Introduction to Gene Therapy in Periodontics

A gene is the basic physical and functional unit of heredity. Genes are made up of DNA consisting of two types of base pairs, that is, AT and GC which can be turned into instruction. Gene therapy can be defined as the genetic modification of cells for therapeutic purposes 1. This therapy aims at transferring the DNA of interest (such as growth factor) into cells, thereby allowing the DNA to be synthesized in these cells and its protein (termed recombinant protein) expressed. The research in the field of gene therapy was initiated initially for patients suffering from Severe Combined Immunodeficiency (SCID) in 1999. Eventually, in the year 2000, first gene therapy “success” resulted in SCID patients with a functional immune system. However, soon after the trials were stopped because two of ten patients in one trial developed leukemia resulting from the insertion of the gene-carrying retrovirus near an oncogene.

Fundamentals of gene therapy

As already stated, gene therapy aims at transferring the DNA of interest into cells, thereby allowing the DNA to be synthesized and proteins of interest formed eventually. This can be achieved by the following methods,

  • The most common technique is by inserting a normal gene into a nonspecific location within the genome to replace a non-functional gene.
  • An abnormal gene could be swapped for a normal gene through homologous recombination.
  • Selective reverse mutation can be used to repair the abnormal gene, which returns the gene to its normal function.
  • The regulation (the degree to which a gene is turned on or off) of a particular gene could be altered.
  • Spindle transfer is used to replace entire mitochondria that carry defective mitochondrial DNA.



Types of gene therapy

The gene therapy can be classified into the following two types,

Germline gene therapy

In germline gene therapy, germ cells (sperm or egg cells) are modified by the introduction of functional genes into their genomes. Modifying a germ cell causes all the organism’s cells to contain the modified gene. The change is therefore heritable and passed on to later generations. Although this technique has never been tested on humans but has been tested on animals. Various techniques used for germline gene therapy include,

  • Gene delivery to the nuclei taken from somatic cells at metaphase stage 2, 3.
  • Ex vivo alteration of egg cells, following in vitro fertilization 4, 5.
  • Manipulation of embryonic stem cells of the mouse during in vitro culture by different gene delivery systems 5, 6, 7.
  • Pronuclear microinjection of exogenous DNA solution by a glass needle 8.
  • Transgenic delivery into sperm cells by direct or indirect injection to the testis or other parts of the genital system 9, 10.

Somatic gene therapy

In the case of somatic gene therapy, the therapeutic genes are transferred into the somatic cells of a patient. Any modifications and effects will be restricted to the individual patient only, and will not be inherited by the patient’s offspring. There are 3 types of somatic gene therapy,

  1. Ex vivo delivery: In this system, the genetic material is explanted from the target tissue or bone marrow, cultivated and mani-pulated in vitro, and then transducted and/or transfected into the target tissue.
  2. In situ delivery: It involves administration of the genetic material directly into the target tissue.
  3. In vivo delivery: It involves the transfer of genetic material through an appropriate vector, which can be a viral or non-viral vector, into the target tissue.

Gene delivery systems

There are various methods that can be used to allow uptake of the gene that has been selected to target the cell. A successful gene delivery procedure involves minimizing potential inhibitory inflammatory response while also overcoming certain barriers at each step of the gene delivery procedure, in order to optimize gene activity 11. There are primarily two systems used for gene delivery,

  1. Viral gene delivery systems.
  2. Non-viral gene delivery systems.

Viral gene delivery systems

Viral vectors are one of the most successful method used for gene delivery. Various viruses used for gene delivery include retrovirus, adenovirus (types 2 and 5), adeno-associated virus, herpes virus, poxvirus, human foamy virus (HFV), and lentivirus 12. Viral systems have advantages such as constant expression and expression of therapeutic genes. However, there are some risks associated with viral vectors which limit their use on humans. These particularly include immunogenicity, toxicity and lack of optimization in large-scale production.

Non-viral delivery systems

These systems have been developed as an alternative to the viral gene delivery systems. One of the most important advantages of these systems is that they develop transfection. The non-viral gene delivery systems are divided into two categories…………………….

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Problems associated with the application of gene therapy

Unpredictability of gene delivery: A successful delivery of the gene into the target cell is not easy or predictable, even for single-gene disorders. For example, the genetic basis of cystic fibrosis is well known, but the delivery of gene therapy is still difficult because of the presence of mucus in the lungs.

Short-lived nature of gene therapy: The primary requirement of successful gene therapy is that the therapeutic DNA introduced into target cells must remain functional and the cells containing the therapeutic DNA must be long-lived and stable. Because of rapidly dividing nature of some cells and problems associated with the integration of the therapeutic gene into the target genome, prevent gene therapy from achieving any long-term benefits. Multiple rounds of gene therapy have to be performed to achieve the desired results in a patient.

Activation of the immune response: As already stated, the viral vector may be recognized as antigen and leads to activation of the immune response. The result may precipitate in the form of a sever undesirable immunogenic response.

Risk of malignancy: The insertion of therapeutic DNA at a wrong position in the genome may elicit uncontrolled cell division and may cause malignancy. As already stated, this outcome was observed in patients with SCID patients treated with gene therapy.

Safety of vector: The viral vectors, although provide us a very effective tool for gene therapy, but they may also cause multiple problems associated with the carrier virus including toxicity, immune and inflammatory responses, and undesirable gene expression.

Multigenic disorders: There are various diseases and conditions that have a multigenic inheritance such as heart disease, high blood pressure, Alzheimer’s disease, arthritis, and diabetes. These diseases and conditions are difficult to treat with gene therapy.

Expensive: Gene therapy is costly and very expensive procedure.

Present status of gene therapy in periodontics

Since the introduction of gene therapy, attempts have been made to regenerate the tooth itself and tooth-supporting periodontal structures. As already discussed in “Tissue engineering in periodontics”, there are three approaches of tissue engineering in periodontics,

Protein-based approach: Application of growth and differentiation factors such as TGF-β, BMP-2,6,7,12, bFGF, VEGF and PDGF for the regeneration of periodontal tissues 13.

Cell-based approach: Using the mesenchymal stem cells for regeneration of the lost periodontal structures, including periodontal ligament, cementum and bone 14.

Gene delivery approach: It involves the integration of therapeutic DNA into the target cell to achieve periodontal regeneration. This approach has been utilized to overcome the short half-lives of growth factor peptides in vivo. So far, two main strategies for gene vector delivery have been applied to periodontal tissue engineering. The in vivo approach utilizes the gene delivery system to target the desired cell type in the patient using various methods described above. As these approaches are based on somatic gene therapy, therefore the genetic alteration will not be transmitted to the next generation. In many countries, human germline gene therapy is considered unethical or even illegal. The ex-vivo gene therapy is performed by transfecting or infecting patient-derived cells in culture with vector DNA and then re-implanting the transfected cells into the patient.



Literature review on gene therapy in periodontics

Application of gene therapy in periodontics has been a major focus of research. Various studies have been done to utilize the potential of this technology to improve periodontal regeneration. The research done so far in the field of regenerative periodontal therapy can be discussed under the following headings,

Bone grafts: Discussed in detail in chapter 67 “Bone grafts in periodontics”.

Barrier membranes: Discussed in detail in chapter 66 “Guided tissue regeneration”.

Biologics

The most extensively studied method to achieve periodontal regeneration is the application of various biological mediators to achieve periodontal regeneration. The mediators can be broadly categorized into…………………….

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Growth factors and other bioactive mediators

PDGF is an important growth factor which plays a vital role in periodontal tissue repair by promoting PDL, fibroblast, and cementum proliferation. PDGF-BB is approved for periodontal regeneration and is commercially available (GEM21®) . Lin et al. (2015) 16 in a systematic review stated that there is good evidence that supports a long term improvement in treatment outcomes in periodontal regeneration with the use of PDGF. Nevins et al. (2013) 17 in a randomized controlled trial demonstrated the clinical and radiographic improvement of localized periodontal defects.

        FGF-2 is an important growth factor that participates in wound healing. It has been observed that it inhibits osteogenic differentiation of PDL cells, maintaining their differentiation potential and increasing cellular proliferation 18. Kitamura et al. (2011) 19 in a clinical trial reported a significant improvement in periodontal regeneration and bone fill in intrabony defects.

        BMPs have been studied extensively for their regenerative potential in periodontal defects. Kirker et al. (2000) 20 in a review stated that BMP-2, 4 and -7 can be effectively used in periodontal regeneration. In an experimental study on rats, a direct gene therapy using an adenoviral gene delivery system for BMP-2 with β-TCP scaffold was done. The results of the study demonstrated a significant increase the mandibular bone repair and new bone formation 21. In another study, ex vivo BMP-2 gene delivery using canine periodontal ligament stem cells (PDLSCs) was evaluated for regeneration in peri-implantitis defects. Firstly peri-implantitis was induced by ligature placement in six beagle dogs, then, regenerative procedures were performed; HA particles and collagen gel with autologous canine PDLSCs were used. The results of the study demonstrated that ex vivo BMP-2 gene delivery using PDLSCs enhanced new bone formation and re-osseointegration in peri-implantitis defects 22. The commercially available preparation containing BMP-2 is Infuse®.

       TGF-β is a multifunctional growth factor structurally related to bone morphogenetic protein, but is functionally quite different. This growth factor has also been evaluated for periodontal regeneration. It has been proposed to play an important role in inducing fibroblastic differentiation of PDL stem/progenitor cells and in maintaining the PDL apparatus under physiological conditions 23.

        EMDs (enamel matrix derivatives) were introduced into clinical dentistry because of their property of improving periodontal regeneration 24. Recently, they have been shown to improve the periodontal regeneration in suprabony defects 25.

Stem cell therapy:

It is the cell-based regenerative approach in which a disease is treated by introducing new cells into a tissue. For cell-based techniques in tissue engineering, both somatic cells and stem cells can be used. The process of stem cell therapy involves harvesting the desired cells, culturing them and finally delivering them at the site where regeneration is desired. Various periodontal cells have been evaluated for their regenerative potential, including PDL fibroblasts, cementoblasts, and dental follicle cells. These cells have demonstrated their regenerative potential 26. One emerging and promising technique of stem cell therapy is using the induced pluripotent stem cells (iPSCs). In this technique, the somatic cells are harvested and subjected to gene therapy. These cells are then reprogrammed into a multipotent or pluripotent state (iPSCs), followed by differentiation of iPSCs into a homogenous population of patient-specific terminally differentiated cells 27. However, these therapies are still in their infancy and a lot of research is required before they are introduced into routine clinical practice.

Gene therapy:

Because of the short half-life of the growth factors in vivo, newer techniques were required to overcome this problem. Gene therapy has been used to overcome this problem. As already stated, in this therapy, genetically modified cells are delivered at the site of interest where they release specific doses of a bioactive protein for a sustained period. Viral and non-viral methods are used in this technique. In non-viral methods, plasmids, which are small circular DNA structures that can replicate in the cell independently of chromosomes, can be used. However, they are less effective. Gene therapy studies have been done on PDGF using vectors for delivery of PDGF. A sustained and biologically active expression, as well as improved alveolar bone and cementum regeneration, has been reported 28. In addition, Chang et al. (2009) 29 reported that AdPDGF-B used with a collagen matrix in periodontal osseous defects can be considered safe for possible use in human clinical studies. Recently, Driscoll et al. (2015) 30 developed nonintegrating lentivirus vectors and used them for reprogramming fibroblasts into iPSCs. In another study by Chen et al. (2013) 31, localized adenovirus-mediated system was designed to immobilize Ad-BMP7 on titanium discs, resulting in attachment and differentiation of osteoblasts. Gene therapy is still in infancy in periodontics and in future, we will see many developments in this field.

References

References are available in the hard-copy of the website.

 

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Suggested reading

  1. Rios HF, Lin Z, Oh B, Park CH, Giannobile WV. Cell‐and gene‐based therapeutic strategies for periodontal regenerative medicine. Journal of periodontology. 2011 Sep;82(9):1223-37.

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