RNA interference (RNAi) is a gene-silencing technique that inhibits gene expression by causing the intracellular degradation of mRNA molecules. Since first reported in 1998,1 RNAi has sparked a revolution in molecular biology and has been employed in a myriad of biological contexts for the systematic evaluation of gene function. The ability to selectively regulate the activity of specific genes within a given biological location represents a methodology by which researchers can upregulate or downregulate protein expression.
Controlling gene expression through RNAi typically involves the use of double-stranded RNA molecules that are between 20–25 base pairs long. Dubbed “small interfering” or “silencing” RNA (siRNA), these oligonucleotides interfere with native gene expression by binding to complementary strands of messenger RNA (mRNA) to form complexes that are then degraded by the cell’s natural machinery before protein translation can occur. While the exact mechanism of siRNA-mediated gene suppression is complex, siRNAs can potentially be used to selectively disrupt the expression of any gene within a given genome. Recently, RNAi-based approaches have been gaining traction as promising modalities to inhibit viral infection and cancer proliferation in humans.2, 3 However, while targeted gene suppression by siRNA has shown promising clinical results for certain applications, consistent delivery of siRNA to specific targets in vivo has remained a difficult challenge.
Intracellular Delivery of siRNA
The in vivo application of siRNA is severely limited by the effectiveness of the delivery system. Oligonucleotides are naturally anionic and, therefore, not readily cell permeable. The plasma membrane, itself anionic, provides a substantial barrier for delivery of siRNA-based therapeutics to the intracellular environment. To circumvent this issue, researchers have turned to developing positively charged lipophilic molecules, such as liposomala, polyethylenimineb, diethylaminoethyl cellulose, and other cationic polymers that are capable of complexing “naked” oligonucleotides, thus neutralizing their anionic character and allowing them to be delivered to cells. Such “transfection reagents” have been shown to significantly enhance the delivery of oligonucleotides, including siRNAs, and are now well-known tools in genomics research. Unfortunately, the in vivo delivery of siRNAs using traditional liposomal delivery agents has been hindered by low levels of delivery and low gene suppression, although recent optimization strategies directed at lipid formulations have indicated that liposomal delivery may remain an appropriate approach for gene delivery in vivo.4 Nevertheless, the issue of delivery has drastically impeded advancements in the field of gene therapy and has necessitated the development of alternative delivery techniques to transport potentially therapeutic siRNAs to their intracellular targets.
Topical Delivery of siRNA
While environmental factors can significantly impact the health and appearance of human skin (nurture), the aging process is undoubtedly linked to an individual’s genes (nature).5 As human skin ages, it becomes thinner and more easily damaged—effects that are augmented by the decreasing ability for skin to heal itself. Topical delivery of siRNA-based therapeutics that selectively suppress certain genes may provide a new method of skin care. Unfortunately, as noted, penetration of oligonucleotides across the epidermal barrier remains a major technological challenge. Negatively charged moieties, such as siRNAs, require positively charged ancillary ingredients or skin penetration enhancers to promote cellular internalization. In the absence of such ingredients, “naked” siRNA molecules would not be expected to penetrate skin cells at concentrations required for sufficient activity.
In an effort to enhance the epidermal permeability of siRNAs, Mirkin et al. generated spherical nucleic acid nanoparticle conjugates (SNA-NCs) that demonstrate enhanced skin penetration.6 These siRNA-based nanoparticle constructs are formed by covalently linking therapeutic siRNAs to the surface of spherical gold nanoparticles that are approximately 13 nm in diameter (see Figure 1). The resultant supramolecular nanoparticles display an overall net anionic charge and, surprisingly, penetrate keratinocytes in vitro, mouse skin and human epidermis at near quantitative levels. Notably, Mirkin’s team was the first to demonstrate that commercially available delivery vehicles such as a combination of petrolatum, mineral oil, ceresin and lanolin alcohola can be used to deliver the SNA-NCs in vivo and regulate gene activity in epidermal cells.
SNA-NCs selective for the epidermal growth factor receptor (EGFR), an important target associated with many malignant cancers, were dispersed in the aforementioned vehiclec and applied three times per week to the skin of mice. After three weeks, EGFR protein levels were significantly suppressed in mice treated with the SNA-NCs. Specificity for the target gene was confirmed by additional biochemical analyses. Following success in rodents, Mirkin’s team successfully demonstrated the benefit of the SNA-NC delivery approach in human skin equivalents, using organotypic raft cultures with intact lipid and well-differentiated protein epidermal barriers. Importantly, the researchers did not report any apparent toxicity of the SNA-NCs at efficacious concentrations.
Using an alternate delivery method, Kaspar et al. demonstrated the benefit of siRNA-loaded dissolvable microneedle (5 μm tip) arrays to inhibit CD44 gene expression in mice harboring human skin xenografts.7 The CD44 family contains transmembrane proteins that bind hyaluronan, a glycosaminoglycan that is widely distributed throughout connective tissues of the epidermis. Here, the authors utilize protrusion array devices (PADs) composed of 20% polyvinyl alcohol polymer on a poly(methyl methacrylate) substrate coated with siRNAs specific for CD44. The human skin equivalent xenografts were treated with up to 20 siRNA-coated PADs for a total of 10 consecutive days. Subsequent inspection of the xenografts found that a significant portion (30–50%) of the PADs were lost to subcutaneous dissolution. The authors point out that this finding is consistent with insertion, hydration, deposition and erosion of the needles. Results from these experiments showed a 40% reduction in CD44 expression over control xenografts. Importantly, these results demonstrate that siRNA delivery via microneedle arrays can efficiently inhibit endogenous genes that are uniformly distributed throughout the epidermal strata.
siRNA in Skin Lightening
Skin pigmentation in humans is regulated by melanocytes, which express enzymes that produce melanin. In mammals, melanin pigments are typically derived from the amino acid tyrosine. Tyrosinase (TYR) is the primary enzyme involved in the synthesis of melanin (melanogenesis), and depigmenting agents often inhibit TYR to control melanin production in skin. Many synthetic and naturally occurring tyrosinase inhibitors, such as kojic acid, tropolone, L-mimosine, hydroquinone, retinoic acid, arbutin and glabridin are known in the pharmaceutical and cosmetic industries, and are used in commercially available skin-lightening products. However, the ability of these compounds to safely lighten human skin is limited, as their use can result in putative cytotoxic metabolites, unwanted depigmentation, irritant dermatitis and ochronosis. Indeed, as the market for skin-lightening products continues to grow, cosmetic scientists need innovative strategies for developing potent compounds that safely inhibit melanin formation and induce hypopigmentation. Accordingly, RNAi has emerged as a promising method to suppress melanin biosynthesis, leading to a new class of products that effectively regulate melanin production at clinically safe concentrations.
Several groups have reported using siRNA technology to effectively inhibit melanin formation by targeting genes within the melanogenesis biosynthetic pathway. However, the majority of these studies rely on using traditional transfection reagents to deliver the siRNA payload into cells. As discussed, most lipid-based transfection reagents are incompatible with the topical delivery of siRNA molecules, prompting researchers to develop alternative techniques to deliver siRNAs into the skin of living organisms. Recently, bioactive peptides, such as oligoarginine and transdermal peptides capable of condensing oligonucleotides and penetrating human skin (TD1), have gained considerable attention as effective siRNA delivery vectors. These studies have demonstrated that such peptide conjugates can be used to effectively deliver siRNAs into the epidermis and dermis of mammals. Lara and fellow researchers successfully utilized a formulation of R7 and TD1 to topically deliver siRNAs specific for microphthalmia-associated transcription factor (MITF) to epidermal melanocytes, inhibiting melanin production upstream of TYR.7 Their results showed that hyperpigmented facial lesions of siRNA-treated subjects were significantly lighter following 12 weeks of therapy, and that overall improvement was noticed within the first four weeks of treatment. At the end of their treatment regimen, the authors used clinical and colorimetric evaluations, demonstrating a > 90% lightening of the siRNA-treated lesions toward normal skin color. This specific technology has been developed into a commercially available siRNA cosmetic productd.
siRNA and Skin Care: The Future
The potential impact that siRNA technology can have on the multi- billion dollar skin care market cannot be overstated. The epidermis is the body’s largest organ and, by some assessments, the most accessible when using topical delivery agents. Overall, the field of gene therapy has been plagued by inefficient delivery of potent gene-silencers and off-target effects, drastically limiting the development of therapeutic siRNAs. Despite these setbacks, this situation represents a unique opportunity for the cosmetic scientist to develop revolutionary products that are capable of preventing or treating debilitating skin disorders. For example, hyaluronidases are a family of enzymes that are largely responsible for the in vivo degradation of hyaluronan. Potentially, the regulation of these enzymes could provide a promising new method for anti-aging treatments and anti-wrinkle therapies. Moreover, siRNA technology should allow researchers to target other genes, such as cofactors and transcription factors within specific biochemical pathways that cannot be targeted by traditional small molecules. Using siRNAs in this context could lead to suppression of genes that were previously considered “undruggable,” opening the door for many new targets for skin care.
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- A Fire et al, Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans, Nature 391(6669) 806–811 (1998)
- GR Devi, siRNA-based approaches in cancer therapy, Cancer Gene Ther 13(9) 819–829 (2006)
- M Jiang and J Milner, Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference, Oncogene 21(39) 6041–6048 (2002)
- D Bhavsar et al, Translational siRNA therapeutics using liposomal carriers: Prospects and challenges, Curr Gene Ther 12(4) 315–332 (2012)
- R Osborne et al, Application of genomics to breakthroughs in the cosmetic treatment of skin ageing and discoloration, Br J Dermatol 166 Suppl 2 16–19 (2012)
- D Zheng et al, Topical delivery of siRNA-based spherical nucleic acid nanoparticle conjugates for gene regulation, Proc Natl Acad Sci USA 109(30) 11975–11980 (2012)
- MF Lara et al, Inhibition of CD44 gene expression in human skin models, using self-delivery short interfering RNA administered by dissolvable microneedle arrays, Hum Gene Ther 23(8) 816–823 (2012)