The Evolving Landscape of Gene Therapy in Plastic Surgery

Abstract

With the rapid rise of personalized genomic sequencing and clustered regularly interspaced short palindromic repeat (CRISPR) technology, previous gaps in gene therapy are beginning to be bridged, paving the way for increasing clinical applicability. This article aims to provide an overview of the fundamentals of gene therapy and discuss future potential interventions relevant to plastic surgeons. These interventions include enhancing tissue regeneration and healing, as well as modifying disease processes in congenital anomalies. Though clinical applications are still on the horizon, a deeper understanding of these new advances will help plastic surgeons understand the current landscape of gene therapy and stay abreast of future opportunities.

Gene-based therapies have demonstrated applicability in the treatment of a spectrum of diseases, from severe combined immunodeficiency (SCID) to cystic fibrosis to various malignancies. Gene therapy describes an experimental technique in which gene insertion, replacement, or inactivation is performed to better equip a patient with the intracellular mechanisms for combating disease. The advent of specific DNA sequence recombination and alteration with subsequent transfer into either normal or diseased cells has revolutionized the landscape of medical research and clinical practice. Current gene therapy strategies include enhancing the immune system's antitumor response, facilitating programmed cell death pathways, identifying and amplifying critical cell biological functions, introducing cytotoxic suicide genes, and correcting aberrant gene expression.

While a rapidly growing area of research and clinical investigation, gene therapy invariably poses a technical challenge for clinicians. Initial gene delivery vehicles were contingent on codelivery of a marker gene conferring some added biologic advantage to the recipient cell. ¹ For example, antibiotic resistance genes, when transferred alongside a particular gene of interest, permit differential expression of transformed cells on media selective for resistance to that particular antibiotic. In further improving efficiency of mammalian gene transfer, recombinant retroviral gene delivery systems emerged. These genetically modified retroviruses carried genes of interest instead of viral structural genes, and these genes could subsequently be introduced into mammalian cells via viral infection. ¹ Viral gene delivery systems greatly expanded the clinical applicability of gene therapy and are notable in a category of delivery methods, including liposomes and the gene gun.

Developments in gene therapy have proven useful in bone formation, nerve regeneration, soft-tissue repair, cranial suture development, and varied other applications in plastic and reconstructive surgery. ² Successful application of gene-based therapies necessitates safe and effective approaches, in addition to feasible modes of gene transfer. Improvements in DNA transfer to cells locally and at distant sites, enhanced gene expression levels, and appropriate antagonism of inhibitory immune responses toward transferred genes all contribute to present efforts in refining the use of gene therapy.

Along this vein of investigation are advances in the understanding of genetic imprinting and consequent clinical implications. These efforts aim to enhance knowledge of how patients' genotypic content can result in unique phenotypic manifestations. Polymorphisms identified at single gene loci can both guide patient care and inform oncologic management. In recognizing patient-specific genetic processes and the wide scope for the application of gene therapy in plastic surgery, the following review identifies both current gene transfer techniques and ongoing investigative development in future clinical applications.

Gene Transfer Techniques

Delivery Systems

Following the delivery of a gene and its associated regulatory elements into the recipient cell nucleus, the transferred gene product is expressed at either physiological or therapeutic levels. The expression of a deficient gene involves maintenance of the transferred gene in the cell nucleus for replication and multiple rounds of cellular division, whereas the expression of a therapeutic gene is more transient and does not need host cell genome integration. Vectors for gene delivery vary depending on the target cells and tissues and act to facilitate uptake of genes through the host cell membrane. These vectors can be classified as either viral or nonviral systems, each confirming unique advantages.

Viral vectors are frequently preferred to nonviral vectors because of their increased in vivo transduction efficiency. ³ A viral vector is a genetically altered virus in which deleted regions are inserted with therapeutic genes. ⁴ With the ability to infect host cells and utilize host cell machinery for genome replication, infecting viruses facilitate replication of therapeutic genes along with retained viral genetic material. The viruses most commonly used in gene therapy include adenovirus, retrovirus, adeno-associated virus (AAV), and herpes simplex virus (HSV).

Adenoviral vectors have a broad spectrum of infectivity and can be introduced into both dividing and nondividing cells. In particular, high capacity or “gutless” adenoviral vectors can accept large DNA (greater than 30 kb), thus allowing high levels of expression of genes of interest. ⁵ Following uptake into host cells, adenoviral contents have an extrachromosomal location and rarely integrate into the host cell genome. This confers a safety advantage to adenoviral vectors over other viral forms. However, the extrachromosomal location of adenoviruses limits the duration of target gene expression, and residual viral antigens can provoke a host immune response to clear virally transduced cells. ³

Retroviral vectors include Moloney murine leukemia virus (MoMLV), avian C-type retroviruses (oncoretroviruses), lentiviruses (HIV and other immunodeficiency viruses), and spumavirus. Retroviral vectors are created through replacement of the viral genes gag (encoding viral core proteins), pol (encoding reverse transcriptase and integrase), and env (encoding viral envelope glycoprotein) with exogenous DNA. ³ The double-stranded retroviral DNA derived from reverse transcription can permanently integrate in the host genome, inactivate tumor suppressor genes, and activate oncogenes. ^{6 7} Retroviral transferred genes consequently have more sustained replication in the host.

Adeno-associated virus vectors are human parvoviruses that were initially identified as a contaminant in adenovirus preparations and require a helper virus to cause a productive infection. ⁸ Following replacement of the rep (encoding replication) and the cap (encoding structural proteins) genes, up to 5 kb of therapeutic gene DNA can be introduced into AAVs. ³ After gene transfer, AAVs promote a slow rise in gene expression and can infect nondividing cells, much like their adenoviral counterparts. ⁹ However, unlike adenoviruses, AAVs do not provoke a host inflammatory response as they do not express native viral genes.

Herpes simplex virus is a DNA virus with unique capacity for latency that permits long-term gene expression without integration into the host cell genome. With the ability to infect several cell and tissue types, as well as transfer up to 30 kb of foreign DNA, HSV is a notable viral vector. ¹⁰ HSV vectors have demonstrated applications in animal model treatment of cancer, neurodegenerative diseases, and pain. In particular, HSV amplicons are plasmids with only the genes necessary for replication and packaging, thereby reducing adverse effects of viral gene expression. ³

Nonviral vectors include liposomal formulations, proteins, polymers, and physical delivery techniques. However, these vectors are limited by poor in vivo transfection efficiency because nonviral vector DNA interacts with blood plasma proteins, undesirable cells, and the extracellular matrix. ¹¹ As nonviral vectors do not integrate into the host genome, their conferred gene expression is not sustained. Physical delivery techniques such as electroporation involve electrical pulse generation of cytoplasmic membrane pores. Electrosonoporation is the combination of electroporation with ultrasound guided deployment of desired plasmids; this technique has shown promise in murine models. ¹²

Applications of Gene Therapy in Plastic Surgery

Gene therapy has permitted advances in reconstructive approaches to bone formation, nerve regeneration, wound and tissue healing, cranial suture development, and treatment of malignancy. Bone growth is mediated by a host of cytokines, including fibroblast growth factors (FGFs), transforming growth factor-β (TGF-β), and bone morphogenetic proteins (BMPs). ^{13 14} Viral vectors can be used to facilitate bone regeneration, particularly adenoviral vectors which can transfect osseous tissue and infect osteoblasts, periosteal cells, endosteal cells, and soft tissues surrounding the injection site. ^{15 16} Virally delivered genes can promote growth plate thickening and bone ossicle formation. ¹⁷ Adenoviral vectors can additionally be used to promote dense trabecular bone formation through introduction of BMP-2; mice transfected with BMP-2–carrying adenoviral vectors demonstrate improved mechanical bone strength. ¹⁸

In addition to adenoviral vectors for bone formation, retroviral and nonviral techniques have demonstrated potential. Retroviral vectors carrying BMP-2 can promote bone formation, much like adenoviral vectors, and BMP expression following retroviral transfection is sustained. ¹⁹ A nonviral delivery technique, gene-activated matrix has shown to improve bone regeneration following gene transfer of human parathyroid hormone (hPTH) and BMP-4. ²⁰

In addition to facilitating bone growth, gene therapy strategies have permitted the delivery of neurotrophic factors for nerve regeneration. Unlike central nerves which possessing impressive capacity for regeneration, peripheral nerves regenerate slowly and often result in an inferior final product. Schwann cells and muscle secrete neurotrophic factors such as brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neutrophin-4/5 (NT-4/5), and nerve growth factor (NGF) that neurons take up and transport to their dorsal root ganglia or spinal motor neurons for regeneration. ² Adenoviral vectors carrying neurotrophic factors have demonstrated favorable outcomes such as increased neuronal survival, and in mice with progressive motor neuropathy, factor delivery via these vectors allows for decreased muscle atrophy, improved electromyography parameters, and decreased myelinated fiber loss. ²¹

An integral focus in regenerative efforts, and an area in which gene therapy offers particular promise, wound and tissue healing is the backbone of plastic surgery. With an interlay of soluble mediators, blood cells, extracellular matrix, and additional environmental mediators, wound healing follows the cellular processes of hemostasis and inflammation, tissue formation and proliferation, and tissue remodeling. ²² Growth factors involved in wound and tissue healing regulate chemotaxis, proliferation, matrix synthesis and breakdown, and inflammation. ²³ Plastic surgeons can appreciate improved patient results when using gene therapy to enhance tissue growth and repair of skin, nerve, muscle, blood vessels, and cartilage. Viral transfection of the growth factors involved in the process of wound healing include platelet-derived growth factor (PDGF), vascular endothelial growth factors (VEGFs), fibroblast growth factor (FGF), insulin-like growth factor-1 (IGF1), tumor necrosis factor (TNF), and the interleukins. ³ While adenoviral gene delivery has previously been cautioned given the risk for instigating a host inflammatory response, this inflammatory response has a favorable role in augmenting the natural inflammatory phase of wound healing. ²⁴ Moreover, transfer of growth factors via viral vectors can promote the formation of granulation tissue and neovascularization.

As with other aspects of wound healing, tendon healing involves the formation of an initial base upon which remodeling can take place. Vascularization within the tendon sheath promotes intrinsic healing, and the appropriate placement of stress enables accelerated healing and acquisition of former tensile strength. ²⁵ Adenoviral and retroviral vectors have been able to promote gene expression of LacZ and BMP-12, promoting tendon healing with both increased tensile strength and stiffness. ²⁶

Additionally, within the scope of gene therapy in reconstruction, surgical cranial vault remodeling is performed in the treatment of craniosynostosis or the premature fusion of cranial sutures. Genetic mutations identified in familial craniosynostosis syndromes have enabled the creation of adenoviral vectors capable of expressing growth factors such as FGF, leading to improved cranial suture fusion in mice models. ²⁷ While craniosynostosis has often been understood as a disease of increased bone mineralization, multiple mouse models have demonstrated concomitant diminished cranial bone volume and density. ²⁸ Other studies have shown the control of tissue mineralization by tissue nonspecific alkaline phosphatase (TNAP), an enzyme used as a necessary catalyst for successful hydroxyapatite crystal formation. ^{29 30} Furthermore, mouse models have shown that fibroblast growth factor receptor-2 (FGFR2) is associated with craniosynostosis and can lead to aberrant mineralization. ³¹ Consequently, a lentiviral vector carrying TNAP has demonstrated success in treating FGFR2-associated craniosynostosis in mice models. ²⁸ As lentivirus requires only a single injection for sustained delivery and ensures stable integration of the viral genome into the host genome for sustained expression, transfected TNAP lentivirus can produce an increase in serum alkaline phosphatase levels and diminish cranial suture fusion. ²⁸

An emerging area for gene therapy application, the treatment of head and neck squamous cell carcinoma (HNSCC) can be approached with techniques such as corrective gene therapy, cytoreductive gene therapy, and gene editing. The tenth most common cancer worldwide, HNSCC disproportionately affects patients in less-developed regions of the world and is frequently diagnosed at advanced stages because of late detection. ³² Plastic surgeons are frequently involved in tumor resection as well as surgical defect reconstruction. Targeted epidermal growth factor receptor (EGFR) therapy treats overexpression of an oncogene identified in >90% of HNSCC cases. ³³ EGFR-targeted oncolytic measles virus has been injected intratumorally into mice implanted with primary HNSCC xenografts, and in conjunction with cytosine deaminase/uracil phosphoribosyltransferase (CD/UPRT), viral therapy has significant antitumor activity in vivo. ^{33 34} Furthermore, gene therapy targeting HNSCC mutations in p53, p15, p27, and the retinoblastoma (Rb1) tumor suppressor genes can help prevent tumorigenesis and increase tumor sensitivity to radiation. ^{35 36}

Gene editing techniques for the treatment of malignancies, including HNSCC, have been refined over the past decade and continue to be developed as zinc finger nucleases, clustered regularly interspaced short palindromic repeat (CRISPR)–associated Cas9 nucleases, and transcription activator-like effector nucleases are further elucidated. ³⁷ In HPV-positive HNSCC, CRISPR can be used to cleave integrated HPV viral DNA and guided RNA sequence therapy specific for HPV viral oncogenes (E6 and E7) can lead to restoration of normal cell cycle function. ^{33 38} Additional applications of CRISPR are reviewed below.

Gene Therapy in Future Clinical Applications

As has been demonstrated with viral delivery of genes intended to correct, inactivate, or amplify host gene activity, tissue engineering similarly focuses on the development of biologic substitutes with tissue-level activity. The field of plastic surgery has noted advances with tissue engineering applied to skin transplantation, tendon healing, and the production of necessary regenerative substrates such as cartilage, bone, adipose tissue, and blood vessels. ³ The segmentation and transplantation of tissue, with preserved blood supply, to fill defects is the essence of reconstructive flaps. Given the potential for compromise of arterial and venous perfusion, especially in patients with vasculopathy, diabetes, or those receiving corticosteroid therapy, there is a growing focus on strategies to enhance flap blood supply and viability. ³

Given the undesirable occurrence of myonecrosis with flap procedures, strategies to improve blood supply have been explored, in particular in transverse rectus abdominis myocutaneous (TRAM) flaps. Upregulated angiogenesis has been observed with the introduction of VEGF into muscle flaps in rats. ³⁹ As VEGF both stimulates angiogenesis and increases vascular permeability, this growth factor is important for neovascularization. ⁴⁰ Via electroporation, VEGF can be introduced into flaps in a manner that increases gene expression significantly when compared with simple DNA injection. ⁴¹ VEGF gene therapy is advantageous over VEGF protein therapy as the former ensures VEGF activity restricted to a specific area of cells that receive the vector, longer duration of VEGF production by transfected cells, and production of human VEGF rather than recombinant bacterial VEGF. ⁴² Intradermal injection of VEGF via electroporation has demonstrated effectiveness in reducing unipedicle TRAM flap necrosis in rat models. ³⁹ Future studies are needed to better characterize the safety profile of this gene therapy procedure and refine the technique through which gene transfer can take place.

While much of plastic surgery focuses on reconstructive principles, recent studies have focused on therapeutic approaches in which gene-modified muscle tissues function as a source of gene products to halt tumorigenesis and promote wound healing. ^{3 43 44} Recombinant AAV vectors can be created and conveyed via autologous fat grafting into muscle tissues, resulting in sustained gene protein production. ⁴⁵ Notably, use of autologous fat graft as a vehicle for viral vector delivery is more efficacious than direct injection of the vectors into muscle tissues. Use of fat grafting in gene therapy is in its early stages, and future exploration of this method is encouraged.

More recent progress in gene therapy applications in reconstructive surgery has been noted in the use of CRISPR/Cas, a system for gene editing, knockout, transcriptional activation and inhibition, epigenetic modification, and genetic screening. ⁴⁶ The CRISPR mechanism of gene modification employs the practice of personalized medicine, sequencing target genes of clinical interest and then modifying gene expression through in vitro, ex vivo, or in vivo approaches. Plastic and reconstructive surgery has many recognized opportunities for incorporation of CRISPR/Cas gene therapy, such as the treatment of craniofacial disorders, wound healing, tissue engineering, and creation of vascular composite allografts. During the 1990s, gene therapy in plastic surgery was proposed as a strategy for enhancing wound healing, regenerating nerve and muscle, and preventing or treating vessel thrombosis. ⁴⁷ The advent of CRISPR technology has made many of these goals more accessible.

Rapid personalized genomic sequencing has enabled the identification of missense variants and other mutations in critical genes. CRISPR/Cas offers an opportunity to correct congenital mutations causing craniofacial disorders both in an in vivo and germline manner, preventing the development of anomalies in animal models and even in the developing embryo. ^{48 49} CRISPR additionally can facilitate genetic modification of autologous cells that can subsequently be engrafted and used to repopulate tissues, stimulate endogenous cells, and modulate immune functions. ⁴⁶ CRISPR enables autologous cells to be enhanced and thereby aid stem cell differentiation into lineages conducive for reconstruction, promote the recruitment of repair signals, and allow for release of growth factors. Recently, CRISPR/Cas has been used to create therapeutic skin grafts derived from human epidermal progenitors with modifications to help control obesity and diabetes in mouse models. ⁴⁸

The dynamic interlay of factors involved in wound healing can be enhanced with CRISPR induction of PDGF and other genomic targets. ⁵⁰ These targets include growth factors and cytokines that accelerate wound healing, such as metalloproteinases, VEGF, hypoxia inducible factor-1 (HIF1), and hepatocyte growth factor. ⁵¹ Future developments in CRISPR systems will aim to differentially control expression and timing of factors involved in wound healing as well as expand the spectrum of critical targets that can be modified. ⁴⁶

The scope of CRISPR in aesthetic surgery remains to be defined with ongoing research focused on understanding the molecular pathways of aging, hair loss, and targets amenable to genetic modification. Nonetheless, there are some ethical concerns associated with wider application of CRISPR/Cas systems. The future costs of CRISPR technology are poorly characterized and vary considerably depending on the efficacy of a single treatment. ⁵² The use of CRISPR to genetically modify human somatic cells is frequently debated, particularly in relation to editing genes for the purpose of germline disease eradication. In light of the advantages and precision afforded by CRISPR therapy, it is important to consider the ethical and judicious use of this technology in caring for patients.

Clinical Implications of Genetic Imprinting

Although not strictly gene therapy, the phenomenon of genetic imprinting is an aspect of genetic medicine that merits consideration. Genetic imprinting involves the chemical modification of genes passed from parent to offspring. Each parent contributes a unique set of chemically modified genes, which are subsequently maintained in the offspring. Thus, even if the genetic sequence in the child is identical to that found in the parents, the DNA inherited from the father will invariably differ from the DNA inherited from the mother. For example, Prader–Willi syndrome (PWS) and Angelman syndrome (AS) are very different diseases that result from the same mutation in the long arm of chromosome 15; the individual's phenotype depends on which parent contributes the mutation. Additionally, the gene insulin-like growth factor-2 (IGF2), a critical growth factor in development, can be subject to a similar form of genetic imprinting. Faulty imprinting in the IGF2 locus, when inherited from the mother, results in an overgrowth disorder known as Beckwith–Wiedemann syndrome (BWS). Conversely, inheritance of faulty imprinting in the IGF2 locus from the father causes a form of dwarfism known as Russell–Silver syndrome (RSS). These diseases are relatively rare and may not be treated by a plastic surgeon. However, common variants in the IGF2 locus have been shown to modify the growth patterns of infantile hemangioma, a common pediatric tumor treated by plastic surgeons.

In the case of infantile hemangiomas, a common C/T polymorphism in the IGF2 region appears to modify tumor aggressiveness. The T allele, when inherited from the father, contributes to a strong growth phenotype. When inherited from the mother, the same T allele does promote growth but less strongly. The C allele appears to be neutral with respect to growth. Thus, TT lesions are highly aggressive and often ulcerate and threaten critical structures, while CC lesions remain small. Of note, heterozygous individuals who receive a T allele from their father have more aggressive hemangioma growth than heterozygous individuals who receive a T allele from their mother.

New genomic methods that are able to detect the presence of imprinting can determine a child's genetic status regarding the IGF2 gene (TT, CC, or TC) as well as identify which parent contributed the allele in the case of heterozygotes. From this genetic context, patients can be stratified into one of four risk categories at birth, when the child's hemangioma is first detected. ⁵³ TT individuals should be considered for immediate or early intervention. On the other hand, CC individuals are safely treated with expectant management. The risk in heterozygotes can be determined based on both the location of the lesion as well as if the father contributed the T allele, potentially placing the patient in the early intervention group. A proposed clinical test to identify imprinting in the IGF2 locus and allelic inheritance, with consequent consideration of the cellular and phenotypic consequences, is in the approval process and may be available in the near future. ⁵³ It is clear that even the most esoteric aspects of the human genome can affect the practice of plastic surgeons.

Conclusion

In summary, the applications of gene therapy in plastic and reconstructive surgery have experienced promising growth over the past decade. Newer approaches to reconstructive procedures such as cleft palate repair, wound healing, flap biology and transplantation, and restoration of form and function after tumor resection can be realized by the surgical community through further exploration and refinement of gene delivery and editing. The favorable union of gene therapy and tissue engineering additionally offers an opportunity to practice personalized medicine and consider individual nuances in patient care. The precise consideration of genetic imprinting and allelic inheritance can inform patient oncologic risk stratification. CRISPR has notably emerged as a major technological advancement in the use of gene therapy in plastic surgery. Dialogue between plastic surgeons and other stakeholders in this technology will enable careful consideration of systemic and unintended effects of CRISPR gene editing, the US Food and Drug Administration (FDA) regulation environment for use of this technology, the cost parameters to consider when delivering this technology on a larger scale, and the patient-centered approach that can help navigate ethical gray areas in genetic editing of human cells. With potential for great impact in the treatment of disease and injury, gene therapy has gained recognition as a disruptive technology that will empower greater innovation in the landscape of plastic surgery.