5. Future of genetic manipulation of tooth movement

With the rise of advanced technology in biomedical engineering and medicine, gene therapy is no longer a science fiction. Several gene therapies have been approved to treat many conditions and deformities not only in the United States but worldwide [111]. In the past decade, gene targeting using endogenous microRNA (miRNA) has emerged as a powerful tool for targeted gene delivery. miRNAs are short, noncoding and highly conserved RNA sequences that tightly regulate the expression of genes by binding to their target sequence in the corresponding mRNAs [112, 113]. Majority of miRNA biogenesis involves transcription by RNA polymerase II to generate primary microRNA (pri-miRNA) followed by Drosha (RNase III enzyme) processing, which produces precursor miRNA (pre-miRNA). The pre-miRNA is transported to the cytoplasm via exportins/RanGTP complex. In the cytoplasm, the pre-miRNA is cleaved by another RNase III enzyme called Dicer to generate mature miRNA. The mature miRNA then forms a microRNA associated RNA-induced silencing complex (miRISC) with Argonaute proteins. The complex is steered to the target mRNA via base pairing with the target sequence of the miRNA. The degree of perfect complementarity at nucleotides 2–8 (binding sequence) in the 5<sup>0</sup> end of the miRNA is essential for a successful action of the RISC complex. Depending on the extent of complementarity with the target sequence, gene expression is repressed either by inhibition of translation or by cleavage of the corresponding mRNA [114]. The process of gene therapy using endogenous miRNAs involves selection process of miRNA candidates, design of expression cassettes if constant expression is needed, selection of delivery carrier, and evaluation of system in cells, animal models and clinical trials [114]. Several miRNAs have been reported for their expression and roles in PDL and alveolar bones [115–118]. Under loading, several miRNAs in PDL and alveolar bone respond to the loading force and orientation of forces in different pattern of expression [119–121]. miRNA-21 has been shown to have critical roles in PDL, osteoblasts and osteoclasts [120, 122–127]. In addition, miRNA-21 deficient mouse demonstrated delayed tooth movement compared to the control mice via inhibition of osteoclastogenesis [127]. miRNA-29 was reported as a crucial miRNA for alveolar bone remodeling during tooth movement due to its expression under different orientation of loading forces and its expression profile in crevicular fluid during tooth movement in human [121, 128]. miRNA-29 expression in human PDL was up-regulated under compression but down-regulated under stretch force orientation [121] and its expression on crevicular fluid increased along the course of tooth movement [128]. Moreover, miRNA-29 sponge transgenic mice demonstrated delayed tooth movement due to the decreased numbers of osteoclasts [129]. These microRNAs could be a target candidate for gene therapy for orthodontic tooth movement. There are viral and nonviral delivery systems in clinical trials for gene therapy. Among viral vector system, lentiviral vector-based system has been developed and tested for its safety for more than 10 years. Non-integrating lentiviral vector have been investigated as a means of avoiding insertional mutagenesis. However, there is a disadvantage of this approach regarding the short-lived of the vectors in dividing cells [130]. Nonviral gene delivery systems (nVGDS) have great potential for therapeutic purposes and have several advantages over viral delivery including lower immunogenicity and toxicity, better cell specificity, better modifiability, and higher productivity. However, there is no ideal nVGDS; hence, there is widespread research to improve their properties [97]. The nVGDS system includes chemicals, peptides, liposomes, and polymers [97]. Exosomes are small (30–150 nm in diameter) extracellular vesicles that are formed in multivesicular bodies and are released from cells as the multivesicular bodies fuse with the plasma membrane. The exosomes were proposed to be used for delivery of miRNAs, protein and oligonucleotide complex [131], and were found to be cell secreted from osteoclasts [131] and in gingival crevicular fluid during the course of tooth movement [128].

of RANKL protein was maintained in the gene therapy group but not in the corticotomy group. The number of osteoclasts in the RANKL gene therapy group was significantly higher at day 10 with or without tooth movement compared to the tooth movement only group. The tooth movement distance was 2 times more in the RANKL gene therapy group and 1.5 times in the corticotomy group; however, the rate of tooth movement slowed down in the corticotomy and controls groups but was constant in the RANKL gene therapy group. It was concluded that gene therapy was an alternative treatment for corticotomy to accelerate tooth movement and the efficacy of treatment was higher than corticotomy to accelerate tooth movement [106]. The OPG gene transfer experiment was performed by another group of investigators using the same viral envelope packaging and delivery system to investigate the inhibition of orthodontic relapse in rats. The first molars in the rats were moved mesially for 3 weeks then the springs were removed to generate orthodontic relapse in the rats. The rats received OPG gene therapy then were observed for 2 weeks. The results showed that relapse was significantly inhibited 2 times compared to the mock and control groups. The bone mineral density and bone volume fraction of alveolar bone were significantly increased in the gene therapy group compared to the mock and control groups. No difference of bone mineral density and bone volume fraction of tibia was found among groups. The investigators stated that local OPG gene therapy to periodontal tissues could inhibit relapse after orthodontic tooth movement via osteoclastogenesis inhibition [110]. The same group of investigators further investigated the effect of local OPG gene therapy on orthodontic root resorption with the same design of experiment. They utilized a microcomputed tomogram and histological analyses. The result showed no difference between root resorption at the beginning and the end of tooth movement in the OPG gene therapy group. However, the repair of root resorption in the gene therapy group was higher than other control groups [107]. Another study investigated the effect of local OPG gene therapy using mesenchymal stem cells as carriers for plasmid containing OPG mRNA. This cell mediated OPG gene transfer was generated by insertion of plasmid containing OPG mRNA into the mesenchymal stem cells and the cells were injected into the animals. The result showed that the cells containing OPG package grew in the animals' PDL and the number of osteoclasts, level of RANKL and bone resorption were reduced significantly after single injection. The level of OPG was highest in the gene

Gene therapy is a promising treatment option for a number of diseases (including inherited disorders, some types of cancer, and certain viral infections). This approach is still in the developing process as an alternative approach to treat deformity or disease that conventional method could not achieved. Although many clinical trials have shown the efficacy of the treatment, the technique remains risky and is still under processes of investigation to make sure that it will be safe and do not elicit any systemic or hereditary effects for the patients.

With the rise of advanced technology in biomedical engineering and medicine, gene therapy is no longer a science fiction. Several gene therapies have been approved to treat many conditions and deformities not only in the United States but worldwide [111]. In the past decade, gene

5. Future of genetic manipulation of tooth movement

therapy group [108].

60 Current Approaches in Orthodontics

Another genome editing system that has recently gained attention in research and clinical application is CRISPR/Cas9 system. The CRISPR/Cas9 system is based on CRISPR (clustered regularly interspaced short palindromic repeats) sequence and CRISPR associated (Cas) gene mechanism that are crucial for innate defense mechanism in bacteria and archaea enabling the organisms to respond to and eliminate invading genetic materials from their phages [132]. The CRISPR/Cas9 system consists of two key molecules that introduce a mutation into the DNA. First, Cas9 is an enzyme that acts as a pair of DNA scissor. It cuts the two strands of DNA at a specific location in the genome so the genome editing could be performed either addition or removal. The other molecule is guide RNA (gRNA) which consists of a small piece of predesigned RNA sequence (20 bases long) located within a long RNA scaffold. The long RNA scaffold binds to DNA and the gRNA sequence guides Cas9 to edit the specific part of genome. gRNA sequence is designed to be complementary to the target DNA sequence in the target gene in the genome. gRNA sequence consists of short palindromic repeats and the sequences that complement with the target genes. The target sequences should be present close to protospacer adjacent motif (PAM) sequence which increases the specificity of Cas9. After Cas9 nuclease enzyme site specifically cleaves double stranded DNA activating double-strand break repair machinery. If the DNA repair template is provided, the piece of DNA repair template will be inserted into the sequence of target genes [133, 134]. With this mechanism, the plasmid containing gRNA, Cas9 sequences, TracrRNA (transactivating CRISP RNA) and DNA repair template sequence can be introduced into cells or embryo of the animals by viral or nonviral delivery system [135]. Until now, there is no CRISPR/Cas9 experiment involving orthodontic tooth movement, however, this technology has been implemented in recent mineralized tissue research [136–139]. Future directions of gene therapy include the enhancement of the lentiviral vector-based approaches, fine tuning of the conditioning regimen, and the design of safer vectors or nonviral vector delivery system. In orthodontic field, the gene therapy approach will need several fundamental cell culture and animal experiments to demonstrate the safety and efficacy of the treatment concept. Clinical trials are required as the next step to ascertain the clinicians and patients for efficacy of the treatments.

Author details

United States

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Phimon Atsawasuwan1

\* and Sajjad Shirazi2,3

1 Department of Orthodontics, University of Illinois at Chicago, Chicago, Illinois,

3 Dental and Periodontal Research Center, Tabriz University of Medical Sciences, Iran

2 Department of Oral Biology, University of Illinois at Chicago, Chicago, Illinois, United States

Advances in Orthodontic Tooth Movement: Gene Therapy and Molecular Biology Aspect

http://dx.doi.org/10.5772/intechopen.80287

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