RESUMEN
Trastuzumab improves overall survival for HER2+ breast cancer, but its short half-life in the cerebrospinal fluid (~2-4 days) and delivery limitations restrict the ability to target HER2+ central nervous system (CNS) disease. We developed an adeno-associated virus (AAV) vector expressing a codon-optimized, ubiquitin C (UbC)-promoter-driven trastuzumab sequence (AAV9.UbC.trastuzumab) for intrathecal administration. Transgene expression was evaluated in adult Rag1 knockout mice and rhesus nonhuman primates (NHPs) after a single intracerebroventricular (ICV) or intra-cisterna magna (ICM) AAV9.UbC.trastuzumab injection, respectively, using real-time PCR, ELISA, Western blot, in situ hybridization, single-nucleus RNA sequencing, and liquid chromatography-mass spectrometry; antitumor efficacy was evaluated in brain xenografts using HER2+ breast cancer cell lines (BT-474, MDA-MB-453). Transgene expression was detected in brain homogenates of Rag1 knockout mice following a single ICV injection of AAV9.UbC.trastuzumab (1 × 1011 vector genome copies [GC]/mouse) and tumor progression was inhibited in xenograft models of breast-to-brain metastasis. In NHPs, ICM delivery of AAV9.UbC.trastuzumab (3 × 1013 GC/animal) was well tolerated (36-37 days in-life) and resulted in transgene expression in CNS tissues and cerebrospinal fluid at levels sufficient to induce complete tumor remission in MDA-MB-453 brain xenografts. With AAV9's proven clinical safety record, this gene therapy may represent a viable approach for targeting HER2 + CNS malignancies.
Asunto(s)
Neoplasias Encefálicas , Dependovirus , Receptor ErbB-2 , Trastuzumab , Trastuzumab/administración & dosificación , Trastuzumab/farmacología , Trastuzumab/uso terapéutico , Dependovirus/genética , Animales , Humanos , Ratones , Receptor ErbB-2/metabolismo , Receptor ErbB-2/genética , Femenino , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/secundario , Neoplasias Encefálicas/patología , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Neoplasias de la Mama/genética , Neoplasias de la Mama/tratamiento farmacológico , Ratones Noqueados , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Macaca mulatta , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico , Antineoplásicos Inmunológicos/administración & dosificación , Ensayos Antitumor por Modelo de Xenoinjerto , Sistema Nervioso Central/metabolismo , Línea Celular TumoralRESUMEN
For successful vector-based gene therapy manufacturing, the selected adeno-associated virus (AAV) vector production system must produce vector at sufficient scale. However, concerns have arisen regarding the quality of vector produced using different systems. In this study, we compared AAV serotypes 1, 8, and 9 produced by two different systems (Sf9/baculovirus and HEK293/transfection) and purified by two separate processes. We evaluated capsid properties, including protein composition, post-translational modification, particle content profiles, and in vitro and in vivo vector potency. Vectors produced in the Sf9/baculovirus system displayed reduced incorporation of viral protein 1 and 2 into the capsid, increased capsid protein deamidation, increased empty and partially packaged particles in vector preparations, and an overall reduced potency. The differences observed were largely independent of the harvest method and purification process. These findings illustrate the need for careful consideration when choosing an AAV vector production system for clinical production.
Asunto(s)
Proteínas de la Cápside , Cápside , Humanos , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Cápside/metabolismo , Células HEK293 , Vectores Genéticos/genética , Dependovirus/genética , Dependovirus/metabolismoRESUMEN
Epilepsy is characterized by spontaneous non-provoked seizures, yet the mechanisms that trigger a seizure and allow its evolution remain underexplored. To dissect out phases of ictogenesis, we evoked hypersynchronous activity with optogenetic stimulation. Focal optogenetic activation of putative excitatory neurons in the mouse hippocampal CA1 reliably evoked convulsive seizures in awake mice. A time-vs-time pulsogram plot characterized the evolution of the EEG pulse response from a light evoked response to induced seizure activity. Our results depict ictogenesis as a stepwise process comprised of three distinctive phases demarcated by two transition points. The induction phase undergoes the first transition to reverberant phase activity, followed by the second transition into the paroxysmal phase or a seizure. Non-seizure responses are confined to either induction or reverberant phases. The pulsogram was then constructed in seizures recorded from a murine model of temporal lobe epilepsy and it depicted a similar reverberance preceding spontaneous seizures. The discovery of these distinct phases of ictogenesis may offer means to abort a seizure before it develops.