I. Introduction
RNA interference (RNAi) is a method of regulating endogenous gene expression. RNAi is one of three mechanisms that can be used to silence a specific target gene, which functions by interfering with translation of mRNA and, subsequently, protein production. The RNAi process (illustrated in Figure 1) is initiated by the introduction of long, doublestranded RNA molecules (dsRNA) to a cell. Next, Dicer, a dsRNA-specific endoribonuclease, recognizes and cleaves these aberrant dsRNAs, producing short interfering RNA (siRNA). siRNAs are dsRNA molecules of 21 nucleotides with 3' overhangs of 2 nucleotides that form a ribonucleotide-protein complex known as the RNAinduced silencing complex (RISC). siRNAs are unwound at RISC and a single-stranded RNA molecule remaining attached to RISC binds to a complementary sequence on a target mRNA. Finally, the target mRNA is cleaved and degraded; thus, expression of the gene is interrupted. Although RNAi is a naturally occurring cellular process it is thought to function in defending the genome from transposons and viruses RNAi has been implicated in therapeutic gene silencing and the downregulation of disease-causing proteins.
Figure 1: An overview of the RNAi process. In addition to RNAi, antisense oligonucleotides (ODNs) and ribozymes can also be employed in gene silencing, however both methods are associated with significant drawbacks. ODNs are short, single-stranded DNA or RNA molecules that are complementary to target mRNAs such that hybridization to mRNA ultimately blocks expression of the target gene via mRNA cleavage. These results have only been demonstrated in vitro, as in vivo study has been impeded by difficulties associated with delivering ODNs. Furthermore, ODNs are less efficient and specific than siRNA in inhibiting gene expression. Ribozymes are RNA molecules with catalytic activity enabling
2 cleavage of single-stranded RNA, however ribozymes are only stable for a few minutes in serum. Therefore, RNAi and delivery of siRNA have become the focus of gene silencing applications.
- Results in smallest nanoparticles (less than 10nm in size), susceptible to enzymatic degradation Cationic Polymer Larger in size which is 100-300nm. It increases circulation time and allows specific tissue/cell interaction. Example, PEI (Polyethylenimine). Positively charges on the polymer (i.e PEI) electrostatically interacts with the negatively charges on the backbone of siRNA Polycation with positive surface charge is useful for in vitro gene transfer. However, when it is introduced in vivo via intravenous injection, it aggregates and forms larger structure by non-specific association with charged serum protein. It eventually leads to rapid clearance from the plasma. Therefore, polycation is undergone further chemical modification by conjugating blood-compatible functional polymers to enhance siRNA delivery efficiency. Examples of chemical modification: a. PEI-PEG b. Cyclodextrin-containing polycation c. Polylysine d. Natural polymer (such as chitosan) Cationic peptides (such as CADY and MPG-8) also complex siRNA efficiently. CADY:i. A secondary amphipathic peptide. ii. Has helical conformation in solution (regardless of pH). iii. Enters cell through mechanism which is independent of major endosomal pathway iv. Delivers siRNA into cytoplasm selectively and rapidly.
4 v. Promotes a significant siRNA associated knockdown of the target A novel CPP-modified protein was able to shield siRNA from degradation and deliver it to several target sites upon binding. Cationic Lipids Lipid vectors work more efficiently with siRNA than polymer vector due to its weak interaction with siRNA, which eventually leads to faster decomplexation in cytosol. Examples: Lipofectamine, Oligofectamine, Lipofectin, siPORT NeoFX and etc Only a limited number of cationic lipids used as carrier for in vivo siRNA delivery due to their poor colloidal stability and toxicity concerns. The first lipid formulations for in vivo siRNA delivery: 1,2-Dioleyl-3trimethylammonium propane (DOTAP) The chemical modification of solid lipid nanoparticle (SLN) (i.e low density lipoprotein(LDL)-mimicking nanoparticle) surface with PEG provides stabilization to SLN/siRNA-PEG polyplex micelles. Natural Liposomes Less than 200nm in size Least toxicity if compared to cationic polymers/lipids Unilammelar structure of liposomes (hydrophilic core & hydrophobic surfaces) protects encapsulated siRNA from degradation by surrounding RNases and facilitating internalization via membrane fusion Example of Drugs CALAA-01 Calando Pharmaceuticals leading drug candidate Is combination of RONDEL (RNAi/Oligonucleotide Nanoparticle Delivery) and a patented siRNA targeted M2 subunit of ribonucleotide reductases. The foundation of RONDEL is cyclodextrin-containing polymer Ribonucleotide reductase is essential in catalyzing conversion of ribonucleosides to deoxyribonucleosides and is needed for DNA synthesis and replication. Both siRNA and CALAA-01 shows anti-proliferative activity over multiple types of cancer cells. Is currently undergoing Phase I Trial at UCLA Medical Centre in Los Angeles. Interim result has demonstrated that CALAA-01 is well tolerated and has shown preliminary proof of RNAi activity in patient with high dose. Mediates specific gene inhibition as shown by mRNA and protein knockdown at tumor sites. Benefits of RONDEL More effective delivery: RONDEL binds to and self-assembles with siRNA in order to form uniform colloidal shaped siRNA (>100nm in size), which ease the accumulation at tumor sites. Increased stability: siRNA with RONDEL is more stable under physiological condition Fewer immune reaction: RONDEL allows repeating dosing without causing any immune response (such as interferon response that occurs in lipid delivery system)
5 Future: RNA interference can be a revolutionary method of therapeutic gene silencing. Triple negative breast cancer is when breast cancer cells dont have the estrogen, progesterone, and Her2/neu; this type of cancer is aggressive and less responsive to drugs. Mice had cancerous cells implanted into the mices mammary fat pad. The mice were injected with siRNA targeting triple negative breast cancer cells. The growth of cancerous cells was reduced and there was no metastasis (spread of cancer) into the lungs, liver, intestines and stomach.
Sample questions 1. Compare and contrast the three methods that can be used to silence specific target genes. Which one is the focus of therapeutic applications and why? 2. Describe two of the four delivery strategies discussed in the article (include any limitations if applicable). 3. Why is chemical modification of cationic polymers important?