RESUMEN
The therapeutic effectiveness of oncolytic viruses (OVs) delivered intravenously is limited by the development of neutralizing antibody responses against the virus. To circumvent this limitation and to enable repeated systemic administration of OVs, here we develop Synthetic RNA viruses consisting of a viral RNA genome (vRNA) formulated within lipid nanoparticles. For two Synthetic RNA virus drug candidates, Seneca Valley virus (SVV) and Coxsackievirus A21, we demonstrate vRNA delivery and replication, virus assembly, spread and lysis of tumor cells leading to potent anti-tumor efficacy, even in the presence of OV neutralizing antibodies in the bloodstream. Synthetic-SVV replication in tumors promotes immune cell infiltration, remodeling of the tumor microenvironment, and enhances the activity of anti-PD-1 checkpoint inhibitor. In mouse and non-human primates, Synthetic-SVV is well tolerated reaching exposure well above the requirement for anti-tumor activity. Altogether, the Synthetic RNA virus platform provides an approach that enables repeat intravenous administration of viral immunotherapy.
Asunto(s)
Neoplasias , Viroterapia Oncolítica , Virus Oncolíticos , Picornaviridae , Animales , Anticuerpos Neutralizantes , Inmunoterapia , Liposomas , Ratones , Nanopartículas , Neoplasias/terapia , Virus Oncolíticos/genética , ARN Viral/genética , Microambiente TumoralRESUMEN
Development of next-generation oncolytic viruses requires the design of vectors that are potently oncolytic, immunogenic in human tumors, and well tolerated in patients. Starting with a joint-region deleted herpes simplex virus 1 (HSV-1) to create large transgene capability, we retained a single copy of the ICP34.5 gene, introduced mutations in UL37 to inhibit retrograde axonal transport, and inserted cell-type-specific microRNA (miRNA) target cassettes in HSV-1 genes essential for replication or neurovirulence. Ten miRNA candidates highly expressed in normal tissues and with low or absent expression in malignancies were selected from a comprehensive profile of 800 miRNAs with an emphasis on protection of the nervous system. Among the genes essential for viral replication identified using a small interfering RNA (siRNA) screen, we selected ICP4, ICP27, and UL8 for miRNA attenuation where a single miRNA is sufficient to potently attenuate viral replication. Additionally, a neuron-specific miRNA target cassette was introduced to control ICP34.5 expression. This vector is resistant to type I interferon compared to ICP34.5-deleted oncolytic HSVs, and in cancer cell lines, the oncolytic activity of the modified vector is equivalent to its parental virus. In vivo, this vector potently inhibits tumor growth while being well tolerated, even at high intravenous doses, compared to parental wild-type HSV-1.
RESUMEN
Oncolytic viruses induce local tumor destruction and inflammation. Whether virotherapy can also overcome immunosuppression in noninfected tumor areas is under debate. To address this question, we have explored immunologic effects of oncolytic herpes simplex viruses (oHSVs) in a genetically engineered mouse model of isocitrate dehydrogenase (IDH) wild-type glioblastoma, the most common and most malignant primary brain tumor in adults. Our model recapitulates the genomics, the diffuse infiltrative growth pattern, and the extensive macrophage-dominant immunosuppression of human glioblastoma. Infection with an oHSV that was armed with a UL16-binding protein 3 (ULBP3) expression cassette inhibited distant tumor growth in the absence of viral spreading (abscopal effect) and yielded accumulation of activated macrophages and T cells. There was also abscopal synergism of oHSVULBP3 with anti-programmed cell death 1 (anti-PD-1) against distant, uninfected tumor areas; albeit consistent with clinical trials in patients with glioblastoma, monotherapy with anti-PD-1 was ineffective in our model. Arming oHSV with ULBP3 led to upregulation of antigen processing and presentation gene sets in myeloid cells. The cognate ULBP3 receptor NKG2D, however, is not present on myeloid cells, suggesting a noncanonical mechanism of action of ULBP3. Overall, the myeloid-dominant, anti-PD-1-sensitive abscopal effect of oHSVULBP3 warrants further investigation in patients with IDH wild-type glioblastoma.
Asunto(s)
Antineoplásicos Inmunológicos/uso terapéutico , Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Péptidos y Proteínas de Señalización Intercelular/inmunología , Viroterapia Oncolítica/métodos , Virus Oncolíticos/inmunología , Simplexvirus/inmunología , Animales , Presentación de Antígeno/genética , Antineoplásicos Inmunológicos/farmacología , Encéfalo/patología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/mortalidad , Línea Celular Tumoral , Terapia Combinada/métodos , Modelos Animales de Enfermedad , Femenino , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/inmunología , Regulación Neoplásica de la Expresión Génica/inmunología , Glioblastoma/genética , Glioblastoma/inmunología , Glioblastoma/mortalidad , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/inmunología , Estimación de Kaplan-Meier , Masculino , Ratones , Ratones Transgénicos , Virus Oncolíticos/genética , Cultivo Primario de Células , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Simplexvirus/genética , Regulación hacia ArribaRESUMEN
Gene therapy currently in development for hemoglobinopathies utilizes ex vivo lentiviral transduction of CD34+ hematopoietic stem and progenitor cells (HSPCs). A small-molecule screen identified prostaglandin E2 (PGE2) as a positive mediator of lentiviral transduction of CD34+ cells. Supplementation with PGE2 increased lentiviral vector (LVV) transduction of CD34+ cells approximately 2-fold compared to control transduction methods with no effect on cell viability. Transduction efficiency was consistently increased in primary CD34+ cells from multiple normal human donors and from patients with ß-thalassemia or sickle cell disease. Notably, PGE2 increased transduction of repopulating human HSPCs in an immune-deficient (nonobese diabetic/severe combined immunodeficiency/interleukin-2 gamma receptor null [NSG]) xenotransplantation mouse model without evidence of in vivo toxicity, lineage bias, or a de novo bias of lentiviral integration sites. These data suggest that PGE2 improves lentiviral transduction and increases vector copy number, therefore resulting in increased transgene expression. As a result, PGE2 may be useful in clinical gene therapy applications using lentivirally modified HSPCs.
Asunto(s)
Dinoprostona/metabolismo , Vectores Genéticos/genética , Células Madre Hematopoyéticas/metabolismo , Lentivirus/genética , Transducción Genética , Anemia de Células Falciformes/genética , Anemia de Células Falciformes/metabolismo , Animales , Antígenos CD34/metabolismo , Línea Celular , Biblioteca de Genes , Técnicas de Transferencia de Gen , Terapia Genética , Globinas/genética , Humanos , Antígenos Comunes de Leucocito/metabolismo , Ratones , Transgenes , Trasplante Heterólogo , Internalización del Virus , Talasemia beta/genética , Talasemia beta/metabolismoRESUMEN
A previously published clinical trial demonstrated the benefit of autologous CD34(+) cells transduced with a selfinactivating lentiviral vector (HPV569) containing an engineered ß-globin gene (ß(A-T87Q)-globin) in a subject with ß thalassemia major. This vector has been modified to increase transduction efficacy without compromising safety. In vitro analyses indicated that the changes resulted in both increased vector titers (3 to 4 fold) and increased transduction efficacy (2 to 3 fold). An in vivo study in which 58 ß-thalassemic mice were transplanted with vector- or mock-transduced syngenic bone marrow cells indicated sustained therapeutic efficacy. Secondary transplantations involving 108 recipients were performed to evaluate long-term safety. The six month study showed no hematological or biochemical toxicity. Integration site (IS) profile revealed an oligo/polyclonal hematopoietic reconstitution in the primary transplants and reduced clonality in secondary transplants. Tumor cells were detected in the secondary transplant mice in all treatment groups (including the control group), without statistical differences in the tumor incidence. Immunohistochemistry and quantitative PCR demonstrated that tumor cells were not derived from transduced donor cells. This comprehensive efficacy and safety data provided the basis for initiating two clinical trials with this second generation vector (BB305) in Europe and in the USA in patients with ß-thalassemia major and sickle cell disease.
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Anemia de Células Falciformes/terapia , Terapia Genética/métodos , Vectores Genéticos , Lentivirus/genética , Talasemia beta/terapia , Anemia de Células Falciformes/genética , Animales , Antígenos CD34/metabolismo , Biología Computacional , Daño del ADN , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Técnicas de Transferencia de Gen , Trasplante de Células Madre Hematopoyéticas , Masculino , Ratones , Ratones Endogámicos C57BL , Talasemia beta/genéticaRESUMEN
In the area of cancer treatment, immunotherapy with vaccines has suffered in the last five years, due to many clinical trial failures. One must keep in mind however, that many of the clinical trials conducted in the past decade were performed without the benefit of sound regulatory guidance or validated and compliant manufacturing processes. This has clearly been the case for patient specific, tumor cell vaccine therapy. The safety concerns that emanated within the regulatory agencies from the Somatic Cell Therapy concepts, translated to active specific immunotherapy with vaccines. Fortunately, in the past five years advances in understanding the immune system, improved design of clinical trials, improvement and compliance of manufacturing processes provided opportunities to significantly improve efficacy and safety. Clearly, the vaccine research establishment has learned the importance of not just selecting antigens but the requirement of tumor associated immunogens that can stimulate a functional immune response. Also, it has become clear that immunotherapy works best in situations of minimal residual disease. Finally, more realistic endpoints in clinical trials have been recognized and accepted by oversight review committees. This commentary describes the "trials and tribulations" of developing a patient specific, autologous tumor cell vaccine for therapy of Stage II colon cancer.
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Vacunas contra el Cáncer/uso terapéutico , Neoplasias del Colon/terapia , Inmunoterapia Activa , Adulto , Anciano , Anciano de 80 o más Años , Vacunas contra el Cáncer/efectos adversos , Neoplasias del Colon/inmunología , Neoplasias del Colon/mortalidad , Neoplasias del Colon/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estadificación de NeoplasiasRESUMEN
Gene delivery represents a revolutionary therapeutic approach with the potential for sustained protein production by the human body, leading to a convenient and effective method for systemic delivery of protein drugs. In this review, advantages of an orally administered DNA formulation, Gene Pill, are presented. Unlike previously described DNA delivery approaches, the Gene Pill enables DNA delivery in a non-invasive manner, leading to the secretion of therapeutic proteins into a patient's blood, supplanting the need for injection of therapeutic protein products. The Gene Pill also has potential for the development of oral DNA vaccination through expression of protein antigens in the gut lymphoid tissue. This approach limits the biodistribution of the delivered DNA to the gut and retains all of the safety advantages of non-viral gene delivery, including repeat dosing. Development of an oral DNA formulation involves overcoming several challenges, including depurination by low pH in the stomach, enzymatic degradation by DNases in the gut, crossing the physical barrier imposed by the mucus layer, cell entry, intracellular trafficking and nuclear uptake. The advantages of the Gene Pill technology, as well as challenges for its development, are presented in this review.
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ADN/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Drogas en Investigación/administración & dosificación , Terapia Genética/métodos , Administración Oral , Animales , Química Farmacéutica , ADN/genética , ADN/metabolismo , Drogas en Investigación/farmacocinética , Humanos , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genética , Vacunas de ADN/metabolismoRESUMEN
Adenoviral vectors are widely used to express transgenes in vitro and in vivo. A major obstacle to the generation of adenoviral vectors is the manipulation of the large (35 kb) adenoviral genome. We developed a hybrid yeast-bacteria cloning system for the creation of novel adenoviral vectors. The adenovirus 5 (Ad5) genome was cloned into a shuttle vector that contains both yeast and bacterial elements for replication and therefore functions as both a yeast artificial plasmid (YAP) and as a plasmid artificial chromosome (PAC). Any sequence can be introduced into any region of the adenoviral genome via the highly efficient homologous recombination in yeast and then these recombinants are rapidly amplified in bacteria. Adenoviral vectors are generated by introduction of the PAC into the appropriate complementing mammalian cell without the need for plaque purification. Vectors were constructed with deletions in the E1, E3, and/or E4 regions. We have generated more than 100 vectors with a number of different transgenes and regulatory elements. In addition, the YAP/PAC vector was used to capture a DNA fragment encompassing the human factor IX gene, demonstrating the utility of this system to clone and analyze genomic DNA. This novel cloning strategy allows the rapid and versatile construction of adenoviral vectors for gene expression and gene therapy applications.