chapter  5
Spatiotemporal Genetic Control of Cellular Systems
Pages 42

Figure 5.1 Constitutive gene expression systems. (a) Placement of a transgene of interest downstream of a constitutive promoter results in spatially and temporally independent expression of the transgene. (b) Placement of an shRNA construct downstream of a constitutive promoter results in constitutive transcription of the shRNA, which causes silencing of target gene expression through RNA interference.Cartilage is perhaps the simplest tissue in the human body in that it contains only one cell type (chondrocytes) and is avascular and aneural. However, despite this morphological simplicity, cartilage tissue engineering is complicated by the tissue’s role as a low-friction, load-bearing material within the body. For chondrocyte implantation, patient chondrocytes can be isolated and expanded ex vivo [6]. This expansion process provides the opportunity to genetically modify the cells before they are implanted back into

the patient. In one study, chondrocytes were transduced with a retroviral vector encoding a constitutively expressed gene for bone morphogenic protein-7 (BMP-7) to expedite the cartilage repair process post-implantation [7]. BMP-7 is normally expressed in articular cartilage [8] and is important for limb and joint formation [9]. Modified or unmodified chondrocytes were suspended in fibrinogen, and this mixture was polymerized in situ in an adult cartilage defect repair horse model. After 4 weeks, implanted BMP-7-expressing chondrocytes exhibited a round, chondrocyte-like morphology and integrated with host subchondral bone, whereas unmodified control chondrocytes were flatter and did not integrate well with host subchondral bone. After 8 months, the appearance, morphology, and integration of treated and control cells were equivalent. Thus, although the end result was the same using genetically modified or unmodified chondrocytes, cells that over-expressed BMP-7 accelerated cartilage tissue repair.Although it is possible to correct smaller defects in situ and generate cartilage tissue in vitro through expansion of patient-derived chondrocytes, other methods are being explored due to the low availability of chondrocytes and their tendency to dedifferentiate during in vitro culture. These alternative methods are necessary for the repair and replacement of larger defects. There is particular interest in the use of patient-derived adult mesenchymal stem cells (MSCs) for cell-based cartilage repair because they are relatively easy to isolate and expand, and their multipotency enables them to differentiate into multiple connective tissue types [10,11]. There are many factors that govern the differentiation of MSCs into chondrocytes, including transforming growth factor-β (TGF-β) superfamily members, BMPs, Wnt proteins, and fibroblast growth factors (FGFs). Differentiation also depends on the presence of cartilage-specific matrix materials such as type II collagen, aggrecan, and fibronectin [10]. MSCs can be genetically engineered to over-express these factors or deposit these materials to facilitate their differentiation into chondrocytes and improve local cartilage repair [12-14]. For instance, isolated murine muscle-derived stem cells were transduced with retrovirus encoding BMP-4 and LacZ and transplanted into a full thickness articular cartilage defect murine model [15]. Modified cells locally delivered BMP-4, which enhanced chondrocyte differentiation and improved articular cartilage

repair as compared to unmodified cells for 24 weeks following cell transplantation. Genetically modified cells are also being tested for the treatment of debilitating diseases such as rheumatoid arthritis. In a phase I

clinical study, autologous synovial fibroblasts were engineered to constitutively express IL-1 receptor antagonist (IL-1Ra), which has been shown to alleviate symptoms of rheumatoid arthritis in mice [13]. These modified cells or control cells were injected into the metacarpophalangeal joints of postmenopausal women suffering from rheumatoid arthritis. After 1 week, joints treated with genetically engineered cells were positive for IL-1Ra mRNA, and the synovial fluid of patients that received intermediate and high doses of treated cells produced high levels of IL-1Ra protein. Future clinical studies are needed to assess the clinical efficacy of the IL-1Ra transgene expression.In addition to expedited engraftment and healing and treatment of diseases, genetically modified cells can also be used to improve the functionality of implanted constructs and grafts. While simple tissues have been shown to function when implanted as monocellular constructs, functionality can be improved by promoting the recruitment of other cell types once implanted in the body. For example, epidermal keratinocyte skin grafts have been shown to successfully cover wounds, enhance patient survival, and relieve pain due to burns [16], vascular leg ulcers [17], epidermolysis bullosa [18], and other ailments [19], but full, native-like functionality and integration of skin grafts requires the presence of other skin cell types such as mast cells and Langerhans cells. Instead of culturing these cell types along with keratinocytes in vitro before implantation, genetic engineering could be used to release factors that promote migration and infiltration of host cells into the implanted construct. Consequently, improved graft incorporation and functionality may be achieved. This type of modification is short-term such that the treatment lasts only during the wound healing process. Gene expression can be temporarily modified within grafted keratinocytes by immobilizing plasmid DNA or non-integrating virus (i.e., adenovirus) packaged with a desired gene to the scaffold surface. Controlled release of the DNA or virus from the scaffold results in transfection or transduction, respectively, of grafted keratinocytes. Benefits of this approach

include local gene delivery only to implanted cells or cells that infiltrate the wound site, protection of the genetic material from proteases present in the healing wound environment, and reduced immunogenicity of the material due to its isolation from host immune cells [20-22].