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”
Colaboración en el proyecto de investigación
TIME TO MOVE: Terapia celular en lesión medular
“Nuevas estrategias de tratamiento de la LM”
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
A la búsqueda de soluciones: TIME TO MOVE
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%.
Origen de las causas de la Lesión Medular
Accidentes
deportivos
11%
Accidentes
laborales
10%
Caidas
casuales
34%
Autolisis
8%
Accidentes
tráfico
37%
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
La finalidad de
TIME TO MOVE es aplicar
nuevas estrategias para
el tratamiento de las
lesiones medulares,
agudas y crónicas.
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.
Hipótesis de trabajo
El trasplante celular puede sustituir a las células
perdidas, y a la vez generar un ambiente propicio
para la supervivencia y buen funcionamiento de los
axones supervivientes y lesionados, aliviando el
proceso de degeneración,
favoreciendo la
regeneración endógena, espontánea, en la fase
crónica de la lesión.
La eliminación de la cicatriz glial y la supresión de
los inhibidores del crecimiento axonal puede ser un
método eficaz para favorecer la reparación de la
médula, en la fase crónica.
El uso de las plataformas biotecnológicas,
herramientas innovadoras y útiles para liberar
compuestos de forma controlada y local, permitiría
usar fármacos y otras sustancias capaces de reparar
la médula y regenerar los axones, creando de este
modo un puente sobre el que pueda generarse el
crecimiento axonal, en la fase crónica.
La
utilización
de
polímeros
terapéuticos
(nanoconstrucciones híbridas) puede ser un sistema
de transporte intracelular y extracelular específico,
para conseguir terapias de aplicación clínica, en fase
crónica y aguda.
La liberación y actuación de fármacos dirigidos
mediante nanotecnología sobre el lugar de la lesión,
permitiría limitar y reducir la inflamación. En fase
aguda, podría ser una terapia eficaz, evitando los
efectos nocivos de la lesión secundaria, y en fase
crónica, crear un ambiente propicio a la
regeneración
Determinar los biomarcadores, tanto a nivel clínico
como experimental, necesarios para conocer el
estado y grado de la lesión, y poder predecir su
evolución en el tiempo.
Antecedentes
La médula espinal es un cordón nervioso que se
extiende desde la base del cerebro hasta la región
lumbar. Está protegida por la columna, por las
vértebras, y funciona como una línea eléctrica de
doble recorrido, de ida y vuelta, que transmite las
órdenes del cerebro al resto del cuerpo y viceversa.
Cuando esta conducción nerviosa se interrumpe por
una lesión –en este caso traumática, sus consecuencias
son más o menos graves según la localización de la
lesión. A nivel cervical (tetraplejia) provoca pérdida o
disminución de la sensibilidad, de la movilidad de
extremidades superiores e inferiores y de todo el
tronco, y de la funcionalidad de las vísceras torácicas
y abdominales. A nivel torácico y lumbar (paraplejia)
provoca pérdida de la sensibilidad, de la movilidad de
las extremidades inferiores y de parte del tronco, y de
la funcionalidad de las vísceras abdominales. A nivel
lumbar bajo (cola de caballo) provoca parálisis de las
extremidades inferiores.
La mayoría de LM traumáticas no presentan una sección completa de la médula, sino que la lesión
se 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 la
médula, que en gran medida es consecuencia de un proceso secundario a la lesión, en el que
intervienen múltiples factores: estrés oxidativo por síntesis excesiva de óxido nítrico; activación
de la microglía; inflamación local; microcirculación alterada; disfunción de la barrera
hematoencefálica; y, según se ha descubierto recientemente, un mecanismo de muerte celular
diferida. Todo ello sucede durante los primeros minutos, horas, días o incluso meses después del
trauma 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 de
los ya existentes o a partir de una población de células madre multipotente que existen en la
médula espinal (células ependimarias).
Las consecuencias de este proceso secundario son
desastrosas: muerte accidental (necrosis) y
muerte programada (apoptosis) de las neuronas;
formación de cicatrices y quistes; pérdida de la
mielina (aislante y protector de los axones);
alteración de la morfología y funcionalidad de los
conductos nerviosos. Los mecanismos por los que
se producen son además complejos y poco
conocidos. Existe asimismo poco conocimiento y
escasa evidencia acerca de los mecanismos,
espontáneos o inducidos, de regeneración y/o
recuperación de la plasticidad de las neuronas.
Hasta el momento actual se han llevado a cabo
multitud de aproximaciones para tratar la LM
traumática 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 los
radicales libres y para inhibir la toxicidad de los glutamatos, tratamientos
antiinflamatorios, antiapoptóticos y antidesmielinizantes, pasando por
sustancias para promover la regeneración axonal y estrategias de reparación
celular y con biomateriales, hasta diversas y numerosas técnicas de terapias
físicas.
Sin embargo, ninguno de los estudios anteriores ha abordado de forma
conjunta un proyecto realizado en animales de experimentación, con un
desarrollo en laboratorio de investigación básica y de desarrollo de sistemas
de biomateriales híbridos biodegradables para la liberación de sustancias y
con 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 cinco
grupos de investigación trabajarán simultáneamente y de forma coordinada en los objetivos
siguientes:
9 Abordar el trasplante celular por método quirúrgico de inyección o mediante punción en el
espacio subaracnoideo.
9 Estudiar biomateriales biodegradables para crear nichos que puedan alojar trasplantes
autólogos (células madre propias).
9 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 la
regeneración celular axonal
9 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 la
lesión).
9 Estudiar el perfil de expresión génica (celular) y el tiempo de respuesta en fase aguda, subaguda y crónica.
9 Estudiar los biomarcadores tras la terapia celular in vivo, a partir de muestras de LCR
(líquido cefalorraquídeo) extraídas de los animales tratados.
9 Contrastar los resultados de biomarcadores obtenidos de las muestras clínicas de los
pacientes y de las de animales de experimentación.
9 Obtener información de los cambios espacio-temporales, en relación con la inflamación y
muerte celular tras la lesión.
9 Aplicar y comparar efectos de factores inmunosupresores endógenos y generales.
9 Analizar el efecto de fármacos (i.e. simvastatina, condroitinasas) que modulan las balsas de
lípidos, en relación a la inflamación y muerte celular.
9 Analizar los efectos de fármacos neuroprotectores y de terapias celulares en relación a las
motoneuronas y los oligodendrocitos, en la lesión crónica.
9 Estudiar el efecto combinado de terapias farmacológicas y celulares en fase crónica.
9 Analizar biomarcadores en líquidos biológicos humanos (LCR, sangre y orina) como
herramienta de pronóstico de gravedad y como predictores de cambios, en fase aguda y
crónica, y comparar sus posibles diferencias.
9 Desarrollar y aplicar biomateriales (microcápsulas, microesferas y/o liposomas) para la
liberación controlada de fármacos.
9 Generar una familia de nanomedicinas con capacidad de paliar o aminorar los daños
celulares 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 trasladados
a los pacientes con lesión medular. La experimentación clínica
se llevará a cabo tanto en fase aguda como en fase crónica de
LM.
Fase lesión
medular
aguda
Fase lesión
medular
crónica
; Usar biomarcadores de inflamación, a partir de muestras de LCR,
como herramienta pronostica de la profundidad de la lesión
medular.
; Utilizar fármacos neuroprotectores mediante nanotecnología, en
relación a las motoneuronas y los oligodendrocitos.
; Aplicar familias de nanomedicinas con capacidad para paliar o
aminorar los daños celulares asociados a la lesión medular.
; Utilizar terapias de regeneración celular, en relación a las
motoneuronas y los oligodendrocitos. ; 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 madre
propias).
; 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 lesión
medular. Vall d’Hebron Institut de Recerca (VHIR) El proyecto, innovador, se ubica en el Hospital Universitari Vall
d’Hebron (HUVH), centro terciario destacado en España. Además
de la Unidad de Lesión Medular de dicho centro, participan
cinco 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 Lesión
Medular del HUVH, así como entre los usuarios de las instalaciones de rehabilitación especializada
de la Fundación Step by Step.
Experiencia del Equipo Investigador y Medios Disponibles
7El VHIR tiene por objeto la investigación biomédica en el ámbito del diagnóstico, la terapéutica y
el tratamiento de los problemas relacionados con la salud humana. En los últimos años ha
evolucionado hasta ocupar las mejores posiciones en Catalunya y España. Sin duda ello ha sido
posible gracias a la estrecha relación entre el Hospital Universitari Vall d’Hebron (HUVH) y los
investigadores, entre la clínica y el laboratorio y viceversa (bedtobenchtobed). La vinculación de la
actividad investigadora en el HUVH es una oportunidad singular e inmejorable, para potenciar la
traslación y la innovación centradas en la salud de la
población. El VHIR tiene en la actualidad 1.200 personas dedicadas a la investigación, 10 áreas con más de
300 líneas de investigación abiertas y cerca de 700
ensayos clínicos activos. Potenciamos la
investigación clínica y la innovación en todos los
ámbitos de las ciencias de la salud. Al mismo
tiempo, promovemos activamente la participación y
colaboración con otros centros, entidades y
empresas. Durante el año 2010, el VHIR es la fundación de
investigación asociada a hospitales que ha recibido más subvenciones del Instituto de Salud Carlos
III (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 los
antagonistas 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). Grupo de
Señalización
Celular y
Apoptosis - VHIR
El grupo, dirigido por el Dr. Joan X. ComellaCarnicé,
posee una amplia experiencia en el estudio de los
mecanismos apoptóticos en modelos neuronales. El
principal objetivo es caracterizar los mecanismos de
acción de los antagonistas de receptores de muerte
(FAIM-L, Lifeguard y FLIP) en el sistema nervioso, lo
cual podría permitir desarrollar nuevas estrategias
terapéuticas para enfermedades del sistema nervioso
muy
diversas,
tales
como
enfermedades
neurodegenerativas,
procesos
tumorales,
o
traumatismos, en los que la apoptosis juegue un papel
primordial.
La Unidad de Lesionados Medulares (ULM) del Hospital Universitario Vall d’Hebron fue creada en
1976. Desde entonces presta servicio ininterrumpidamente a un promedio de 100 pacientes con
lesión medular aguda por año, siendo centro de referencia en el tratamiento de lesiones agudas
para Catalunya, Baleares, Andorra y en ocasiones zonas del sur de Francia.
La Unidad está dirigida por el Dr. Miguel A. González
Viejo, quien posee una amplia experiencia en Medicina
Física y Rehabilitación de las lesiones medulares. El
equipo lo componen la Dra. Lucrecia Ramírez
Garcerán, adscrita a la ULM desde hace más de 18 años
y experta en la atención al lesionado medular agudo, y
la Dra. Lluïsa Montesinos Magraner, que se incorporó en
el año 2010 a la Unidad después de su estancia en el
Texas InstituteforRehabilitation de Houston (EE.UU.).
Cuenta con un equipo multidisciplinarformado por
personal médico especialista en rehabilitación de
lesión medular, cirugía ortopédica, medicina intensiva,
psiquiatría, psicología, así como personal sanitario
especializado en cuidados de enfermería, fisioterapia,
Unidad de
Lesión Medular
Hospital
Universitario
Vall d’Hebron
terapia ocupacional, trabajo social y tecnología ortopédica y del soporte de los
diferentesespecialistas del Hospital Universitari Vall d’Hebron, necesarios en momentos puntuales
en la atención de estos pacientes. Dispone del equipamiento específico y de los habitáculos
especialesnecesarios para la asistencia integral durante el período agudo y subagudo.
En el período 1997-2010 se han atendido 1.147 pacientes, de los cuales 917 eran hombres y
230 mujeres, con una estancia media de 67 días y
alto 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
Violencia
Deportivo
últimos años: disminución del porcentaje
Autolisis
2%
11%
5%
de accidentes de tráfico, aunque con
Caidas
Laboral
lesiones
más
severas
por
19%
16%
politraumatismos, sin variación en los
accidentes deportivos, aumento de los
intentos de autolisis (suicidio) en las
mujeres, e incremento de caídas
Tráfico
fortuitas en personas de edad avanzada.
47%
Centro de Investigación Príncipe Felipe (CIPF) Su finalidad es investigar soluciones para la mejora de
la salud humana, aplicando las tecnologías más
avanzadas para desarrollar nuevas terapias y/o
métodos de diagnóstico, e impulsar la ciencia básica
biomédica con el fin de avanzar en la comprensión de
las bases moleculares de patologías humanas, que
requieran nuevos procedimientos diagnósticos y
clínicos para su identificación y tratamiento. Y en
particular, el desarrollo de terapias avanzadas para el
tratamiento de patologías humanas. En el campo de las células madre, la misión del CIPF es
ser un centro europeo de referencia en terapias
celulares, potenciando la investigación interdisciplinar en células madre adultas y embrionarias, con el objeto de regenerar órganos dañados, y
compartir el conocimiento con investigadores y clínicos. Asimismo, ser el integrador de datos
"ómicos" de referencia en Europa y computación para el modelado cuantitativo de sistemas
biológicos, potenciando las áreas de Genómica estructural, Regulación genómica y Genómica
funcional.
El CIPF desarrolla métodos (bio) químicos y genéticos para descifrar redes de señalización
complejas, así como agentes terapéuticos potenciales mediante la investigación (ciencia
básica). En el campo de desarrollo de nuevas terapias, el CIPF aspira a convertirse en un
centro de referencia internacional, mediante el desarrollo de terapias bio y nano-tecnológicas
(nanomedicina) que permitan avanzar significativamente en los aspectos clínicos de patologías
humanas graves.
Laboratorio de Regeneración Neuronal
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 la
participación de células troncales comrecientemente que el trasplante en fase aguda de
células ependimarias con capacidad multipotente, procedente de individuos adultos que han
sufrido una lesión, en un modelo de contusión en la médula espinal, restaura la función
neurológica y la actividad locomotora perdida de forma significativa. Mejorar el
conocimiento de los procesos moleculares y celulares que ocurren en una lesión del sistema
nervioso contribuirá a la búsqueda de herramientas farmacológicas, que favorezcan el
escenario más propicio para la regeneración in vivo, con efectos sinérgicos sobre la función
neuronal perdida.
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.
Laboratorio de Polímeros Terapéuticos
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.
Fundación Step by Step (SbS)
La Fundación Step by Step es una organización
sin ánimo de lucro dedicada al lesionado
medular. Su Centro de Rehabilitación está
especializado en tratamientos innovadores de
rehabilitación para lesionados medulares y ha
sido 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 en
aplicar este avanzado método de recuperación de lesionados medulares, implantado con éxito en
su centro pionero en esta metodología. SbS cuenta con un Patronato Rector, presidido por Frederic Crespo, y formado por
profesionales de la medicina e investigadores de gran prestigio, que otorgan gran credibilidad
al proyecto de la fundación:
‘ Dr. Miguel Ángel González Viejo, Jefe de la Unidad de Lesionados Medulares del
Hospital Universitari Vall d’Hebron, Barcelona.
‘ Dra. MercèAvellanet, Jefe
del
NostraSenyoraMeritxell, Andorra .
Servicio
de
Rehabilitación
del
Hospital
‘ Dra. Anna Veiga, Directora Banco de Líneas Celulares del Centro de Medicina
Regenerativa 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.
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:
resupuesto proyecto investigación
Terapia Celular en Lesión Medular (TIME TO MOVE) Capítulo Año 1 Año 2
274.500,00 €
274.500,00 €
Contratación personal investigador y técnico Material inventariable 102.500,00 €
0,00 €
Material fungible 201.000,00 €
201.000,00 €
Viajes y dietas 7.500,00 €
9.000,00 €
Otros gastos 10.000,00 €
14.000,00 €
Canon (% costes indirectos) 151.719,00 €
143.584,00 €
Gestión y coordinación 100.000,00 €
100.000,00 €
Subtotal 847.219,00 €
742.084,00 €
Total Total financiación solicitada Año 3
274.500,00 €
0,00 €
221.000,00 €
9.500,00 €
22.000,00 €
150.584,00 €
100.000,00 €
777.584,00 €
823.500,00 €
102.500,00 €
623.000,00 € 26.000,00 €
46.000,00 €
445.887,00 €
300.000,00 €
2.366.887,00 € “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.
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.
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
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
La discapacidad
se traduce en un
elevado coste
económico
Colaboración y Contraprestaciones
Apoyar la investigación en salud es una inversión en el futuro del país y contribuye a
generar riqueza, añade valor a la atención sanitaria y crea las bases de una sociedad
mejor, en la que personas que ahora son subsidiadas, puedan convertirse en
contribuyentes natos.
La involucración y complicidad de todos los sectores y agentes resulta imprescindible. La
colaboración en este proyecto significa la oportunidad para “transformar la vida de las
personas con lesión medular”.
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 remodelingcontrolling 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 neurofilamentlabeled 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-(2hydroxypropyl)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: polymerdrug 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 (PGApaclitaxel 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, neuroimmunomodulation 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 intraperitoneal 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-maskingUnMasking- 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 “endogenousactivation” 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/reabsorbable 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 selfrenewal 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
2
3
4
1 year
PI_3, 1
PI_2,3,1
PI_1
PI_2,1,3
2 year
PI_3,1
PI_2,3,1
PI_1,3
PI_2,1,3
3 year
PI_3,1
PI_2,3, 1
PI_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 proregenerative 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
Capítulo Presupuesto proyecto investigación
Año 1 Año 2
Contratación personal investigador y técnico Material inventariable Material fungible Viajes y dietas Otros gastos Gestión y coordinación Subtotal Canon (% costes indirectos) Total financiación solicitada Año 3
274.500 274.500 274.500 823.500 102.500 0 0 102.500 201.000 201.000 221.000 623.000 7.500 10.000 100.000 9.000 14.000 100.000 9.500 22.000 100.000 26.000 46.000 300.000 695.500 151.719 598.500 143.584 627.000 150.584 1.921.000 445.887 Total PRESUPUESTO POR GRUPO DE INVESTIGACIÓN
2.366.887 € GRUPO VALL D’HEBRON
PRESUPUESTO ANUAL Año 1
Año 2
Año 3
PERSONAL MATERIAL INVENTARIABLE MATERIAL FUNGIBLE VIAJES y DIETAS OTROS GASTOS SUBTOTAL Costes indirectos/Canon 95.000
52.500
70.000
2.000
5.000
224.500
49.005
95.000
0
70.000
4.000
10.000
179.000
40.870
95.000
0
90.000
4.000
18.000
207.000
47.870
TOTAL 273.505
219.870
254.870
Año 4‐5 Ensayo Clínico TOTAL
285.000
52.500
230.000
10.000
33.000
610.500
137.745
450.000 1.198.245 €
GRUPO CENTRO INVESTIGACIÓN PRINCIPE FELIPE (Lab. Regeneración)
PRESUPUESTO ANUAL Año 1
Año 2
Año 3
TOTAL PERSONAL MATERIAL INVENTARIABLE MATERIAL FUNGIBLE VIAJES y DIETAS OTROS GASTOS SUBTOTAL Costes indirectos/Canon 85.000
30.000
36.000
500
1.000
152.500
32.025
85.000
0
36.000
1.000
1.000
123.000
25.830
85.000
0
36.000
1.500
1.000
123.500
25.935
255.000 30.000 108.000 3.000 3.000 399.000 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 MATERIAL INVENTARIABLE MATERIAL FUNGIBLE VIAJES y DIETAS OTROS GASTOS SUBTOTAL Costes indirectos/Canon 66.000
20.000
40.000
500
1.000
127.500
26.775
66.000
0
40.000
1.000
2.000
109.000
22.890
66.000
0
55.000
1.500
3.000
125.500
26.355
198.000 20.000 135.000 3.000 6.000 362.000 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 VIAJES y DIETAS OTROS GASTOS SUBTOTAL Costes indirectos/Canon 28.500
3.500
2.000
34.000
49.444
28.500
3.000
1.000
32.500
49.444
28.500
3.500
1.000
33.000
49.445
85.500 10.000 4.000 99.500 148.333 TOTAL 83.444
81.944
82.445
247.833 €