RESUMO
Chimeric antigen receptor (CAR) T cells are engineered cells used in cancer therapy and are studied to treat infectious diseases. Trafficking and persistence of CAR T cells is an important requirement for efficacy to target cancer. Here, we describe a CAR RNA FISH histo-cytometry platform combined with a random reaction seed image analysis algorithm to quantitate spatial distribution and in vivo functional activity of a CAR T cell population at a single cell resolution for preclinical models. In situ, CAR T cell exhibited a heterogenous effector gene expression and this was related to the distance from tumor cells, allowing a quantitative assessment of the potential in vivo effectiveness. The platform offers the potential to study immune functions of genetically engineered cells in situ with their target cells in tissues with high statistical power and thus, can serve as an important tool for preclinical assessment of CAR T cell effectiveness.
Assuntos
Heterogeneidade Genética , Hibridização in Situ Fluorescente , RNA/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Linfócitos T/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Imunofluorescência , Humanos , Imuno-Histoquímica , Imunofenotipagem , Imunoterapia Adotiva , Hibridização in Situ Fluorescente/métodos , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/terapia , Especificidade de Órgãos/genética , Reação em Cadeia da Polimerase , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Chronic hepatitis B virus (HBV) infection is a major public health problem. New treatment approaches are needed because current treatments do not target covalently closed circular DNA (cccDNA), the template for HBV replication, and rarely clear the virus. We harnessed adeno-associated virus (AAV) vectors and CRISPR-Staphylococcus aureus (Sa)Cas9 to edit the HBV genome in liver-humanized FRG mice chronically infected with HBV and receiving entecavir. Gene editing was detected in livers of five of eight HBV-specific AAV-SaCas9-treated mice, but not control mice, and mice with detectable HBV gene editing showed higher levels of SaCas9 delivery to HBV+ human hepatocytes than those without gene editing. HBV-specific AAV-SaCas9 therapy significantly improved survival of human hepatocytes, showed a trend toward decreasing total liver HBV DNA and cccDNA, and was well tolerated. This work provides evidence for the feasibility and safety of in vivo gene editing for chronic HBV infections, and it suggests that with further optimization, this approach may offer a plausible way to treat or even cure chronic HBV infections.