The oncolytic adenovirus VCN-01 promotes anti-tumor effect in primitive neuroectodermal tumor models

Last advances in the treatment of pediatric tumors has led to an increase of survival rates of children affected by primitive neuroectodermal tumors, however, still a significant amount of the patients do not overcome the disease. In addition, the survivors might suffer from severe side effects caused by the current standard treatments. Oncolytic virotherapy has emerged in the last years as a promising alternative for the treatment of solid tumors. In this work, we study the anti-tumor effect mediated by the oncolytic adenovirus VCN-01 in CNS-PNET models. VCN-01 is able to infect and replicate in PNET cell cultures, leading to a cytotoxicity and immunogenic cell death. In vivo, VCN-01 increased significantly the median survival of mice and led to long-term survivors in two orthotopic models of PNETs. In summary, these results underscore the therapeutic effect ofVCN-01 for rare pediatric cancers such as PNETs, and warrants further exploration on the use of this virus to treat them

Oncolytic adenoviruses as a therapeutic approach for osteosarcoma: A new hope

Osteosarcoma is the most common bone cancer among those with non-hematological origin and affects mainly pediatric patients. In the last 50 years, refinements in surgical procedures, as well as the introduction of aggressive neoadjuvant and adjuvant chemotherapeutic cocktails, have increased to nearly 70% the survival rate of these patients. Despite the initial therapeutic progress the fight against osteosarcoma has not substantially improved during the last three decades, and almost 30% of the patients do not respond or recur after the standard treatment. For this group there is an urgent need to implement new therapeutic approaches. Oncolytic adenoviruses are conditionally replicative viruses engineered to selectively replicate in and kill tumor cells, while remaining quiescent in healthy cells. In the last years there have been multiple preclinical and clinical studies using these viruses as therapeutic agents in the treatment of a broad range of cancers, including osteosarcoma. In this review, we summarize some of the most relevant published literature about the use of oncolytic adenoviruses to treat human osteosarcoma tumors in subcutaneous, orthotopic and metastatic mouse models. In conclusion, up to date the preclinical studies with oncolytic adenoviruses have demonstrated that are safe and efficacious against local and metastatic osteosarcoma. Knowledge arising from phase I/II clinical trials with oncolytic adenoviruses in other tumors have shown the potential of viruses to awake the patient´s own immune system generating a response against the tumor. Generating osteosarcoma immune-competent adenoviruses friendly models will allow to better understand this potential. Future clinical trials with oncolytic adenoviruses for osteosarcoma tumors are warranted

Oncolytic viruses as therapeutic tools for pediatric brain tumors

In recent years, we have seen an important progress in our comprehension of the molecular basis of pediatric brain tumors (PBTs). However, they still represent the main cause of death by disease in children. Due to the poor prognosis of some types of PBTs and the long-term adverse effects associated with the traditional treatments, oncolytic viruses (OVs) have emerged as an interesting therapeutic option since they displayed safety and high tolerability in pre-clinical and clinical levels. In this review, we summarize the OVs evaluated in different types of PBTs, mostly in pre-clinical studies, and we discuss the possible future direction of research in this field. In this sense, one important aspect of OVs antitumoral effect is the stimulation of an immune response against the tumor which is necessary for a complete response in preclinical immunocompetent models and in the clinic. The role of the immune system in the response of OVs needs to be evaluated in PBTs and represents an experimental challenge due to the limited immunocompetent models of these diseases available for pre-clinical research

Delta-24-RGD combined with radiotherapy exerts a potent antitumor effect in diffuse intrinsic pontine glioma and pediatric high grade glioma models

Pediatric high grade gliomas (pHGG), including diffuse intrinsic pontine gliomas (DIPGs), are aggressive tumors with a dismal outcome. Radiotherapy (RT) is part of the standard of care of these tumors; however, radiotherapy only leads to a transient clinical improvement. Delta-24-RGD is a genetically engineered tumor-selective adenovirus that has shown safety and clinical efficacy in adults with recurrent gliomas. In this work, we evaluated the feasibility, safety and therapeutic efficacy of Delta-24-RGD in combination with radiotherapy in pHGGs and DIPGs models. Our results showed that the combination of Delta-24-RGD with radiotherapy was feasible and resulted in a synergistic anti-glioma effect in vitro and in vivo in pHGG and DIPG models. Interestingly, Delta-24-RGD treatment led to the downregulation of relevant DNA damage repair proteins, further sensitizing tumors cells to the effect of radiotherapy. Additionally, Delta-24-RGD/radiotherapy treatment significantly increased the trafficking of immune cells (CD3, CD4+ and CD8+) to the tumor niche compared with single treatments. In summary, administration of the Delta-24-RGD/radiotherapy combination to pHGG and DIPG models is safe and significantly increases the overall survival of mice bearing these tumors. Our data offer a rationale for the combination Delta-24-RGD/radiotherapy as a therapeutic option for children with these tumors. SIGNIFICANCE: Delta-24-RGD/radiotherapy administration is safe and significantly increases the survival of treated mice. These positive data underscore the urge to translate this approach to the clinical treatment of children with pHGG and DIPGs

The oncolytic virus Delta-24-RGD elicits an antitumor effect in pediatric glioma and DIPG mouse models

Pediatric high-grade glioma (pHGG) and diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors in desperate need of a curative treatment. Oncolytic virotherapy is emerging as a solid therapeutic approach. Delta-24-RGD is a replication competent adenovirus engineered to replicate in tumor cells with an aberrant RB pathway. This virus has proven to be safe and effective in adult gliomas. Here we report that the administration of Delta-24-RGD is safe in mice and results in a significant increase in survival in immunodeficient and immunocompetent models of pHGG and DIPGs. Our results show that the Delta-24-RGD antiglioma effect is mediated by the oncolytic effect and the immune response elicited against the tumor. Altogether, our data highlight the potential of this virus as treatment for patients with these tumors. Of clinical significance, these data have led to the start of a phase I/II clinical trial at our institution for newly diagnosed DIPG (NCT03178032)

Generación de una adenovirus Ad5/52s pseudotipado con la proteína fiber corta del Ad52 para su caracterización in vitro e in vivo como vector de terapia génica

La terapia génica consiste en la manipulación y utilización de material genético para el tratamiento de patologías. No obstante, esta estrategia requiere el uso de vectores de terapia génica para transportar el material genético al tejido diana de forma eficiente. Los vectores de terapia génica más comunes son los basados en el adenovirus humano de serotipo 5 (Ad5), porque, respecto a otros serotipos, el Ad5 tiene como ventaja su bioseguridad y facilidad de producción. Sin embargo, para que una célula sea transducida por el Ad5 ha de expresar un receptor reconocible por el Ad5, principalmente la proteína CAR, al que se une a través de su proteína fiber. El fiber de otros serotipos de adenovirus se une a receptores diferentes, permitiendo así la transducción de tipos celulares alternativos a los transducidos por el Ad5. De esta manera, una de las estrategias utilizadas para obtener un vector Ad5 con el tropismo de un serotipo diferente, es la pseudotipación del adenovirus, que consiste en substituir el fiber del Ad5 por el de otro serotipo. De entre los serotipos de adenovirus humanos, uno de los más recientemente descritos es el Ad52. Este serotipo contiene dos fiber de diferentes longitudes, lo cual es un rasgo compartido con los adenovirus humanos intestinales de la especie F. Como el Ad52 fue identificado a partir de muestras de heces de pacientes con gastroenteritis, se le atribuye también un tropismo intestinal, aunque no se ha podido demostrar. En el caso de los adenovirus de la especie F, su fiber largo reconoce el receptor CAR y, por lo tanto, se asume que su tropismo intestinal es mediado por el fiber corto. Dada la cercanía evolutiva entre el Ad52 y los adenovirus de la especie F, se asume que su fiber largo también reconocerá el receptor CAR, mientras que el fiber corto lo hará a otro receptor, dándole así al virus un tropismo independiente de CAR. Así pues, en esta tesis se ha generado un vector quimérico Ad5 pseudotipado con el fiber corto del Ad52 (Ad5/52s) con la finalidad de estudiar el rol específico de esta proteína fiber y, a la vez, caracterizar este adenovirus como vector de terapia génica. La primera parte de la tesis consiste en la generación del genoma del adenovirus Ad5/52s y la producción del vector, lo que incluye un estudio de su ciclo viral para optimizar la producción. El resultado del ciclo viral indica que éste se encuentra moderadamente retrasado respecto al del Ad5. Una vez producidos los vectores, se comparó su tropismo con el del Ad5 en cultivos in vitro, demostrando que la substitución del fiber comporta un cambio en el tropismo del Ad5 hacia líneas celulares, así como también aumenta su transducción en células de Schwann primarias, aunque no aumenta el tropismo del Ad5 en modelos intestinales in vitro. Ya caracterizado el virus in vitro, se demostró que el fiber corto del Ad52 no tiene afinidad por el receptor CAR, y su unión al receptor es dependiente del dominio knob del fiber. Posteriormente, el estudio comparativo de su distribución in vivo por administración intravenosa respecto al Ad5, mostró que el Ad5/52s tan solo transduce ligeramente pulmón a pesar de no ser secuestrado en hígado. Una vez descartados problemas de estabilidad, se observó que la baja eficiencia de infección del Ad5/52s se debe a una mayor inactivación de este virus en sangre, probablemente por la interacción con algún factor plasmático como la trombina. Finalmente, se modificó el fiber corto para limitar esta interacción, lo que permitió aumentar la supervivencia del Ad5/52s en sangre.Gene therapy is a biomedical approach, which consists of manipulating and delivering genes to treat a wide range of diseases. Nevertheless, a successful treatment also requires a vector to carry the therapeutic gene towards the targeted tissue and thus to increase itstransduction efficiency. Human adenovirus 5 derived vectors (Ad5) are the most commonly usedin gene therapy strategies due totheir higher biosafety and productivity. However, the Ad5 mediated transduction is restricted to those cells expressing appropriate viral receptors (mainly the CAR protein), recognized by the fiber protein. The fiber of other adenovirus serotypes binds to different receptors, allowing transduction of alternative cell types of those transduced by Ad5. Thus, one strategy to modify the natural Ad5 tropism is adenovirus pseudotyping, which consists of Ad5 fiber replacement by the fiber protein of another serotype. Among the different human adenovirus serotypes, one of the most recently described is Ad52. This serotype contains two fiber of different lengths, which is a common trait shared with enteric Ad40 and Ad41 human adenovirus. The Ad52 was initially found in stool samples obtained from patients with gastroenteritis and, as a consequence, it is supposed to be also an enteric virus, although it has not been demonstrated yet. In the specie F adenoviruses the long fiber recognizes the CAR receptor, and therefore, their enteric tropism is assumed to be mediated by the short fiber. Given the evolutionary relationship between the Ad52 and the species F adenoviruses, it is assumed that its long fiber also recognizes the CAR receptor, while the short fiber will recognize a different receptor, thus giving the virus a CAR-independent tropism. So, in this thesis we have generated a chimeric Ad5 vector pseudotyped with the Ad52 short fiber (Ad5/52s) protein, in order to study the specific role of the fiber protein, in turn, characterize this adenovirus as a vector for gene therapy. The first part of the thesis consists in the generation of adenovirus Ad5/52s genome and its production, which includes a study of its viral cycle to optimize production. The results show that the Ad5/52s viral cycle is moderately delayed compared to Ad5. Once the vectors were produced, the tropism of Ad5/52s and Ad5 were compared in in vitro, demonstrating that fiber replacement induces a switch in the Ad5 tropism in different cell lines, as well as an increased transduction in primary Schwann cells, but not in intestinal models in vitro. Then, it was also demonstrated that short fiber from Ad52 doesn’t recognizes the CAR receptor, and also that its binding is mediated by the fiber knob domain. Further studies to compare Ad5 versus Ad5/52 in vivo biodistribution after intravenous administration showed only a slight transduction in lung with Ad5/52s, despite not being trapped in liver. Once discarded stability problems, it was observed that the low levels of transduction achieved by the Ad5/52s were caused by a faster inactivation in plasma, probably by an interaction with a plasmatic factor, such as thrombin. Finally, to minimize this interaction, the short fiber was mutated, which enhanced the Ad5/52s survival in blood

Oncolytic adenoviruses as a therapeutic approach for osteosarcoma: A new hope

Osteosarcoma is the most common bone cancer among those with non-hematological origin and affects mainly pediatric patients. In the last 50 years, refinements in surgical procedures, as well as the introduction of aggressive neoadjuvant and adjuvant chemotherapeutic cocktails, have increased to nearly 70% the survival rate of these patients. Despite the initial therapeutic progress the fight against osteosarcoma has not substantially improved during the last three decades, and almost 30% of the patients do not respond or recur after the standard treatment. For this group there is an urgent need to implement new therapeutic approaches. Oncolytic adenoviruses are conditionally replicative viruses engineered to selectively replicate in and kill tumor cells, while remaining quiescent in healthy cells. In the last years there have been multiple preclinical and clinical studies using these viruses as therapeutic agents in the treatment of a broad range of cancers, including osteosarcoma. In this review, we summarize some of the most relevant published literature about the use of oncolytic adenoviruses to treat human osteosarcoma tumors in subcutaneous, orthotopic and metastatic mouse models. In conclusion, up to date the preclinical studies with oncolytic adenoviruses have demonstrated that are safe and efficacious against local and metastatic osteosarcoma. Knowledge arising from phase I/II clinical trials with oncolytic adenoviruses in other tumors have shown the potential of viruses to awake the patient´s own immune system generating a response against the tumor. Generating osteosarcoma immune-competent adenoviruses friendly models will allow to better understand this potential. Future clinical trials with oncolytic adenoviruses for osteosarcoma tumors are warranted. Keywords: Oncolytic adenovirus, Virotherapy, Osteosarcoma, Bones, Cancer, Tumo

Oncolytic virotherapy for the treatment of pediatric brainstem gliomas

Diffuse intrinsic pontine glioma (DIPG) is the most frequent brainstem glioma and the most lethal brain tumor in childhood. Despite transient benefit with radiotherapy, the prognosis of children with this disease remains dismal with severe neurological morbidity and median survival less than 12 months. Oncolytic immunovirotherapy is emerging as a potential therapeutic approach in neuro-oncology. The oncolytic adenovirus Delta-24-RGD has shown efficacy in adult patients with recurrent GBM. Our group has demonstrated that Delta-24-RGD has oncolytic activity and triggers immune response in preclinical models of DIPG, and has a synergistic effect with radiotherapy in animal models of this disease. In this scenario, we conducted a first-in-human phase 1 clinical trial to evaluate the safety and efficacy of intratumoral injection of Delta-24-RGD in pediatric patients with newly diagnosed DIPG prior to standard radiotherapy. The study confirmed the feasibility of this treatment with an acceptable safety profile and encouraging efficacy results. Correlative analyses showed a biological activity from Delta-24-RGD in DIPG. Further advanced trials are needed to validate these results. Meanwhile, plenty of opportunities to increase the potential contribution of oncolytic viruses in the management of devastating tumors with no current effective treatment such as DIPG need to be explored and exploited

Viroinmunoterapia para el tumor rabdoide/ teratoide atípico (AT/RT): caracterización del efecto antitumoral del adenovirus DELTA-24-RGD

El tumor AT/RT es considerado un tumor raro y de los más agresivos del SNC que afecta a pacientes pediátricos. El pronóstico de estos niños sigue siendo malo, sobre todo para los pacientes menores de 3 años, donde los tratamientos convencionales (radioterapia, cirugía y quimioterapia) se ven limitados por las graves secuelas que pueden llegar a producirse. De esta manera, la mediana de supervivencia de los niños afectados por AT/RT no supera los 10 meses. Este trabajo se centra en caracterizar el efecto del adenovirus oncolítico Delta-24-RGD como nueva herramienta terapéutica para el tratamiento de los AT/RT. El objetivo final es encontrar estrategias alternativas eficaces y seguras para estos pacientes que, a día de hoy, no tienen opciones terapéuticas viables. El Delta-24-RGD (DNX-2401 en clínica) es un adenovirus oncolítico diseñado específicamente para infectar, replicar y eliminar directamente las células cancerosas, así como para provocar una respuesta inmunitaria antitumoral más amplia. Basándonos en los prometedores datos clínicos de DNX-2401 en glioblastoma adulto y en glioma pontino intrínseco difuso pediátrico (DIPG), evaluamos su actividad en AT/RT. Estos datos preclínicos demuestran la eficacia in vivo de DNX-2401 en modelos establecidos que recapitulan características importantes de estos tumores, incluyendo modelos de lesiones diseminadas de AT/RT y en ratones inmunocompetentes humanizados. Los datos subrayan el potencial terapéutico de DNX-2401 y proporcionan una base sólida para su traslado al ámbito clínico para estas indicaciones

Generación de una adenovirus Ad5/52s pseudotipado con la proteína fiber corta del Ad52 para su caracterización in vitro e in vivo como vector de terapia génica

La terapia génica consiste en la manipulación y utilización de material genético para el tratamiento de patologías. No obstante, esta estrategia requiere el uso de vectores de terapia génica para transportar el material genético al tejido diana de forma eficiente. Los vectores de terapia génica más comunes son los basados en el adenovirus humano de serotipo 5 (Ad5), porque, respecto a otros serotipos, el Ad5 tiene como ventaja su bioseguridad y facilidad de producción. Sin embargo, para que una célula sea transducida por el Ad5 ha de expresar un receptor reconocible por el Ad5, principalmente la proteína CAR, al que se une a través de su proteína fiber. El fiber de otros serotipos de adenovirus se une a receptores diferentes, permitiendo así la transducción de tipos celulares alternativos a los transducidos por el Ad5. De esta manera, una de las estrategias utilizadas para obtener un vector Ad5 con el tropismo de un serotipo diferente, es la pseudotipación del adenovirus, que consiste en substituir el fiber del Ad5 por el de otro serotipo. De entre los serotipos de adenovirus humanos, uno de los más recientemente descritos es el Ad52. Este serotipo contiene dos fiber de diferentes longitudes, lo cual es un rasgo compartido con los adenovirus humanos intestinales de la especie F. Como el Ad52 fue identificado a partir de muestras de heces de pacientes con gastroenteritis, se le atribuye también un tropismo intestinal, aunque no se ha podido demostrar. En el caso de los adenovirus de la especie F, su fiber largo reconoce el receptor CAR y, por lo tanto, se asume que su tropismo intestinal es mediado por el fiber corto. Dada la cercanía evolutiva entre el Ad52 y los adenovirus de la especie F, se asume que su fiber largo también reconocerá el receptor CAR, mientras que el fiber corto lo hará a otro receptor, dándole así al virus un tropismo independiente de CAR. Así pues, en esta tesis se ha generado un vector quimérico Ad5 pseudotipado con el fiber corto del Ad52 (Ad5/52s) con la finalidad de estudiar el rol específico de esta proteína fiber y, a la vez, caracterizar este adenovirus como vector de terapia génica. La primera parte de la tesis consiste en la generación del genoma del adenovirus Ad5/52s y la producción del vector, lo que incluye un estudio de su ciclo viral para optimizar la producción. El resultado del ciclo viral indica que éste se encuentra moderadamente retrasado respecto al del Ad5. Una vez producidos los vectores, se comparó su tropismo con el del Ad5 en cultivos in vitro, demostrando que la substitución del fiber comporta un cambio en el tropismo del Ad5 hacia líneas celulares, así como también aumenta su transducción en células de Schwann primarias, aunque no aumenta el tropismo del Ad5 en modelos intestinales in vitro. Ya caracterizado el virus in vitro, se demostró que el fiber corto del Ad52 no tiene afinidad por el receptor CAR, y su unión al receptor es dependiente del dominio knob del fiber. Posteriormente, el estudio comparativo de su distribución in vivo por administración intravenosa respecto al Ad5, mostró que el Ad5/52s tan solo transduce ligeramente pulmón a pesar de no ser secuestrado en hígado. Una vez descartados problemas de estabilidad, se observó que la baja eficiencia de infección del Ad5/52s se debe a una mayor inactivación de este virus en sangre, probablemente por la interacción con algún factor plasmático como la trombina. Finalmente, se modificó el fiber corto para limitar esta interacción, lo que permitió aumentar la supervivencia del Ad5/52s en sangre.Gene therapy is a biomedical approach, which consists of manipulating and delivering genes to treat a wide range of diseases. Nevertheless, a successful treatment also requires a vector to carry the therapeutic gene towards the targeted tissue and thus to increase itstransduction efficiency. Human adenovirus 5 derived vectors (Ad5) are the most commonly usedin gene therapy strategies due totheir higher biosafety and productivity. However, the Ad5 mediated transduction is restricted to those cells expressing appropriate viral receptors (mainly the CAR protein), recognized by the fiber protein. The fiber of other adenovirus serotypes binds to different receptors, allowing transduction of alternative cell types of those transduced by Ad5. Thus, one strategy to modify the natural Ad5 tropism is adenovirus pseudotyping, which consists of Ad5 fiber replacement by the fiber protein of another serotype. Among the different human adenovirus serotypes, one of the most recently described is Ad52. This serotype contains two fiber of different lengths, which is a common trait shared with enteric Ad40 and Ad41 human adenovirus. The Ad52 was initially found in stool samples obtained from patients with gastroenteritis and, as a consequence, it is supposed to be also an enteric virus, although it has not been demonstrated yet. In the specie F adenoviruses the long fiber recognizes the CAR receptor, and therefore, their enteric tropism is assumed to be mediated by the short fiber. Given the evolutionary relationship between the Ad52 and the species F adenoviruses, it is assumed that its long fiber also recognizes the CAR receptor, while the short fiber will recognize a different receptor, thus giving the virus a CAR-independent tropism. So, in this thesis we have generated a chimeric Ad5 vector pseudotyped with the Ad52 short fiber (Ad5/52s) protein, in order to study the specific role of the fiber protein, in turn, characterize this adenovirus as a vector for gene therapy. The first part of the thesis consists in the generation of adenovirus Ad5/52s genome and its production, which includes a study of its viral cycle to optimize production. The results show that the Ad5/52s viral cycle is moderately delayed compared to Ad5. Once the vectors were produced, the tropism of Ad5/52s and Ad5 were compared in in vitro, demonstrating that fiber replacement induces a switch in the Ad5 tropism in different cell lines, as well as an increased transduction in primary Schwann cells, but not in intestinal models in vitro. Then, it was also demonstrated that short fiber from Ad52 doesn't recognizes the CAR receptor, and also that its binding is mediated by the fiber knob domain. Further studies to compare Ad5 versus Ad5/52 in vivo biodistribution after intravenous administration showed only a slight transduction in lung with Ad5/52s, despite not being trapped in liver. Once discarded stability problems, it was observed that the low levels of transduction achieved by the Ad5/52s were caused by a faster inactivation in plasma, probably by an interaction with a plasmatic factor, such as thrombin. Finally, to minimize this interaction, the short fiber was mutated, which enhanced the Ad5/52s survival in blood