este colectivo.

Económico: la utilización de las terapias farmacológicas y celulares mejorará el

pronóstico y funcionalidad de la lesión medular. En consecuencia se espera una reducción de: los costes directos sanitarios (hospitalización en fase aguda, rehabilitación y atención a las complicaciones crónicas derivadas de la lesión medular), los costes directos no sanitarios (asistencia social y/o familiar) y los costes indirectos derivados de la discapacidad permanente (ayudas técnicas para los desplazamientos, modificación del entorno, ayudas para la discapacidad urinaria, cuidados generales). 

Sistema sanitario: mostrar el coste-beneficio de la atención tras la aplicación de nuevos procedimientos, que acortan la estancia hospitalaria y reducen la asistencia socio sanitaria de estos pacientes puede abrir nuevas modalidades de tratamiento en los sistemas de salud.

Capacitación profesional: se acrecentará la

investigación en lesión medular crónica, muy prevalente, y en la aguda, ambas de origen traumático. Este conocimiento podrá

Apoyar la investigación en salud es una inversión en el futuro del país y contribuye agenerar riqueza, añade valor a la atención sanitaria y crea las bases de una sociedadmejor, en la que personas que ahora son subsidiadas, puedan convertirse encontribuyentes natos.

La involucración y complicidad de todos los sectores y agentes resulta imprescindible. Lacolaboración en este proyecto significa la oportunidad para “transformar la vida de laspersonas con lesión medular”.

La discapacidad se traduce en un

elevado coste económico

Colaboración y Contraprestaciones

Se propone un estudio para desarrollar la Terapia Celular en Lesión Medular (TIME TO MOVE), partiendo del conocimiento y experiencia acumulados por el grupo de investigadores, clínicos y rehabilitadores de Vall d’Hebron, Centro de Investigación Príncipe Felipe y Fundación Step by Step, para el que se solicita la colaboración de entidades públicas y privadas, así como de particulares que estén interesados en aportar su granito de arena al avance de la investigación y la mejora de la calidad de vida del lesionado medular.

En reconocimiento de la colaboración con el proyecto TIME TO MOVE, se destacará su presencia y participación en:

‐ Todos los elementos de comunicación relacionados con el proyecto ‐ En el espacio propio de la página web del proyecto ‐ La presentación individual de los investigadores principales de cada grupo ‐ Notas de prensa y noticias generadas por los resultados del proyecto ‐ Reseña específica en la memoria del proyecto

Asimismo, se rendirán cuentas de forma periódica y regular del avance del proyecto hasta su finalización.

ANEXOS

ANEXO 1: FICHA TÉCNICA

PROJECT TITLE: TRANSPLANTATION FOR SPINAL CORD INJURY REPAIR STRUCTURED ABSTRACT  Background/Main objective: Currently, there are no effective therapies to reverse spinal cord injury (SCI)-induced paralysis. The difficulty results in that SCI is not a simple disorder. The trauma causes segmental loss of all types of required cells including interneurons, motorneurons, oligondendrocytes and neural precursors which implies restricted therapeutic options. The inflammatory influx and its related products, micro-astro-glia activation and the fast and progressive apoptosis with inevitable axonal damage and the subsequent tissue degeneration and the formed reactive scar limits the potential re-growing axons. Considering all these damage cascades, transplantation of spinal cord derived and functional compatible ependymal cells result mandatory for tissue repair. However, and based on the multifaceted lesion that occurs, a combinatory therapeutic approach is needed. To solve this complex pathological scenario a mandatory synergistic formula by combining the expertise from at least three groups is proposed, including investigators, clinicians, and the patients. This proposal is focus on cell replacement, neuroprotection and axonal growth promotion to favor re-establishment of ascending/descending tracts and local synapses in an experimental model. For a translational approach, a human ependymal cell bank in GMP conditions will be generated. Methodology: Intramedullar and intrathecal ependymal spinal cord-derived progenitor cells transplantation will be performed in acute, subacute and chronic phases after SCI. In chronic phase cell transplantations will be implemented with biomaterials (scaffolds or hydrogels). Intrathecal administration of newly design nano-conjugates to block inflammation and apoptotic process and to reinforce axonal regrowth. Expected results: We expect to find a combinatory formula (cell transplantation + nanoconjugates associated treatment) for functional neuronal rescue after SCI. We expect to find better efficiency on acute and sub-acute transplantation approaches. Nevertheless we expect to induce axonal re-growth and partial rescue of the neuronal function even in chronic phases, in this case including biomaterials in the strategy. We expect to developed a functional transplantation strategy and new nano-conjugates with potential clinical value in SCI.

BACKGROUND AND STATE OF THE ART, INCLUDING RELEVANT BIBLIOGRAPHY Considerable research has been performed in the last 20 years using experimental models to detail the spinal cord injury (SCI) process and to apply potential therapeutic tools, in many cases with therapeutic success. Then, why the stabilized paraplegia results irreversible? A big gap is still on the need of connections between the experimental data and the clinical daily practice. It is time to give to the patients the potential option to take advantage of the work done in the lab. This is an ambitious proposal that strongly wants to move from the bench to the bedside. Let´s start with the numbers: only in Spain, more than a thousand people suffer a traumatic spinal cord injury EVERY year that is about 3 people for every medium sized city. These are cumulative numbers with so far more than 40 thousand patients with the need of daily health care assistance. In Europe, the statistics talk about 330.000 people affected by any SCI with 11.000 new cases every year. It is time to break the statistics. To do so, we have to do the global effort to collect the valorous data produced mainly by the researcher in experimental models and put it together with the necessities on the clinic. But, what is a Spinal Cord Injury? The SCI is not a simple disorder. Whether you are a person living with SCI or a scientist studying SCI, there is nothing simple about what you are faced with every day. The spinal cord is a very complex organ, after all it, in combination with the brain, controls every single aspect of our body. Hence, when the spinal cord sustains damage people lose more than just the ability to walk. Aside from the loss of movement and sensation, almost every single SCI results in the loss of bladder and bowel control. Depending on where in the spinal cord the injury occurs, there may be additional impairments in sexual function, loss of the ability to regulate body temperature and blood pressure reduced breathing and coughing capacity, and inefficient metabolism of food. Is here mandatory to review the physio-pathological events that concatenate after spinal cord injury and drives into paralysis. The SCI pathological process can be described into two parts: the primary and secondary injury phases (reviewed in 1,2). Normally the primary injury phase is due to either contusion (caused by shattered vertebral bones) or compression (caused by an increased pressure to the spinal cord). The cervical and lumbar spine is commonly the most affected areas of SCI. Damage to upper motoneurons results in hyperreflexia, hypertonia and muscle weakness. In contrast, the insults to lower motoneurons cause hypotonia, hyporeflexia and muscle atrophy. From seconds to minutes after the trauma an immediate massive necrotic cell death occur releasing potassium ions and protons altering the ion balance and creating an acid environment. Lipid peroxidation, and glutamate release are additional products of dead cells3. SCI also lead to acute local ischemia, after a vascular net loss with edema and disruption of the blood-spinal cord barrier (BSCB) which also contribute to secondary degeneration4. The platelet aggregation/coagulation cascade and the adhesion and vasospasm by activation of haemostatic mechanism decrease in fact the oxygen and glucose availability5. Following the initial trauma, on the next hours through next weeks, the secondary injury phase can be described as complex damage that occurs at the cellular level. This phase can be broken down into several pathological events: 1) activation of microglia6: specialized phagocytes but also very reactive cells, releasing reactive oxygen species, prostaglandins, thromboxanes, cytokines and chemokines, which acts as attractant signals of the inflammatory response; 2) Influx of monocytes/macrophages, neutrophyls and lymphocytes to clean up the cell death debris and 3) continues release of inflammatory-related products7. The reactive oxygen species damage the surrounding cells and the cytokines re-actives the microglia and the remote astrocytes. The chemokines attract again more inflammatory cells into the lesion. 4) Apoptosis8: the high levels of glutamate and the released products of inflammatory cells induce fast and progressive apoptosis into oligodendrocytes, astrocytes, microglia and precursor cells consequently with an axonal damage. Cytokines and inflammatory mediators like IL1�, IL-1�, IL-6 or TNF-�� increase within 6 to 12 hours after lesion. IL-6 and its related family (some member are Leukemia inhibitory factor, LIF and ciliary neurotrophic factor (CNTF) are also known to induce and stimulates neural stem cell differentiation in vivo by activating JAK/STAT pathway in the acute phase after injury9. However it also stimulates astroglia with induction of proteoglycans which as we mention block axonal growth10. Fas and FasLigand bellows to TNF-alpha superfamily and mediates induction of apoptosis11. The expression of Fas is noted within the first 72 hours after injury12. NFkappaB mediates at the transcriptional level an important part of the apoptotic-

induced stimulus with, for instance inflammatory component13. In the other hand, lipid rafts disruption results in neural protection against exocitotoxic-induced apoptosis14. Proteases like tissue plasmigen activator, better known as a thrombolytic agent, is involved in the myelin sheaths breakdown in a multiple activation of the matrix metalloproteinase (MMP) cascade15. Nowadays, MMP-2 has been shown to promote functional recovery after SCI by remodeling-controlling the extension of the glial scar16. In the chronic phase, since weeks to months after the injury, the most evident process is the demyelization of the axonal tracts by oligodendrocyte apoptosis death. Consequently, the axons are weaker to the new lymphocytes attack. The additional neural death and axonal degeneration with loss of neuronal circuits is the base of the Wallerian degeneration1,17. Later on, since months to years, a reactive scar is formed by activation of pedycites and astrocytes to isolate the injured area, forming an impenetrable wall with extracellular matrix proteins and chondroitin sulphate18. The cavities conformed by the glial scar result in siringomyelia. Importantly, the products produce by and the surrounding scar, proteoglycans and/or the products of demielinization process (NOGO, MAG, OMPG) block the axonal growth and collapse the growth cones killing the regenerative potential of those axons enable to reconnect with the distal segments19. A synergic platform of therapeutic strategies for efficient translational treatment of SCI. Going beyond the State-of-the-Art. Considering all these damage cascades, any potential method for functional restoration must incorporate all, but first (1) tissue or specific cell replacement/transplantation, and also (2) neuroprotection and (3) axonal growth promotion strategies for the remaining alive tissue with re-establishment of ascending/descending tracts and local synapses. In the last decade notable progress in SCI restoration has been achieved in multiple animal models, and some of the experimental approaches are currently in clinical trials, mainly for acute or sub-acute stages of SCI. However, there are currently no studies which cover all three criteria. The groundbreaking nature of this proposal is based on the combinatory therapy with innovative design of pharmacological and cell-replacement approaches in a bio-/nano-technological novel context.The present grant with its synergistic characteristics will provide us a great opportunity and will allow us to efficiently work from the individual approaches to progressively and efficiently combine experience in the experimental model to further move into the clinic. In fact, the selected bio/nano-conjugates and a human adult spinal cord-related stem-cell bank, generated in GMP conditions will be further translate the experimental approaches into the clinical practice and to move forward from the bench to the bedside. TRANSPLANTATION/Cell replacement therapy: The post-traumatic environment has been previously shown to cause apoptosis and damage to the surrounding functional neurons, consequently a very important loss of cells limits the neuronal function. Any potential method for functional restoration must incorporate both cell substitution and neuroprotective strategies for the remaining alive tissue with re-establishment of ascending/descending tracts and local synapses. Cell-restitution based therapies constitute then a rational approach. A diverse cell types on tissue transplantation had shown sustained success on experimental models. The differentiation stage classify in to two the source of already tested cells: 1) mature cells like olfactory ensheathing cells (OEG)20, Schwann cells (SC)21 and 2) non-completely differentiated cells (stem cells): neural precursor cells from adult neuronal tissue22 or directed-differenciated from embryonic stem cells23 o inducible pluripotent stem cells24, oligodendrocyte precursor cells25 or mesenchymal stem cells (MC)26. These transplanted cells modulate the inflammatory response27 and proteoglycan expression by reactive astrocytes, and promote axonal regeneration and functional recovery after a complete spinal cord injury 28, including when are transplanted at 45 days postinjury29. Stem cell biology is a rapidly growing field including in SCI repair. The stem cell transplantation for cell replacement is a promising strategy to bridge the lesion site either in the acute an more even in the chronic phases. The implanted cells create an environment in which remyelination, axon elongation, and formation of new circuits may occur as it has been probed in rodents. The recruitment of endogenous or transplantation of neuronal precursors with limited multipotenciality but with “neuronal memory” is getting more adepts in the experimental side. Adult neural precursor cells (NPC) can be easily propagated in culture and efficiently renders both oligodendrocytes and motoneurons in vitro30. Rather than the embryonic or pluripotent cells NPC have showed less tumorigenic complications. The subventricular zone, hypocampus or the rostral migratory stream are known sources of NPC in

the adult brain31. In the spinal cord lining the central canal there also are stem cells, ependymal cells (epSPC) which show multipotency. These cells reside in the adult spinal cord almost quiescent but after SCI the epSPC proliferate and are recruited by the injured zone, being modulated by innate and adaptive immune responses32. In fact, transplantation of epSPC “activated” after injury rescue loss motoneuronal function in the rat after traumatic SCI33. Now we know that after SCI, the epSPC proliferate 10 times faster in vitro than those ones derived from non-injured donors and display enhanced self renewal properties. Genetic profile analysis revealed an important influence of inflammation on signaling pathways in epSPC after injury, including the upregulation of the Jak/Stat and mitogen activated protein kinase pathways. Even do, the activated epSPC by the lesion differentiates efficiently to oligodendrocites and functional spinal motoneurons showing better yield than before activation. The transplanted cells migrate long distances from the rostral and caudal regions to reach the neurofilament-labeled axons in and around the lesion zone. The epSPC transplanted animals always shown fewer cavities and smaller scar area33. The stronger hypothesis on the regenerative potential of the epSPC is focus on its neuroprotective and neuro-immunomodulation effect. The central canal is extending from the third ventricle through the spinal cord and finishing at the filum terminale, almost composes by epSPC. The cells isolated from this structure (including in humans) shows multilineage differentiation capacity in vitro 34, showing an ideal source for ependymal cells isolation in allo- or auto-transplants35 for human approaches. Small compounds, i.e favoring the stemnes stage for amplification either the endogenous or the transplanted precursor population will conforms a good coadjutant for epSPC strategies36. In the other hand, in vivo modulation of neuronal differentiation programs will allow the precursor cells to reconstruct the neuronal loss network. Neuroprotection and axonal growth protection: The are interesting studies done with non-classical anti-inflammatory treatments like the compound FK 506, which administration protects the damaged spinal cord37. The experimentation with drugs already in the clinical use, for instance, in the case of valproic acid (VPA), prescribed for a different purpose, but working in SCI open the avenue for its therapeutical use in SCI. VPA administration partially prevent cord tissue, myelin and axonal loss, preserve the survival rates of oligodendrocytes in the damaged spinal cord38. After initial insult, astrocytes, microglia, macrophages, migrated fibroblasts and Schwann cells actively produce chemokines and cytokines such as TNF-α and IL-1β, which in turn, mediate the recruitment of inflammatory cells to the injury site. TNF- α and its cascade represents a good strategy to reduce the secondary damage. The used strategies include antibodies, soluble receptors, recombinant TNF-binding proteins, TNF receptor fusion proteins, and non-specific agents like thalidomide (reviewed in 39). Recently, FLIP, Lifeguard and FAIM-L have been described to have a potential role in neurite growth, neurogenesis and neuronal differentiation together with its antiapoptotic role11,40, opening the door for new strategies to target programmed cell death through inhibition of apoptotic events in a neural-specific fashion preserving then the neuronal activity. The trauma disrupts the blood-spinal cord barrier (BSCB) and the microvascular system changes lead to reduction of blood supply, it is actually a hallmark of spinal cord secondary injury causing further deterioration of the lesion area with edema and ischemic-related cell death. Loss of BSCB makes more difficult the administration of any drug. Although the cord is encrypted into the vertebrates segments, the space were the CSF flow –subarachnoid space- serve as a direct local administration space of bioactive conjugates41. The nano-size delivery systems will allow us to play with a controlled release of the drugs and to prolong stability after release -due to the shielding properties of the polymer carrier-42. Innovative alternatives to the canonical pharmacological approaches: Nano-sized drug delivery systems: Advances in polymer chemistry and nanoscience over the last decade are providing a new basis for the development of innovative delivery and imaging techniques with significant potential to bring benefits to patients and open new markets to pharmaceutical industry43,44. Polymer therapeutics are amongst the most successful nanomedicines43. Polymer Therapeutics comprises a variety of complex macromolecular systems, whose common feature is the presence of a rationally designed covalent chemical bond between a water-soluble polymeric carrier (with or without inherent activity) and the bioactive molecule(s). Drug conjugation to a polymer not only enhances its aqueous solubility but also changes drug pharmacokinetics at the whole organism level and even at cellular level with the possibility to clearly enhance drug therapeutic value. This family can be subdivided in five general categories: polymeric drugs, polymer-protein

conjugates, polyplexes, polymeric micelles and polymer-drug conjugates. They are considered as ‘new chemical entities’ (NCEs) rather than conventional drug-delivery systems or formulations that simply entrap, solubilise or control drug release without resorting to chemical conjugation45. Due to their intrinsic characteristics at the nanoscale (conjugate size < 25 nm, potential for spatially controlled multifunctionality and architecture, and presence of bioresponsive elements), this class of nanopharmaceuticals can be carefully engineered to exhibit unique advantages (i) they are able to get to places that other larger 'nanocarriers' cannot reach, (ii) they are more able to cross biological barriers and can display architecture specific intracellular trafficking and (ii) they allow a greater control on drug pharmacokinetics due to the use of bioresponsive chemical conjugation. Translational research in polymer therapeutics has already transferred 13 products into the market. These include those polymeric drugs46, polymer-protein47 and polymer-aptamer48 conjugates currently in routine clinical use. PEGylated proteins, antibodies and most recently aptamers have been among the most successful. Improvement on PEG chemistry and protein/peptide conjugation strategies, including sophisticated enzyme-mediated as well as recombinant techniques, has importantly contributed to this success49. Most polymer-drug conjugates in the clinics use N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers, PEG or more recently polyglutamic acid (PGA) as carriers50. Biopersistent carriers (PEG, HPMA) present disadvantages if chronic parenteral administration and/or high doses are required as there is the potential to generate 'lysosomal storage disease' syndrome. Preclinical evidence of intracellular vacuolation with certain PEG-protein conjugates51 is raising awareness of the potential advantage of biodegradable polymers regarding potential safety benefit apart from the possibility to use higher molecular weight carriers allowing pharmacokinetic optimisation52. The potential benefits of controlled polymer chemistry (including architecture) have also emerging with new insights into the effect of shape and deformability on biodistribution and cell trafficking52. For instance, an enhanced rate of transfer across biological barriers has been seen for dendrimers and hybrid dendritic architectures53. Due to molecular complexity of human pathologies multiple drugs in combination are often administered simultaneously to hit different pharmacological targets and thus improve efficacy and decrease resistance. This is the rationale behind the so called polymer-based combination therapy. Polymer-drug conjugates are excellent tools for developing this interesting concept. Four main types of systems have been included under the term “Polymer-drug conjugates for combination therapy”, which includes: polymer-drug conjugates + free drug (type 1), polymer-drug conjugate + polymer-drug conjugate (type 2), single polymer carrying a combination of drugs (type 3) and, polymer-directed enzyme prodrug therapy (PDEPT, type 4). A growing number of polymer conjugates are already being used/tested clinically in combination with drugs or radiotherapy, such as, the combination of OpaxioTM (PGA-paclitaxel conjugate) with platinates or radiotherapy (in phase II-III) for non-small cell lung cancer (NSCLC) and ovarian cancer patients49. Using one polymer to carry multiple drugs (type 3) is an even more exciting prospect supported by first clinical data coming from a liposomal anticancer approach in Phase II trials Celator Technologies Inc54. Polymer conjugates have the added advantage of release rate control that can be tailored for each drug carried, moreover can guarantee simultaneous delivery of both drugs to the same site of action that can enable synergism. With first clinical proof of concept for polymer therapeutics there is a fantastic opportunity to apply the concept in new therapeutic areas of clinical importance such as Spinal Cord Injury50. Synthetic biomaterial, scaffolds and matrix for neuronal-network sustaining: a three dimensional polymeric scaffold for placement into a spinal cord after injury has been the interest of a handful of groups during the last decade. Many experts agree that the greatest hope for treatment of spinal cord injuries will involve a combinatorial approach that integrates biomaterial scaffolds, cell transplantation, and molecule delivery. To enhance the regenerative capacity of these two scaffold types, researchers are focusing on optimizing the mechanical properties, cell-adhesively, biodegradability, electrical activity, and topography of synthetic and natural materials developing mechanisms to use these scaffolds to deliver cells and molecules. The scaffold biocompatibility within the spinal cord environment has been already probed (reviewed in55). Scaffold permeability allows access to various molecular sizes, and the access to oxygen and nutrients is crucial as well as the removal of metabolic wastes. Stiffness, permeability, swelling, strength and degradation will be specific to a particular polymer, then, the optimal conditions for axonal regeneration in every context have to be tested. Due to the

highly debilitating nature of SCI and the loss of functional cells, this pathology has provided inspiration for the design of new strategies providing a combination of approaches for better cell survival, integration, differentiation and function are expected in the combinational use of functional cells and biodegradable biomaterials. These biomaterials include CLMA, chitosan, hyaluronic acid and poly(ethyl-acrylic acid) that have been previously reported to show no any glial reaction in vivo. Pororusly scaffolds, hydrogels, even micro-channels for axon guidance.. the possibilities on structural design are open to many application, with a hifh future in transplantantion approaches. Chitosan is a polysaccharide of natural origin, with biocompatible and biodegradable properties. Hyaluronic acid is another polysaccharide with a many physical and chemical possibilities. It is the unique non-sulfated glycosaminoglycan present in most tissues. Recently Cho et al, have shown a potent neuroprotective role of hyaluronic acid in rodents throughfunctional locomotor recovery after in vivo implantation after SCI56. The poly(ethyl-acrylic acid) is a stable synthetic material and has been used for different purposes in biomedicine. Overall, a three dimensional polymeric scaffolds (biodegradable on time)57 would allow to bridge and in terms substitute the repulsive environment for axonal growth and reconnection58 more importantly in the chronic stage of the SCI. Following these indications, herein is aimed to develop novel technology platforms with controlled architecture and high versatility that allow to obtain polymer conjugates and hybrid polymeric systems with high therapeutic value for the treatment of acute and chronic SCI.

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PRESENTATION OF THE HYPOTHESIS AND DESCRIPTION OF THE PROJECT OBJECTIVES Here we propose an ambitious project, well coordinated in its execution through a fruitful exchange between all the experts from the clinic (clinicians and patients) and from academia in SCI therapeutics. The proposed strategy in the consortium is a convergent approach from individual efforts focusing on axonal regeneration and/or neural/glial protection, neuro-immunomodulation by applying tissue specific cell transplantation approachs in order to identify those combinations that offer the best clinical benefits for SCI patients. First, we will discuss individual approaches and then deal with combinatory strategies. Next, we will cover translational approaches and how to combine classical ideas with innovative bio-/nano technology. To do so, we will work in basic science with animal models within the already known mechanisms involved in SCI as well the newly proposed mechanism of action, making use of a complex pharmacological arsenal and cell-based transplantation therapy. Novel nano-sized drug delivery technologies, in particular Advanced Polymer Therapeutics, will be developed to improve the activity of the desired pro-regenerative drugs. Mainly in the chronic scenario, an additional support for transplant engraftment is given by diverse biomaterial structures with modulated properties for releasing molecules and favouring axon growth. Similarly, the acute and chronic phase of SCI should be dealt with separately. For the first time, here we will describe -in detail- neural degeneration along the lesion after a traumatic injury in voluntary patients. A multimodal convergent study will be done correlating the neurophysiological activity and progress associated also to the neural degeneration, with the active rehabilitation treatment in the explored acute/sub-acute and chronic patients. This grant will provide us, a synergistic group of experts in basic research and clinical treatment of SCI and will offer a great opportunity to move forward from the bench to the bedside. Herein, clinicians and researchers propose to conduct the basic science into potential and efficient treatment/s in the more translational way for the treatment of SCI no associated with adverse effects that would regret the benefits versus any incompatibility. To develop such a complex recipe a highly complementary and therefore synergistic consortium as the one proposed here is required, which will approach the proposed ambitious goal through 4 main objectives each covering a complex Work Package (WP) to be developed up to advanced pre-clinical phase (left), in experimental models and in a clinical study (right), in both acute and chronic stages, summarized in the scheme and break down below: (The arrows indicates the synergy among the WP, first between the pre-clinical (in rodents) objectives and data and then into the clinical studies)

 

  

                

   METHODOLOGY Spinal cord contusion and cell transplantation: Surgery procedure, female Sprague Dawley rats (~200g) will be pre-medicated with morphine (2.5mg/kg) and Baytril (enrofloxacine, 5mg/kg, Bayer, Germany) and subsequently anesthetized with isofluorane. A laminectomy at the T8-T9 level will be performed in all the groups. The contused animals, at this level of the laminectomy, will be contused by applying 250 kdyn using the "Infinitive Horizon Impactor" (IHI, Precision Systems, Kentucky, IL, USA). Records out of range on the impact magnitudes and tissue displacement will be used for discarding individuals out of the analysis. The precursor cells for the transplanted groups will be obtained from homozygote transgenic rats (SD-eGFP+/+ (SD-Tg(GFP)2BalRrrc), with constitutive expression of eGFP 69 and previously “activated” by a moderate lesion (150 kdy impact) one week before spinal cord dissection. The transplanted groups by intramedullar injections, half a million cells will be implanted by using a stereotaxic flanking the lesion area, 2 mm rostral and caudal to the lesion in 4 positions, from anterior to posterior. For intrathecal administration the minimal volume containing a million cells will be applied using the nanolitter device through a catheterization (see bellow). The applied post-surgery care of injured rats (fluidotherapy, antibiotherapy, analgesia) and manually drain of the bladder, three times a day, until autonomous functioning will be restored. All groups will be subjected to rehabilitation procedures consisting initially of passive mobilization through a full range of movement to maintain joint flexibility and reflexes in the hind limbs for 15 min every day. Subsequently, animals will be subjected to active rehabilitation by forcing limb movement on a treadmill at constant speed (8-15 cm/sec), plus five minutes of exercise in a warm swimming pool. Intrathecal administration: First, anatomical identifcation of the structure tuberosidad isquiatica, between the L6 and the first sacral segment (S1), could be notice because its posses a higher intervertebrate space of the lumbar part. The incision of around 1cm length will allow us to visualized the muscular fascia. Then, after disection of the fascia and the muscles that covers the L6 and S1 a partial laminectomy of L6 should be done (for at least 3mm open space). We measure the distance from the open access in L6 to the lessioned area at T8 to cut the catheter. 22G needle should be used to open the duramadre to introduce the cathether (previously filled with a 0.9% of a saline solution). Slowly and smoothly we move in the cathether into the lesioned segments (there is a tale reflex movement), and saw it by a simple point to set the end side of the catheter to the fascia to avoid any furher movement and slipping out of the intrathecal space. Then we procced to close and to saw the muscles. Then, we fix the apropiate osmotic pump (Alzet Corp. Germany; previously filled and incubated overnigh at 37ºC in a saline solution) into the catheter and then located into the next subcutaneos space. In case of puntual daily infussion using nanoliter device, an empty pump will be used to keep a closer circuit but allowing to access the end of the catheter to fix it into the nanoliter Hamilton syringe. Behavioural tests for functional recovery analysis: Basso, Beattie and Bresnahan scale, prior to surgery, the animals will be trained for 2 weeks and baseline measurements will be recorded. During the first 14 days alternating recording for fast recovery will be monitories and later in weekly intervals for up to 60 days after surgery. Open-field locomotion will be evaluated by using the 21-point Basso, Beattie and Brenham (BBB) locomotion scale. In each testing session, the animals will be individually videotaped for 4 min for scored it blind by two unbiased observers. Narrow beam crossing test, animals will be forced to walk on a 1 m-long horizontal metal beam elevated 30 cm from the

ground. Analysis will be performed by identifying the animals that will be able to walk through the beam. Foot print analysis, this analysis will be done using CatWalk® (Noldus, Leesburg, VA ) video-based system for automated gait analysis. Paw contact will be quantified by counting high-intensity pixels as the mean of at least three rounds per analysis. Electrophysiology measurements in vivo: The motor potentials will be evoked and recorded according to a prior study 70. Instead and standard cranial screw implantation a needle electrode will be used. According to the anaesthetics study of Oria et al 71, intravenous administration as a bolus dose of 10 mg/kg of propofol will be used. For the recording of evoked potential [MEP and compound motor action potential (CMAP)] one needle electrode will be placed in the tibialis anterior muscle (cathode) and one needle electrode subcutaneously at the foot pad level (anode). For the induction of CMAP following peripheral nerve stimulation, one electrode will be placed in the muscle (cathode) and another subcutaneously (anode), both near the sciatic nerve. For the induction of MEP (after central stimulation) one needle electrode will be placed subcutaneously at the level of the lower jaw (anode) and a needle electrode (cranial level) will be for the cathode. For ground, an electrode will be placed subcutaneously in the lumbar region. The electrophysiological recordings will be performed with an electromyographer (Medtronic Keypoint Portable, Denmark) or similar (to be acquired). The recordings will started by measuring the maximum amplitude of the CMAP. It will be achieved by stimulating the sciatic nerve with a single pulse of supramaximal intensity. In order to induce MEP, a stimulation of 25 mA intensity will be applied at the needle electrode (cranial level). Spinal cord tissue isolation and spinal cord stem/progenitor cell culture: Neural precursors from spinal cord will harvested from female Sprague Dawley adult transgenic SD-eGFP+/+ rats (~200 g) for cell-transplantation experiments, and SD-eGFP-/- will be used for in vitro assays, 1 week after moderate contusion lesion at T8-9. Once the overlying meninges and blood vessels are removed, the dissected tissue can be cut into 1 mm3 pieces and homogenized without enzymatic treatment. Neural precursors will be cultured as neurospheres-forming cells as previously shown 20. For human ependymal cell bank, NCAM-1 antibody bind to magenetic beads to isolate the neural precursors will be performed to avoid cell culture in non-fre animal condicitons. In parallel, a protocol for clinical application will be set up. The tissue will be collected under steril conditions from the organ donors in the surgery aand then the cell isolation and freexing process will be perfomed in the GMP facilities. Morphological and Immunostaining analysis: Two months after surgery, the animals will be transcardially perfused with a 0.9% saline solution followed by 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS), and 2 days incubation time on 30% sucrose before Tissue-Teck® OCT blocks (Miles, Elkhart, IN, USA) preparation. Sagittal and coronal 20 µm criosections will be performed for immunoassays. Every seventh section was collected for phosphotungstic acid haematoxylin (PAH) staining to determine the cavitation/cystic area. The area of cyst/cavities (pixels) will be quantified from coronal sections using the Metamorph Offline software (Molecular Devices, USA). PAS staining will be performed for infiltration detection. At least 2 animals per group will be intra-peritoneal injected with BrdU (50mg/kg body weight/day) during 1 week after injury to trace cell division. Tissue sections will be fixed with 4% PFA at room temperature for 10 min, permeabilized with 0.1% Triton X-100 and subsequently blocked with 1% fetal bovine serum in PBS before primary antibodies (for all the mentioned targets in the objectives) overnight incubation. Signals will be visualized by Confocal Microscopy (Leica, Wetzlar, Germany). Total RNA isolation and Real Time qPCR: Total RNA from all purified cells will be extracted with miRNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s

instructions. RNA purity will be examined by spectrophotometric determination at 260/280 and 260/230 nm and under electrophoretic analysis. Briefly, 10 ng of total RNA from each sample will be reverse-transcribed to cDNA using TaqMan® RNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) and the selected genes primers should be first design. The validation experiments will be carried out in at least three different replicates. A no-template control will be used as a negative control. Critical threshold (CT) values will be determined using ABI Prism 7000 SDS Software (Applied Biosystems). The relative quantity of each gene will be related to GAPDH expression and will be described using 2−ΔCT, where ΔCT=(CT gene-CT GAPDH). Biomaterials generation: The Biomaterials will be produced by the Biomaterial Lab leaded by Dr Monleon in The Polytechnic University (Valencia) and in the LEPAMAP Lab, leaded by J.A. Méndez in the Polytechnic University (Gerona). Bi-dimensional copolymeric networks of synthetic materials (PEA and CLMA) will be produce by radical copolymerization reactions. Chitosan and HA films will be develop using the solvent casting evaporation technique. In order to fabricate the three-dimensional porous structures (scaffolds) from synthetic and natural polymers we will use porogen leaching technique and an innovative method that combines freeze gelation/extraction and porogen leaching techniques respectively. Filaments of different diameters will be use as porogen to create uni-dimensional channels. Nano-sized drug delivery systems: It is proposed to use combination therapy33 in two different approaches: (i) the development of hybrid systems (scaffold(hidrogel)-polymer conjugate) that would provide a local controlled release of the selected bioactive agent with a prolong stability after release due to the shielding properties of the polymer carrier. This concept also includes an innovative strategy named Polymer-masking-UnMasking- Protein Therapy (PUMPT)33(ii) The combination of more than one bioactive agent either in the same polymer carrier34, 35 or in rationally designed conjugates in combination always looking for synergistic effects (combining several of these effects: neuroprotective, anti-inflammatory, antiapoptotic or neuroregenerative). WORK PLAN, BROKEN DOWN BY TASK AND INVESTIGATORS, AND TIMETABLE This project has been design to favours the maximal synergy among the three groups, with active participation of all groups, in all the four main objectives, nevertheless very group will be in charge of specific working packages (WP). The groups will be indicated as: PI-1_Coordinator (cell therapy and transplantation, experts on SCI experimental models); PI-2 (nanomedicine expert group) and PI-3 (complementary group of clinicians and researchers, experts on human SCI and the inflammatory related signals, and the patients). The physical interactions in this consortium will progressively grow. The investigators will integrate their strategies based on the clinical necessities. Frequent visits between the three groups will be organized to communicate results and for further future experimental design (specific synergies between groups for every specific aim are break-down below). WP1 and WP2_SCI characterization, in rodents and in humans for a specific pharmacological approach: the inflammatory process after lesion in the spinal cord constitutes a physiological mechanism for tissue repair. However, the temporal extension and the self-amplification of this process along the time transform it in an aggressive phenomenon more pathologic than actually the primary lesion produced by the traumatism. PI-3 and PI-1 groups will coordinately study the spatial-temporal changes on expression of those known ligands, receptors and co-receptors related to intracellular signaling pathways involved in the inflammatory response and its consequence on the cell death after SCI. The analysis of this targets, on a contusion spinal cord injury model, in

rodents, at 1, 3, 6, 12, 18 hours and 1, 3, 7, 10, 14, 21 and 28 days after injury will give us information about the proper timing to re-define the pharmacological strategies to be apply focus on this pathways or even to disclose new ones. Characterization and quantification of the motoneurons and oligodendrocyte cell death grade. No data has been yet described in this respect. Exhaustive histological analysis of HB9 (motoneurons) and Olig1/2/RIP (oligodendrocytes) will be performed at all time points (14, 21, 30, 45, 60, 90, 120 and 150 days after SCI). WP2. To modulate and/or inhibit inflammation and cell death: PI-3 in close relation with PI-2 will be mainly in charge of this one objective. This objective will allow us to discern onto the best pharmacological treatments with mechanisms related to the inflammation and cell death. The MMP inhibitors for instance will improve vessels stabilization diminishing in this way the extravasations of inflammatory cells into the injured cord. Nowadays it will be potentially improve our knowledge on the intracellular signaling closer related to the protectoral face of those anti-inflammatory and/or anti-cell death applied drugs. WP1 & 2 will also have the aim to characterize the temporal window of the proper pharmacological application in order to do not completely block but diminished the inflammatory process associated to the SCI. The clinical studies in concordance with the experimental ones show a profile defined by two picks of maximum inflammatory activity, first 1-3 days after acute lesion and then 7-10 days later. The first influx plays a more reparative role, mainly cleaning the cellular debris and the later ones results more aggressive. In this context the application of the previously described pharmacological arsenal along the first 10 days after lesion will try to address this question. WP3 SCI-cell based treatments in SCI Transplantation: PI-1 will be in charge of this WP. The central concept of the use of neural precursor cells from the adult spinal cord, so called ependymal stem progenitor cells (epSPC), is to take advantage of the intrinsic information of this resident and specialized type of cells coming from the same microenvironment in which they will be hosted. In order to find the most accurate transplantation strategy, different approaches will be performed. It is very important for the clinical application of cell transplantation to take into account how is the real scenario when a patient arrives to the hospital after SCI. Acute methylprednisolone administration is included into the immediate protocol procedure62. Within this protocol it is not possible to include any additional therapeutic strategy earlier than 72 hours (since the patient has arrived to the hospital). Moreover once the spinal cord has been externally stabilized a new laminectomy in the injured area earlier than 8-9 months after stabilization is too risky. Therefore, we will set up alternatives for the transplantation procedures done so far in the experimental model (by intramedullar injections) to imply new possibilities to the diverse clinical scenarios; Allosteric epSPC will be transplanted into the intramedullar tissue by controlled microinjection immediately after lesion or 1 day or 3 days o 5 days or 7 days after injury (from acute to sub-acute stages) and one, two or four months after lesion (for chronic phases). In this way we would evaluate the influence of the inflammatory response on the efficiency of the transplantation, mainly focus on the better survival of the hosted cells. In these groups, the influence of both, the inflammation and the transplanted cells into the endogenous cell survival will be also quantified; Allosteric intrathecal transplantation of epSPC will be performed at the same time points. Lumbar access with single apophysis laminectomy will allow us to approach the catheter into the injured zone (thoracic segments T8-9); Autologous and allosteric intrathecal transplantation of epSPC will be performed 7 days after lesion and filum terminale dissection. Functional neurological examination will be done to analyze and the impact of filum terminale dissection. In vitro screening platform of drugs for improve epSPC “activation”. We have been shown the importance of “activation” of the epSPC population in vivo, conferring to those a more efficient regenerative property. In order to improve the “endogenous-activation” of the neural precursors either transplanted or the remaining endogenous

ones, a battery of selected targets will be analyzing fist in vitro and then in vivo, alone or in combination with epSPC transplanted cells after SCI: Fasudil, Rhock inhibitor, FM19G11, rapamycin, Rolypram, AMPc, FGF, EGF, GDNF, BDNF, NT3. For this purpose the epSPC in culture will be transformed with Stat3/5 promoter enhancer controlled by a luciferase reporter gene system. WP4. New Therapeutic approaches for clinical applications: Development of new nanoconjugates for controlled and sustained drug delivery in the SCI in acute phase. PI-2 will be in charge to develop this WP. Due to the complex molecular mechanisms of SCI in order to achieve an efficient therapy, the use of combinatory therapy capable to modulate simultaneously various signaling pathways is required. Combination therapy is already fully established in the clinics for the treatment of diseases such as cancer. However, combination therapy by means of drug delivery systems is just now starting to demonstrate its therapeutic benefit. Only a Canadian company, Celator Technologies Inc., is currently developing a systemic methodology to design liposomal nanosystems, currently in Phase II, for the treatment of cancer (http://www.celatortechnologies.com). The property of multivalency provided by the use of polymeric carriers allows the conjugation of several bioactive agents in the same backbone in such a way that for SCI, the combination of a neuroprotector (antimmflamatory or antiapoptotic drug) with a neuroprotector agent (growth factor or NFkB inhibitor), other active principle (i.e. able to permeate the extracellular matrix or glial scar) or a targeting vector could markedly enhance the therapeutic value of these macromolecules as the arrival to the damage is secured for both active principles at the same time or in a programme time scheduled as per rational design, favoring in this way possible synergism. Another very useful concept within this project is the so-called ‘Polymer Unmasked-Masked protein therapy’ (PUMPT). The main idea here is to protect the active principle (peptide, protein or growth factor) by means of a polymer that mask its activity until the presence of an specific physiological feature (change in pH, presence of specific enzymes, etc) triggers polymer degradation and therefore the recovery in a controlled manner of the therapeutic activity. This concept has been already proven in ‘Wound healing’ in vivo models using EGF as active principle. In particular, the design of nanopharmaceuticals for SCI in acute phase will be based in two parallel strategies: Combination nanoconjugates for systemic administration: Polyglutamates with a variety of synthesized architectures will be used as carrier for drug(s) combination. Neuroprotective (antiapoptotic or antiimmflataory) and neuroregenerative drugs (small Mw drugs or growth factors) will be combined in the same polymer carrier. Another alternative will be the combination of 2 neuroprotective agents (antiapoptotic plus antiimmflamatory). Polymer-drug and polymer-protein linkers will be optimized to achieve selective conjugation and the correct drug release kinetics to achieve the desired synergism. As inflammation is present in an acute injury is expected the conjugates design will accumulate in the damage are by the After the identification of biomarkers by means of metabolomics studies performed by PI_3 and 4, these biomarker will be use as targeting vectors for the conjugates designs offering also an active targeting mechanisms and enhancing even more their therapeutic value. Combination nanoconjugates for local/intrathecal administration. Here hydrogels will be developed (poly-�-glutamic, hyaluronic acid, acrylates, its combinations or molecular gels will be used) to served as homogeneous scaffolding for the designed nanoconjugates. Not only similar combination nanoconjugates as those described above for passive targeting could be used here, but also conjugates following the PUMPT concept. The treatment of SCI by sytemic administration has not been highly explored yet, however, an important advance in this field has been achieved by means of local controlled drug delivery systems. Mainly biomaterials as scaffolds have been used as support for cell therapy (also to be explored in this proposal) o by means of hydrogels/nanogels (as that proposed herein) (polylaminines, tiol-acrylates-PEG, self-assembly peptides and functionalised

‘nanowires’). Herein, the idea is to obtain an homogeneous solution that would allow an intrathecal injection and once in the spinal cord injury to form a biodegradable/re-absorbable gel that will release locally the nanoconjugate in a controlled manner. The presence of the nanogel will also help to stop the progression of the disease. Two main designs could be used: a.The combination of agents able to diminish the glial scar formation (i.e. curcumin, IL-6 or tamoxifen as demonstrated in wound healing applications) encapsulated in the hydrogel and neuroprotective/neurorregenerative drugs (growth factors, antiimmflamatory, antiapoptotic agents, etc.) in a polyglutamate carrier and relying on the polymer-drug linker for controlling release kinetics; b.The combination of agents able to diminish the glial scar formation (.e. curcumin, IL-6 or tamoxifen as demonstrated in wound healing applications) encapsulated in the hydrogel and neurorregenerative agents (growth factors, etc.) masked using PUMPT approach.WP2_3_4: The Combinatory therapy: At the best time, dose and way of administration the cell Transplantation experiments in acute SCI will be implemented with the pharmacological arsenal already tested for the control of inflammation and/or apoptosis. In addition axonal growth support by nanoconjugates will be also implemented. Solid or semi-solid biodegradable biomaterials for in situ implants: cell recipients and drug delivery. PI-1 group will be in charge of this WP. Development of in vitro cellular assays to disclose the potential reparative activities of different biomaterials with diverse physics-chemical properties: Polymeric scaffolds of natural and synthetic origin, in hydrogel forms or sponges-like scaffolds or channels-like geometrical structures will be performed. CLMA, Chitosan, hialuronic acid and and poly(ethyl-acrylic acid) will be the chosen biomaterials. Studies of the SCI in humans: Developed mainly by PI-3.WP1.1 Functional analysis of residual motoneurons on patients with chronic SCI. In this clinical assay patients with homologous lesions will be selected and divided into two groups depending on the physiotherapeutic rehabilitation treatment. The functional magnetic resonance images acquired in both groups will allow us, first to characterize the grade of motoneurons survival at chronic stages, second, to extrapolate the neuronal activity on the motor tract to motoneuronal survival rates, and finally, we will be able to find a relationship between active rehabilitation-motoneuron survival-neuronal functional. The results obtained here would be translated into a diagnostic prove to decide if a cell transplantation for motoneuron reconstitution will improve the neuronal activity.In order to improve the value and advantages of this new application of the functional magnetic resonance technique, with non-invasive or aggressive approaches for the patients, research on experimental animals will be performed. All the conclusions obtain in the human will be validated in the lab before go to any further invasive step. Search of new biomarkers for SCI diagnosis (PI-3, PI-1 and PI-2): For the first time we are here proposing to describe biomarkers from the biological fluids, cerebrospinal fluid, blood and urine for the SCI prognostic along all stages, acute, sub-acute and chronic. A global magnetic resonance spectrum will be explored. Although particular attention will be focus on the detection of inflammatory cytokines (IL2, IL8, IL6, TNF-a), chemokines (MCP1), Reactive Protein C, proteins related to axonal degeneration (tau, neurofilaments), proteins related to oligodendrocyte/myelin degradation (NOGO, S100) and proteins related to the reactive astroglia (GFAP, vimentin). After analysis, from every patient, we will determine a potential profile of biomarkers and its correlation with the individual neurological activity. SCI-cell based treatments: Performed by PI-1 and 3. To isolate human epSPC from spinal cord dissection post-mortem from organ donates to characterize the human epSPC culture properties in vitro (in term of self-renewal and differentiation potential). And evaluate its regenerative activity by cell transplantation in the experimental model of acute and chronic SCI. Generation of a cell bank of this type of cell for allosteric transplantation in GMP conditions.Chronogram:  

  

WP 1 year 2 year 3 year 1 PI_3, 1 PI_3,1 PI_3,1 2 PI_2,3,1 PI_2,3,1 PI_2,3, 1 3 PI_1 PI_1,3 PI_1,3 4 PI_2,1,3 PI_2,1,3 PI_2,1,3

   DETAILED EXPLANATION OF THE COORDINATION BETWEEN THE DIFFERENT SUBPROJECTS PI_1 team, in tight coordination with PI_3 will collect and create for the first time an ependymal cell bank in GMP conditions for allosteric patient transplantation from the filum terminale structure. In rodents, alternative transplantation strategies will be studied according to the diverse scenarios found at all stages after SCI. PI_3, will work on neuroprotective therapies and focus on inflammatory-related signals and apoptosis and will use the same in vivo experimental model than PI-_1 to evaluate pharmacological agents. Novel nano-sized drug delivery technologies, in particular Advanced Polymer Therapeutics, will be developed by PI_2 to improve the activity of the desired pro-regenerative drugs assayed by PI_1 group. The desired targets from PI_1 and 3 (tested first by canonical administrations) will be developed and test in vivo PI_1. Biomaterials provide support for tissue transplant engraftment and allow for the release of molecules that support axon growth (also assayed by PI_3). PI_1 with the support of biomaterial experts will develop avenues in cell transplantation to favour axon growth and inhibit glial scaring. For the first time, in voluntary patients, we will describe the neural degeneration along the lesion after a traumatic injury (PI_3). Finally, the search for biomarkers as early predictors of injury, will serve as a powerful tool for diagnosis and prognosis. The profiles of cerebrospinal fluid of human volunteers (PI_3) compared to that of rodents (PI_1,3) will serve as a predictive tool. The physical interactions in this consortium will progressively grow. The investigators will integrate their strategies based on the clinical necessities. Frequent visits to perform experiments and communicate results will improve future experimental design. PI_1 will implement cell replacement strategies from the pharmacological targets tested by PI_3. The mechanism of action of these in vivo experiments will be determined and shared by PI_1 and PI_3. Herein, clinicians and researchers propose to conduct the basic science from individual approaches into a combinatorial recipe, first developed in a pre-clinical phase in acute and chronic SCI models in rodents to be ready for the patient. The coordinator, PI-1, will be in charge to organized periodic meetings, every three months (or more frequents where it will be required).              

   

ANEXO 2: PRESUPUESTO

  PRESUPUESTO TOTAL  

TIME TO MOVE

Presupuesto proyecto investigación Total financiación solicitada Capítulo  Año 1  Año 2 Año 3Contratación personal investigador y técnico 

274.500   274.500   274.500   823.500  

Material inventariable 

102.500   0   0   102.500  

Material fungible 

201.000   201.000   221.000   623.000  

Viajes y dietas   7.500   9.000   9.500   26.000  Otros gastos  10.000   14.000   22.000   46.000  Gestión y coordinación 

100.000   100.000   100.000   300.000  

Subtotal   695.500   598.500   627.000   1.921.000  Canon (% costes indirectos) 

151.719   143.584   150.584   445.887  

Total           2.366.887 €     PRESUPUESTO POR GRUPO DE INVESTIGACIÓN GRUPO VALL D’HEBRON  

PRESUPUESTO ANUAL  Año 1 Año 2 Año 3 Año 4‐5  TOTALEnsayo Clínico 

PERSONAL  95.000 95.000 95.000    285.000MATERIAL INVENTARIABLE  52.500 0 0 52.500MATERIAL FUNGIBLE  70.000 70.000 90.000    230.000VIAJES y DIETAS  2.000 4.000 4.000 10.000OTROS GASTOS  5.000 10.000 18.000    33.000SUBTOTAL  224.500 179.000 207.000 610.500Costes indirectos/Canon    49.005 40.870 47.870    137.745

TOTAL   273.505 219.870 254.870 450.000  1.198.245 €    

     GRUPO CENTRO INVESTIGACIÓN PRINCIPE FELIPE (Lab. Regeneración)  

PRESUPUESTO ANUAL  Año 1 Año 2 Año 3 TOTAL 

PERSONAL  85.000 85.000 85.000 255.000 MATERIAL INVENTARIABLE  30.000 0 0 30.000 MATERIAL FUNGIBLE  36.000 36.000 36.000 108.000 VIAJES y DIETAS  500 1.000 1.500 3.000 OTROS GASTOS  1.000 1.000 1.000 3.000 SUBTOTAL  152.500 123.000 123.500 399.000 Costes indirectos/Canon    32.025 25.830 25.935 83.790 

TOTAL   184.525 148.830 149.435 482.790 €   GRUPO CENTRO INVESTIGACIÓN PRINCIPE FELIPE (Lab. Nanotecnología)  

PRESUPUESTO ANUAL  Año 1 Año 2 Año 3 TOTAL 

PERSONAL  66.000 66.000 66.000 198.000 MATERIAL INVENTARIABLE  20.000 0 0 20.000 MATERIAL FUNGIBLE  40.000 40.000 55.000 135.000 VIAJES y DIETAS  500 1.000 1.500 3.000 OTROS GASTOS  1.000 2.000 3.000 6.000 SUBTOTAL  127.500 109.000 125.500 362.000 Costes indirectos/Canon    26.775 22.890 26.355 76.020 

TOTAL   154.275 131.890 151.855 438.020 €   FUNDACIÓN STEP BY STEP (Gestión y Coordinación Proyecto)  

PRESUPUESTO ANUAL  Año 1 Año 2 Año 3 TOTAL 

PERSONAL  28.500 28.500 28.500 85.500 VIAJES y DIETAS  3.500 3.000 3.500 10.000 OTROS GASTOS  2.000 1.000 1.000 4.000 SUBTOTAL  34.000 32.500 33.000 99.500 Costes indirectos/Canon    49.444 49.444 49.445 148.333 

TOTAL   83.444 81.944 82.445 247.833 €