Delivery of therapeutic nucleic acids in general and small interfering RNAs (siRNAs) in particular although a relatively nascent area of research, has already overcome several technical and regulatory hurdles over past few years. Some lead formulations undergoing clinical evaluation against wide range of diseases from age-related macular degeneration (AMD) to hypercholesteremia, amyloidosis and cancer. Roughly 4000 diseases are associated with malfunctioning genes in a particular cell type. Gene therapy provides opportunity to modify (inhibit or enhance) the expression of disease-causing genes at the cellular level. Gene correction at the DNA level poses risks of inciting mutations or teratogenicity in comparison with RNA interference (RNAi) approach, which works at the transcription level leading to desired outcomes at the protein production stage such as therapeutic gene silencing of a cancer-causing gene.
Currently, the key molecules employed in executing RNAi strategies are short hairpin RNA (shRNA), small interfering RNA (siRNA) or endogenous microRNA, of which siRNA forms the major mode presently under clinical evaluation. siRNA molecules are easy to synthesize and do not need to be delivered into the nucleus as they do not require genome integration or the usage of DNA promoters. They act in a sequence-specific manner against a target messenger RNA (mRNA), which avoids unintended off-target effects. The RNA-induced silencing complex (RISC), which drives RNAi, protects siRNA from degradation by cytosolic nucleases thereby recycling the molecule for many rounds of cleavage activity against the target mRNA. This implies that even few siRNA molecules if delivered stably into the cell cytosol are sufficient to trigger long-lasting gene silencing.
In order to reach the desired organ/tissue and subsequently cross the cell membrane, these therapeutic siRNAs need to overcome several physiological barriers such as renal clearance, serum degradation and cell-specific uptake. Following crossing the cell membrane, intracellular barriers in the form of saturation of endogenous RNAi machinery or endosomal escape postendocytosis can reduce the efficiency of the delivered siRNA molecules. Small negatively charged siRNAs can also lead to immunotoxicity and over the past years efforts have been dedicated to develop an efficient carrier modalities to overcome these systemic barriers and toxicity issues, which broadly falls into viral and nonviral strategies. Due to safety concerns on usage of viral vectors, nonviral techniques have taken the center-stage in siRNAs delivery.
Delivery vehicles such as liposomes, lipid-based nanoparticles (LNPs) or polymeric nanoparticles are biodegradable, have relatively low toxicity, are stable in storage, easy-to scale up and have low production costs – traits favored from an industrial perspective. Moreover, most components used in the development of nanostructures in the form of lipids or polymers are approved by the regulatory agencies for human use. From a technical standpoint, delivering siRNAs in nanocarriers offers high encapsulation rate, potentially a controllable sustained release option or burst release and stability against degradation in the biological environment. In the case of cancer care, tumor physiology may aid in the accumulation of nanocarriers in the desired tissue or solid tumor via enhanced permeability and retention effect in the case of carcinoma but this needs to be tested on case-by-case studies and may not be an ultimate mechanism for all types of solid tumors.
Beginning with chemical modification of the siRNA molecules to stably sustain the harsh biological environment such as 2′o-Me or 2′o-Flu in specific sequences, much advancement has been achieved also by using various delivery vectors. Mostly classical cationic lipids (DOTAP, DOTMA) or cationic polymers (PEI, chitosan) have been used in formulations, conjugates or complexes to form lipoplexes or polyplexes. Exosomes are also being explored for gene delivery purposes. One of the most characterized platforms for siRNA delivery is the stable nucleic acid lipid particles (SNALPs), which represent approximately 70–120 nm nanocarriers. Many formulations under clinical investigations from Tekmira Pharmaceuticals and Alnylam Pharmaceuticals are formulated using SNALPs, which use ionizable lipids such as the DLin-DMA-based amino lipids as their prime component. A common feature of most carriers is PEGylation (coating of polyethylene glycol [PEG]), which provides both steric stability and protection from systemic uptake by macrophages and other types of leukocytes. Further modifying the nanocarrier surface with molecules that can impart/enhance functional activity are for example the use of targeting moieties to endow these carriers’ high selectivity toward cell surface receptors.