1 Introduction

The degenerative processes that lead to skin breakdown and possibly form into chronic wounds is nontrivial. To add to the complexity of addressing the wound itself, patients with chronic wounds frequently have complicating factors which can add to this challenging localized event. Some of these factors may include diabetes, resistant infection, immunologic suppression, retained foreign bodies, obesity, tobacco product use, excessive biomechanical stress, inadequate venous and/or arterial flow, peripheral neuropathy, etc. [1,2,3,4,5,6,7]. Treatment of only one of these complications (lower extremity complications of diabetes) now constitutes greater direct costs in the USA than the five most costly cancers [8, 9]. Consequently, the solutions derived to address these aberrant processes have progressively been refined to help address these complicating factors.

In practice, wound healing approaches traditionally have targeted controlling environmental aspects of the wound and neighboring tissues. Common clinical management can include revascularization surgery, mechanical compression, mechanical off-loading, sharp debridement of the wound bed, inflammation/infection control, moisture control via wound dressings, negative-pressure wound therapy, advanced biological agent application for growth stimulation and/or reepithelialization, etc. [10, 11]. These methods represent critical therapeutic elements for chronic wounds as well as provide vital areas of investigation as researchers seek to understand the fundamental behavior of wounds and how to heal them efficiently.

To build upon that common goal, genetic engineering researchers have sought to take a different approach. Instead of attempting to control the environmental aspects and heal the wound from the outside-in as is done with more traditional therapies, the genetic engineering approach is to modify the wound itself to heal the wound from the inside out. Highlighted in this chapter are biotechnologies that have been used to enhance wound healing as well as emerging technologies that can overcome previous barriers. It is intended that the reader will gain both an appreciation for previous genetic engineering work as well as understand the direction of research and intended clinical application as it relates to wound healing enhancement and genetic engineering.

2 Traditional Viral Transduction

Viral transduction is the classical model that has been used for gene modification and generally consists of using viral machinery that has been reprogrammed to deliver a designed genetic payload. Primarily, adenovirus, adeno-associated virus, and (less commonly) Retroviridae family viruses (retrovirus and lentivirus) have been used in cutaneous targets [12].

Adenoviruses (ADV) are frequently used in cutaneous modification experimentation for several reasons. Unlike some viruses, ADV does not insert into the host cell’s genome, which eliminates concerns for insertional mutagenesis that can occur when DNA is inserted randomly (as is the case with non-functionalized genetic loads using non-viral transfection methods) [13, 14]. This also has the added benefit of operating in the cell regardless of current cell cycle stage [15]. Furthermore, the ADV facilitates entry into the cell via receptor-mediated endocytosis, which allows for a degree of cell tropism, and transports the genetic load to the nucleus, bypassing the possible degradation that can occur during transit. These logistical benefits are significant barriers to non-viral transfection methods for gene delivery.

However, ADV do have challenges that can limit their utility. Host cell entry requires the use of the ADV capsid. Unfortunately, the ADV capsid proteins may incite inflammatory responses. In fact, it is estimated that 90% of people have formed antibodies to these capsid proteins and thereby challenge its broad use [16].

The other commonly used virus is adeno-associated virus (AAV), which is similar in function to ADV (Fig. 1). Some of the advantages of AAV include being less immunogenic than ADV [15], less likely to be deactivated by heat [17], able to target many cell types [18], and can transduce nondividing cells [15]. Unfortunately, AAV have even more modest capacity to carry engineered genes than ADV (4–5 vs. 8.5 kbp) [19]. Also, AAV viral particles require high multiplicity of infection to be effective, which can be time-consuming to generate [15].

Fig. 1
figure 1

Mechanism of action for ADV and AAV, two most commonly used viral transduction methods used in cutaneous experimentation. Both viruses employ receptor-mediated endocytosis for cell membrane entry. However, for nuclear membrane entry, ADV mechanism of entry is unknown, while for AAV entry occurs as the viral capsid binds to the nuclear pore complex and genetic contents are imported into the cell. It should also be noted that in rare cases, ADV-mediated transduction can have random host genome inserts

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Viral-mediated genetic modification in animal models has shown to be effective in terms of several key signaling proteins, which include vascular endothelial growth factor (VEGF), platelet-derived growth factor-B (PDGF-B), inducible nitric oxide synthase (iNOS) [20,21,22,23,24], as well as efficacy in ErbB3 receptor modulation [25]. While discussed here in brief, a comprehensive listing of animal experimentation with viral transduction can be found in [12]. In terms of ADV transduction, Romano Di Peppe et al. [22] reported a 3.7-fold increase in blood vessel concentration 1 week after applying a topical ADV vector that was designed to target VEGF upregulation in diabetic mice with impaired healing. In another study, a similar ADV vector targeting VEGF was delivered via microneedling to a porcine model, which did also increase VEGF levels, although not enough to induce measurable neovascularization [23]. In terms of AAV transduction and VEGF upregulation, two studies separately showed more reliable wound healing than ADV studies on the basis of being able to stimulate neovascularization, protein scaffold formation, and reepithelialization [26, 27].

One element to these early ADV studies that has been hypothesized as being a weakness in clinical application is that dysfunctional healing is not a result necessarily of there being a lack of growth factors present but rather a lack of receptors to be acted upon. Okwueze et al. [25] provides evidence for this hypothesis in terms of ADV transfection targeting the ErbB3 receptor gene, a key receptor in signaling reepithelialization. By supplying a topical EGF-like ligand topically, this work showed that significant healing maturation occurred relative to non-treated controls.

3 Emerging Biologic Tools for Genetic Modification

Viral transduction has historically experienced several limitations that has made broad application difficult [18, 19, 28,29,30,31,32,33,34]. However, many of these downfalls have been addressable, and workable solutions have been found, as evidenced by the animal experimentation. The one major exception to this generalized statement is the fact that viruses have a limited carrying capacity and lack mechanisms to actively promote the production of the engineered insert in a consistent manner. Consequently, with reduced capacity and precision of expression, researchers have needed to explore non-viral transfection modalities to achieve the same job of gene delivery without the same limitations. Some of these non-viral transfection methods include micro-seeding [35,36,37], particle bombardment [38, 39], liposomal reagents [40], electroporation [41,42,43,44,45], conjugated nanoparticles, nano-wires, microfabricated needles, and multi-electrode arrays. These non-viral transfection methods have produced a wide range of results in terms of the current context of chronic wounds. However, despite the progress that non-viral methods have made, viral delivery efficiency rates are considered to be the benchmark for all methods, reaching modification rates consistently in the 80–90% range. (Note: Although viral transduction can have high modification efficiencies, it should be recognized that some of the non-viral technologies can achieve similar rates [46].) While these technology solutions are beyond the scope of the current discussion on biologic tools used for genetic engineering, it is worth mentioning their development has been stimulated as a way to bypass historic problems faced by viral methods. For a more complete discussion on this topic, the reader is directed to [12].

Returning to viral transduction, it should be apparent to the reader that viruses can be effective at gene delivery [47,48,49,50]. However, viruses do not necessarily have the machinery to reliably implement expression of the genetic construct that they transport—thus greatly diminishing the potential for engineering biologic outcomes—that is until the recent and exciting development of clustered regularly interspaced short palindromic repeats (CRISPR) and the associated Cas9 for genetic engineering.

4 CRISPR/Cas9

The development of programmable CRISPR/Cas9 plasmids have in the last few years lead to a veritable tectonic shifting terms of the ability to reliably and precisely target and modify gene expression [51,52,53,54]. Clinically applicable projects employing CRISPR/Cas9 sweep the gamut and include treating HIV [55,56,57], hepatitis B [58,59,60], Duchenne muscular dystrophy [61,62,63,64,65], β-thalassemia [66], hemophilia A [67], fragile X syndrome [68], Hunter syndrome [68], Friedreich’s ataxia [68], etc.

To place context to why CRISPR/Cas9 is such a pivotal element to the current state of genetic engineering, it is first important to understand why gene editing agents can struggle to genetically modify human cells. In the most basic sense, the eukaryotic cell is described as having a cell membrane which houses several organelles, each with functional contributions that allow the cell to function. Central to the cell is the nucleus which contains genomic DNA, which of course acts as the biologic coding for the cell’s proteins and RNA. Because of the potentially cell-threatening effects of any alteration made to the genome, the nucleus is a highly regulated structure. In fact, the only physical passageway through the nuclear membrane comes via the nuclear pore complex. The complex consists of ~30 individual supramolecular nucleoporin structures that form an octagonal ring and act to scrutinize incoming biologic agents that seek entrance [69]. Without proper signaling elements, a biologic molecule cannot enter the nucleus (unless assisted, like in the case of ADV and AAV). Furthermore, inside the nucleus is a highly regulated histone-DNA chromosome superstructure that tightly wraps the DNA and keeps only limited portions of the genome accessible at a given time.

It is from the basis of the interaction inside the nucleus that CRISPR/Cas9 operates to facilitate gene altering functions. Simplistically described, CRISPR/Cas9 operates with two main components, the single guide RNA (sgRNA) and the Cas9 protein. The sgRNA is a short programmable RNA strand that contains a specific sequence that complimentarily matches a specific locus within the genomic DNA [70, 71]. When the sgRNA is complexed with Cas9, the complex can locate into the genomic DNA and under Cas9 action provide a variety of actions, which include transient transcriptional activation [72,73,74] or repression [72, 75], permanent gene insertion [76, 77] or knockout [78], inducible gene activation or repression, and genomic loci imaging/loci screening (Fig. 2). Additionally, because the sgRNA is a separate component to the Cas9 protein, several unique sgRNA sequences can be used in a single transduction/transfection event, thereby affecting multiple gene loci at a time—a process known as multiplexing [62, 79,80,81,82]. As an example of how this would be applicable in a clinical setting, in a recent study, researchers used a multiplexed CRISPR/Cas9 construct to target multiple “mutational hotspots” in Duchenne muscular dystrophy at exons 45–55. Following editing, dystrophin expression was restored to in vitro myoblasts [62].

Fig. 2
figure 2

The versatility of CRISPR/Cas9 for genetic engineering and genomic diagnostics

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Given the vast array of design options, the combinational use of transporting viral vectors equipped with CRISPR/Cas9 payloads capable of precision editing lends itself well for chronic wound healing applications for several reasons. First, CRISPR/Cas9 plasmids are small and relatively simple to construct, making it possible to use within the carrying capacity limits for viruses. Second, random gene insertion issues faced with some viruses are bypassed and instead can be guided to genomic sites, thus eliminating concerns for insertional mutagenesis. Third, tissue repair occurs in stages, which means at different stages there are different molecular actions that predominate and then regress as other functional proteins act. Thus, having transiently active gene expressions that are externally controllable with molecular triggers is highly desirable—a function that has been already been demonstrated in terms of a doxycycline-regulated Cas9 [83].

5 Antibacterial CRISPR/Cas9 Applications

While there is little research in terms of CRISPR/Cas9 applied to direct wound bed healing, there has been a great deal of research activity in terms of using CRISPR/Cas9 to examine and control bacteria, particularly antibiotic-resistant bacteria, which is highly useful in infected wounds. As noted, CRISPR/Cas9 can be used to delete portions of DNA. Zheng et al. [84] demonstrated recently in Escherichia coli that CRISPR/Cas9 can be used to delete large portions of DNA fragments, a proof of concept that can be extended to vital bacterial components and other bacterial types. In a similar approach, several investigators have used CRISPR/Cas9 to identify antibiotic resistance genes, particularly as they behave in stress response conditions, and in some cases edit them [85,86,87,88]. Additionally, although not mentioned in great deal previously, the Cas9 protein component of the complex can be altered to be deactivated (dCas9). Albeit a CRISPR/dCas9 system lacks editing capabilities, the dCas9 protein can be linked to marker proteins, which allow the CRISPR/dCas9 complex to be used to map specific locations in a bacterial genome (in this case). This has led to many researchers being able to map antibiotic-resistant genes and identify adaptive resistance pathways with the intent to then alter the bacterial fitness to stress conditions [89,90,91]. This represents a critical and exciting component to chronic wound beds because of the ability to sort through heterogenous bacterial populations to identify genetic variation and antibiotic-resistant gene presence and then eliminate those pathogens deleterious to healing.

Conclusions

Chronic wounds constitute a widespread problem with complex conditions to resolve. This chapter provides contextual research in the area of biologic tools that have been and can be used to help treat them. While genetic engineering has only begun to be explored in terms of chronic wounds, the future is promising, and with proper combinational approaches, there can be clinically viable solutions.