Systemic administration of checkpoint inhibitors alone and especially concurrent with intratumoral administration of oncolytic herpesviruses (oHSV) has a major impact on cancer therapy marred by rare failures of healthy organs.Furthermore, tumors vary with respect to susceptibility to oncolytic effects of oHSV. Here we report the construction and properties of 3 families of oncolytic herpes simplex viruses expressing no immunomodulatory genes (T1 series), murine IL-12 (T2 series) or murine or human IL-12 and anti PD-1 antibody (T3 series). We report that insertion of the gene encoding PD-1 Ab significantly augmented the oncolytic activity of oHSV bereft of immunostimulatory genes (T1 series) or expressing IL-12 alone (T2 series). The T3 oHSV expressed IL-12, PD-1 Ab were restricted to the tumor bed whereas the induced IFN-γ accumulated to high levels both in tumor bed and in blood. Furthermore, the T3 oHSV was superior to systemic administration of IL-12 and antibody to PD-1. This report also shows that the oncolytic activity of T3 oHSV in a relatively resistant tumor was enhanced by concurrent intratumoral administration of genetically engineered exosomes carrying miRNA targeting CTLA-4 checkpoint.
hnRNPA2B1, an abundant cellular protein, has been reported to recruit RNAs bearing a specific sequence (EXO motif) into exosomes. We characterized an exosome population averaging 100 ± 50 nm in diameter and containing a defined set of constitutive exosome markers. This population packages microRNAs (miRNAs) and can be directed to block targeted gene expression in a dose-dependent fashion. The objective of this study was to characterize the role of hnRNPA2B1 in the recruitment of miRNA. We report the following four key findings. (i) hnRNPA2B1 is not a component of exosomes produced in HEp-2 or HEK293T cells. Hence, hnRNPA2B1 carries its cargo, at most, to the site of exosome assembly, but it is not itself incorporated into exosomes. (ii) The accumulation of exosomes produced by cells in which the gene encoding hnRNPA2B1 has been knocked out (ΔhnRNPA2B1 cells) was reduced 3-fold. (iii) In uninfected HEp-2 cells, hnRNPA2B1 is localized in the nucleus. In cells infected with herpes simplex virus 1 (HSV-1), hnRNPA2B1 was quantitatively exported to the cytoplasm and at least a fraction of hnRNPA2B1 colocalized with a Golgi marker. (iv) Lastly, in ΔhnRNPA2B1 cells, there was a 2- to 3-fold reduction in virus yield but a significant (>10-fold) reduction in HSV-1 released through the apical surface into the extracellular environment. The absence of hnRNPA2B1 had no significant impact on the basolateral export of HSV-1 from infected to uninfected cells by direct cell-to-cell contact. The results suggest that hnRNPA2B1 plays a key role in the transport of enveloped virus from its site of assembly to the extracellular environment. IMPORTANCE In this report, we show that hnRNPA2B1 is not a component of exosomes produced in HEp-2 or HEK293T cells. In herpes simplex virus 1 (HSV-1)-infected cells, hnRNPA2B1 was quantitatively translocated from the nucleus into the cytoplasm. In infected ΔhnRNPA2B1 cells, Golgi-dependent transport of virus from the apical surface to the extracellular medium was significantly reduced. In essence, this report supports the hypothesis that hnRNPA2B1 plays a key role in the egress of exosomes and HSV-1 from infected cells.
Background Exosomes are small, cellular membrane-derived vesicles with a diameter of 50–150 nm. Exosomes are considered ideal drug delivery systems with a wide range of applications in various diseases, including cancer. However, nonspecific delivery of therapeutic agents by exosomes in vivo remains challenging. Human epidermal growth factor receptor 2 (HER2) is an epidermal growth factor receptor tyrosine kinase, and its overexpression is usually associated with cell survival and tumor progression in various cancers. In this study, we aim to develop novel exosomes with dual HER2-targeting ability as a nanoparticle delivery vehicle to enhance antitumor efficacy in vivo. Results Here, we report the generation of two kinds of exosomes carrying miRNAs designed to block HER2 synthesis, which consequently showed a distinct anti-tumor effect. The 293-miR-HER2 exosomes package and deliver miRNAs targeting HER2 to recipient cells to block HER2 synthesis. The anti-tumor effect of these exosomes on cancer cells dependent on HER2 for survival but do not affect cells that lack HER2 or that are engineered to express HER2 but are not dependent on it for survival. In contrast, 293-miR-XS-HER2 exosomes carry an additional peptide, which enables them to adhere to HER2 on the surface of cancer cells. Consequently, these exosomes preferentially enter these cells with surface expression of HER2 and further displayed a tumoricidal effect. The 293-miR-XS-HER2 exosomes are significantly more effective than the 293-miR-HER2 exosomes in shrinking HER2-positive tumors implanted in mice. Conclusions Collectively, as novel antitumor drug delivery vehicles, HER2 dual-targeting exosomes exhibit increased target-specific delivery efficiency and can be further utilized to develop new nanoparticle-based targeted therapies.
Background: Exosomes are small, cellular membrane-derived vesicles with a diameter of 50-150 nm. Exosomes are considered ideal drug delivery systems with a wide range of applications in various diseases, including cancer. However, nonspecific delivery of therapeutic agents by exosomes in vivo remains challenging. Human epidermal growth factor receptor 2 (HER2) is an epidermal growth factor receptor tyrosine kinase, and its overexpression is usually associated with cell survival and tumor progression in various cancers. In this study, we aim to develop novel exosomes with dual HER2-targeting ability as a nanoparticle delivery vehicle to enhance antitumor efficacy in vivo.Results: Here, we report the generation of two kinds of exosomes carrying miRNAs designed to block HER2 synthesis, which consequently showed a distinct anti-tumor effect. The 293-miR-HER2 exosomes package and deliver miRNAs targeting HER2 to recipient cells to block HER2 synthesis. The anti-tumor effect of these exosomes on cancer cells dependent on HER2 for survival but do not affect cells that lack HER2 or that are engineered to express HER2 but are not dependent on it for survival. In contrast, 293-miR-XS-HER2 exosomes carry an additional peptide, which enables them to adhere to HER2 on the surface of cancer cells. Consequently, these exosomes preferentially enter these cells with surface expression of HER2 and further displayed a tumoricidal effect. The 293-miR-XS-HER2 exosomes are significantly more effective than the 293-miR-HER2 exosomes in shrinking HER2-positive tumors implanted in mice.Conclusions: Collectively, as novel antitumor drug delivery vehicles, HER2 dual-targeting exosomes exhibit increased target-specific delivery efficiency and can be further utilized to develop new nanoparticle-based targeted therapies.
Among 29 distinct miRNAs expressed by the herpes simplex virus-1 (HSV-1) during lytic infection, miR-H11, together with miR-H1 to miR-H8 are reported to locate in the RNAinduced silencing complex (RISC). miR-H11 is encoded within viral origins of replication and lies entirely within the origins of replication. However, the roles of this miRNA derived from lytic infection with HSV-1 remain unclear. Using the advantage of vaccinia virus protein VP55 (VP55)-mediated degradation of miRNAs, we constructed a recombinant virus expressing VP55 (R5502) to demonstrate that: (1) accumulation of miR-H11 from R5502 was reduced by 540-fold versus that in cells infected with wild-type HSV-1, but miR-H1 to miR-H8 which also located in the RISC were not reduced significantly from R5502 compare with wild-type HSV-1; (2) downregulation of miR-H11 from R5502 infected cells results in markedly lower viral DNA synthesis compared with wild-type HSV-1; and (3) downregulation of miR-H11 also restricted viral spreading, and resulted in low accumulation of representative viral proteins and viral yields. The findings were confirmed through either using of a miR-H11 inhibitor or pre-transfection of a plasmid expressing VP55. These data suggest that miR-H11 plays a currently unidentified role in maintaining sufficient viral DNA synthesis during the course of viral infection.
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