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Gene therapy is a field of Biomedicine. With the advent of gene therapy in dentistry, significant progress has been made in the control of periodontal diseases and reconstruction of dento-alveolar apparatus.
Implementation in periodontics include:
-As a mode of tissue engineering with three approaches: cell, protein-based and gene delivery approach.
-Genetic approach to Biofilm Antibiotic Resistance.
Future strategies of gene therapy in preventing periodontal diseases:
-Enhances host defense mechanism against infection by transfecting host cells with an antimicrobial peptide protein-encoding gene.
Gene therapy is one of the recent entrants and its applications in the field of periodontics are reviewed in general here.
Genes are specific sequences of bases present on the chromosomes that form the basic unit of heredity. Each person's genetic constitution is different and the changes in the genes determine the differences between individuals. Some changes, usually in a single gene, may cause serious diseases. More often, gene variants interact with the environment to predispose some individuals to various ailments.
Gene therapy uses purified preparations of a gene or a fraction of a gene, to treat diseases [Figure 1]. A common approach in gene therapy is to identify a malfunctioning gene and supply the patient with functioning copies of that gene. Whichever approach is used, the aim of gene therapy is to introduce therapeutic material into the target cells, where it becomes active and exerts the intended therapeutic effect.
In the mid 1980s, the focus of gene therapy was entirely on treating diseases caused by single-gene defects. However, in the late 1980s and early 1990s, the concept of gene therapy was being increasingly considered for the treatment of a number of acquired diseases.
Remarkable progress has been made in the field of gene therapy, including seven areas relevant to dental practice: bone repair, salivary glands, autoimmune disease, pain, DNA vaccinations, keratinocytes and cancer.
For correcting faulty genes, one of the several approaches may be employed:
A gene inserted directly into a cell usually does not function. Instead, a carrier called a vector is used to introduce the therapeutic gene into the patient's target cells [Figure 2]. The most common vector used is a virus.
Viral vectors are natural infectious agents for transferring genetic information. They are quite efficient, and at present they generally provide more pre-clinical and clinical utility than non-viral vectors, although that gap is diminishing.
Viruses cause diseases in humans by encapsulating and delivering the genes into cells. Some types of viruses, such as retroviruses, integrate their genetic material (which can be manipulated to include the therapeutic gene) into a chromosome in the human cell. Other viruses, such as adenoviruses, introduce their DNA into the nucleus of the cell, but the DNA is not integrated into a chromosome.[8–10,15]
Some of the different types of viruses used as vectors in gene therapy are:
The vector can be given intravenously or injected directly into a specific tissue in the body. Alternatively, cultured cells are exposed to the vector and then reintroduced into the patient.
Besides virus-mediated gene-delivery systems, there are several non-viral options for gene delivery.
a) The simplest method is the direct introduction of therapeutic DNA into target cells.
Disadvantage: It requires large amounts of DNA to bring out the desired effect and hence this technique has restricted use.
b) Another non-viral approach involves the creation of an artificial lipid sphere (a liposome) with an aqueous core. This liposome, which carries the therapeutic DNA, is capable of transporting the DNA through the target cell's membrane. Therapeutic DNA can also be introduced into target cells by chemically linking the DNA to a molecule that will bind to special cell receptors. Once bound to these receptors, the therapeutic DNA constructs are engulfed by the cell membrane and passed into the interior of the target cell.
Disadvantage: This delivery system tends to be less effective than the others
c) Experiments with the introduction of a 47th chromosome (an artificial, human techno-chromosome) into target cells are being carried out. This chromosome would exist autonomously alongside the standard 46th chromosome, without affecting their functions or causing any mutations. It would be a large vector capable of carrying substantial amounts of genetic code and, because of its construction and autonomy, the body's immune system would not attack it.
Disadvantage: Difficulty in delivering such a large molecule into the nucleus of a target cell.
Gene therapy can target somatic (body) or germ (egg and sperm) cells. In somatic gene therapy the recipient's genome is changed, but the change is not passed on to the next generation; with germ line gene therapy, the newly introduced gene is passed on to the offspring.
Successful gene delivery is not easy or predictable, even in single-gene disorders. For example, although the genetic basis of cystic fibrosis is well known, the presence of mucus in the lungs makes it physically difficult to deliver genes to the target lung cells. Delivery of genes for cancer therapy may also be complicated by the disease at several sites.
Some gene therapy approaches aim at long-term effects. Two possible ways of achieving this are to either use multiple rounds of gene therapy or integrate the therapeutic genes so that they remain active for some time. Integrating therapeutic DNA may cause possible undesirable side effects.
For instance, babies with the X-SCID syndrome go on to develop leukemia-like symptoms as therapeutic material might have integrated at a site where it affected another gene which may have produced the rapid growth of cancerous cells.
Other approaches seek more immediate effects where integration is not the aim. For instance, in gene therapy to treat cancer, the aim may be to use ‘suicide’ genes to kill cancerous cells as quickly as possible.
Viral vector may be recognized as ‘foreign’ and mobilize the immune system to attack it. This may hamper the efficacy of gene therapy or induce serious side effects.
Viruses present a variety of potential problems to the patient, e.g., toxicity, immune and inflammatory responses and gene control and targeting issues. In addition, viral vector may recover its ability to cause disease.
First, and thus far only, death as a result of a clinical gene transfer procedure, occurred in 1999.
The tissue engineering approach reconstructs the natural target tissue by combining four elements, namely, the scaffold, signaling molecules, blood supply and cells. Currently, genetic principles are being applied along with tissue engineering for periodontal rehabilitation.
The three basic approaches in tissue engineering are the following
Ex vivo gene delivery: In this approach, cultured cells are transfected (in non-viral delivery systems) or transduced (in viral delivery systems) with gene constructs in vitro before they are transplanted into the tissue defect [Figure 4].
BMP gene delivery for alveolar bone engineering at dental implant defects:
An adenoviral vector with a collagen matrix to immobilize the transgene at the dental implant defect site was used that led to optimal effects in both transduction efficiency and bone repair. In addition, previous studies used a similar dosing. However, future dose levels using direct gene delivery of BMPs need to be explored. This technique efficiently delivered platelet-derived growth factor genes to tooth-supporting structures. Peak gene expression was found one to seven days after delivery, with a subsequent decrease in expression over time. A low level of transgene expression was detected for up to 10-35 days, suggesting that the collagen was capable of immobilizing the virus for extended periods of time.
Biofilms are surface-attached microbial communities with characteristic architecture and phenotypic and biochemical properties distinct from their free-swimming, planktonic counterparts. One of the best-known of these biofilm-specific properties is the development of antibiotic resistance that can be up to 1,000-fold greater than planktonic cells. A genetic determinant of this high-level resistance in the Gram-negative opportunistic pathogen, Pseudomonas aeruginosa was reported. A mutant of P. aeruginosa capable of forming biofilms with the characteristic P. aeruginosa architecture, does not develop high-level biofilm-specific resistance to three different classes of antibiotics. The locus identified ndvB, is required for the synthesis of periplasmic glucans. These periplasmic glucans interact physically with tobramycin suggests that these glucose polymers may prevent antibiotics from reaching their sites of action by sequestering these antimicrobial agents in the periplasm. Biofilms themselves are not simply a diffusion barrier to these antibiotics, but rather that bacteria within these microbial communities employ distinct mechanisms to resist the action of antimicrobial agents.
Host responses to recombinant hemagglutinin B of Porphyromonas gingivalis in an experimental rat model:
Extensive studies[33,34] described the cloning of four P. gingivalis genes and their expression in Escherichia coli. These genes encode for the proteins Hag A, B, C, and D. Hag A and D have about 73.8% identity, whereas Hag B and C are 98.6% homologous. However, neither shows significant homology to Hag A. There are several genes encoding for hemagglutinin molecules, which may be an indication of their importance in virulence. Currently, Hag A and B have been more extensively characterized than Hag C or D. Furthermore, there has been a great deal of interest in the potential utilization of Hag B in vaccine development. For instance, hagB gene was expressed in an avirulent strain of S. typhimurium.
Repair of tooth supporting alveolar bone defects caused by periodontal and peri-implant tissue destruction is a major goal of reconstructive therapy. Oral and craniofacial tissue engineering has been achieved with limited success by the utilization of a variety of approaches such as cell-occlusive barrier membranes, bone substitutes and autogenous block grafting techniques. Signaling molecules such as growth factors have been used to restore lost tooth support because of damage by periodontal disease or trauma. This article reviewed emerging periodontal therapies in the areas of material science, growth factor biology and cell/gene therapy. Results from preclinical and clinical trials have been reviewed using the topical application of bone morphogenetic proteins and platelet-derived growth factor for periodontal and peri-implant regeneration. It concludes with recent research on the use of ex vivo and in vivo gene delivery strategies via gene therapy vectors encoding growth promoting and inhibiting molecules (PDGF, BMP, noggin and others) to regenerate periodontal structures including bone, periodontal ligament and cementum.
I am extremely grateful to Dr. Swapna Mahale, MDS Periodontics and Implantology, Professor and PG Guide, whose suggestions and encouragement have been my mainstay. I gratefully acknowledge Dr. Nitin Dani, MDS Periodontics and Implantology, Professor and HOD, who has been a source of inspiration. Dr. Triveni Kale, MDS Periodontics and Implantology, Lecturer, has provided valuable help with her untiring efforts and Dr. Amit Agarwal, MDS Periodontics and Implantology, Lecturer, provided great assistance at every step. I put on record my gratitude to my seniors especially Dr. Padmaja Jadhav, my colleagues and all teaching and non-teaching staff members. Finally, I wish to acknowledge my family's constant, invaluable support.
Source of Support: Nil
Conflict of Interest: None declared.