Gene therapy in periodontics.

GENES are made of DNA - the code of life. They are made up of two types of base pair from different number of hydrogen bonds AT, GC which can be turned into instruction. Everyone inherits genes from their parents and passes them on in turn to their children. Every person's genes are different, and the changes in sequence determine the inherited differences between each of us. Some changes, usually in a single gene, may cause serious diseases. Gene therapy is 'the use of genes as medicine'. It involves the transfer of a therapeutic or working gene copy into specific cells of an individual in order to repair a faulty gene copy. Thus it may be used to replace a faulty gene, or to introduce a new gene whose function is to cure or to favorably modify the clinical course of a condition. It has a promising era in the field of periodontics. Gene therapy has been used as a mode of tissue engineering in periodontics. The tissue engineering approach reconstructs the natural target tissue by combining four elements namely: Scaffold, signaling molecules, cells and blood supply and thus can help in the reconstruction of damaged periodontium including cementum, gingival, periodontal ligament and bone.

INTRODUCTION

Periodontal diseases, have a broad spectrum of inflammatory and destructive responses, and are thought to be multi-factorial in origin. Genetic variance has been considered as a major risk factor for periodontitis. With the advent of gene therapy in dentistry, significant progress has been made in controlling the periodontal disease and reconstruction of damaged periodontal tissue.

A broad definition of gene therapy is the genetic modification of cells for therapeutic purposes.[1] This approach is becoming possible owing to the increased understanding of the molecular basis for many diseases and the advances in the technology of gene transfer. The goal of gene therapy is to transfer the DNA of interest (for example, growth factor and thrombolytic genes) into cells, thereby allowing the DNA to be synthesized in these cells and its protein (termed recombinant protein) expressed [Figure 1].

Figure 1

Approaches for regenerating tooth supporting structure

Currently genetic principles are being applied along with tissue engineering for periodontal rehabilitation. This article reviews the fundamentals of gene therapy, and its implication in periodontal disease.

HISTORY

The first gene therapy trials on humans began in 1990 on patients with Severe Combined Immunodeficiency (SCID). In 2000, the first gene therapy “success” resulted in SCID patients with a functional immune system. These trials were stopped when it was discovered that two of ten patients in one trial had developed leukemia resulting from the insertion of the gene-carrying retrovirus near an oncogene.

FUNDAMENTALS OF GENE THERAPY

There are a variety of different methods to replace or repair the genes targeted in gene therapy.

A normal gene may be inserted into a nonspecific location within the genome to replace a nonfunctional gene. This approach is most common

An abnormal gene could be swapped for a normal gene through homologous recombination

The abnormal gene could be repaired through selective reverse mutation, 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.[2]

TYPES OF GENE THERAPY[3]

Gene therapy may be classified into the following types:

Germ line gene therapy

In the case of germ line gene therapy, germ cells, i.e., sperm or eggs are modified by the introduction of functional genes, which are ordinarily integrated into their genomes. Therefore, the change due to therapy would be heritable and would be passed on to later generations.

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.

GENE DELIVERY

In general, gene therapy involves the transfer of genetic information to target cells, which enables them to synthesize a protein of interest to treat disease.[456] The technology can be used to treat disorders that result from single point mutations.[7] The preferred strategy for gene transfer depends on the required duration of protein release and the morphology of the target site production.[8] There are various methods for gene delivery:

Viral

A carrier molecule called a vector must be used to deliver the therapeutic gene to the patient's target cells. Currently, the most common vector is a virus that has been genetically altered to carry normal human DNA. Viruses have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner. Scientists have tried to take advantage of this capability and manipulate the virus genome to remove disease-causing genes and insert therapeutic genes.

Some of the different types of viruses used as gene therapy vectors:[9]

Retroviruses: A class of viruses that can create double-stranded DNA copies of their RNA genomes. These copies of its genome can be integrated into the chromosomes of host cells. Human immunodeficiency virus (HIV) is a retrovirus

Adenoviruses: A class of viruses with double-stranded DNA genomes that cause respiratory, intestinal, and eye infections in humans. The virus that causes the common cold is an adenovirus

Adeno-associated viruses: A class of small, single-stranded DNA viruses that can insert their genetic material at a specific site on chromosome 19

Herpes simplex viruses: A class of double-stranded DNA viruses that infect a particular cell type, neurons. Herpes simplex virus type 1 is a common human pathogen that causes cold sores.

Non viral

The simplest method is the direct introduction of therapeutic DNA into target cells. This approach is limited in its application because it can be used only with certain tissues and requires large amounts of DNA

Another non viral approach involves the use of an artificial lipid sphere with an aqueous core. This liposome, which carries the therapeutic DNA, is capable of passing the DNA through the target cell's membrane.

MAJOR DEVELOPMENTS IN GENE THERAPY

In 1999

Trial treatments of SCID have been gene therapy's only success; since 1999, gene therapy has restored the immune systems of at least 17 children with two forms (ADA-SCID and X-SCID) of the disorder.

In 2002

In 2002 a question was raised when two of the ten children treated developed a leukemia-like condition.[10]

In 2006

Scientists have successfully treated metastatic melanoma in two patients using killer T cells genetically retargeted to attack the cancer cells.[11] As well as in March again, scientists announced the successful use of gene therapy to treat two adult patients for a disease affecting myeloid cells.[12]

Italy reported a breakthrough for gene therapy in which they developed a way to prevent the immune system from rejecting a newly delivered gene. Similar to organ transplantation.[13]

In 2007

The world's first gene therapy trial was done for inherited retinal disease.[14] The sub retinal delivery of recombinant adeno associated virus (AAV) carrying RPE65 gene, was found to be safe and yielded positive results.[15]

In 2009

The journal Nature reported that researchers at the University of Washington and University of Florida were able to give trichromatic vision to squirrel monkeys using gene therapy, a hopeful precursor to a treatment for color blindness in humans.[16]

Technical difficulties in using gene therapy

Difficulty in delivering of gene: Delivery of successful gene in gene therapy is not easy or predictable, even for single gene disorders. For example genetic basis of cystic fibrosis is well known but delivery of gene therapy is still difficult because of presence of mucus in lungs.[2]

Short-lived nature of gene therapy: Before gene therapy can become a permanent cure for any condition, the therapeutic DNA introduced into target cells must remain functional and the cells containing the therapeutic DNA must be long-lived and stable. Problems with integrating therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent gene therapy from achieving any long-term benefits. Patients will have to undergo multiple rounds of gene therapy.

Activation of immune response: Viral vector may be recognized as antigen and leads to activation of immune response. This may lead the efficacy of gene therapy and can induce serious side effect.

Chance of inducing a tumor (insertion mutagenesis): If the DNA is integrated in the wrong place in the genome, for example in a tumor suppressor gene, it could induce a tumor. This has occurred in clinical trials for X-linked severe combined immunodeficiency (X-SCID) patients, in which hematopoietic stem cells were transduced with a corrective transgene using a retrovirus, and this led to the development of T cell leukemia in 3 of 20 patients.

Safety of vector: Viruses, the carrier of choice in most gene therapy studies, present a variety of potential problems to the patient toxicity, immune and inflammatory responses, and gene control and targeting issues. In addition, there is always the fear that the viral vector, once inside the patient, may recover its ability to cause disease.

Difficulty to treat multigene disorders: Conditions or disorders that arise from mutations in a single gene are the best candidates for gene therapy. Unfortunately, some of the most commonly occurring disorders, such as heart disease, high blood pressure, Alzheimer's disease, arthritis, and diabetes, are caused by the combined effects of variations in many genes. Multigene or multifactorial disorders such as these would be especially difficult to treat effectively using gene therapy.

Expensive: Gene therapy is costly and very expensive procedure.

IMPLICATIONS OF GENE THERAPY IN PERIODONTICS

There have been tremendous advances in gene therapy relevant to dentistry since 1995. Even in the field of periodontics, it has been studied extensively. Currently genetic principles are being applied along with tissue engineering for periodontal rehabilitation.

Approaches for regenerating tooth-supporting structures (A) Guided tissue regeneration uses a cell occlusive barrier membrane to restore periodontal tissues. (B) Alternatively, an example of gene therapy uses vector-encoding growth factors aimed at stimulating the regeneration of host cells derived from the periodontium [Figure 2].

Figure 2

In vivo

There are three approach of tissue engineering in periodontics:

Protein based approach

Growth and differentiation factors are used for regeneration of periodontal tissues likes TGF-β, BMP-2,6,7,12, bFGF, VEGF and PDGF.[17]

Cell based approach

Several studies using mesengymal stem cell have demonstrated efficient reconstruction of bone defect that are too large to heal spontaneously.[18]

Gene delivery approach[19]

To overcome the short half-lives of growth factor peptides in vivo, gene therapy that uses a vector that encodes the growth factor is utilized to stimulate tissue regeneration. So far, two main strategies of gene vector delivery have been applied to periodontal tissue engineering.

Gene vectors can be introduced directly to the target site (in vivo technique) or selected cells can be harvested, expanded, genetically transduced, and then reimplanted (ex vivo technique).

IN VIVO

Gene therapy is done by targeting the gene delivery system to the desired cell type in the patient using either physical means such as tissue injection (brain tumor) or biolistics (dermal DNA vaccination), or potentially in the future, using systemic infusion of cell-specific receptor-mediated DNA carriers (reconstructed liposome's or viruses). Importantly, neither of these gene therapy strategies involve reproductive germline cells nor therefore the genetic alteration will not be transmitted to the next generation. In many countries, human germ line gene therapy is considered unethical or even illegal [Figure 2].

EX VIVO

Ex-vivo gene therapy is performed by transfecting or infecting patient-derived cells in culture with vector DNA and then reimplanting the transfected cells into the patient. Two types of ex-vivo gene therapies under development are those directed at fibroblasts and hematopoietic stem cells [Figure 3].

Figure 3

Ex vivo

CLINICAL TRIALS USING GENE THERAPY

The application of PDGF-gene transfer strategies to tissue engineering originally was generated to improve healing in soft tissue wounds, such as skin lesions.[20] But, recently various trial have been done PDGF using Plasmid[21] and Ad/PDGF gene deliver,[22] for regeneration of periodontal tissue.

Platelet-derived growth factor gene delivery

Early studies in dental applications using recombinant adenoviral vectors that encode PDGF demonstrated the ability of these vectors constructs to transduce potently the cells isolated from the periodontium (eg osteoblasts, cementoblats, PDL cells, and gingival fibroblasts).[23] continuous exogenous delivery of PDGF-α may delay mineral formation induced by cementoblasts, whereas PDGF clearly is required for mineral neogenesis.[24]

Jin et al., demonstrated in their study that direct in vivo gene transfer of PDGF-B stimulated tissue regeneration in large periodontal defects.[25]

Anusaksathien et al., reported that in an ex vivo investigation, showed that the expression of PDGF genes was prolonged for up to 10 days in gingival wounds.[26]

Giannobile et al., reviewed different mechanisms of drug delivery and novel approaches to reconstruct and engineer oral- and tooth-supporting structures, namely the periodontium and alveolar bone.[27]

Bone morphogenetic protein delivery

Franceschi et al., investigated in vitro and in vivo Ad gene transfer of BMP-7 for bone formation.[28]

Dunn et al., demonstrated that in case of direct in vivo gene delivery of Ad/BMP-7 in a collagen gel carrier promoted successful regeneration of alveolar bone defects around dental implants.[29]

GENE ENHANCED TISSUE ENGINEERING

The general strategy of tissue engineering is to supplement the regenerative site with a therapeutic protein like growth factors. However the problem with the delivery of growth factor is its short life. This is due to protelytic breakdown and receptor mediated exocytosis and solubility of delivery vehicle. To overcome these problems, gene therapy has been developed which provides long term exposure of growth factor to periodontal wound [Figure 4].

Figure 4

Tissue engineering

APPLICATIONS

They are as following:

Gene Therapeutics-Periodontal Vaccination

Genetic Approach to Biofilm Antibiotic Resistance

An In vivo Gene Transfer by Electroporation for Alveolar Remodeling

Antimicrobial Gene Therapy to Control Disease Progression

Designer Drug Therapy in Treating Periodontal Disease

Gene therapeutics-periodontal vaccination

A salivary gland of a mouse when immunized using plasmid DNA encoding the Porphyromonas gingivalis (P. gingivalis) fimbrial gene produces fimbrial protein locally in the salivary gland tissue resulting in the subsequent production of specific salivary immunoglobulin's A, or IgA and immunoglobulin G, or IgG, antibodies and serum IgG antibodies. This secreted IgA could neutralize P. gingivalis and limit its ability to participate in plaque formation.

Scientists have also demonstrated the efficacy of immunization with genetically engineered Strepto-cocci gordoni vectors expressing P. Gingivalis is fimbrial antigen as vaccine against P. gingivalis associated periodontitis in rats.[30]

The gene hemagglutinin which is an important virulence factor of P. gingivalis has been identified, cloned and expressed in Escherichia coli. The recombinant hemagglutinin B (rHag B) when injected subcutaneously in Fischer rats infected with P. gingivalis showed serum IgG antibody and interleukin-2 (IL-2), IL-10, and the IL-4 production which gave protection against P. gingivalis induced bone loss.[31]

Genetic approach to biofilm antibiotic resistance

Researchers have found bacteria growing in biofilms become up to 1,000 fold more resistant to antibiotics as compared to a planktonic counterpart making them hard to control.

Recently Mah et al., identified gene ndvB[32] encoding for glycosyltransferase required for the synthesis of periplasmic glucans in wild form of Pseudomonas aeuroginosa RA14 strain.

This remarkably protected them from the effects of antibiotics biocides, and disinfectant [Figure 5].

Figure 5

Genetic approach to biofilim antibiotic resistance

Using a genetic approach.

Researchers have isolated ndvB mutant of Pseudomonas aeuroginosa still capable of forming biofilm but lacking the characteristic of periplasmic glucans there by render- ing microbial communities in biofilm more susceptible to conventional antibiotic therapy [Figure 6].

Figure 6

Electroporation for alveolar bone

An in-vivo gene transfer by electroporation for alveolar remodeling[33]

Using an in vivo transfer of LacZ gene (gene encoding for various remodeling molecules) into the periodontium and using plasmid DNA as a vector along with electroporation (electric impulse) for driving the gene into cell, has shown predictable alveolar bone remodeling

Step A- Cells obtained from outpatient skin biopsy

Step B- Gene of therapeutic interest is introduced into cells by electroporation

Step C- Genetically engineered cells are propagated and characterized

Step D- Genetically engineered cells are returned back to clinician [Figures 6 and 7].

Figure 7

Electroporation for alveolar bone

Antimicrobial gene therapy to control disease progression[34]

One way to enhance host defense mechanism against infection is by transfecting host cells with an antimicrobial peptide/protein- encoding gene

Researchers have shown when host cells were infected in vivo with β defensin-2 (HBD-2) gene via retroviral vector; there was a potent antimicrobial activity which enhanced host antimicrobial defenses.

Designer drug therapy in treating periodontal disease[34]

If genes necessary for normal development are known, then “designer drug therapies” aimed at one area of the gene or the other can be developed.

These designer drugs will be safer than today's medicines because they would only affect the defect in a gene clearly identified through genetic research.

CONCLUSION

Gene therapy has a promising role in the field of periodontics but it does encompass serious ethical issue to be dealt with. It is evident that gene therapy has emerged from its stage of infancy of mere theoretical and hypothetical quotations to factual scientific researches, which reveals potential hopes. There are still lots of research and details of mechanisms to be understood to include these practically in day to day treatment modalities.