Cutaneous Gene Therapy for Skin and Systemic Disorders

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William F. Buitrago and Dennis R. Roop

Baylor College of Medicine Houston, Texas, U.S.A.


The skin serves multiple functions owing to its unique and complex structure. The skin presents remarkable advantages as a tissue for developing innovative genetic therapeutic strategies. The skin is a readily accessible organ, which facilitates gene delivery, subsequent monitoring of transgene expression, and excision of small or large areas if required (1). Epidermal keratinocytes and dermal fibroblasts can be readily expanded in culture. In addition, keratinocytes have high proliferative potential and inherent biological characteristics that allow them to synthesize mature proteins from a vast array of transgenes (2).

There have been recent advances in the molecular characterization of many skin disorders, vector design, immune modulation, regulation of gene expression, administration, and in other aspects in the field of skin gene therapy. This rapid progress has made the skin a formidable target to develop and test a variety of gene therapy approaches to both cutaneous and systemic diseases.


The skin, or integument, is the most superficial and largest organ of the body. Its functions are essential for the homeo-static balance of the organism. The skin provides protection against ultraviolet (UV) light, mechanic, thermic, and chemical insults, and also prevents excessive dehydration due to its relative water impermeability (3,4). It also acts as a physical barrier against microorganisms and is involved in the coordination of multiple immune responses (5,6). The skin is the major organ involved in sensory perception. For instance, it possesses tactile, pressure, pain and temperature receptors (7,8). In humans and in many other mammals the skin is essential in thermoregulatory responses (6,9,10). For example, heat conservation is aided by the presence of hair and adipose tissue in the hypodermis, and heat loss is increased by the evaporation of water through the skin's surface and by increasing the blood flux through the rich capillary plexus of the dermis (9). The skin also plays a definite role in the body's metabolism as an important store of energy in the form of triglycerides and in the synthesis of vitamin D (6,9).

The skin presents regional variations with regards to thickness, coloration, and presence of adnexa. For instance, the skin is thicker, presents lower levels of coloration, and has no hair follicles in the palms and soles. However, the basic structure of the skin is maintained in all body areas. The external part of the skin, the epidermis, is formed by a stratified squamous epithelium, which is composed in its majority by keratinocytes. Other cell types such as melanocytes, Langerhans cells, and Merkel cells are also found in this layer. The epidermis is divided into 2 major compartments: the basal (proliferative) and suprabasal (differentiation) compartments. The suprabasal compartment is further divided into different layers based on microscopic characteristics: the spinous layer, the granular layer, and the cornified layer (Fig. 1). The basal keratinocytes are attached to the basement membrane (BM) through specialized multiprotein junctional complexes called hemidesmosomes (11). The epidermis, a tissue in constant turnover, is sustained by permanent mitotic divisions in the basal compartment where stem cells reside (12). Recent studies suggest that the skin contains 3 distinct stem cell reservoirs: the interfollicular epidermis and the anagen hair follicle ger

Figure 1 Schematic representation of the skin.

minal matrix where stem cells with a limited differentiation potential exist, and the upper outer root sheath (bulge), which contains potent reserve stem cells that act in the maintenance of not only the epidermis, but hair follicles and sebaceous glands (13). The cells in the basal layer undergo as series of maturational changes, and at some point, a given number move upward to the postmitotic suprabasal compartment where they undergo terminal differentiation. The basal layer is composed of a heterogeneous population of cells that can be classified according to their capacity for sustained growth into 3 subpopulations: (1). the holoclones or stem cells, which have the greatest reproductive capacity; (2). the paraclones or differentiated cells with a short replicative lifespan and limited growth capacity; and (3). the intermediate meroclones, which are the transitional stage between the holoclones and the paraclones (14). The expression of keratins, integrins, and involu-crin is regulated during keratinocyte differentiation. Keratin 5 (K5), keratin 14 (K14), and integrins are expressed by basal cells, their expression is down-regulated as the cells detach from the BM and move upward to the suprabasal compartment where keratins 1 (K1) and 10 (K10) become highly expressed (15,16). Subsequently, as keratinocytes migrate toward the epidermal surface the expression of other markers, such as filagrin and loricrin, is detected (17,18). Most of the cellular organelles are degraded in the stratum granulosum, and the dead keratinocytes form the stratum corneum.

The dermis, a thick layer of fibroelastic dense connective tissue that supports and nourishes the epidermis, is composed of numerous blood and lymphatic vessels, sensory elements, and different cell types such as fibroblasts, macrophages, and lymphocytes. The hypodermis (subcutaneous layer or subcutis) is localized under the dermis, and contains adipose tissue and blood vessels. The adnexal structures such as sweat glands, sebaceous glands, and hair follicles are structures of ectodermal origin that form by invagination of the epidermic epithelium into the dermis, and sometimes the hypodermis (Fig. 1).


A. Main Strategies

The accessibility, visualization, and monitoring that made the skin an appealing target for genetic therapy has also allowed the development and use of strategies for gene transfer not possible in other tissues. The 2 main strategies currently used for gene delivery in skin disease models are the ex vivo and in vivo approaches.

1. The Ex Vivo Approach

In the ex vivo approach, the skin cells from the host are isolated and harvested after removal by biopsy. The cells are then grown in vitro where therapeutic gene transfer is performed. Finally, the altered cells are grafted back into the host (Fig. 2). This method offers some advantages because primary kera-tinocytes, including human, are receptive to gene modification, readily expanded in tissue culture under selective conditions, and easily grafted back into host (19-22, Buitrago and Roop, unpublished data). However, the ex vivo approach is disadvantageous because of its labor intensity and potential scarring.

Figure 2 Ex vivo approach for cutaneous gene transfer.

2. The In Vivo Approach

In this approach, gene transfer is achieved through direct administration of genes by different modalities, such as naked DNA or in vectors of nonviral or viral origins (Fig. 3). The in vivo approach is favored over the ex vivo approach as the need for cell culture and surgery are bypassed, making it technically, clinically, and economically advantageous (19). In spite of these advantages, the direct in vivo approach is still limited by the low levels of transduction frequency for stem cells that leads to transient expression of the transferred gene.

B. Gene Transfer Systems in Cutaneous Gene Therapy

The first step in effective gene therapy is the ability to deliver the corrective gene/genetic material to the correct tissue with high efficiency. The skin can be specifically targeted by transduction in culture, intradermal injection, topical application, or other methods. A variety of specific techniques and vectors for skin gene delivery have been developed, and they can be classified in 2 large categories: viral and nonviral.

1. Viral Gene Transfer Vectors

Currently, viruses provide the most efficient means of delivering genes to target cells. Several classes of viruses have been successfully used for gene delivery and expression in the skin (Table 1). Viral vectors are efficient for gene transfer in cell culture as part of the ex vivo approach. In addition, they have also yielded positive outcomes when administered topically or by direct injection during the in vivo approach (23,24). As reviewed by Ghazizadeh and Taichman (25), there are 6 main factors to consider in a viral system for efficient gene transfer: ability for high titer generation, cargo-carrying capacity, capacity to transduce dividing and/or nondividing cells, integration properties, vector antigenicity, the ease with which clinical grade vectors can be prepared free of replication-competent virus, and the length of time of gene expression required (25). Some disadvantages should also be considered when working with viral vectors. Because the expression of the delivered genes depends heavily on the integration site, it is important to understand that all the integrative viral vectors have the limitation of their lack of true site-specific integration. Furthermore, down-regulation of the introduced genes by epigenetic mechanisms represents another significant obstacle. To overcome these obstacles, several strategies have been developed including use of insulator elements, and the bacterial tetracycline regulatory system (26-28). a. Adenoviruses. These vectors have been widely used for skin gene transfer. Most of these vectors have been rendered replication deficient by deletion of their E1A and E1B essential genes (29,30). The development of ''gutless'' adenoviral vectors in which more viral genes are stripped has increased their cargo capacity and minimized the toxicity of viral products to target cells (31). Adenovirus vectors can carry up to 35 kb of foreign DNA, and the viral particles can be produced at high titers. They can infect a wide variety of cell types, and both replicating and nonreplicating cells. In addition, safety precedents already exist because adenovirus-based vaccines have been used in patients without any major side effect (32).

Adenoviral transgene expression has been detected in all cell types of both dermis and epidermis when injected subcutane-ously (19). However, adenoviruses are highly antigenic, and because they do not integrate into the host genome, only transient expression of the delivered gene is achieved most of the time. Therefore, adenoviral vectors are not ideal for sustained expression in a regenerating tissue such as the epidermis, making their use to correct inherited skin diseases limited. Nonetheless, it has been recently reported that recombinant adeno-virus vectors carrying the xeroderma pigmentosum A (XPA) and C (XPC) genes achieved long-term expression, as well as long-term restoration of biological activity (up to 2 months) in XP-A and XP-C immortalized and primary fibroblast cell lines (33). Adenoviral vectors have been successfully used in applications such as DNA vaccination (34), anticancer therapy (35), and promotion of wound healing (36-38).

b. Adeno-associated viruses. Adeno-associated viruses (AAVs) were once considered poor vectors due to their limiting packaging size (about 4.7 kb). However, the requirement for a helper virus (adenovirus or herpesvirus) for productive infection makes them one of the safest viral vectors (39). Currently, recombinant AAVs have a high safety profile because 96% of the AAV genome has been removed. AAVs can transduce a great variety of human cell types, both in vitro and in vivo (40). AAVs can exist in both integrated and non integrated forms, and are able to transduce replicating and nonreplicating cells (25,41). AAVs do not induce a strong innate immunologic or a cytotoxic T cell response, and long-term expression of the transgene is possible (42). However, they induce an antibody response, and the transfection efficiencies are usually low (42).

c. Retroviruses. These are the most widely used viruses for gene delivery. They are capable of delivering genes to

Table 1 Viral Gene Transfer Vectors for Cutaneous Gene Therapy

Vector Type




Transduce almost all dividing mammalian

Random integration into host genome; do

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