Adeno-associated virus-mediated trastuzumab delivery to the central nervous system for human epidermal growth factor receptor 2+ brain metastasis.

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.

The Dynamic Chromatin Architecture of the Regenerating Liver.

BACKGROUND & AIMS: The adult liver is the main detoxification organ and routinely is exposed to environmental insults but retains the ability to restore its mass and function upon tissue damage. However, extensive injury can lead to liver failure, and chronic injury causes fibrosis, cirrhosis, and hepatocellular carcinoma. Currently, the transcriptional regulation of organ repair in the adult liver is incompletely understood. METHODS: We isolated nuclei from quiescent as well as repopulating hepatocytes in a mouse model of hereditary tyrosinemia, which recapitulates the injury and repopulation seen in toxic liver injury in human beings. We then performed the assay for transposase accessible chromatin with high-throughput sequencing specifically in repopulating hepatocytes to identify differentially accessible chromatin regions and nucleosome positioning. In addition, we used motif analysis to predict differential transcription factor occupancy and validated the in silico results with chromatin immunoprecipitation followed by sequencing for hepatocyte nuclear factor 4α (HNF4α) and CCCTC-binding factor (CTCF). RESULTS: Chromatin accessibility in repopulating hepatocytes was increased in the regulatory regions of genes promoting proliferation and decreased in the regulatory regions of genes involved in metabolism. The epigenetic changes at promoters and liver enhancers correspond with the regulation of gene expression, with enhancers of many liver function genes showing a less accessible state during the regenerative process. Moreover, increased CTCF occupancy at promoters and decreased HNF4α binding at enhancers implicate these factors as key drivers of the transcriptomic changes in replicating hepatocytes that enable liver repopulation. CONCLUSIONS: Our analysis of hepatocyte-specific epigenomic changes during liver repopulation identified CTCF and HNF4α as key regulators of hepatocyte proliferation and regulation of metabolic programs. Thus, liver repopulation in the setting of toxic injury makes use of both general transcription factors (CTCF) for promoter activation, and reduced binding by a hepatocyte-enriched factor (HNF4α) to temporarily limit enhancer activity. All sequencing data in this study were deposited to the Gene Expression Omnibus database and can be downloaded with accession number GSE109466.