Gene Therapy In Dentistry
We have seen already in the preceding sections of this book that diseases are caused by defective gene structure and function. Several strategies have been adopted to treat genetic diseases caused by such defects. These strategies are either direct or indirect interventions directed to correct those defects. Indirect methods apparently try to treat the ‘results’ of the disease whereas direct interventions try to mend the ’cause’ (genetic defects) of these diseases.
Attempts have been made to cure genetic diseases with the correction or replacement of defective genes by molecular tools of genetic engineering. Several methods that have been adopted to treat genetic diseases at different levels have been discussed in the subsequent sections of this chapter.
Common Strategies To Treat Genetic Diseases
Prenatal Treatment
The scope of treating genetic diseases in fetuses inside the uterus is absolutely negligible till date. Positive prenatal detection of any genetic disease mostly results in its spontaneous or voluntary termination due to the absence of the option of a permanent cure at the genetic level. There is of course some hope that prenatal treatment for few diseases may evolve in the near future.
Read and Learn More Genetics in Dentistry Notes
A few disorders like congenital adrenal hyperplasia (CAH) and severe combined immunodeficiency can be treated inside the uterus (in-utero) to some extent. Low doses of dexamethasone are given throughout the pregnancy on detection of congenital adrenal hyperplasia (CAH). The later disorder may be corrected by transfusion of stem cells that give rise to immune precursor cells. In both the cases the intervention or the therapeutic correction is aimed at the level of the gene products and not at the level of the genes.
It is hoped that gene therapy may become a possibility in near future to treat a genetic disease at the level of the genes. This treatment can be extended not only to living patients but to fetuses detected with the disease in-utero. It is hoped that stem cell transplantations in-utero may treat many serious early onset genetic diseases. In-utero gene therapy has successfully treated cystic fibrosis in mouse.
Treatments at the molecular level include transplantation of stem cells (containing normal genes) at the site of defects or comprise replacement of the defective genes from affected cells within the body. Somatic cells can also be taken out of the body, injected with the normal replacement of genes and then put back inside the body.
Postnatal Treatment
Cure for most of the genetic disorders is not available due to our incomplete understanding of links between defective genes and their products. The therapies directed towards genetic disorders today mainly depend upon supplementation of deficient gene products (enzyme, protein, etc.) from extraneous sources.
Compounding our limited knowledge of the dynamics of genetic disorders is the difficulty to deliver gene products into the cell for intracellular metabolism. Replenishment of secretory products of cells into the extracellular milieu is comparatively a better strategy to combat a genetically mediated deficiency. On the other hand corrections have been attempted in the abnormal genes themselves.
Genes in the germ-line as well as somatic cells in affected tissues have been manipulated in order to integrate normally expressing elements in them. Though no single and foolproof therapy exists in treating genetic diseases, strategies as described below have evolved to minimize the disabilities rising thereof.
The following section discusses the strategies that have been conceived and are being worked upon for developing therapies for treating genetic disorders including dental diseases.
Supplementing a Gene Product
Genetic disorders resulting in the deficiency or reduced effectivity of a gene product (enzyme or protein) can be treated with supplementation of the product from outside. Recombinant DNA technology has proven to be a boon in this regard as it can yield large amounts of polypeptides that can be introduced in affected individuals.
Treating with Drugs
Drugs with varied pharmacotherapeutic effects intervene and allay symptoms in a few of the genetic dis- orders of metabolism. Cholestyramine helps to reduce cholesterol levels in familial hypercholesterolemia as does the chelating agent penicillamine in Wilson’s disease (in defective copper metabolism).
Transplantation or Removal of Tissue
Several approaches of reconstructive surgeries (autologous or allograft bone replacement) are usually tried in the patients who suffer from a major loss of alveolar bone loss. It is a priority to preserve the affected tooth and/or restoration of the diseased tooth is favored instead of sacrificing the tooth. The results of conventional therapeutic modalities in treating genetic diseases are mostly unpredictable at best.
Activation of body’s own reconstructive mechanisms can be targeted with gene therapy in such cases to hasten recovery. Manufacturing of tissues like bone has been attempted from within the body rather than from without.
Stem Cell Transplantation
Stem cell transplantation seems to be a viable option in the near future as a strategy directed for treating genetic diseases. Patients suffering from certain genetic disorders involving the blood cells can be injected with precursor stem cells that differentiate into the required population of matured blood cells. Compatibility matching between the donor and the recipient is mandatory except for administration of stem cells derived from fetal umbilical cord or bone marrow derived mesenchymal stem cells.
Concept Of Gene Therapy And Its Applications
Gene therapy is based on intricate principles of genetic engineering that involves correction of defective genes or their replacement with normally functioning genes in cells. Gene therapy may be of two kinds, Germ line gene therapy and Somatic cell gene therapy. Germ line gene therapy involves genetic manipulation of the defective gamete producing cells so that a normal gamete is produced and a corrected haploid complement of chromosome is transferred to the future generations.
This kind of gene therapy is of course associated with its own moral and ethical issues. Somatic cell gene therapy on the other hand the change of a given somatic environment of an inditargets only particular tissues or organs resulting invidual. This kind of therapy is universally accepted.
Treatments with protein delivery systems have been tried for sometime now. Supplementation with growth factor enhancement can be useful in replenishing bone loses in the alveoli of the mandible. These factors increase the turnover of bone production. The effect of externally introduced growth factors is extremely short-lived with the factors getting dissolved or being broken down by proteolysis. Gene therapy can be adopted as an alternative option for sustaining the delivery of such factors for a prolonged period.
The transfer of genes can be achieved in two ways. In one procedure the desired gene and the vector (within which resides the injected gene) is introduced directly into the area of interest or indirectly through the intravenous route. The vectors are taken up by the target cells.
This direct application of target gene is called the in-vivo process. On the other hand genes may be introduced into cells after being taken out of the body (biopsy) in the laboratory, with the help of inoculating vector viruses. These cells are further cultured (multiplied) and then put back into the host. This process is termed as the ex-vivo method of gene transfer.
Gene Therapy Involves the Following Steps
Identification of the Defective Gene
Several molecular techniques are used to detect defective genes (structural genes, promoter genes, etc.) that are responsible for causing disease. Identification of such genes can be done both in somatic as well as in the germ line cells.
Cloning of Normal Healthy Gene
Cloning or duplication of DNA sequences involves copying of structural genes, promoter regions and other segments of DNA that regulate the expression of that gene. The desired gene is generally cloned or copied inside a vector. The vector is capable of penetrating and depositing the foreign or corrected gene into a target cell. Once inside the cell, a structural gene may take the help of promoters that are already present in the cell for its activation.
Identification of Target Cell Tissue/Organ
Target cells or tissues are the ones that are affected by the functioning of the abnormal gene or genes, e.g. alveoli of the mandible that suffer from bone loss. Cells are taken out from the organ/tissue, genetically manipulated and then they are introduced back into the blood stream. The engineered cells ‘home’ at the target regions to resume normal function. Corrected genes may also be introduced directly at a desired location inside the organism.
The Method of Insertion of a Normal Functional Gene in the Host DNA
A physical and chemical method of gene transfer includes microinjection of DNA into the cells by electroporation (permeability of the cell membrane is increased by application of electric current), Calcium- phosphate precipitation where endocytosis of the DNA element is facilitated by precipitating it with calcium-phosphate.
Cationic liposome mediated gene transfer is another technique in which synthetic cationic lipid vesicles encapsulating DNA particles fuse with specific cell membranes and release DNA into the cell. The methods of physical gene transfer techniques have evolved with time and graduated from the most basic direct injection of the DNA (micro- seeding technique), usage of electrically charged aqueous liposomes (bags of lipid associated DNA) that pass through the cell wall, to the more sophisticated processes of gene delivery by the macromolecular conjugate method where a negatively charged target DNA molecule is attached to an oppositely charged chemical substance or antibodies that bind to certain receptors on the cell wall with subsequent endocytosis of the DNA construct to the interior of the cell.
More advanced physical methods include the transfer of genes with the gene activated matrices (GAMS) where naked DNA fragments are carried on polymer matrix tools for gene delivery as they are safer than the viral sponges. Non-viral or physical methods are attractive methods. DNA of relatively large size also can be delivered with the physical technique. The drawback of the physical method is that it is not as efficient as the viral methods because of its complicated designing and application. Though repeated application of gene transfer is possible with the physical technique but the effect of transfer is short-lived.
The most common method used in gene therapy is the viral vector method for gene transfer. Adeno- viruses and retroviruses are the most used vectors. Adenoviruses are DNA viruses and do not integrate their DNA into the host genome. The disadvantage with this vector is that the introduced gene may be unstable. The inserted gene is activated outside the host genome.
Retroviruses are RNA viruses that integrate into the host DNA. The inserted stretch of viral RNA uses the cellular machinery of the host to synthesize selected proteins from the viral genome but multiplication of the entire virus particle is not allowed by deliberately silencing certain regions of the incorporated viral genome. This prevents propagation of the virus themselves.
Though their uses have not been widely reported, lentivirus and herpes simplex virus are some of the other example of viruses used for this purpose. Adeno-associated viruses are also gaining acceptance for their selective benefits for this technique. All said and done, the transferred gene would only function normally when the coding regions for the gene as well as their regulatory elements are present in the host and more so, when they are correctly aligned. The designing of a perfect genomic architecture is the biggest challenge for the scientists.
The selection of types of promoters that influence the expression of introduced genes is an important aspect of gene transfer. The promoters are responsible for persistent, stable and elevated levels of gene expression. Erroneous selection of viral promoters has shown unregulated expression of undesired mammalian host genes. Promoters are varied in function and as such are put to trials before tagging with specific transferrable genes. The application of tissue-specific promoters is gaining popularity as they allow the genes to be expressed only in specified tissues.
Characteristics of Different Viruses used in Gene Transfer
Viral methods are actually nature’s own mode of gene transfer. Scientists have adopted this technique for delivery of genes to the target cells. Though an efficient technique, viral transfers of genes have their own safety concerns. The criteria for selection of a definite type of viral vector depend upon the tissue target, the duration for which the expression of the transferred gene is desired and the size of the concerned gene to be transferred.
Viruses have different characteristics in terms of their replication. Retroviruses infect only dividing cells whereas adenoviruses and adeno- associated viruses infect both dividing and non- dividing cells. Retroviruses can attach into desired region of the host cell DNA leading to a prolonged and stable expression of the gene. The disadvantage with retroviruses is that its application may cause mutations in the host genome by integrating the gene at ‘risky’ regions in the genome.
Adenoviruses on the other hand introduce DNA into the host cell where these DNA remain independent (called Episomes) and do not integrate into the host genome. Thus with each cell division the number of cells that contain the introduced DNA is reduced.
This results in the period of expression of the introduced gene being reduced. However, adenoviruses can be generated in huge numbers and as such the viruses can be introduced in large numbers directly to the desired tissue (in-vivo). Adeno-associated viruses integrate desired genes to sites in the genome that are not ‘risky’ in terms of mutagenesis.
Retroviruses are preferred and used for an ex-vivo type of gene transfer where cells like the blood or bone marrow cells are briefly taken out of the body and infected with the virus. These cells are then reintroduced into the body. The size of the introduced gene is a limiting factor in developing a fully functional vector. Adenoviruses are the tiniest of viruses and can accommodate a foreign DNA that is only a fraction of the size of its own DNA.
An important step before introduction of the vector virus into the host cell is to render it completely harmless and incapable of self replication within the host cell to cause damage or disease. Viruses are rendered deficient in replication by means of deleting certain elements from their genomes that are involved in replication. These viruses can be manipulated to grow only in laboratory settings and not in any settings outside the laboratory.
Ribozymes are certain types of RNA molecules that can act like an enzyme to cleave and destroy mRNA transcripts of cancer producing genes. Experimentally designed ribozymes directed against transcripts of the E6 and E7 genes of the oral cancer producing Human Papilloma Viruses (type 16 and 18) have been shown to cut and destroy the mRNA of those E6 and E7 proteins that cause defects in the cell growth regulation and produce tumors, especially oral cancers.
The DNA encoding such ribozymes can be introduced inside replication-deficient viral vectors and then these vectors could be used to transfer the gene into the oral mucosal cells to stop E6 and E7 translation and prevent unregulated cellular proliferation.
As discussed in the previous chapter as well as in appropriate segments in the book, the advancement in molecular biology has enabled us to understand the nuances of the development of human structure and the importance of several molecules that work in tandem and with immaculate precision to bring forth flawless and wonderful functional structures. The concept of molecular dentistry is fast gaining its due acceptance as research is progressing toward a detailed understanding of dental diseases and their management.
The human genome project, transcriptomics, proteomics and related developments have revolutionized the discipline of basic sciences. Clinical research is facilitating the application of the ideas of basic science to the benefit of the patients. The oral health professional community, of late, has emphasized their commitment to the need of improving standards of oral health care, education and training about research innovations, discoveries and their clinical applications like never before.
The capacity to design and fabricate tissues and organs has been achieved with interdisciplinary research involving material scientists and biologists, and is no longer a distant dream. Revelation of the regulations of molecular biology has enabled scientists to design models that simulate or mimic biological system.
‘Biomimetics’ is a new concept that uses genetics and stem cell biology methods to engineer biomimetic cartilage, bone, muscle and nerve tissues that have been applied to tackle clinical problems. Such an approach can be applied through molecular dentistry to improve soft and hard tissue engineering and towards regeneration of tooth and salivary glands.
It is to the credit of scientific advancement that it has also transformed imaging procedures. Starting from the application of simple dental X-rays, to the use of magnetic resonance imaging (MRI), 360 degrees craniofacial-oral-dental imaging, computer-assisted tomography, ultrasound imagining, digital radiography and innovations such as biomarker reporter molecule detection, usage of these modalities have changed dimensions of medical intervention.
Recent advancement in molecular genetics has not only aided to the diagnostic confirmation of a disease but also have pinpointed to the etiology of a disorder. Molecular techniques have identified the disease causing events or molecules to ultimate perfection and these tools have also given precise insights to the genetic maps and mechanisms dynamically involved in producing the disease.
Genetic as well as environmental factors affect tooth agenesis. Hypodontic individuals may show the characteristic in isolation or as a feature along with other traits of a syndrome. In both the cases though, hypodontia is determined genetically. Non-syndromic hypodontia involve the Msx1, Pax9, and Axin2 genes.
A few important genes associated with early embryonic development like the Shh, Pitx2, Irf6, and p63 have been implicated in several syndromes that induce dental agenesis. Molecular therapies and bioengineering methods can be used along with dental implants and other conventional treatment modalities for treating tooth agenesis.
The interaction between the genetic and environ- mental factors is complex and thus it is more difficult to implicate a single factor in the development of a dental disorder. Yet we can simplify to understand that there are conditions that are simple and result from single gene defects. The more complex conditions result from the interaction between a set of defective genes and environmental influences.
It is also imperative that one understands the mechanisms and events that shape the development of the craniofacial complex. These events are guided under strict molecular control. Details of each of the genetically defined dental anomalies are available in the book. In the subsequent section of the chapter we would preview the potential application of molecular treatment in dental disorders.
Genetic conditions may be simple (single gene regulated) and complex (multiple gene and environmentally regulated) situations. Genetics and molecular events related to single and multiple gene disorders are discussed in the appropriate sections of the book.
Gene theraphy was tried for the first time on a child suffering from ADA deficiency. Absence of the Gene therapy was tried for the first time on a child adenosine deaminase (ADA) enzyme results in inactivation of the white blood cells leading to incapacitation of the immune system. WBC’s culled from the boy were allowed to mix with viral vectors containing normal ADA genes.
The normal genes got transferred into the white blood cells through the vectors. These WBC’s were cultured further and transfused back in large numbers into the patient. Though the patient required repeated transfusions of the same kind at repeated increasing intervals, this effort paved the way for others improved techniques to follow.
Applications Of Gene Therapy In Dentistry
Use in Bone Repair
In-vivo gene transfer technology is utilized with adenovirus acting as vectors to carry the BMP genes to the diseased area. This recombinant adenovirus (Ad- BMP) population is directly injected to the site of the bony lesion. Lesions of periodontal diseases or surgical wounds can be healed and osseous defects can be treated with new bone replacement.
After being delivered, the genes encoding for bone morphogenetic protein-7 (BMP-7) in the virus tend to upregulate the bone forming mechanisms in the local diseased area and heal large wounds around dental implants in the supporting bones. BMP-7 belongs to the family of cartilage and bone producing gene family.
A mixture of the BMP gene and Adeniovirus has been successfully introduced into target cells at the defect. When inside the host cell, BMP-7 genes are seen to be guided near the host genome by the virus to precise locations where they are required to be present. The host cell stimulates the expression of the BMP that peaks in about ten days. The expression gradually tapers with time as the target gene does not get integrated into the target cell genome and do not get multiplied or replicated at the time of cell division. Thus the effect of the gene is temporary and to the advantage of the treatment.
Ex-vivo methods are also used to transfer BMP 2 and BMP 7 to the target cells. These genes are introduced into cultured keratinocytes outside the body and then introduced to the desired affected areas. The genes help to repair bones, ligaments and the cementum. New bone and blood vessels can also be formed from stem cells that are induced to express bone morphogenetic proteins.
Use in Salivary Glands
Gene transfer has successfully been tried in the salivary glands both with the in-vivo and ex-vivo models. The salivary glands are vulnerable to radiations applied to treat cancers of the head and neck regions. These structures also commonly get affected irreversibly with several autoimmune diseases (Sjögren’s syndrome, etc.). Repair in salivary glands has been achieved by inserting the gene that encodes the water channel protein aquaporin-1 or AQP-1 into the ductal cells of the gland.
This results in the nonsecretory cells of the ducts of the glands being converted into secretory cells thereby restoring the function of the gland. In another example of gene therapy, a definite gene for example, the one responsible for synthesis of the polypeptide histatin is delivered into the cells in the gland resulting in increased levels of its production in the saliva. Histatin being a natural anticandidal polypeptide is postulated to be effective in preventing or treating resistant oral candidal infection. Oral candidiasis is common in AIDS and also occurs secondary to dental implants.
Though the salivary glands are exocrine glands, they can be manipulated to act as an endocrine gland by gene transfer. Genes encoding hormones like the growth hormone can be introduced into the salivary gland. The new endocrine secretions from these glands are carried from the acini directly into the blood and serum.
Application of immunomodulatory properties of stem cells can be utilized to combat autoimmune dis- eases. Specific and local activation of certain genes also can act as mediators of immunomodulation and can prove to be good methods for restricting autoimmune diseases related to salivary glands found commonly in dental practice.
Use in Pain Management
The management of pain involves the participation of maximal resources in dental as well as medical practice. As it is well-established that the intrinsic mechanisms in the body to combat pain depends upon the expression of the endogenous opioids and their receptors, gene therapy has emerged as a promising tool for the management of pain at different levels.
Managing or eliminating pain is a major part of dental practice. The use of viral vector mediated gene transfer is being experimented as the technique to achieve expression of specific genes in the host cell.
The genes enhance the expression of endorphins and enkephalins and simultaneously upregulating the expression of the μ, delta and kappa receptors. This activation of opioid systems at the levels of the peripheral and the central nervous system causes delayed conduction of nociception with induction of analgesia.
Use in Periodontal Diseases
The introduction of the Porphyromonas gingivalis (P. gingivalis) fimbrial gene into the salivary glands through plasmids has been tried successfully with adenovirus recombination. This experiment has resulted in two outcomes. The DNA delivered directly into the salivary glands of the mice has lead to the production of immunoglobulins like IgA, IgG in the saliva as well as antibodies IgG in the serum.
The salivary antibodies are able to reduce plaque formation by neutralizing the plaque forming organism P.gingivalis. Researchers have also identified and isolated the fimbrillin gene. The fimbrillin protein is one of the surface proteins of the organism P.gingivalis.
Recombination and transfer of the fimbrillin gene through adenovirus vectors into salivary glands is expected to secrete the protein fimbrillin locally around the gland and in the saliva. The availability of fimbrillin in the saliva would attach to the pellicle elements and thus prevent the harmful P. gingivalis to attach to the pellicle and form plaque.
Periodontal diseases can be controlled by pre- venting the process of tight microbial attachment to the infecting surfaces. The degree of virulence of a pathogen depends to a great extent to the levels of attachment of the pathogen to a surface. Adherence is brought about by the expression of “tight adherence genes” as found in a certain strain of Actinobacillus. Localized and destructive periodontitis results from Actinobacillus.
The strength of adherence adds to the degree of pathogenicity of organisms. A strategy has been evolved that uses application of artificially mutated strains of the organism deficient in the ‘Tight adherence’ gene. These strains when introduced with the virulent strains of organism colonize with them. This cocolonization of the mutated with the virulent strain causes limitation in the extent of pathogenic colonization of the organism. The spread of periodontitis can be prevented with the help of application of this model of genetic engineering.
Similar to the strategy applied to expedite bone growth, osseous defects in the periodontal region can be addressed by the application of in-vivo or ex-vitro gene transfer of BMP 7 and BMP 9 genes with the help adenovirus vectors into affected regions in the oral cavity. BMP 2 can expedite the formation of blood vessels.
Stem cells with specifically activated genes may also differentiate into osseous tissue on application into the defects.
Hard and soft tissue regeneration is distinctly related to the growth factor called the Platelet Derived Growth Factor (PDGF). This factor is a potent sub- stance and has profound action on cellular proliferation. In situations of tissue injury the interactions between the receptors for this molecule and the PDGF is disrupted that limits the activity of the growth factor. Investigators have tried to transfer the PDGF gene through an adenovirus to the injured areas in order to enhance cell signaling and proliferation.
Use in Keratinocytes
Keratinocytes are preferentially used as targets for the study and therapeutic application of gene therapy. This is due to the fact that keratinocytes being epidermal cells are easily accessible. Culture models for keratinocytes are well-founded techniques. The technique of gene transfer as well as their subsequent therapeutic application and monitoring are simpler in keratinocytes.
Researchers have used the ex-vivo method to transfer genes into cultured keratinocytes with retroviral vectors. These viruses insert the foreign gene permanently into the keratinocyte genome. The keratinocytes are then cultured easily in sheets and are applied for treatment in specified areas.
This technique can be used as gene product delivery systems in the oral mucosa and elsewhere. As keratinocytes are well-designed to deliver proteins, epithelial sheets have already been experimentally made that deliver proteins like apolipoprotein E.
Use in Cancers of the Head and Neck
As mentioned elsewhere in this book the role played by the p53 molecule in detecting structural DNA damage is of immense importance. This system of surveillance identifies defective DNA and stops the progress of the cell cycle and instructs either a DNA repair or cellular apoptosis. Efforts are being made to develop adenoviruses that when introduced into the system replicate and destroy only those abnormal cells that contain a mutated p53 gene.
Normal cells remain unaffected and repopulate the tissue. Such a therapy can boost the outcome of treatment in cancers if they are tried along with the conventional modes of cancer therapy. The genomes of these viruses are manipulated in such a way that their propagating machinery is activated only in conditions where it detects an abnormality in the host p53 molecule.
As discussed earlier, the application of the ribozymes to inactivate the Human Papilloma Virus (HPV 16 and HPV 18) proteins E6/E7 that mediate cancerous growth in the oral cavity has led scientists to create recombinants using the DNA coding for those ribozymes from the protein mRNA.
This strategy is under development and investigators hope that its application would not only halt progression of a primary tumor but also help to scavenge dysplastic cells not yet turned malignant.
Use in Growing New Teeth
Though quite futuristic in outlook, the idea of growing teeth in the laboratory and transplantation to edentulous patients has been worked upon for some time now. This feat of bioengineering would create teeth almost with the composition similar to normal teeth but without nerves or blood vessels.
This effort would involve the identification and activation of several genes that are associated with synthesis of over more than 25 proteins constituting dental tissues. The discovery of the role of the master gene PAX 9 will help to understand the sequence of gene activation critical for fabrication of tooth in time to come.
Dental tissues or dissociated dental cells have been used for at least sometime now for tooth engineering purposes as a part of recombination experiment. Recently, of course, certain type of stem cells and types of non-dental cells have been applied in tooth bio- engineering. These cells range from mesenchymal stem cells, bone marrow stromal cells to dental pulp stem cells.
In 2009, researchers at the Akita University in Japan have reported a novel epithelial-mesenchymal interaction experiment. The report explains an attempt of tooth regeneration by recombination of intact dental epithelium with a transformed, continuous dental mesenchymal cell line (see Suggested Readings) called the odontoblast-lineage cells (OLC).
Interestingly, these cell lines were grown on three dimensional, Use in Periodontal Vaccination in-vitro organ culture constructs and also transplanted beneath the renal capsule in mice as an in-vivo experiment. The OLC seem to have shown induction of dental development in both the in-vivo and the in-vitro models.
Other Modalities of Bone Repair with Gene Therapy
The introduction of Bone sialoprotein (BSP) in areas deficient in osseous tissue can trigger alveolar and periodontal bone proliferation. BSP is expressed in the event of bone repair and regeneration. This gene controls cell differentiation. It has also been found that the BSP is under the control of the mastergene Cbfa. Bone sialoprotein is non-collagenous in nature and one of the chief constituents of bone.
The application of the new NTF-hydrogel technology is based upon the delivery of a nonviral gene mixed with a hyaluronic acid-derived, non-immuno- genic gel at the site of an osseous defect. This technique can be used as an adjuvant to conventional therapies.
This method does not invoke any immune reaction and helps in bone regeneration by inducing the resident cells at the neighboring sites of the wound to add new bone to fill the defect.
Vascular endothelial growth factor (VEGF) delivery into rat mandibular condyles involving in-vivo technique have proven to be of help in cases of craniofacial deformities. This growth factor when delivered using adeno-associated virus (rAAV), have shown subsequent increases in certain osteogenetic and chondrogenetic markers accompanied by increase in the size of the mandibular condyle (See Suggested Readings).
Delivery of antiapoptotic genes like the Bcl2 gene to the site of tissue injury could be effective in recovery. This process involves much more localized delivery of the gene. The gene is actually processed with the gene activated matrix (GAM) technology (as done with NTF-Hydrogen) prior to its application.
These “DNA devices,” are the latest concepts in fabrication of special dental implants. Implantable products are made biocompatible by coating them with polymers capable of incorporating intact DNA molecules. The delivery of specific genes at the required sites creates implants with site-specific gene delivery.
Use in Periodontal Vaccination
The immunization of the salivary glands with non- virulent DNA encoding P. gingivalis and its fimbrial protein using plasmids and adenovirus has been discussed in the preceding paragraphs. Vectors like the Streptococcus gordonii have successfully been tested in animal models against the organisms like the P. gingivalis that cause periodontitis.
It has been observed that inoculation of hemagglutinin in a certain variety of rats increases the levels of IgG antibodies as well as enhances the production of interleukins as an immune response. The availability of these immune mediating factors induces protection against attack of P. gingivalis. Since hemagglutinin has been identified as one of the virulence factors of P. gingivalis, the production of antibodies against hemagglutinin provides such a protection.
Genetic Approach to Biofilm Antibiotic Resistance
It is interesting to note that certain microorganisms become resistant manifolds to antibiotics as they start living in microbial colonies attached to surfaces. This phenomenon is called biofilm formation. The reasons for the development of such resistance are not well understood and may be attributed to the activation of definite genes like the ndyB, which is related to the synthesis of the enzyme glycosyl transferase.
Glycosyl transferase is further linked to the production of periplasmic glucans that impart them resistance against disinfectants and antibiotics. Scientists have been able to identify, isolate and replicate a mutated version of the ndvB gene. This gene when introduced into some of the replicating cells in a pseudomonas biofilm, rendered the other members of the biofilm vulnerable to common antibiotics. Such an approach can be adapted for application in dentistry to destroy resistant bacteria in a biofilm.
Use in Alveolar Remodeling
Alveolar remodeling is a natural phenomenon that occurs due to stress, injuries and inflammation of the periodontal tissue. The alveolar structures including the bone undergo active remodeling as a reaction to mechanical stimulation. The process of remodeling can be expedited by enhancing the expression of several factors that induce and maintain alveolar remodeling.
This can be achieved by the transfer of the LacZ gene into the periodontal tissue directly with the help of a plasmid. The integrated gene within the plasmid can be introduced into cells with the application of an electric impulse (electroporation).
Used in Antimicrobial Control Disease Progression
Host defense mechanisms can be boosted with the introduction of genes that contribute to host cell defense against pathogens. This boosting may be done with supplementation of genes encoding certain anti- microbial agents. These factors or genes can be introduced into the host cell through retroviral mediated in-vivo techniques into the host genome at areas susceptible to infections.
Some proprietary products are available that applies the defensin-2 gene for this kind of an effect. The above discussion on the application of gene therapy can be reviewed in terms of the basic designs of gene transfer into the cell. The approach adopted for gene delivery may be an in-vivo technique involving gene constructs trapped in physical or viral agents and delivered into the cell.
The ex-vivo method transfects cells in culture in-vitro and then introduces them into the target cells in the body. The protein-based methods apply the gene products to the required regions and the cell-based approach uses mesenchymal stem cells for activation of tissue repair.
Constraints and Limitations of Genetic Therapy
Though a lot has been written both in favor and against the application of gene therapy, the message is clearly home that a foolproof therapeutic package involving gene therapy still needs some more ground-work to become a practicable reality. The regulatory authorities have been rightfully alarmed by outcomes of certain trials and are skeptical about the safety as well as the feasibility of such therapies.
Planners have reiterated the need of extensive preclinical trials of novel therapies before they become standard modes of treatment. Other issues related to the confidentiality of genetic information, disclosure of susceptibility concerns and the risk statuses of individuals are a few of the ethical aspects that need to be addressed in context of gene therapy.
There are several systemic disorders associated with specific types of periodontal diseases. Treatments in such cases are basically framed on the logic of treating not only the defect within terms of the parameters of dentistry but treating the symptoms of the disorder as a whole.
Chronic and early-onset periodontitis need chemical and mechanical control of bacterial plaque. Severe congenital neutropenia or depletion in IgA levels my cause premature loss of teeth and need antibiotic prophylaxis along with chemical and mechanical control of bacterial plaque.
Conditions associated with hormonal changes and arising due to unresponsive bacteria call for extensive and rigorous bacterial control.
Diseases and traits that are genetically transmitted have been studied extensively and analyzed for their causative molecular defects and the modes of their inheritance. The frequency of occurrences of the coding elements of the genome has been studied along with that of noncoding sequences in the DNA.
Certain disease causing genes have always been found to occur along with certain noncoding sequences. They have been identified always to occur together and the details of this occurrence is analyzed in linkage studies. This phenomenon occurs perhaps due to the close proximity of these two segments in the genome that always segregate together in the gamete.
As stated earlier, molecular research has revealed that specific regions of the non-coding regions are intimately associated with the inheritance of a particular gene. HLA associations of the disease producing genes have also been discussed earlier. Several thousands of similar genes have also been found existing across different organisms. The sequences of these stretches of DNA have not been defiled or disturbed by time and evolution in the organisms.
The genes and allied segments in the genome are said to be highly conserved in terms of structure and function. The origin of these genes and their subsequent distribution in the nature can be studied by analyzing their inheritance and linkage patterns.
The virulence of certain microorganism as well as susceptibility to diseases in an individual is determined by the genetic make-up of the microorganism as well as the individual. Craniofacial birth defects, orthognathic disorders, abnormal tooth size and shape, cancers, temporomandibular joint diseases and several others are linked to outcomes of gene-gene, gene-environment interactions.
Though gene therapy seems to be the panacea for all genetic disorders, it has its own share of limitation and pitfalls. The technique of gene delivery is tedious and difficult. Even if the gene causing a disorder is identified and mutations are well-defined, an attempt to introduce the corrected version of the gene in a cell may not be successful.
The limitations range from difficulty to pinpoint the exact gene responsible for a disease (except for a single gene disorder), developing an ideal vector for a gene, identification of the site of delivery, compatibility of the environment in the host tissues and eventually the normal and desired expression of the inoculated gene in the system.
The success of gene transfer cannot be predicted successfully specially in cancers as there are multiple affected sites in the system, which makes the decision of selection of the target region difficult. Some of these problems can be circumvented with the understanding of the mechanisms of viral replication and gene regulatory pathways.
Issues related to the durability and integration of the transferred gene is of immense significance as the desired period for sustenance of therapeutic benefits from a gene transfer varies with the type of the disease being addressed. Genetic integration of the transferred material into the host genome provides a long-term replication as well as expression of the gene.
Such functional durability comes at the cost of certain risks of undesirable effects. Unwarranted and unexpected integration of genes at different locations may trigger and disastrous consequences. Multiple introduction of gene therapy is possible with physical agencies but frequent repetition of gene transfer using viral vectors is not recommended.
Precise introduction of genes is the prime requisite for delivering nonspecific apoptotic genes that kill cancer cells. These genes don’t need to integrate into the genome but become active anywhere inside the cell. Applications of these ‘suicide genes’ produce more immediate effects irrespective of the site of application.
The success of gene therapy also depends upon the degree of immune responses elicited by the host especially against viral vectors. Viral vectors elicit immune responses in the host against themselves if the host cell recognizes the vector as “foreign”. In fact development of such immune responses is desired in the host immune system if the therapy is directed against cancer cells in treating carcinomas.
Undesired immune responses reduce the efficacy of the therapy. Repeated applications of viral vector mediated gene delivery may cause increased immune mediated destruction of the viral vectors or may result in serious side effects. Usage of viral vectors may be a potential cause of toxicity, immune and inflammatory responses with the very first instance of its application.
Other than perhaps single gene disorders, more commonly occurring diseases like hypertension and diabetes are dependent on more than a single factor and hence the applicability of gene therapy in such situations is debatable. The other more contentious issues with gene therapy are related to ethical considerations like questions regarding the criteria that decide what is ‘normal’ and what defines ‘abnormality’.
‘Whether a disability can be viewed as a disease’ and ‘whether a somatic gene therapy is more ethical than germ line therapy’ are some of the probing questions that remain to be answered. The issues of the feasibility of developing such expensive treatment modalities and the affordability of these regimes by less affluent population are also unanswered. Majority of diseases in dentistry are difficult to treat with single gene transfers.
New interventions that combine gene therapy with other approaches such as stem cell therapy are fast emerging. Gene therapy has the potential to treat diseases such as cystic fibrosis, cancers, heart diseases and human immunodeficiency virus infection. However, no clinical trial of gene therapy has resulted in the development of a commercially available treatment till date.
Unsettled issues in gene therapy also include the effectiveness of delivery, longevity of the therapy and safety of the procedures. While patient groups are largely satisfied with the current disease- based approach to gene therapy research, scientists have called for more studies on vector safety, delivery techniques, identification the molecular causes of diseases and finding the reasons for uncertainty of outcomes of current applications.
Gene Therapy In Dentistry Summary
- Control of genetic diseases have been tried with several strategies applied both as prenatal as well as postnatal treatment modalities.
- Common strategies to treat genetic diseases include supplementing a gene product, treating with drugs, transplantation or removal of diseased tissue and stem cell therapy.
- Therapy at the level of genes is called gene therapy. Gene therapy may be applied to the germ line cells or directed towards somatic cell lines.
- Gene therapy involves the steps of identification of the defective gene, cloning of normal healthy gene, identification of target cell (tissue or organ) and insertion of a normal functional gene into the host DNA.
- Physical and chemical methods as well as viral vectors are used for gene transfer.
- Genes can be transferred directly into affected tissues (in-vivo process) or may be introduced into cells taken out of the body (biopsy) and then put back into the host (ex-vivo process).
- Ribozymes are certain types of RNA molecules that can act like an enzyme to cleave and destroy harmful mRNA transcripts.
- Gene therapy can be used in bone repair, in treating diseased salivary glands, for pain management and in conditions of periodontal diseases. Applications are also being tried to treat cancers of the head and neck region and for active alveolar remodeling.