Ishiguro, Kaori

By exploiting their biological functions, the use of biological nanoparticles such as extracellular vesicles can provide an efficient and effective approach for hepatic delivery of RNA‐based therapeutics for the treatment of liver cancers such as hepatocellular cancer (HCC). Targeting liver cancer stem cells (LCSC) within HCC provide an untapped opportunity to improve outcomes by enhancing therapeutic responses. Cells with tumor‐initiating capabilities such as LCSC can be identified by expression of markers such as epithelial cell adhesion molecule (EpCAM) on their cell surface. EpCAM is a target of Wnt/β‐catenin signaling, a fundamental pathway in stem‐cell growth. Moreover, mutations in the β‐catenin gene are frequently observed in HCC and can be associated with constitutive activation of the Wnt/β‐catenin pathway. However, targeting these pathways for the treatment of HCC has been challenging. Using RNA nanotechnology, we developed engineered biological nanoparticles capable of specific and effective delivery of RNA therapeutics targeting β‐catenin to LCSC. Extracellular vesicles isolated from milk were loaded with small interfering RNA to β‐catenin and decorated with RNA scaffolds to incorporate RNA aptamers capable of binding to EpCAM. Cellular uptake of these EpCAM‐targeting therapeutic milk‐derived nanovesicles in vitro resulted in loss of β‐catenin expression and decreased proliferation. The uptake and therapeutic efficacy of these engineered biological nanotherapeutics was demonstrated in vivo using tumor xenograft mouse models. Conclusion: β‐catenin can be targeted directly to control the proliferation of hepatic cancer stem cells using small interfering RNA delivered using target‐specific biological nanoparticles. Application of this RNA nanotechnology–based approach to engineer biological nanotherapeutics provides a platform for developing cell‐surface molecule–directed targeted therapeutics.

AbstractExtracellular vesicles are endogenous biological nanoparticles that have potential for use as therapeutic nanoparticles or as delivery vehicles for therapeutic agents. Milk nanovesicles (MNV) are extracellular vesicles isolated from bovine milk that have been explored for use as delivery vehicles for RNA therapeutics such as small interfering RNA (siRNA). We performed in vivo toxicological studies of MNV or therapeutic MNV (tMNV) loaded with siRNA as a prelude to their clinical use. Development toxicity was assessed in zebrafish embryos. Acute toxicity was assessed in both mice and zebrafish whereas safety, biochemical, histological and immune effects after multiple dosing were assessed in mice. Zebrafish embryo hatching was accelerated with MNV and tMNV. While acute toxicity or effects on mortality were not observed in zebrafish, developmental effects were observed at high concentrations of MNV. There was a lack of discernable toxicity, mortality and systemic inflammatory or immunological responses in mice following administration of either MNVs or tMNVs. The tolerability and lack of discernable developmental or systemic in vivo toxicity support their use as biological nano‐therapeutics. Adoption of a standardized protocol for systematic analysis of in vivo safety and toxicity will facilitate preclinical assessment of EV based formulations for therapeutic use.

Hepatocellular carcinoma (HCC) is a leading cause of cancer‐related death worldwide. Although HCC can respond to immune checkpoint inhibitors, such as monoclonal antibodies against programmed death 1 (PD‐1), many patients fail to respond or develop secondary resistance. Activation of Wnt/β‐catenin signaling can contribute to immune evasion. Mutations in β‐catenin are among the most frequent mutations associated with HCC. Thus, our aim was to directly target β‐catenin to enhance the therapeutic response to immune checkpoint inhibition. A synthetic transgenic mouse model of experimental HCC induced by tyrosine‐protein kinase Met/β‐catenin expression and extracellular vesicles (EVs) as a therapeutic delivery agent was used to evaluate the efficacy of directly targeting β‐catenin on the response to anti‐PD‐1. These studies showed that (1) oncogenic β‐catenin could be therapeutically targeted using a biological nanoparticle‐based delivery approach, (2) targeting β‐catenin using small interfering RNA (siRNA) delivered within EVs can reduce tumor growth, and (3) the therapeutic response to anti‐PD‐1 can be enhanced by concomitantly targeting β‐catenin using therapeutic EVs. These preclinical studies establish the efficacy of the use of biological nanoparticles as an endogenous delivery vehicle for therapeutic RNA delivery and support the use of therapeutic strategies targeting tumor‐intrinsic β‐catenin as an adjunct to anti‐PD‐1‐based therapy.Conclusion:Combination therapy with anti‐PD‐1 and β‐catenin siRNA delivered using biological nanoparticles provides an effective strategy for the treatment of HCC. This strategy could be further exploited into targeted approaches for immune potentiation by countering oncogene‐mediated resistance to immunotherapies.

Aberrant activation of β-catenin signaling is frequently observed in hepatocellular cancer. Although Wnt/β-catenin signaling can be targeted by vitamin D, therapeutic use of vitamin D for this purpose is not currently established. We evaluated the therapeutic use of vitamin D or its analogs using a synthetic transgenic mouse of hepatocarcinogenesis induced by mutant β-catenin, and MET overexpression in which 75% of mice develop well-differentiated HCC within 8 weeks in the absence of fibrosis. Vitamin D receptor expression was similar in both tumoral and nontumoral tissue. There was no significant difference in overall survival, or in tumor progression assessed by imaging, biochemical, or tumor cell burden assessments in mice receiving a vitamin D-supplemented diet containing 12.0 IU VD/g (HVD) compared with a standard diet (SD) containing 2.3 IU VD/g. Furthermore, systemic treatment with calcitriol [vitamin D analog 1α,25(OH) 2 D 3 ] or EB1089 (synthetic vitamin D analog) by intraperitoneal injection for 4 weeks prolonged median survival but did not increase overall survival compared with controls. Although tumor formation was delayed in males compared with that in females, there was no difference in overall survival between males and females. In conclusion, although 1α,25(OH) 2 D 3 is reported to inhibit β-catenin signaling, as well as proliferation, migration, and differentiation in cancer cells, neither dietary supplementation with vitamin D nor treatment with vitamin D analogs altered the formation or growth of HCC associated with β-catenin activation. These results conclusively demonstrate the lack of utility of targeting vitamin D for therapy of HCC in this setting.