In vivo engineering of lymphocytes after systemic exosome-associated AAV delivery.

Ex-vivo gene therapy using stem cells or T cells transduced by retroviral or lentiviral vectors has shown remarkable efficacy in the treatment of immunodeficiencies and cancer. However, the process is expensive, technically challenging, and not readily scalable to large patient populations, particularly in underdeveloped parts of the world. Direct in vivo gene therapy would avoid these issues, and such approaches with adeno-associated virus (AAV) vectors have been shown to be safe and efficacious in clinical trials for diseases affecting differentiated tissues such as the liver and CNS. However, the ability to transduce lymphocytes with AAV in vivo after systemic delivery has not been carefully explored. Here, we show that both standard and exosome-associated preparations of AAV8 vectors can effectively transduce a variety of immune cell populations including CD4+ T cells, CD8+ T cells, B cells, macrophages, and dendritic cells after systemic delivery in mice. We provide direct evidence of T cell transduction through the detection of AAV genomes and transgene mRNA, and show that intracellular and transmembrane proteins can be expressed. These findings establish the feasibility of AAV-mediated in vivo gene delivery to immune cells which will facilitate both basic and applied research towards the goal of direct in vivo gene immunotherapies.

Virus vector-mediated genetic modification of brain tumor stromal cells after intravenous delivery.

The malignant primary brain tumor, glioblastoma (GBM) is generally incurable. New approaches are desperately needed. Adeno-associated virus (AAV) vector-mediated delivery of anti-tumor transgenes is a promising strategy, however direct injection leads to focal transgene spread in tumor and rapid tumor division dilutes out the extra-chromosomal AAV genome, limiting duration of transgene expression. Intravenous (IV) injection gives widespread distribution of AAV in normal brain, however poor transgene expression in tumor, and high expression in non-target cells which may lead to ineffective therapy and high toxicity, respectively. Delivery of transgenes encoding secreted, anti-tumor proteins to tumor stromal cells may provide a more stable and localized reservoir of therapy as they are more differentiated than fast-dividing tumor cells. Reactive astrocytes and tumor-associated macrophage/microglia (TAMs) are stromal cells that comprise a large portion of the tumor mass and are associated with tumorigenesis. In mouse models of GBM, we used IV delivery of exosome-associated AAV vectors driving green fluorescent protein expression by specific promoters (NF-κB-responsive promoter and a truncated glial fibrillary acidic protein promoter), to obtain targeted transduction of TAMs and reactive astrocytes, respectively, while avoiding transgene expression in the periphery. We used our approach to express the potent, yet toxic anti-tumor cytokine, interferon beta, in tumor stroma of a mouse model of GBM, and achieved a modest, yet significant enhancement in survival compared to controls. Noninvasive genetic modification of tumor microenvironment represents a promising approach for therapy against cancers. Additionally, the vectors described here may facilitate basic research in the study of tumor stromal cells in situ.