**1. Gene suppression therapies in cancer: an overview**

Gene therapy, which was initially developed for the treatment of genetic (primarily monogenic) diseases, has mainly focused on cancer therapy, so that more than 65% of all gene therapy trials worldwide (**Figure 1**) are aimed at the treatment of solid and hematological malignancies [1]. As a consequence, cancer gene therapy is a predominant field of basic research, as well as of clinical activities (**Table 1**) [2].

Various strategies at different molecular levels (**Figure 2**) have been employed to treat malignant diseases in recent decades, such as specific drug inhibitors acting at the protein level,

**Figure 1.** Gene therapy trials worldwide.


**Table 1.** Gene therapy trials worldwide.

gene suppression therapies at the mRNA level, and genome-editing nucleases at the DNA level [3].

The ability of several drugs to inhibit the activity of a targeted oncoprotein has been exploited as a therapeutic approach for a variety of malignancies, the best example being imatinib mesylate, a tyrosine-kinase inhibitor (TKI) indicated for the first-line treatment of chronic myeloid leukemia (CML). The advent of imatinib mesylate at the end of the twentieth century has revolutionized CML prognosis, yielding an overall survival (OS) rate of 88% after 5years, whereas previous nonspecific treatments produced an OS rate of only 57% [4]. Unfortunately, despite the increased efficacy and better clinical responses, many patients receiving targeted drugs have a poor initial response, develop resistance, or undergo relapse after initial success. Except for a subgroup of patients who achieve a deep and sustained molecular response, TKI

CRISPR-ERA for Switching Off (Onco) Genes 9 http://dx.doi.org/10.5772/intechopen.80245

**Figure 2.** Different strategies to block oncogene effects.-

therapies would need to be continued indefinitely because TKIs do not completely eliminate the leukemia stem cells (LSCs), but they remain even during effective TKI treatment [5].

An alternative oncoprotein inhibition approach emerges from the ability of some small RNAs to fold into three-dimensional structures that can then bind to proteins and thereby inhibit them in a manner similar to protein antagonists [6]. This is the logic behind the use of RNA "decoys" or RNA aptamers. Recent preclinical and clinical data support the potential activity of a 45-nucleotide-long RNA aptamer (NOX-A12) that specifically antagonizes the CXC chemokine ligand 12/stromal cell-derived factor-1 (CXCL 12/SDF-1), which is a regulatory chemokine essential for the migration of leukemic stem cells into the bone marrow [6]. This inhibition of the binding of SDF-1 to its receptors can prompt the leukemic stem cells to reenter the cell cycle and become vulnerable to chemotherapeutic attack.-

Other gene suppression therapies focus on the intervention of gene transcription and translation, which are vital elements for cancer growth, spread, survival, and therapy resistance.- Ribozymes, antisense oligodeoxynucleotides (AS-ODNs), and short-interfering RNAs (siRNAs)- are an emerging class of targeted DNA-based pharmaceuticals. Ribozymes, a subset of catalytic- RNAs, can be artificially synthesized and used to specifically suppress gene function. They can- also be used to validate disease-related genes as potential targets for new therapeutic interventions. Their ability to cleave mRNA to prevent protein synthesis enables them to be applied in- cancer and virology. Transcripts of genes of different function have been targeted by AS-ODN- gene therapies such as c-myb, c-raf, c-fos, H-ras, Her2/neu, bcl-2, VEGF, and Ang-1. The useof AS-ODNs was shown to successfully inhibit gene expression in association with tumor- growth inhibition, radiosensitization, or chemosensitization [7–9]. The use of siRNA technology provides another novel approach for targeted sequence-specific suppression of target gene- expression. In this system, siRNA stability and proper delivery are key factors for successful- application. *In vitro* and *in vivo* studies with siRNA targeting PKN3 mRNA have been successful- at inhibiting tumor progression and metastasis in lung and mammary carcinoma models [10]. Nonetheless, inefficient/complete silencing and transient effects present major challenges to cancer gene therapy mediated by ribozymes, AS-ODNs, or siRNAs [2]. Other important challenges- that need to be addressed for the successful translation of these approaches are their delivery to- the site of action, the choice between direct delivery or the use of a vehicle, mass production at- low cost, more clearly defined pharmacokinetics, and the ability to produce sustained long-term- effects, immunogenicity, and toxicity (including inappropriate or excessive expression).-

With the recent explosion of genome editing tools, including clustered, regularly interspaced short palindromic repeats and their nuclease-associated protein Cas9 (CRISPR/Cas9), the landscape of suppression techniques has dramatically changed. Although CRISPR/Cas9 is similar in- action and efficacy to protein-based targeted nucleases, such as zinc finger nucleases (ZFNs) and- transcription activator-like effector nucleases (TALENs) [11], the ease with which these reagents can be designed and tested through the construction of single-guide RNAs (sgRNAs) has made- gene editing available to a wider variety of users and for a broader range of applications.-

CRISPR/Cas9 works at the DNA level and has the advantage of providing permanent and full gene knockout, while AS-ODNs and siRNAs only silence genes transiently because they working at the mRNA level [12, 13]. CRISPR/Cas9 cuts DNA in a sequence-specific manner with the possibility of interrupting coding sequences, thereby making it possible to turn off- cancer drivers in a way that was not previously feasible in humans [14, 15]. This notable application of permanent gene disruption is based on the cellular mechanisms involved in double-stranded break (DSB) repair.-
