RESUMEN
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
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.