After cellular introduction, sgRNA binds target DNA sequences and recruits Cas9, the molecular scissor. The end result is a DNA double-strand break repaired by either nonhomologous end joining or homology-directed repair. Nonhomologous end joining is used in generating functional gene knockouts, whereas homology-directed repair is used to rewrite DNA sequences and produce precise genetic modifications such as correcting pathogenic gene mutations.
CRISPR systems are not limited to single-gene editing. Multiplexed gene modifications are possible with introduction of multiple sgRNAs. In addition, Cas9 can serve as a platform to recruit other factors to the target DNA. Using nuclease-deactivated Cas9 fused with transcriptional activators and inhibitors, gene expression activation (CRISPRa) and inhibition (CRISPRi) regulation can be accomplished.
CRISPR can also be used with recently discovered Cas13 to specifically target RNA for either knockdown or RNA editing.14 Thus, RNA can be edited without permanently altering the genome. Importantly, this can be used in nondividing cells, is transient in nature, and can be multiplexed.
CRISPR components are delivered into cells directly. Cas9 and sgRNA are introduced as nucleic acids or directly as protein and RNA, respectively. The main delivery methods are nonviral and viral delivery. Nonviral methods include electroporation, lipid-mediated particles, nanoparticles, and microinjection; whereas viral delivery methods use lentivirus, adenovirus, and adenoassociated virus vectors. Advantages and disadvantages to each method are beyond the scope of this review but are detailed in comprehensive reviews.15,16
The primary therapeutic approaches using CRISPR are ex vivo and in vivo. Ex vivo applications remove cells from the body and genetically modify them before reintroduction. In vivo applications directly deliver CRISPR components into cells within the body. In general, nonviral delivery systems are applied to in vitro or ex vivo systems, whereas viral delivery systems can work in complicated in vivo conditions. There have been many advances in protocols and delivery systems focused on optimizing these methods.
CRISPR applications include gene editing/knockout, transcriptional activation/inhibition, epigenetic modifications, genetic screens, and animal model generation. CRISPR has altered genomes of bacteria, viruses, plants, mammalian cells, zebrafish, mice, pigs, and primates.17–19 CRISPR/Cas9 can also edit human embryos, an area of research under intense scrutiny.4,20,21
CRISPR/Cas9 in vivo gene editing demonstrates promise in “curing” human diseases. Specific examples include Duchenne muscular dystrophy and human immunodeficiency virus. In Duchenne muscular dystrophy patient cells and mouse models, CRISPR/Cas9 restored normal dystrophin expression by deleting exons of the Duchenne muscular dystrophy gene and improved muscle strength.22,23 CRISPR/Cas9 excised latent human immunodeficiency virus DNA in vivo from genomes of human immunodeficiency virus–infected mice to shut down human immunodeficiency virus replication and eliminate further infection.24 There are active investigations targeting diseases amenable to CRISPR/Cas9 gene editing including cystic fibrosis, β-thalassemia, retinitis pigmentosa, sickle cell anemia, and hemophilias.12
Many specialties have realized the potential for use of CRISPR/Cas9’s to study and treat human disease. Leaders in hepatology, cardiology, hematology/oncology, ophthalmology, neurology, rheumatology, and dermatology are using CRISPR/Cas9 within their specialties.25–31 The therapeutic use of CRISPR/Cas9 in humans is coming to fruition, as there is a National Institutes of Health–approved clinical trial to engineer human T cells using CRISPR/Cas9 to attack cancer.32 Worldwide, many CRISPR-based clinical trials are underway.33
POTENTIAL APPLICATIONS IN PLASTIC AND RECONSTRUCTIVE SURGERY
Plastic and reconstructive surgery encounters clinical and surgical problems requiring understanding of various types of tissue and wound healing with vascular physiology. Many of the problems that the plastic surgeon confronts are amenable to various gene therapy applications, including craniofacial disorders; wound, tendon, and bone healing; cell and tissue engineering; reconstructive flaps; and vascular composite allografts. All of these areas within plastic surgery are amenable to the benefits of CRISPR/Cas9 (Fig. 2).
The application of gene therapy to plastic surgery problems is a topic with long-term interest.15,34,35 During gene therapy’s infancy in the 1990s, “gene therapy [was thought to] become the standard treatment for enhancing wound healing and nerve and muscle regeneration and for preventing or treating vessel thrombosis, areas critical to the plastic surgeon and the patient.”35 Although gene therapy has yet to fulfill this prophecy, it continues to be actively pursued by several laboratories. A 2013 review of gene therapy in plastic surgery provides details of studies that have been performed using gene therapy in wound healing; burns; scar treatment; and regeneration of bone, cartilage, tendon, and nerves.36 The review’s conclusion was that translation of gene therapy strategies into clinical trials would be difficult and expensive, with challenges in production and cost-effectiveness of therapies, and that we needed novel, reliable, safe, efficient techniques. Fortunately, as described above, CRISPR technology potentially makes the application of gene therapies more affordable, attainable, and feasible.
CONGENITAL CRANIOFACIAL MALFORMATIONS AND DEVELOPMENTAL BIOLOGY
CRISPR/Cas9 has been used to understand craniofacial development and pathogenesis of orofacial clefts and craniofacial syndromes. CRISPR/Cas9 use for single and multiplexed knockouts, high-throughput mutagenesis screens, and conditional knockout/knock-ins in zebrafish18,37–39 and mice40–42 has enhanced knowledge of craniofacial developmental pathways. The senior author’s (E.C.L.) craniofacial development laboratory has used CRISPR/Cas9 in zebrafish to illuminate pathways in craniofacial development. The laboratory generated mutant zebrafish lines using CRISPR/Cas9 to define key roles of Wnt pathway genes in palate morphogenesis.38,39 Several projects in human functional genomics are in progress to investigate human genes implicated in various craniofacial anomalies and understand the functions of those genes in craniofacial development.
With rapid personalized genomic sequencing, identification and modeling craniofacial disorders has become more commonplace. Individuals with clinical phenotypes of interest can easily undergo rapid DNA sequencing to identify individual gene mutations involved. This information is used to create disease animal models with CRISPR/Cas9 to elucidate molecular mechanisms for a given disease phenotype43 (Fig. 1). For example, a recent study involving the senior author identified the first genetic basis for arhinia (congenital absence of the nose) in 40 subjects, a rare malformation of unknown cause. Sequencing these subjects revealed a missense variant in the structural maintenance of chromosomes flexible hinge domain containing-1 (SMCHD1) gene, which was then altered in zebrafish through CRISPR/Cas9 to create a disease model to investigate the molecular basis of arhinia.44
In vivo and germline correction of congenital mutations causing craniofacial disorders is a long-term gene therapy goal. Many craniofacial anomalies that arise from single-gene mutations have been identified and reproduced in animal models.43,45 CRISPR/Cas9 has been used to generate these animal models with increased efficiency and potential to correct mutations in vivo as seen with Duchenne muscular dystrophy46,47 and retinitis pigmentosa.48 In these studies, disease-causing gene variants are corrected by CRISPR/Cas9. With human embryo germline editing feasible with CRISPR/Cas9,4 the ability to correct pathogenic mutations and prevent the development of craniofacial anomalies in the developing embryo may become reality.
WOUND HEALING AND TISSUE REPAIR
Gene therapy enhancement of wound healing has produced positive preclinical results and is the focus of clinical investigations and trials.49–52 Genes encoding growth factors/cytokines have shown potential for wound healing acceleration.53–55 Targets include platelet-derived growth factor, tissue inhibitor of metalloproteinases-2, vascular endothelial growth factor, hypoxia-inducible factor-1, and hepatocyte growth factor.56 The wound healing microenvironment poses an ideal environment for targeted gene therapy, as skin and wounds are easily accessible to transfect and monitor. High cell turnover with active cellular proliferation makes ideal conditions for gene transfer methods.
In addition to cutaneous wounds, repair and regeneration of bone, cartilage, nerve, and muscle all have potential to be augmented with CRISPR/Cas9. For example, previous animal studies demonstrated the efficacy of gene therapy for tendon healing through expression of fibroblast growth factor, vascular endothelial growth factor, or bone morphogenic proteins, whereas inhibiting transforming growth factor beta-1 prevents tendon adhesions.57,58 Multiplexing these multiple growth factors using CRISPR may be synergistic in the approach to enhanced tendon repair.
CRISPR can induce permanent or inducible expression of desired factors through direct gene insertions or transcriptional regulation. A recent abstract used CRISPR to overexpress platelet-derived growth factor-B in human mesenchymal stem cells, resulting in accelerated wound healing in mice.59 A distinct advantage of using CRISPR is the ability to modify multiple genomic targets with a single treatment, which may be beneficial within the complex wound healing milieu that occurs in tissue repair and regeneration. Inducible and thus transient in vivo gene editing with CRISPR is easily achievable with either drugs or light in an optogenetics system.60,61 This “on/off switch” could be used to transiently increase cytokine or growth factor expression to facilitate desired wound healing. Theoretically, using inducible CRISPR systems triggered by alternate drugs and/or light could differentially control expression in time of multiple factors. Furthermore, although attempts are being made to translate chemical modulators of wound healing signaling pathways to clinical therapies, many critical targets such as transcription factors are often considered undruggable but would be amenable to precise modulations with CRISPRa and CRISPRi in precisely specified cell types within the wound healing microenvironment.
CELL THERAPY AND TISSUE ENGINEERING
Cell therapy with autologous stem cell and fat transfer are relatively new concepts within our field that may benefit from CRISPR. Genetically modified autologous cells can be used to engraft and repopulate aged or damaged tissues, stimulate endogenous cells, and/or modulate immune functions. They may also release targeted growth factors, synthesize extracellular matrix, or send recruitment signals for repair. Stem cells may differentiate in situ into lineages needed for reconstruction. Using ex vivo gene modification by means of CRISPR, these autologous cells can be enhanced to fulfill roles that they have been previously incapable of achieving.
Recently, CRISPR was used to create therapeutic skin grafts, demonstrating the feasibility and potential of genetically modified autologous cells for therapy. This study used CRISPR/Cas9-engineered skin grafts derived from human epidermal progenitors modified to secrete factors to control obesity and diabetes in a mouse model.62 The authors used CRISPR/Cas9 and inserted an expression cassette for controllable release of their target protein, glucagon-like peptide-1. This allowed them to turn on/off gene expression as needed, an important control feature to have in gene editing cells for therapeutic purposes. This technique of creating skin grafts with therapeutic potential would have widespread impact in reconstructive surgery.
FLAP BIOLOGY AND VASCULARIZED COMPOSITE ALLOGRAFTS
Creating genetically modified flaps to enhance the recipient site to which they are transferred has been investigated by several laboratories. This included ex vivo modification of free flaps to deliver cancer treatment,63–65 promote bone healing,66 and treat chronic wounds or infections.67 CRISPR would facilitate this concept by making this process more cost-effective, efficient, and multiplexed.
Vascularized composite allografting has had promising outcomes, and refinements have made technical execution feasible.68 However, chronic immunosuppression is the major determinant of success and a major barrier. Recipient chimerism is the holy grail of immunotolerance and may be facilitated by mesenchymal stem cell therapies that provide prolonged immunomodulatory, restorative, and regenerative changes.69 As multiplexing of gene editing with CRISPR becomes more commonplace, achieving immune tolerance may be possible by genetically reprogramming donor vascularized composite allografting tissue before transplantation or using cell therapy to modulate allograft survival and tolerance.
Xenotransplantation of skin, soft tissue, and organs from transgenic pigs may become more prevalent in reconstructive surgery because of recent CRISPR achievements. A xenotransplantation resurgence has resulted from CRISPR/Cas9 generation of donor pigs with multiple protective genetic modifications; what once took years to achieve can now be performed in months, with much greater precision and scope.70 CRISPR/Cas9 eradicated porcine endogenous retroviruses, a major step forward toward safely using porcine tissues and organs in humans.71 Furthermore, CRISPR/Cas9 could be used to disrupt the formation of certain tissues in pigs and facilitate blastocyst complementation where human tissues can be grown in replacement of porcine tissues in animal donors. These tissues can be harvested and directly transplanted, all within a comparatively short clinical time window.
Gene therapy has historically been characterized as trying to create “designer” babies or enhance cosmetics. Although these ideas are still far-fetched, with CRISPR there are more tangible and attainable applications in aesthetic surgery. As with many new technologies, ethical considerations lag behind feasibility. Humanization of donor animals with CRISPR could be used to create novel and less immunogenic fillers while reducing cost. Furthermore, as we understand more of the molecular pathways driving aging skin and hair loss, genetic targets could be easily modified by CRISPR systems. For example, a recent discovery that a specific collagen (COL17A1) deficiency in hair follicle stem cells has been shown to drive hair loss in mammals that is prevented by maintenance of COL17A1 expression.72
CURRENT LIMITATIONS AND ETHICAL CONCERNS
One area of ongoing improvement in CRISPR/Cas9 is reducing off-target effects of Cas9. These off-target effects could potentially alter gene expression and function, or create mutagenic and oncogenic transformations. Several strategies have been used to improve Cas9 precision, and there are computational algorithms dedicated to search for potential off-target sites. Combined, these strategies have already improved precision and reduced off-target effects.73
Improvements in controlling the efficiency of DNA repair mechanisms would improve CRISPR/Cas9 applicability in genetic therapy. Strategies to induce homology-directed repair in nondividing and postmitotic cells or targeted gene corrections by means of nonhomologous end joining are needed. A recent study using CRISPR/Cas9 with a homology-independent targeted integration strategy allowed for robust DNA knock-in in both dividing and nondividing cells in vitro and in vivo.48
U.S. Food and Drug Administration regulation and undetermined costs of CRISPR-based therapeutics are important considerations. In the United States, there are numerous applications filed with the U.S. Food and Drug Administration to test therapies in humans and limited trials. The U.S. Food and Drug Administration’s Center for Biologics Evaluation and Research has a well-established program and policies evaluating gene therapy products. At this time, it is difficult to predict future costs of CRISPR-based therapeutics. Other gene-therapy products currently on the market can range from $450,000 to upward of $1 million for single treatments that have effectively cured a lifetime of disease. If a single treatment can potentially cure disease, new models of pricing may support such high one-time costs.74
Germline editing has generated disease models with which to study molecular underpinnings of specific gene functions. Concern for ethical use of CRISPR in human cells spans somatic cell modification, basic and preclinical use, and germline cell modification. Recently, genetic modification of human embryos by CRISPR/Cas9 to correct a pathogenic gene mutation has brought intense scrutiny. The National Academy of Sciences and the American Society of Human Genetics issued consensus statements urging caution but also promoting funding of human embryonic gene editing research.75 Nevertheless, there will continue to be public debate as germline disease eradication becomes more feasible. The scientific community understands that regulation and oversight are necessary but that research efforts and funding need to continue. Federal and state policymakers should be aware of the power and opportunities CRISPR germline editing brings to human life. The American Society of Plastic Surgeons and its members should become stakeholders as well, and participate in future debates on the ethical use of CRISPR for the betterment of our patients.
CRISPR gene editing is revolutionizing the potential of gene therapy because of its simplicity, specificity, efficiency, low cost, and versatility. Potential applications of CRISPR are numerous and will certainly impact plastic and reconstructive surgery. CRISPR is a major technological advance revolutionizing broad areas of oncology, wound healing, immunology, and craniofacial genetics, such that plastic surgeons should gain familiarity with this disruptive technology, and become active contributors and leaders in applying CRISPR to our respective areas of expertise. New rungs of the reconstructive ladder may one day be modified to include molecular enhancement of tissue repair with CRISPR gene editing, administration of CRISPR-edited cells with enhanced survival, or even transplantation of humanized tissues from CRISPR-edited animals (Fig. 3). In this way, we can harness CRISPR to create innovative treatment strategies to better the lives of our patients.
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