Despite decades of effort, gene therapy( GT) has failed to deliver clinically significant anticancer treatment, owingi np art to lows electivity,l ow efficiency,a nd poor accessibility of folded RNAt argets.H erein, we propose to solve these common problems of GT agents by using aD NA nanotechnology approach. We designed ad eoxyribozymebased DNAm achine that can i) recognizet he sequence of ac ancer biomarker with high selectivity,i i) tightly bind as tructured fragment of ah ousekeeping gene mRNA, and iii)cleave it with efficiency greater than that of at raditional DZ-based cleaving agent. An important advantage of the DNA nanomachine over other gene therapya pproaches (antisense, siRNA, and CRISPR/cas) is its ability to cleave ahousekeeping gene mRNAa fter being activated by ac ancer marker RNA, which can potentially increase the efficiency of anticancer gene therapy. The DNAm achine could become ap rototype platform for anew type of anticancer GT agent.

Oligonucleotide gene therapy (OGT) agents (e. g. antisense, deoxyribozymes, siRNA and CRISPR/Cas) are promising therapeutic tools. Despite extensive efforts, only few OGT drugs have been approved for clinical use. Besides the problem of efficient delivery to targeted cells, hybridization specificity is a potential limitation of OGT agents. To ensure tight binding, a typical OGT agent hybridizes to the stretch of 15-25 nucleotides of a unique targeted sequence. However, hybrids of such lengths tolerate one or more mismatches under physiological conditions, the problem known as the affinity/specificity dilemma. Here, we assess the scale of this problem by analyzing OGT hybridization-dependent off-target effects (HD OTE) in vitro , in animal models and clinical studies. All OGT agents except deoxyribozymes exhibit HD OTE in vitro , with most thorough evidence of poor specificity reported for siRNA and CRISPR/Cas9. Notably, siRNA suppress non-targeted genes due to (1) the partial complementarity to mRNA 3'-untranslated regions (3'-UTR), and (2) the antisense activity of the sense strand. CRISPR/Cas9 system can cause hundreds of non-intended dsDNA breaks due to low specificity of the guide RNA, which can limit therapeutic applications of CRISPR/Cas9 by ex-vivo formats. Contribution of this effects to the observed in vivo toxicity of OGT agents is unclear and requires further investigation. Locked or peptide nucleic acids improve OGT nuclease resistance but not specificity. Approaches that use RNA marker dependent (conditional) activation of OGT agents may improve specificity but require additional validation in cell culture and in vivo .

Soon after their discovery, RNA‐cleaving deoxyribozymes (RCDZ) were explored as anticancer gene therapy agents. Despite low toxicity found in clinical trials, there is no clinically significant anticancer RCDZ‐based therapy. Some of the reported disadvantages of RCDZ agents include poor accessibility to folded nucleic acids, low catalytic efficiency inside cells, and problems of intracellular delivery. On the other hand, structural DNA nanotechnology provides an opportunity to build multifunctional nano‐associations that can address some of these problems. Herein we discuss the possibility of building RCDZ‐based multifunctional DNA nanomachines equipped with RNA unwinding, cancer marker recognition, and RCDZ‐based RNA‐cleavage functions. An important advantage of such “nanomachines” is the possibility to cleave a housekeeping gene mRNA in a cancer‐cell‐specific manner. The proposed design could become a starting point for building sophisticated DNA‐based nanodevices for cancer treatment.

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Despite decades of effort, gene therapy (GT) has failed to deliver clinically significant anticancer treatment, owing in part to low selectivity, low efficiency, and poor accessibility of folded RNA targets. Herein, we propose to solve these common problems of GT agents by using a DNA nanotechnology approach. We designed a deoxyribozyme‐based DNA machine that can i) recognize the sequence of a cancer biomarker with high selectivity, ii) tightly bind a structured fragment of a housekeeping gene mRNA, and iii) cleave it with efficiency greater than that of a traditional DZ‐based cleaving agent. An important advantage of the DNA nanomachine over other gene therapy approaches (antisense, siRNA, and CRISPR/cas) is its ability to cleave a housekeeping gene mRNA after being activated by a cancer marker RNA, which can potentially increase the efficiency of anticancer gene therapy. The DNA machine could become a prototype platform for a new type of anticancer GT agent.