MicroRNA199a-Based Post-transcriptional Detargeting of Gene Vectors for Hepatocellular Carcinoma

Abstract

A gene therapeutic platform needs to be both efficient and safe. The criterion of safety is particularly important for diseases like hepatocellular carcinoma (HCC), which develop in a background of an already compromised liver. Gene vectors can be constructed either by targeting HCC or by detargeting liver and/or other major organs. miRNA-based negative detargeting has gained considerable attention in recent times due to its effectiveness and the ease with which it can be adapted into current gene delivery vectors. In this study, we provide a proof-of-concept using miRNA199a as a negative targeting agent. We introduced vectors harboring reporters with miRNA199a binding sites in cells expressing high endogenous levels of miRNA199a and compared the reporter expression in HCC cells with low endogenous miRNA199a. We observed that the expression of reporters with miRNA199a binding sites is significantly inhibited in miRNA199a-positive cells, whereas minimal effect was observed in miRNA199a-negative HCC cells. In addition, we created a post-transcriptionally regulated suicide gene therapeutic system based on cytosine deaminase (CD)/5-fluorocytosine (5-FC) exploiting miRNA199a binding sites and observed significantly lower cell death for miRNA199a-positive cells. Furthermore, we observed a decrease in the levels of miRNA199 in 3D tumorspheres of miRNA199a-positive Hepa1-6 cells and a reduction in the inhibition of reporter expression after transfection in these 3D models when compared with 2D Hepa1-6 cells. In summary, we provide evidence of miRNA199a-based post-transcriptional detargeting with relevance to HCC gene therapy.

Journey to the Center of the Cell: Tracing the Path of AAV Transduction

“…Additionally, the field of AAV vectorology has now advanced to a stage where the capsid can be tailored to transduce specific cell types [9,10]. Capsid engineering combined with AAV genome modification using cell type-specific gene regulatory elements like enhancers/promoters for transcriptional targeting [11] and cell/tissue-specific miRNAs for post-transcriptional targeting [12,13] provides diverse tools to reduce off-target effects [14].…”

Novel Combinatorial MicroRNA-Binding Sites in AAV Vectors Synergistically Diminish Antigen Presentation and Transgene Immunity for Efficient and Stable Transduction

“…miRNA-mediated regulation of transgene expression by engineering miR-BSs in rAAV expression cassettes has proven to reduce transgene-specific immune responses. Binding sites against miR-122, miR-199, miR-1, miR-183, and miR-206 can successfully detarget transgene expression from tissues like liver, heart, dorsal root ganglia and skeletal muscles (35,36,(95)(96)(97)(98). We previously demonstrated that miR-142BS elements can blunt CTL activation and can confer sustained transgene expression in transduced mouse TA muscle (42).…”

Involvement of microRNAs and their potential diagnostic, therapeutic, and prognostic role in hepatocellular carcinoma

“…MiRNA‐based therapeutic strategies may alter the expression networks of HCC, thereby significantly affecting cell behavior and making it possible to cure HCC. Virus‐based delivery system or non‐viral systems including nanoparticles, lipid‐based vesicles, exosomes, and liposomes can deliver miRNAs 112–115 . Delivery of miR‐26a using an exosomes‐based nanosystem inhibited proliferation of HCC 113 .…”

“…Virus-based delivery system or non-viral systems including nanoparticles, lipid-based vesicles, exosomes, and liposomes can deliver miRNAs. [112][113][114][115] Delivery of miR-26a using an exosomes-based nanosystem inhibited proliferation of HCC. 113 Targeted delivery of miR-199a-3p using self-assembled dipeptide nanoparticles or exosomes reduced HCC efficiently.…”