Ribonucleic acid interference induced gene knockdown.

Despite major advances in periodontal regeneration over the past three decades, complete regeneration of the lost periodontium on a regular and predictable basis in humans has still remained elusive. The identification of stem cells in the periodontal ligament together with the growing concept of tissue engineering has opened new vistas in periodontal regenerative medicine. In this regard, ribonucleic acid interference (RNAi) opens a new gate way for a novel RNA based approach in periodontal management. This paper aims to summarize the current opinion on the mechanisms underlying RNAi, in vitro and in vivo existing applications in the dental research, which could lead to their future use in periodontal regeneration.

INTRODUCTION

Periodontitis is an inflammatory disease that causes pathological alterations in the tooth-supporting tissues, potentially leading to loss of periodontal structure and eventually tooth loss. The treatment of periodontal disease points to the need for more effective and efficient management of this condition, in an attempt to restrict its impact on general health and patient quality-of-life for which a wide range of treatment strategies have been in use with variable success to repair or regenerate the periodontal tissues to reduce the likelihood of tooth loss. Current efforts to reproduce a natural periodontium are based on the principles of tissue engineering.

Tissue engineering is a term that describes the application or use of cells, scaffolds and growth factors to restore, maintain or enhance tissue function. Various therapeutic approaches have been proposed in the recent times to enhance the regenerative potential of the periodontium in addition to the conventional procedures which include (i) conductive therapeutics, (ii) inductive therapeutics, (iii) cell based approach, (iv) protein based approach and (v) gene based approach. However, ribonucleic acid (RNA) based approach is a novel therapeutic modality, which provides a therapeutic platform for regeneration of the lost periodontium as well as inhibition of disease progression. RNA interference (RNAi) is an efficient natural mechanism for controlling gene expression based on gene silencing. In recent years, RNAi has become one of the most powerful tools for probing gene functions and rationalizing the drug design. It has been used as a potential therapeutic agent for a wide range of disorders, including cancer, infectious diseases, systemic and metabolic disorders.

DISCOVERY OF RNAi

A large number of small RNA molecules work in conjunction with proteins in ribonucleoprotein (RNP) complexes, which help in gene expression. These various RNP particles are now being extensively studied in an attempt to understand their specific roles in the gene expression and protein synthesis. In the early 1980s, investigators revealed that small RNA molecules (about 100 nucleotides (nt) in length) can bind to a complementary sequence in messenger RNA (mRNA) and inhibit translation in Escherichia coli.[12] Until date, about approximately 25 cases of regulatory trans-acting antisense RNAs are discovered in E. Coli.[3] However, this regulation of translation by antisense RNA's is also found in eukaryotes as was first demonstrated in 1993. It was found in a few experiments that a transgene could not only induce or stimulate gene activity, but could also inhibit the expression of homologous sequences. This phenomenon was called homology-dependent gene silencing. However, it was evident that RNA played a key role in gene silencing, the phenomenon remained enigmatic until the discovery of RNAi[4] provided a most unexpected explanation with many profound consequences. The discovery of RNAi[5] has opened the door to RNA-based therapeutics that may prove to be an effective tool for the treatment of a large variety of diseases and for tissue regeneration, including periodontal regeneration.

RNAi MECHANISMS

RNAi is a classical mechanism of regulation of gene expression. The phenomenon is also known as co-suppression in plants and quelling in fungi. The RNAi world includes a variety of small RNA molecules such as small interfering RNA (siRNA), micro RNA (miRNA), short-hairpin RNA (shRNA), ribozymes etc., which are capable of gene suppression. These entire molecules knock down the expression of the targeted genes, i.e. they do not entirely abolish the gene expression, but lessen active gene product temporarily. The RNAi technique is thus referred to as gene knock down procedure to distinguish it from gene knockout procedures. The silencing mechanisms can occur at different levels of gene expression, i.e., transcription,[6] post-transcription[78] and translation.[9] RNAi can also cause augmentation of gene expression due to direct effects on the translation.[9]

POST-TRANSCRIPTIONAL RNAi

The RNAi pathway is present in cells of virtually every living organism. It is postulated that RNAi may have evolved as an innate immune response to combat organisms against double-stranded RNA (dsRNA) viruses.[78] Briefly, dsRNA when introduced into a cell gets chopped up by the enzyme Dicer, which is a member of the ribonuclease III family. The enzyme Dicer plays two biochemically distinct roles in the RNAi mechanics. It functions to generate siRNA molecules and also plays an important role in loading one of the two siRNA strands onto RNA-induced silencing complex (RISC) complex. This enzymatic cleavage degrades the RNA to 19-23 bp duplexes, each with 2-nucleotide 3′ overhangs. siRNA then binds to the RISC and is unwound in an adenosine triphosphate (ATP)-dependent manner. The assembly of RISC is an ATP dependent process reflecting the requirement for energy driven unwinding of the siRNA duplex or any conformational or compositional changes of the pre-assembled RNA duplex containing RNP. The major constituents of RISC are the single-stranded siRNA and any one of a number of different proteins of the Argonaute (Ago) family. Eight Ago proteins have been typified in humans, among this family of proteins; Ago2 protein has been implicated to be associated with RISC complex. The Ago protein is considered as the catalytic engine or the signature component of the RISC of RNAi, which cuts mRNA targets guided by siRNA via its endonuclease nick named “slicer.” The active RISC further promotes unwinding of siRNA through an ATP-dependent process and the unwound antisense strand guides active RISC to the complementary mRNA. The targeted mRNA is cleaved by RISC at a single site that is defined with regard to where the 5’ end of the antisense strand is bound to mRNA target sequence. The RISC cleaves the complimentary mRNA in the middle, 10 nucleotides upstream of the nucleotide paired with the 5’ end of the guide siRNA. This cleavage reaction is independent of ATP. However, multiple rounds of mRNA cleavage, which require the release of cleaved mRNA products, are more efficient in the presence of ATP [Figure 1].

Figure 1

At the initiator step of post-transcriptional gene silencing, long double-stranded ribonucleic acid, which can be produced by endogenous genes, invading viruses, transposons or experimental transgenes, are cleaved by the enzyme Dicer, which generates 21-23 nucleotide duplex RNAs with overhanging 3’ends, called small interfering RNAs. Next, siRNAs are incorporated into the RNA-induced silencing complex, which directs RISC to recognize target messenger RNAs and cleave them with complementary sequences to the siRNA

TRANSLATIONAL GENE SILENCING

RNAi gene inhibition at the level of translation also involves Dicer, which produces 21-23 nt miRNA's from 60-70 nt stem-loop precursor miRNAs (pre-miRNAs).[9] The complex of the activated RISC and miRNA binds the 3’untranslated region of specific mRNAs, which triggers cleavage by perfect base-pairing recognition or translational repression by partial base-pairing recognition.[10]

TRANSCRIPTIONAL GENE SILENCING AND GENE ACTIVATION

Studies have shown that the RNAi machinery is located in the cytoplasm and therefore acts on mature rather than nuclear precursor mRNA.[11] However, promoter-directed siRNA s can also mediate transcriptional gene silencing in mammalian cells when delivered to the nucleus.[1213] This silencing is associated with deoxyribonucleic acid (DNA) methylation of the targeted sequence.[12] Moreover, miRNAs complementary to promoter regions have been observed using the RNAi pathway to activate genes in the nucleus.[1415]

The gene silencing mechanism starts within hours of its application and lasts for up to 7 days, whereas activation of genes takes few days to appear and last for about a month. However the exact mechanism behind gene activation is not yet known.

RNAi DELIVERY METHODS

Injection of in vitro synthesized dsRNA

Soaking in dsRNA

Feeding dsRNA producing bacteria

Transfection of synthetic siRNA

Transgenes that produce dsRNA molecules (hairpins)

Viruses producing dsRNA molecules.

Although various small RNA molecules have been proposed for use in gene silencing mechanism, siRNA molecules are the most commonly used synthetic or naturally produced molecules that cause RNAi. The various alternative forms of both, miRNAs, piwi-interacting RNAs, shRNAs and small modulatory RNAs have been shown to be useful in successful gene silencing.

siRNA DELIVERY

siRNA's are the most commonly used and the most effective forms of small RNA's tried in in vitro clinical trials for gene silencing. However, the challenge of siRNA delivery is to overcome the various barriers, which prevent the host in achieving efficient target cell delivery. Studies by Pouton and Seymour[16] have shown that siRNA and DNA have difficulty in circulating in the blood stream, passing across cellular membranes and escaping from endosomal-lysosomal compartments. Various carrier systems have been developed to increase the delivery of siRNA, including both the carrier and non-carrier systems. The use of viral vectors to deliver siRNA's based on retrovirus, adenovirus or adeno-associated viruses has shown effective gene silencing in both in vitro and in vivo.[171819]

Non-viral delivery systems, using for instance cationic liposomes and polycation-based carriers such as polyethylenimine, have been developed for siRNAs. However, these systems exhibit in vivo toxicity and activate the immune system.[17] This has led to the development of more efficient carrier systems. Chitosan is one such material, which is widely used in drug delivery systems, especially for DNA-mediated gene therapy. It has been shown that a chitosan/siRNA nano particle delivery system silences genes. Moreover, chitosan has been shown to be biocompatible, non-inflammatory, non-toxic and biodegradable.[17]

APPLICATIONS OF RNAi

RNAi is an effective and efficient natural mechanism for regulation of gene silencing. RNAi is also regarded as a natural defense mechanism against mobile endogenous transposons and invasion by exogenous viruses, which have dsRNA as an intermediate product.[20] With this defense mechanism, organisms maintain genetic integrity and hinder infection. In recent years, RNAi has been employed as a potential therapeutic agent for combating a wide range of disorders and systemic diseases including cancer, infectious diseases and metabolic disorders. Over the past 10 years, a plethora of in vitro and in vivo studies have showed that practically every human disease with a loss of function of one or more of the genes can become a target for therapeutic RNAi. These studies have been extensively reviewed in detail in the recent literature and are briefly listed [Table 1].

Table 1

In vivo disease treatments using RNAi

Yet in recent months, there are also increasing studies on the various therapeutic modalities and exploitation of the types of RNAi. A recent concern with RNAi in therapeutic use is that when used to target pathogenic viruses, it might face the same problems as that of other monotherapies. The biggest challenge is to control viral mutants, which rapidly arise and escape due to the often high viral mutation rate. This is especially true for RNA viruses, such as hepatitis C virus. Thus, the superior specificity of RNAi can turn into a disadvantage as a single nucleotide mismatch between transgenes incorporated and the target mRNA can abrogate recognition and thus hinder the silencing process. A clever solution to the problem of viral variability is to target their genomes with a cocktail of siRNAs using combinatorial RNAi or with vectors expressing multiple shRNAs.[36]

APPLICATION OF RNAi IN DENTISTRY

Song et al., in 2006 developed a strategy using a lentivirus- mediated RNAi approach to silence gene expression in dental mesenchymal cells and assess gene function in tooth development.[37] The authors showed that knock down of Ms × 1 or Dl × 2 expression in the dental mesenchyme faithfully recapitulate the tooth phenotype of their targeted mutant mice. Silencing of Bar × 1 expression in the dental mesenchyme causes an arrest of tooth development at the bud stage, demonstrating a crucial role for Bar × 1 in tooth formation. Thus, they established are liable and rapid assay using RNAi that would permit large-scale analysis of gene function in mammalian tooth development. Girard et al., in 2011 used RNAi in the construction of cellular models to understand the functional consequences of the causative mutations in SH3TC2 in charcot marie tooth disease and reported that RNAi is useful to understand the cellular consequences of a loss of function in autosomal recessive diseases and is complementary to the construction of the corresponding knock out models.[38]

Hef et al., in 2009 investigated the effects of Notch-Delta1 RNAi on the proliferation and differentiation of human dental pulp stem cells (DPSC's) in vitro.[39] The deficient notch signaling inhibits the self-renewal capacity of DPSCs and tends to induce DPSCs differentiation under odontoblast differentiation inducing conditions. These findings suggested that DPSC's/Delta 1-RNAi might be applicable to stem cell therapies and tooth tissue engineering. Tumor necrosis factor-α-targeted siRNA can suppress osteolysis induced by metal particles in a murine calvaria model, opening the way to the application of RNAi in orthopedic and dental implant therapy [Table 2].

Table 2

Oral and dental applications of RNAi

Applications in periodontal regeneration

In terms of bone regeneration, Gazzerro et al., have demonstrated that down regulation of gremlin by RNAi inST-2 stromaland MC3T3 osteoblastic cells increases the bone morphogenetic protein-2 stimulatory effect on alkaline phosphatase activity and on Smad 1/5/8 phosphorylation, enhances osteocalcin and Runx-2 expression and increases Wnt signaling, with the potential to increase bone formation in vivo.[44] Wang et al., in 2010 reported that the delivery of siRNA targeting receptor activator of nuclear factor-kappaB (RANK) to both RAW 264.7 and primary bone marrow cell cultures produces short term repression of RANK expression without off-targeting effects and significantly inhibits both osteoclast formation and bone resorption.[45] Moreover, data support successful RANK knock-down by siRNA specifically in mature osteoclast cultures. Taken together, these studies prove that RNAi, when adequately used, can foster tissue regeneration and inhibit bone resorption [Figure 2].

Figure 2

The cytokine receptor activator of nuclear factor-kappaB ligand binds to the receptor protein RANK in an interaction antagonized by osteoprotegerin-RANK-L binding. Upon receptor stimulation by RANK-L, the pre-fusion osteoclasts differentiate into multinucleated osteoclasts and get activated to form active osteoclast, which enhances bone resorption leading to loss of supporting tissues. Delivery of transgenes (small interfering ribonucleic acid) targeting RANK results in repression of RANK expression and thus inhibits osteoclast formation and bone resorption

Off-target effects

When considering using siRNAs as therapeutic drugs, it is also important to investigate the sequence specificity of RNAi and the risk of off-target effects.[46] For instance, it is vital to ensure that only the targeted mRNA is degraded because otherwise essential genes may be blocked. It seems that siRNAs can have off-target effects as a result of one of three mechanisms:[46] (1) Since both shRNAs (pre-siRNAs/pre-miRNAs) and siRNAs contain strings of dsRNA, they can activate non-specific cellular innate immune responses such as the interferon response. (2) Transfected or expressed siRNAs might have other non-specific effects. For example, artificial siRNAs or shRNAs could saturate the cell's RNAi machinery and there by inhibit the function of endogenous miRNAs. (3) Although mature siRNAs are designed to be fully complementary to a single mRNA transcript, they may inadvertently show considerable complementarities to other non-target mRNAs. Studies have shown that an interferon response is induced by dsRNAs more than 30 bp in length, but also perfect dsRNAs as small as 11 bp in length can produce a weak induction.[47]

CONCLUSION

The challenges in regenerative periodontal therapy lie in the ability to induce the regeneration of a complex apparatus composed of different tissues such as bone, cementum and periodontal ligament. Despite yeomen advances in periodontal therapy, a complete regeneration of the damaged periodontium is still unattainable. RNAi is a cellular process that is revolutionizing scientific research and there is considerable excitement about using RNAi for therapy. The technique has an upper hand over the traditional approaches to treating disease, including broad applicability, therapeutic precision and selectivity avoiding side-effects. Thus the wide spread applicability, coupled with relative ease of synthesis and low cost of production makes RNAi an attractive new class of drugs in periodontal regeneration.