**5. RNA interference**

Most of the strategies of cancer gene therapy discussed so far have involved introduction of therapeutic transgenes. An alternative strategy that is gaining considerable attention in the cancer gene therapy field involves inhibiting expression of problematic genes. Inhibition of gene expression can be facilitated by RNA interference (RNAi) that binds to mRNA. RNAi, discovered by Fire and Mello in 1998 [128], can be defined as a mechanism of gene-silencing produced by small RNAs. These RNAs include endogenous miRNA and exogenous siRNA or shRNA and their gene silencing activity is highly dependent on gene sequence [129]. These small RNAs then recruit cellular proteins, such as the RNA-induced silencing complex (RISC), to elicit their effect either through degradation of the mRNA or blocking the translation of mRNA [130, 131]. RNAi interference is therefore a highly attractive approach to cancer gene therapy and is currently a major research focus.

#### **5.1. siRNA and shRNA**

the construction of a vector combining a hypoxia response promoter with the CMV promoter (HRE-CMV) that resulted in a 2-fold increase in apoptotic gene expression compared to

**Figure 2.** Targeted therapeutic transgene expression using affibodies and a tumour-specific promoter.

Transcrip6on 
 fails 
 Transcrip6on 

Tumour 
 cell 
 targeted 
 by 
 affibody 
 ( 

 )/ ligand 
 ( 

 ) 
 interac6on. 
 Vector 
 endocytosed & 
 DNA 
 transported 
 to 
 nucleus. 

Transformed 
 cells' 
 nuclei 
 express 
 TFs 
 aberrantly. 
 Overexpression 
 of 
 TFs 
 can 
 be 
 exploited 
 to 
 drive 
 transgene 
 expression 
 using 
 tumour-‐specific 
 promoters. 

TFs 
 overexpressed 
 in 
 tumours 
 form 
 transcrip6on 
 complexes 
 at 
 tumour-‐specific 
 promoters. 
 RNA 
 polymerase 
 ( 

 ) 

 executes 
 transcrip6on. 

> Expression 
>  of 
>  transgene 
>  produces 
>  therapeu6c 
>  effects 
>  in 
>  cancer 
>  cells.

Normal 
 cells 
 lack 
 cell 
 surface 
 ligand. 
 Occasional 
 non-‐specific 
 endocytosis 
 occurs 
 & 
 DNA 
 transported 
 to 
 nucleus. 

74 Gene Therapy - Principles and Challenges

Nuclei 
 of 
 non-‐transformed 
 cells 
 express 
 normal 
 complement 
 of 
 transcrip6on 
 factors 
 (TFs). 

Non-‐transformed 
 6ssue 
 lacks 
 TFs 
 required 
 for 
 transcrip6on 
 complex 
 forma6on. 
 Transcrip6on 
 fails. 

No 
 transgene 
 expression 
 in 
 non-‐ transformed 
 6ssue, 
 sparing 
 toxicity. 

> siRNA is a short (usually 21-bp) double-stranded RNA with phosphorylated 5' ends and hydroxylated 3' ends with two overhanging nucleotides. siRNA exerts its effect by directly

incorporating into RISC, where its guide-strand binds to and cleaves the complementary mRNA with a perfect match. The cleaved mRNA is subsequently released and the siRNA guide-strand-bound RISC is free to bind to another mRNA and start a new round of cleavage [132]. However, the short half-life of siRNA has resulted in production of shRNA, which has been developed as an alternative RNA molecule. Transcription of shRNA occurs in the nucleus from an expression vector that bears a short double-stranded DNA sequence with a hairpin loop. This shRNA transcript is then processed by RNase enzymes and incorporated into RISC in the cytoplasm [133].

The use of siRNA and shRNA to silence unfavourable genes that are overexpressed in cancer has gained much attention. Multidrug resistance (MDR) genes, responsible for resistance to chemotherapeutics have been problematic in the treatment of cancer and associated with poor prognosis. By silencing these genes using siRNA, it has been possible to improve response to conventional treatments. For example, Chen et al. used siRNA to silence the MDR1 gene in doxorubicin resistant MCF-7 breast cancer cells, which resulted in 85–90% reduction in MDR1 gene expression and subsequently sensitisation of 70% of cells to doxorubicin [134]. Another approach is to target and silence pro-angiogenic genes such as the Notch pathway. Yang et al. used a non-viral delivery system to deliver siRNA for silencing the Notch-1 gene in breast cancer and found that transfected MDA-MB-231 cells exhibited significantly decreased expression of Notch-1, inhibited cell proliferation, and increased cell apoptosis [135]. One advantage of using siRNA to down-regulate overexpressed proteins is that non-specific delivery is often less toxic than the delivery of plasmid DNA that encodes genes such as IL-2 and TNF-alpha. However, to limit any toxicity that does exist, many groups have added targeting ligands to the delivery systems to increase tumour specificity [136].

#### **5.2. MicroRNA**

MicroRNAs (miRNA) are highly conserved short non-coding RNAs that negatively regulate a wide range of physiological processes at the post-transcriptional level including apoptosis, proliferation, and migration [137]. Initially, miRNA is transcribed in the nucleus as a primary transcript (pri-miRNA), which is processed to give a two-nucleotide overhang at its 3' and is termed a pre-miRNA. Pre-miRNA is subsequently exported to the cytoplasm where it is further cleaved and mature miRNA is loaded into RISC to elicit its effect [138]. miRNAs can be either oncogenic or tumour suppressive in nature and as a result, may be overexpressed (e.g., miR-132, miR-20, and miR-17-92 family) or underexpressed (e.g., miR-34a and miR-126) in cancer cells making them targets for cancer gene therapy. A vast amount of information has been obtained in recent years on many different miRNAs and their role in cancer and with cancer stem cells, and by characterising their function, it may be possible to exploit them in cancer gene therapy [139, 140].

A single miRNA may have several varied targets to which it could bind and bring about gene silencing. miRNA-34a, known to be down-regulated in various cancers, has been shown to be a potent tumour suppressor that has various targets including the Notch pathway, BCL-2, survivin, c-Myc, and c-Met transcription factors [141]. Hu et al. demonstrated the value of miR-34a-mediated tumour suppression with the in vivo systemic administration of a non-viral miR-34a delivery system. Nanoparticles were used to deliver the miR-34a using a tumourtargeting and penetrating bifunctional CC9 peptide (CRGDKGPDC) conjugated to β-cyclo‐ dextrin-polyethylenimine in a PANC-1 pancreatic cancer xenograft model; the miR-34aloaded particles significantly inhibited tumour growth and induced cancer cell apoptosis [142]. Conversely, the inhibition of some miRNAs using complementary miRNA antagonist oligonucleotides (anti-miRNAs) can be an attractive gene therapy strategy to neutralise miRNA function. miR-132 acts as an angiogenic switch at the endothelium, inducing tumour neovascularization. Anand et al. reported the systemic administration of anti-miR-132 containing liposomes incorporating an integrin αvβ3-targeting cyclic RGD peptide to inhibit angiogenesis [143]. The authors demonstrated that anti-miR-132 blocked the action of mi-132 on angiogenesis induced by a VEGF-secreting ID-8 ovarian carcinoma in mice, and signifi‐ cantly reduced tumour burden and angiogenesis in an MDA-MB 231 xenograft model of human breast carcinoma when compared to treatment with scrambled miR-132. There is a huge potential of miRNA therapeutics for cancer. However, miRNA gene therapy is still in its infancy and more research is required to elucidate the exact pathways and possible targets available.

The active targeting of cancer gene therapy is hugely important for efficiency and safety. Yet despite the plethora of characteristics that can be targeted, active targeting remains elusive in many non-viral gene delivery systems. A move towards a combination of targeting strategies in one delivery system may hold promise for improved specificity using non-viral vectors.
