Gene Therapy in Oral Tissue Regeneration

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Gene Therapy in Oral Tissue Regeneration

Fernando Suaste Olmos,1,* Patricia González-Alva,2 Alejandro Luis2 and Osmar Alejandro2


 

 

 

Introduction to Gene Therapy

Definition

Gene therapy is an experimental strategy initially developed for the treatment of a variety of genetic disorders. This approach pursues the re-establishment of a defective gene copy (which could be affected by single nucleotide substitutions, insertions or deletions) and its products by the introduction of a functional copy in trans (Siddique et al. 2016).

Tissue Regeneration (TR) is defined as a process of reproduction and reconstitution of the architecture of tissue loss, which requires three main elements: scaffolds, cell sources and tissue-inducing factors. Stem cells are the main element in tissue regeneration because its self-renewable and totipotential capacity to differentiated into multiple cell lineages. Additionally, growth factors are molecular signals that control the fate of these mesenchymal stem cells under differentiation. In tissue engineering, gene therapy has emerged as an alternative, supporting both cell therapy and therapy that relies on the use of signaling factors.

History

In tissue engineering, different approaches have been conducted to carry out tissue reconstmction and repair. These treatments aim to guide and induce the regeneration of damaged tissue, using specialized scaffolds that allow the rearrangement and proliferation of the cells that reshape the affected region. Likewise, scaffolds can function as triggered vectors that could delivery progenitor cells, growth factors, genes, or more recently, RNA molecules (Intim 2010).

Cell therapy has been constituted as a base strategy for the reconstmction of the tissues of the oral cavity, due to the cellular plasticity that progenitor cells possess to differentiate to multiple cellular lineages. Within the oral cavity, different types of mesenchymal cells have been isolated: Dental Pulp stem Cells (DPSCs), Stem cells from Human Exfoliated Deciduous teeth (SHED), Stem Cells from Apical Papilla (SCAP), Periodontal Ligament Stem Cells (PDLSCs), Dental Follicle Progenitor Cells (DFPCs) and Gingiva-derived Mesenchymal Stem/stromal Cells (GMSCs) (Botelho et al. 2017).

All these cell types are able to self-renew, as well as to differentiate into osteoblasts, adipocytes, odontoblasts and neural cells. While this methodology proposes restoring the mesenchymal cells derived from the bone marrow, oral cavity or other tissue in the wound area could trigger the repair of damaged tissues. The process of differentiation into a particular lineage is highly complex since it involves the space-time expression of growth and transcriptional factors that act in a hierarchical manner triggering signaling pathways, which in turn activates a pattern of gene expression that results in determining cell identity fate.

In addition to this process, the deposition of specific components of the extracellular matrix also contributes synergistically to the establishment and differentiation of each of these cell types. All these elements generate a limiting factor in cell therapy because not all the factors necessary for the differentiation of mesenchymal cells are produced simultaneously at the site of the injury.

The growth factors are a group of small polypeptides involved in the stimulation of different cellular signaling pathways through its association with specific membrane receptors and promoting its phosphorylation in tyrosine, threonine or serine amino acid residues, which in turn activates a complex system of transcriptional regulation inside of the cell.

Skeletal cells produce a variety of biological factors associated with bone remodeling, i.e., PDGF, BMPs, VEGF, FGF, TGF-beta, IGF, which are not exclusive from skeletal cells. In oral cavity regeneration, different signaling factors have been tested to improve the clinical outcomes: Platelet-Derived Growth Factor (PDGF), Bone Morphogenetic Proteins (BMPs), Fibroblast Growth Factor (FGF), Transforming Growth Factor-beta (TGF-beta) (Ohba et al. 2012).

Gene therapy has generated an alternative parallel to cell therapy, since its main component is the delivery of genes, which encode mainly for growth and transcription factors, allowing the activation of the differentiation process of undifferentiated mesenchymal cells present at the site of injury to the corresponding cell lineage. The primary limitations that this methodology faces are associated with the type of vector used for the release of the gene or genes of interest.

The genes used in gene therapy are released through the use of viral vectors such as adenovirus, adeno-associated viruses, retroviruses and lentivimses. These vectors can integrate stablely and in a random way to the host genome (lentivirus and retrovirus), generating mutations that can derive in severe genetic disorders. Alternatively, in the adeno-associated viruses and adenoviruses that have a very low integration efficiency to the host genome, they generate a robust immune response when producing viral proteins (Jooss and Chirnule 2003).

Currently, methodologies such as PCR (Polymerase Chain Reaction) and genetic cloning have made it possible to manipulate DNA sequences in vitro both coding regions (genes) and regulating elements of gene expression (promoters, enhancers, insulators, etc.). Through this method it has been possible to determine the role of specific genes on the cell homeostasis. Genetic manipulation is also associated with the reprogramming the functioning of a cell.

In prokaryotes, this approach has made it possible to generate organisms that contribute to the synthesis of biomolecules for therapeutic purposes, or to modify organisms that carry out bioremediation processes (Davies 2019).

In bacteria, the delivery of genetic material has been done through the use of plasmids, which are circular DNA molecules that replicate and segregate independently on the host bacterial chromosome (episomes); this allows that the sequences cloned in these vectors are expressed stablely in the bacterial cytosol. Bacterial transformation emerged as one of the first approaches to introduce genetic material into a cell. In this strategy the permeability of the cell membrane is disturbed either by thermal shock or by the use of an electric field (electroporation). Additionally, cellular mechanisms such as conjugation and phage-mediated transduction were used to transfer genetic elements from one cell to another.

Vectors

In mammalian cells, unlike prokaryotes, there are no naturally occurring episomal vectors. The strategy to express target DNA sequences has been based on the development of viral vectors. The delivery of genetic material by this process is known as ‘Transfection’, from which two types are derived: stable transfection and transient transfection (Kim and Eberwine 2010).

In stable transfection the viral vector along with the target DNA sequence is integrated into the genome, and its expression is sustained for an extended period of time; whereas in transient transfection there is no integration into the host cell genome and its expression of the target DNA sequence occurs for a limited period of time (Kim and Eberwine 2010).

Several limitations have been generated from both types of transfection. For example, in stable transfection, the integration of the viral vector is random, which can create insertions in coding DNA sequences (ORFs) resulting in the silencing of a gene or genes and altering cell viability. Otherwise, the gene integrated into the genome is subject to transcriptional and epigenetic regulation controls, which can alter the chromatin state of surrounding genes, causing defects in its expression (Anguela and High 2019).

Transfection is, therefore, a method designed for the introduction of genetic elements (coding and non-coding DNA sequences) that control the expression of a gene, either by increasing its expression or suppressing it. In the former, transfection can be used in gene therapy for the treatment of diseases or induce changes in cell reprogramming by introducing transcription factors into mesenchymal cells (cell renewal). Moreover, transfection can be used to reduce the expression of a gene, through RNAi, or by performing genetic editing to correct errors in the sequence or eliminating them through the CRISPR/Cas system (Glorioso and Lemoine 2017; Anguela and High 2018).

Different types of viral vectors have been investigated for gene therapy, and several characteristics have to be considered for their use: the type of genome (RNA or DNA), its ability to infect dividing cells, the amount of genetic material that can be packaged in its capsid, activation of the immune response (toxicity), its ability to integrate into the genome and the expression of transgene in a long or short term (Anguela and High 2018).

Transfection Methods

In mammal cells, three types of strategies have been developed for the introduction of genetic material into a cell (transfection methods): Biological, chemical and physical. In the first instance, viral vectors have been the primary strategy due to their ability to infect different cell types by passing through the cell membrane and reaching the core by the endocytic pathway, and then releasing their genetic material into the nucleus, where its expression is controlled by endogenous transcriptional machinery (Table 12.1).

Another characteristic that distinguishes these type of vectors is their ability to evade the immune response of the cell. Chemical transfection is based on the use of cationic polymers that aim to change the net loaded charge of DNA, this results in an interaction with the plasmatic membrane, and then its introduction has been suggested to mediate by cellular processes such as endocytosis/ phagocytosis Finally, physical methods involve the use of electric current pulse (electroporation), direct microinjection of the genetic material to the target cell or the use of nanoparticles introduced by bio-ballistics (Kim and Eberwine 2010).

Table 12.1. Viral Vectors for Gene Therapy.

Viral vector Viral genome Packing range Target cell infection Transfection
Adenovirus (Ads) DNA 8 kb Dividing and non-dividing cells Transiently transfection
Adeno-associated Virus (AAV) DNA 5 kb Dividing and non-dividing cells Transiently and stable transfection
Lentivirus RNA 8 kb Dividing and non-dividing cells Stable transfection
Retrovirus RNA 8 kb Dividing cells Stable transfection
Herpes simplevirus DNA 30-40 Kb Dividing cells Transiently transfection

The features of suitable vectors for gene therapy, are associated with several, types of the treatment to which it is directed (genetic disorders—hereditary and acquiring—or tissue engineering); the transfection method (biological, chemical and physical), and the mechanism through which the vector is delivered to the target cell (in vivo and ex vivo). For in vivo gene transfer, the vector is introduced directly into the region of the affected tissue, while in the ex vivo mechanism, cells in cell culture are transfected with the vector of interest and then transplanted into the organism with the affected tissue (Siddique et al. 2016) Figure 12.1.

Viral vectors for genetic manipulation in human cells are designed with defects in their replication mechanisms, and this is done by separating their structural and functional components into different plasmids (Van Tendeloo et al. 2001). On the one hand, a plasmid has the structural components of the virus, but the packaging signal for the encapsulation of the viral RNA is excluded.

On the other hand, the gene of interest is found in another vector with the packaging signal and the LTR (Long Terminal Repeats) sequence (for lentivirus), necessary for the integration of the virus in the genome. In practice, this process has been made more efficient by establishing a protocol for the generation of viral particles. In this process, the plasmid that carries the gene of interest along with the viral packaging signals, as well as the LTR integration sequences, is introduced to a cell line that expresses the structural components of the viral vector (capsid envelope proteins, transcriptase, integrase). Obtaining these viral particles ensures that a single transduction event is generated (Scow and Wood 2009).

Regarding the mechanisms that could be used by viral vectors to cross through the cell membrane, three examples have been well documented with adenovirus, adeno-associated virus and lentivirus (Waehler et al. 2007; Warnock et al. 2011). Adenoviruses which are non-envelop viruses, their capsid is structured in the form of an icosahedron, formed by a set of proteins denoted as hexon and penton, which make up the sides of the icosahedron and its vortices respectively.

Besides, the pentons extend a structure in the form of fiber ending in a knob domain. The genome of the adenovimses is divided by a set of genes of early-stage, and late-stage comprising 40 kb, which contain Inverted Terminal Repeats (ITR) at their terminal ends. The introduction of this type of viral vector is generated by the interaction of the knob domain with the CAR receptor, followed by the union of a second receptor, the integrin that interacts with the penton and then the integration of the AVs is directed by endocytosis (Waehler et al. 2007; Warnock et al. 2011).

The Adeno-Associated Viruses (AVV) are DNA viruses without envelopes, surrounded by a protein cover that forms an icosahedron; its genome is constituted by two open reading frames that encode four proteins involved in its replication denoted Rep and three structural proteins of the capsid (VP1-VP3). The VP3 capsid protein binds to the membrane receptor proteoglycan heparan-sulfate (HSPG) and then to a coreceptor such as integrin, and finally the virus is internalized via endocytosis.

Finally, lentiviruses, which are a subcategory of retroviruses, are enveloped viruses. They have an RNA genome, with three common genes: gag, pol, and env; and six additional genes that fulfill both regulatory and structural functions. The most common lentivirus is the human immunodeficiency virus type I (VIH-I). The introduction of this type of vector to the host cell is given by the fusion of membranes, which is determined by the surface receptors of the virus envelope (glycoproteins) (Waehler et al. 2007; Warnock et al. 2011).

fig12_1_C.jpg

Figure 12.1. Strategies used for the introduction of viral vectors. (A) In ex vivo transfection, mesenchymal cells are removed from the tissue damage and then the culture in specialized scaffolds. The cells are then reprogrammed by introducing transcription factors employing viral vectors and subsequently transferred to the original niche; (B) in vivo transfection, the delivery vehicle is directly injected in the affected zone. In both strategies, the principal route to be internalized into the cell is the endocytic pathway.

Gene therapy in the field of dental sciences, conducted its first studies on salivary gland cells, keratinocytes and cancer cells, from these early attempts, research in this field has been growing constantly (Baum et al. 2002; Siddique et al. 2016).

Target Genes, Growth Factors for Regeneration in the Oral Cavity

Gene therapy possesses numerous advantages over traditional treatments, such as greater sustainability than that of a single protein or compound application (Padial-Molina and Rios 2014). In this regard, gene therapy is a two-step process; the first one involves a human genetic coding for the therapeutic protein; the coding must be cleaved and inserted into the genome of a carrier vector. The second part of the process includes the entry of the modified vector to the target human cells; then, the DNA sequence is released and becomes integrated within the chromosome. Ideally, the cells with the new genetic design begin forming the required therapeutic proteins (Siddique et al. 2016).

Coupled with tissue engineering strategies, gene therapy offers strong potential for three-dimensional tissue regeneration at the tooth-ligament-bone interface (Padial-Molina and Rios 2014).

An expanding area of clinical importance in dentistry which applies gene therapy is related to bone repair and regeneration, regeneration of tooth-supporting structures, implants, treatment of salivary gland disorder, among others. In this regard, gene therapy represents an ideal approach towards enhancing bone regeneration (Gupta et al. 2015). However, in the periodontium, regenerative treatment has been confronted with the morphological and functional specificities of each component of tooth-supporting tissues (Kaigler et al. 2006).

Notably, the healing of osseous tissues is a highly regulated process, in which growth factors and cytokines participate in a sequence of overlapping events similar to cutaneous wound repair (Kaigler et al. 2006). Systems for regulating the magnitude and temporal expression of the osteoblastic phenotype has been studied as a tool of genetic engineering in dentistry (Gupta et al. 2015).

The replacement of damaged bone with new bone mimics embryonic bone development. This process is driven by a cellular and molecular mechanism controlled by the Transforming Growth Factor-beta (TGF-13) superfamily of genes, also encoding a large number of extracellular signaling growth factors (Kaigler et al. 2006).

Next we provide a brief description of the most common study growth factor used in gene therapy (Table 12.2).

Bone Morphogenic Proteins

A unique family within the TGF-I3 superfamily are the Bone Morphogenic Proteins (BMPs). The BMPs play an essential role in the regulation of bone formation and repair; they can induce the formation of both bone and cartilage by stimulating the cellular events of mesenchymal progenitor cells (MSCs) (Kaigler et al. 2006; Rao et al. 2013).

Also, BMP-2, -4, -7 and -9 are the only growth factors that can individually induce de novo bone formation both in vitro and at heterotopic sites (Gupta et al. 2015). Moreover, BMPs -2, -4, -7 and -12 have been studied for periodontal and peri-implant bone regeneration. The most extensively research BMP for bone and periodontal regenerative treatment is BMP-2 (Kaigler et al. 2006).

In a direct gene therapy application, Chang et al. provided evidence that membranous bone repair with autologous tissue-engineered strategy could be achieved when using MSCs with adenovirus-BMP-2 gene transfer (Chang et al. 2003).

Moreover, Park et al. demonstrated that ex vivo BMP-2 gene delivery using periodontal ligament stem cell enhance new bone formation and re-osteointegration in peri-implantitis defect (Park et al. 2015).

Preclinical studies with BMP-2 have been conducted, and they demonstrated improvement of alveolar bone regeneration in different types of periodontal defects after treatment with BMP-2 via different vectors. However, ankylosis between tooth and alveolar bone is also occasionally reported, apparently associated with rapid osteogenesis (Kaigler et al. 2006).

In the case of BMP-7, direct in vivo gene delivery of adenovirus-BMP-7 transduced cells in a collagen gel carrier showed successful regeneration of alveolar bone defects around dental implants (Franceschi et al. 2000). However, preclinical and pilot human clinical studies made with rhBMP-7 and bone grafts for maxillary bone defects showed no significant differences between conventional treatments (Kaigler et al. 2006; Van den Bergh et al. 2000).

Table 12.2. Applications of growth factors and the vector use for their delivery

Growth factors Applications Vector Reference
BMP’s

Repair critical-sized mandibular defects.

Inducers of bone and bone marrow regeneration.

The osteoinductive potential makes them clinically valuable as alternatives to bone grafts. fts

Bone and cement regeneration.

Adenoviral vector.

Biological matrices: demineralized bone matrix, collagen, fibrin.

Synthetic matrices: polylactic acid or polyglycolic acid.

Inorganic matrices: hydroxyapatite, tricalcium phosphate, other bioceramics.

Autologous bone marrow.

Akeel et al. 2000; Franceschi et al. 2000; Chang et al. 2003
PDGF

Only PDGF has FDA-approval and commercialized as Regranex.

Enhance wound closure kinetics and re-epithelialization on full-thickness excisional skin wounds.

Lesions treated with Ad-PDGF-B tended to display increased evidence of new blood vessel formation, especially in the PDL region.

Ad-PDGF-B gene transfer not only enhanced the alveolar bone fill by two- to threefold but also increased cementum formation by higher than threefold, compared to controls.

Adenoviral vector.

Anusaksathien et al. 2003; Cho et al. 1995; Jin et al. 2004
VEGF

Plays an essential role in bone growth via the endochondral ossification pathway.

More bone was produced in calvaria defects that were treated with containing both VEGF and BMP genes than when either VEGF or BMP-transduced cells were implanted alone.

Adenoviral vector VEGF + BMP.

Scheller et al. 2012; Pass neau 2017
TGF-b

Improvement wound closure kinetics, re-epithelialization and angiogenesis on full-thickness excisional skin wounds,

Treatment of critical size, supra-alveolar periodontal defects in beagle dogs may be of limited clinical benefit.

Plasmid electroporation.

Hydroxy-ethyvisco- elastic gel with CaCO3 particles.

Lee et al. 2012; Wikesjö et al. 2004; Nakashima et al. 2004
bFGF

The increased periodontal ligament, bone formation on class II furcation on dog.

Dose-dependent bone and cement regeneration on class II furcation; nonhuman primate.

Enhanced alveolar bone height, periodontal ligament regeneration on 74 patients, Phase II randomized controlled trial, 2-3 wall intrabony defects; clinical.

Topical application.

Gelatin hydrogel.

Hydroxypropyl cellulose hydrogel.

Kaigler et al. 2003; Shi et al. 2010
IGF

No periodontal regeneration was shown in chronic periodontitis in a primate model.

PDGF-B/IGF showed increased bone fill on 38 patients, phase I/II randomized controlled trial, bilateral intrabony, and furcation defects; clinical.

Methylcellulose gel.

Gelatin.

Giannobile et al. 2001; Shi et al. 2010
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Aug 7, 2022 | Posted by in General Dentistry | Comments Off on Gene Therapy in Oral Tissue Regeneration
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