Extracellular vesicles (EVs) are under evaluation as therapeutics or as vehicles for drug delivery. Preclinical studies of EVs often use mice or other animal models to assess efficacy and disposition. However, as most EVs under evaluation are derived from human cells, they may elicit immune responses which may contribute to toxicities or enhanced EV clearance. Furthermore, EVs from different cell sources or EVs comprising various cargo may differ with respect to immunogenicity or toxicity. To assess EV-induced immune response and toxicity, we dosed C57BL/6 mice with EVs intravenously and intraperitoneally for 3 weeks. EVs were harvested from wild type or engineered HEK293T cells which were modified to produce EVs loaded with miR-199a-3p and chimeric proteins. Blood was collected to assess hematology, blood chemistry, and immune markers. Spleen cells were immunophenotyped, and tissues were harvested for gross necropsy and histopathological examination. No signs of toxicity were observed, and minimal evidence of changes in immune markers were noted in mice dosed with engineered, but not with wild type EVs. This study provides a framework for assessment of immunogenicity and toxicity that will be required as EVs from varying cell sources are tested within numerous animal models and eventually in humans.

Persistent HPV16 infection is a major cause of the global cancer burden. The viral life cycle is dependent on the differentiation program of stratified squamous epithelium, but the landscape of keratinocyte subpopulations which support distinct phases of the viral life cycle has yet to be elucidated. Here, single cell RNA sequencing of HPV16 infected compared to uninfected organoids identifies twelve distinct keratinocyte populations, with a subset mapped to reconstruct their respective 3D geography in stratified squamous epithelium. Instead of conventional terminally differentiated cells, an HPV-reprogrammed keratinocyte subpopulation (HIDDEN cells) forms the surface compartment and requires overexpression of the ELF3/ESE-1 transcription factor. HIDDEN cells are detected throughout stages of human carcinogenesis including primary human cervical intraepithelial neoplasias and HPV positive head and neck cancers, and a possible role in promoting viral carcinogenesis is supported by TCGA analyses. Single cell transcriptome information on HPV-infected versus uninfected epithelium will enable broader studies of the role of individual keratinocyte subpopulations in tumor virus infection and cancer evolution.

Extracellular vesicles (EVs) hold great potential as novel systems for nucleic acid delivery due to their natural composition. Our goal was to load EVs with microRNA that are synthesized by the cells that produce the EVs. HEK293T cells were engineered to produce EVs expressing a lysosomal associated membrane, Lamp2a fusion protein. The gene encoding pre-miR-199a was inserted into an artificial intron of the Lamp2a fusion protein. The TAT peptide/HIV-1 transactivation response (TAR) RNA interacting peptide was exploited to enhance the EV loading of the pre-miR-199a containing a modified TAR RNA loop. Computational modeling demonstrated a stable interaction between the modified pre-miR-199a loop and TAT peptide. EMSA gel shift, recombinant Dicer processing and luciferase binding assays confirmed the binding, processing and functionality of the modified pre-miR-199a. The TAT-TAR interaction enhanced the loading of the miR-199a into EVs by 65-fold. Endogenously loaded EVs were ineffective at delivering active miR-199a-3p therapeutic to recipient SK-Hep1 cells. While the low degree of miRNA loading into EVs through this approach resulted in inefficient distribution of RNA cargo into recipient cells, the TAT TAR strategy to load miRNA into EVs may be valuable in other drug delivery approaches involving miRNA mimics or other hairpin containing RNAs.