Recent gene therapy studies by using encapsulated transgenic cells
A major challenge to emerging cell-based medicine including gene therapy is the host immune rejection of transplanted donor cells or engineered tissue. One way to address this problem is to use drugs to achieve immunosuppression. However, suppressing the patient’s immune system may put the patient at risk for many other diseases. An alternative is to encapsulate living cells in macro/microcapsules to achieve immunoisolation of the cells, thereby increasing cell viability in the patient’s body following transplantation. The capsule’s membrane protects the encapsulated cells from being damaged by both the host’s immune system and mechanical stress while allowing free diffusion of nutrients and metabolic waste for the cells to survive. Moreover, the membrane could be designed to achieve controlled and/or sustained release of therapeutic products produced by the encapsulated transgenic cells to treat a variety of diseases such as cardiovascular disorders, anemia, wounds, bone fractures, and cancer.
- Cell microencapsulation
- Gene therapy
- Cell-based medicine
Cell encapsulation is the process of entrapping cells into a matrix. In general, the matrix is spherical in shape and in the form of a polymeric hydrogel. Cell encapsulation technology has shown great promise for immunoisolation and controlled release of therapeutic products towards gene therapy. Figure 1 demonstrates the mechanism of encapsulated transgenic cells for gene therapy.
1.1. Encapsulation materials
Both natural and synthetic polymers have been utilized for cell encapsulation. Natural polymers that have been used include alginate, agarose, collagen, and hyaluronic acid, while synthetic polymers, including poly(vinyl alcohol), poly(lactic-co-glycolic acid), polyacrylates, HEMA-MMA-MAA, polyphosphazines, and polyepoxides, have been studied. Natural polymers are more commonly used because of their biocompatibility and are easily accepted by the public. However, their product quality and characteristics can vary greatly between companies and batches compared to synthetic polymers. Alginate, agarose, and polylactide-co-glycolide (PLGA), the most commonly used encapsulation materials, are introduced here.
Alginates, polysaccharides, are linear block polymers consisting of α-l-guluronic acid (G) and β-d-manuronic acid (M) blocks (Figure 2). Divalent cations, such as Ca2+, Ba2+, and Sr2+, can link alginate molecules together (i.e. through ionic cross-linking) forming alginate hydrogel capsules while encapsulating cells inside. The G and M contents of the alginate molecules can affect the gel properties including mechanical strength, biocompatibility, and permeability.[2–6] Recently, it has also been shown that oligochitosan could be used as a cross-linker for polysaccharide-based gel formations.
Agarose, a thermal-responsive polymer, consists of β-d-galactopyranose and 3,6-anhydro-α-l-galactopyranose units which can undergo a sol–gel transition upon cooling (i.e. through thermal cross-linking) (Figure 3). Some agarose products have a transition temperature close to body temperature, making it a good candidate for cell encapsulation.
1.2. Encapsulation technologies
Different technologies have been used for preparing macro/microcapsules, which include air-jet encapsulation, electrostatic spray, laminar jet breakup, and microfluidic channel/nozzle. Among them, electrostatic spray and microfluidic channel/nozzle are two of the most frequently used encapsulation approaches.
1.2.1. Electrostatic spray method
The electrostatic spray method has a significant appeal due to its ease of operation, scale-up capabilities, negligible damage to cells, and allowance for sterile operation conditions. The mechanism of cell encapsulation by using the electrostatic pray method is shown in Figure 5A. In general, a cell polymer mixture is extruded through a nozzle by using a pump or compressed air. The droplets are broken down into smaller ones under electrostatic force and/or other introduced forces (e.g. vibration). Once the droplets reach the gelling bath containing the cross-linkers, the cell-loaded hydrogel capsules form immediately through various forces, such as ionotropic reaction between divalent ions and alginate molecules. Moreover, the system could be modified to prepare the core-shell structure hydrogel capsules, as depicted in Figure 5B.
1.2.2. Microfluidics channel/nozzle method
Microfluidics devices can be used to generate micrometer-scale droplets with a narrow size distribution and controlled morphology.[12–14] This method shows great promise for cell encapsulation, especially for single cell encapsulation. In general, capsules are formed by allowing a core fluid to be surrounded by a flowing sheath stream. Recently, these devices have also been successfully applied for the generation of cell-loaded core-shell capsules (Figure 6). Besides the relatively low encapsulation efficiency, a significant drawback of the current microfluidic technologies is that the oil used for shearing may leave a residual adhesive oil layer on the capsule which affects subsequent coating processes.[10,17]
2. Recent progress on transgenic cell encapsulation for gene therapy
Encapsulation of genetically modified cells has been conducted for the treatment of central nervous system diseases, cardiovascular disorders, mucopolysaccharidosis type VII (MPSVII) disease, wounds, bone fractures, and cancer.[18–30] Considering most genetically engineered cells are from allogeneic or xenogeneic sources, immunoisolation is a critical factor when using these cells.
2.1. Bone-related diseases
Bone morphogenic protein-2 (BMP-2) is a member of the transforming growth factor-β (TGF-β) superfamily and has been widely reported to have osteoinductive activity. Ding et al.  studied the behaviour of BMP-2 gene-transfected bone marrow-derived mesenchymal stem cells in alginate-poly-l-lysine-alginate (APA) microcapsules. The results showed that encapsulated transfected cells could secrete BMP-2 proteins for at least 30 days and the APA microcapsules could be used for immunoisolation. Olabisi et al.  investigated microencapsulation of AdBMP-2-transduced MRC-5 cells (human diploid fetal lung fibroblasts) in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. After injecting the encapsulated cells intramuscularly, the volume of the bone formed was about twice that of the control group (unencapsulated cells). Recently, rapid heterotrophic ossification by using cryopreserved PEGDA encapsulated BMP-2 expressing mesenchymal stem cells (MSCs) was also observed (as shown in Figure 7). Additionally, human calcitonin delivered by microencapsulated recombinant myoblasts showed potential for allergenic gene therapy for postmenopausal osteoporosis.  Furthermore, transplantation of fibrin glue-compounding hepatocyte growth factor-transgenic MSCs is a promising novel method for avascular necrosis of the femoral head (ANFH) therapy.
Both mouse myoblasts (C2C12 cells) and human embryonic kidney 293 (HEK293) cells were engineered to continuously secrete angiostatin, and were encapsulated into alginate-based microcapsules for cancer treatment. The in vivo experimental results demonstrated the potential for angiostatin-mediated cancer therapy by using an encapsulated transgenic cell-based approach.[35,36] Considering immunotherapies have been proven to be alternative strategies for malignancy treatment, combined immunotherapy (an interleukin 2 fusion protein, sFvIL-2) and antiangiogenic therapy (angiostatin) were tested. It was shown that transplantation of angiostatin expression and sFvIL-2-expressing C2C12 cells encapsulated in APA microcapsules improved the survival rate of experimental animals. Recently, microencapsulation of therapeutic antibodies producing cells in APA microcapsules was tested for cancer treatment.  Additionally, with the advancement of stem cell research, there is an increased potential for cancer therapy by using encapsulated stem cells.
2.3. Neural diseases
Parkinson’s disease (PD) belongs to a group of conditions called motor system disorders, resulting from the loss of dopamine-producing brain cells. This disease could be amenable to gene product replacement strategies including implantation of encapsulated transgenic cells. There are several publications regarding encapsulated cell biodelivery of glial cell line-derived neurotrophic factor (GDNF) for PD treatment; GDNF has been proven to have neuroprotective and neurotrophic properties on dopaminergic neurons.[26,43,44] Furthermore, encapsulated transgenic cells could be utilized in brain tumour treatment.[45,46]
Small capsules (<200 µm) have been developed for the delivery of gene products, secreted by encapsulated transgenic cells, to the brain, bypassing the blood–brain barrier (BBB). To date, several alginate-based microcapsule systems, Ca-alginate, APA, and alginate-chitosan-alginate (ACA), have been reported.[10,47,48] Encapsulation of transgenic cells has also been used for other disease treatments, such as mucopolysaccharidosis VII and myocardial infarction. Table 1 summarizes the recent gene therapy studies based on encapsulated transgenic cells, with the exception of bone-related and neural diseases and cancer treatment.
|Disease||Therapeutic Product(s)||Cell Type||Encapsulation System||Ref.|
|Fabry disease||α-Galactosidase A||Chinese hamster ovary cells||Semipermeable Polymer Fiber|||
|Mucopolysaccharidosis VII||β-Glucuronidase||Mouse 2A-50 fibroblasts||Alginate-poly-l-lysine|||
|Human amniotic epithelial cells||Polymer (polysulfon) Hollow fibers|||
|Myocardial infarction and wound||Glucagon-like peptide-1||Human mesenchymal stem cells||CellBeadsTM|||
|Vascular endothelial growth factor||Chinese hamster ovary cells||Alginate-Poly-l-Lysine-Alginate Microcapsules|||
|Adipose stem cells||AP-PLL-brPEG microcapsules|||
|NIH3T3 cells||Alginate-barium microcapsules|||
|Human umbilical cord mesenchymal stromal cells||Alginate-barium microcapsules|||
|Human umbilical cord mesenchymal stem cells||Alginate-barium microcapsules|||
|Polycythemic diseases||Erythropoietin||Mouse C2C12 myoblasts||Semipermeable polyethersulf hollow fibers|||
|Hypertension and/or congestive heart failure||Atrial natriuretic peptide||Chinese hamster ovary cells||Polycaprolactone tubes|||
|Acute skin flap ischemia||Basic fibroblast growth factor (FGF-2)||Mouse C2C12 myoblasts||Microporous polyethersulfone hollow fibers|||
|Hemophilia B||Factor IX||Mouse C2C12 myoblasts mouse C2C12 myoblasts||Alginate-poly-l-lysine-alginate microcapsules|||
|Alginate-poly-l-lysine-alginate and alginate-poly-l-arginine-alginate microcapsules|||
|Laron syndrome||Recombinant human IGF-1||Pig Sertoli cells||Alginate microcapsules|||
3. Challenges and future direction
Recent clinical trials regarding gene therapy by using encapsulated transgenic cells are summarized in Table 2. For eventual clinical applications of encapsulated transgenic cells for gene therapy, however, there are still some issues that need to be addressed.[62,63]
Protrusion of encapsulated cells
Cell growth leads to protrusion of cells over time, which may cause the failure of immunoisolation following in vivo transplantation. Bhujbal et al. reported a novel multilayer immunoisolating encapsulation system aiming to prevent cell protrusion without compromising cell survival (Figure 8).
Scaling-up cell microencapsulation
Cell encapsulation processes are usually performed at the lab scale. For successful clinical applications, massive production of encapsulated cells following good manufacturing practices (GMP) standardized procedures  for transplantation is critical. Different designs have been reported for scaling-up cell encapsulation. One design based on a 3D microfluidic approach, which contains a 3D air supply and multinozzle outlet, has been reported recently.
Monitor and control the encapsulated transgenic cells
Once the therapy has reached its goal or when undesirable deleterious effects occur, noninvasive monitoring and deactivation/elimination of the encapsulated cells are critical for clinical practice. Recently, Shen et al.  reported the encapsulation of recombinant cells by using a magnetized ferrofluid alginate for in vivo monitoring by magnetic resonance imaging (MRI). Moreover, magnetic field-controlled gene expression in encapsulated cells, coencapsulated with magnetic nanoparticles, has been reported. The cells were modified to produce therapeutic products under the control of a heat-inducible promoter. Heat induction could be achieved by elevating the temperatures of the capsules through coencapsulated magnetic nanoparticles subjected to a magnetic field (Figure 9). Catena et al. reported an interesting and smart system which shows potential for monitoring encapsulated cells and selectively eliminating them at a specific moment by using the SFGNESTGL triple reporter system.
|Project||Therapeutic Product(s)||Target Disease(s)||Phase||Status|
|A study of encapsulated cell technology (ECT) implant for patients with late stage retinitis pigmentosa||Ciliary neurotrophic factor (CNTF)||Late-stage retinitis pigmentosa||II and III||Completed|
|A study of encapsulated cell technology (ECT) implant for participants with early stage retinitis pigmentosa||Ciliary neurotrophic factor (CNTF)||Early stage retinitis pigmentosa||II and III||Completed|
|A Study of an Encapsulated Cell Technology (ECT) Implant for Patients With Atrophic Macular Degeneration||Ciliary neurotrophic factor (CNTF)||Macular degeneration||II||Completed|
|Pilot immunotherapy trial for recurrent malignant gliomas||Insulin-like growth factor receptor-1||Malignant glioma of brain||I||Completed|
|GLP-1 CellBeads® for the treatment of stroke patients with space-occupying intracerebral hemorrhage||Glucagon-like peptide-1||Intracerebral hemorrhage (ICH)||I and II||Terminated|
|CNTF implants for CNGB3 achromatopsia||Ciliary neurotrophic factor (CNTF)||Eye disease achromatopsia||I and II||Active|
|Retinal imaging of subjects implanted with ciliary neurotrophic factor (CNTF)-releasing encapsulated cell implant for early-stage retinitis pigmentosa||Ciliary neurotrophic factor (CNTF)||Early stage retinitis pigmentosa or Usher syndrome (type 2 or 3)||II||Recruiting|
|A phase 2 multicenter randomized clinical trial of CNTF FOR MacTel||Recombinant human ciliary neurotrophic factor||Macular telangiectasia type 2||II||Recruiting|
|MVX-ONCO-1 in patients with solid tumours||Irradiated autologous tumour cells||Solid tumour cancer||I||Recruiting|
|Study of the intravitreal implantation of NT-503-3 encapsulated cell technology (ECT) for the treatment of recurrent choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD)||Anti-VEGF therapy||Macular degeneration||I and II||Not yet recruiting|
|Encapsulated cell biodelivery of nerve growth factor to Alzheimer´s disease patients||Nerve growth factor (NGF)||Alzheimer’s disease||I||Unknown|
The author thanks Dr. Eryn Hassemer, Dr. Jung Lee, Alexander Dyble, Kendra Lehman, Rebecca Majewski, and Michael Wedemeyer for proofreading this book chapter. Also, the author thanks Dr. Jung Lee for drawing some of the figures. The author is grateful for funding provided by the Faculty Summer Development Grant at the Milwaukee School of Engineering.