*4.2.1 Discussion*

*Antisense Therapy*

*4.1.1 Discussion*

MiRNA inhibition therapy is used to suppress the expression of oncomiRs that are frequently overexpressed in human cancers and reestablish the normal expression of tumor-suppressor genes that are targeting directly (**Figure 2**). The therapy for miRNA inhibition includes the following agents: antisense anti-miR oligonucleotides (AMOs), locked nucleic acid (LNA) anti-miRs, antagomiRs, miRNA sponges, and small molecule inhibitors of miRNAs (SMIRs) [27]. The principle of this therapy consists of an isolation of the endogenous miRNAs in an unrecognizable configuration, leading to inactivating and excluding the mature miRNAs from the RISC. AMOs are single-stranded, chemically modified antisense oligonucleotides of about 17–22 nucleotides that are complementary to a miRNA of interest [28]. These antisense oligonucleotides anneal to the complementary mature miRNAs and

LNA anti-miRs represent an example of a modified antisense anti-miR oligonucleotide [29]. LNA-modified oligonucleotides present a higher thermal stability and affinity for their miRNA target molecules, as well as a higher aqueous solubility

The antagomiRs are single-stranded RNA molecules of about 23 nucleotides in length complementary to miRNA targets that are chemically modified to increase the stability of the RNA and protect it from degradation [31]. One of the most important aspects of using these agents is due to their lack of inducing any immune response. miRNA sponges represent a class of RNAs that include multiple artificial binding sites similar to those found in the endogenous miRNA targets. The expression vectors represent the source of miRNA sponge transcription, thus reducing the miRNA's effects and increasing the expression of the miRNA's native targets [32]. SMIRs are small molecules that suppress the miRNA biogenesis or block the interaction between a miRNA and the target. The inhibition therapy using SMIRs is an encouraging one due to the reduced time of production, approval, and cost [33].

Krützfeldt et al. [31] demonstrated that intravenous administration of several antagomiRs toward miR-16, miR-122, miR-192, and miR-194 leads to a significant

Moreover, an important positive effect observed in this study was that after the administration of antagomiR-122, the cholesterol levels in plasma have decreased. Due to the fact that, so far, the therapy using antagomiRs did not induce a significant immune response, it is worth into consideration the development of a promis-

One of the main advantages of using locked nucleic acid (LNA) anti-miRs is that they present a higher thermal stability, high-affinity Watson-Crick hybridization with their RNA target molecules, higher aqueous solubility, and increased metabolic stability for *in vivo* delivery. Overexpression of miR-21 is a common place in glioblastomas, and Griveau et al.'s [34] study was able to silence miR-21 in U87MG glioblastoma cell line, using a LNA conjugated to lipid nanocapsules (LNC). Another advantage of using LNA-LNC complexes in combination with external beam radiation is represented by the improvement of cell sensitivity to treatment.

**4.2 miRNA replacement therapies for tumor-suppressor miRNAs**

Also defined as miRNA restoration therapy, the replacement therapy with miRNAs includes the following agents: small molecules, synthetic miRNA mimics, and DNA plasmids encoding a miRNA gene that epigenetically alters endogenous

inhibit their interaction with specific mRNA targets.

and increased metabolic stability for *in vivo* delivery [30].

reduction in the corresponding endogenous miRNAs.

ing antisense therapy based on antagomiRs.

**36**

expression of miRNAs [35].

One of the main challenges of miRNA replacement therapy is represented by finding the most suitable, efficient, and specific delivery system. The efficacy of this therapy is significantly decreased by an unsuitable size of the vector or by gene expression. Since miRNAs can be introduced into cells using a similar technique to small interference RNAs [36], it is recommended to improve those techniques based on the insertion of synthetic miRNA mimics, DNA plasmids, and small molecules, as well as to improve the quality of molecules used for this type of therapy.
