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Tissue Engineering - John P. Fisher

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14.3.3 Physical Delivery Methods

A highly efficient way of transferring naked DNA into a select cell or group of cells is via physical transfection methods. Microinjection by a skilled operator offers nearly perfect delivery efficiency on a cell-by-cell basis. This technique can be used to transfer oligonucleotides to cell cytoplasms or nuclei [58], or entire nuclei into enucleated eggs, as is the case with cloning [59,60]. However, the use of microinjection to transfect hundreds or thousands of somatic cells in vivo would be an infeasible venture, both in terms of getting to and visualizing the cells of interest, and the amount of time and effort required for the procedure. There exist alternative physical delivery methods that can be used to deliver genetic material directly into a larger number of cells, albeit in a slightly less precise fashion. These methods include electroporation and the gene gun.

Since 1982, electroporation has been a promising approach for both in vitro and in vivo gene delivery [61]. Electroporation causes disruption of the plasma membrane and formation of membraneassociated DNA aggregates, which enter the cytoplasm through transient pores [62]. These complexes have been shown to enter the cytoplasm 30 min after administration of current, and proceed to perinuclear locations by 24 h postadministration [62]. Electroporation has shown promising results in the administration of complexes to skeletal muscles, indicating a possible role for the technique in future in vivo muscular applications [63]. Electroporation can be combined with chemical transfection methods, including dendrimers, to increase the overall efficiency of transfection [64]. A similar approach, termed nucleofection, utilizes an electrical current in conjunction with specific solutions to deliver DNA not only into the cell but also into the cell nucleus [65].

The gene gun provides a ballistic means of transporting genetic material into cells. A typical application of the gene gun would entail attaching DNA to gold particles that are on the order of 1 µm in diameter. The coated gold is then loaded onto a cartridge or onto a disc and propelled down the barrel of the gun via pressurized helium at a force on the order of 200 to 300 psi. The end of the gun barrel is too small for the disk to exit, so its motion is halted abruptly before exit. The coated gold colloids detach from the disc and continue their path, passing through or becoming embedded within cells. When passing through cell membranes, cytoskeletons, and other structures, some of the DNA can become dislodged and remain within the cells for processing. This method provides good transfection of tissue surface layers and offers an advantage over microinjection in that many cells in a specified area are quickly transfected. However, cell damage and depth of gold penetration are two limiting factors of this method. The gene gun has been used successfully in vivo to transfect murine tumors with cytokines, successfully restricting cell growth [66]. Direct gene gun transfer of gold-adhered DNA particles has also been used in successful transfections of beating hearts with genes encoding the green fluorescent protein [67].

14.4 Intracellular Pathways

The mechanisms governing the transport of nonviral gene delivery complexes into the cytoplasm and on into the nucleus vary between different vectors. Endocytosis is the primary means for cellular import of nonviral gene carriers, which makes complex modification through the addition of specific ligands beneficial. Endocytosed complexes enter the cytoplasm in endosomes, which mature from early to late endosomes before meeting up with lysosomes to form endolysosomes. As this vesicle maturation occurs, membrane bound H+/ATPases create an acidic environment within the compartments. It is this acidic environment which allows lysosomal enzymes (acid hydrolases) to remain active and potentially degrade the endocytosed material. The endolysosome therefore presents a major obstacle to nonvirally mediated gene therapy, as evidenced by increases in transfection efficiency in the presence of the lysosomotrophic agent chloroquine [68–70]. Research into lysosomal disruption has produced a peptide that can degrade the membrane of the late endosome, based on its cholesterol content. This method has reportedly increased the transfection efficiency of parenchymal liver by 30-fold [71].

PEI has been hypothesized to serve as a proton acceptor within endolysosomes, serving to buffer the pH of the endolysosome while additional H+ is pumped in. According to this hypothesis, Clions will

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leak into the vesicles to alleviate the electric gradient, thereby creating an osmotic gradient which will be offset by an influx of water molecules into the endolysosomes. The result will be swelling and bursting of the endolysosomes [45]. PEI has been shown to enter cells via endocytosis, and the endosomes have been shown to swell within a period of under 6 h [72]. However, there exists a question of the degree of lysosomal involvement during PEI-mediated transfection. PEI/DNA-containing endosomes have been imaged while passing through lysosomal beds without lysosomal interaction [73]. In addition, PEI has been reported to utilize microtubules of the cytoskeleton as a direct pathway to the nuclear envelope, thereby eluding lysosomes [74]. It is perhaps the large excess of positive charges in PEI/DNA complexes that help direct the complexes in a seemingly atypical fashion during cellular processing. More research is required to elucidate the complete cellular mechanism of PEI-mediated transfection.

If the delivered genetic material is DNA, and the desired outcome of the transfection is expression of the delivered gene, then the next major hurdle to successful transfection is getting the delivered DNA into the cell nucleus for transcription. Many studies have monitored the effect of cell cycle stage upon transfection efficiency. Some viral vectors show strong transduction during all phases of the cell cycle because of their strong nuclear-importation machinery [75]. However, polyplex and lipoplex carriers show optimal transfection if they are administered during the G1 or S phases of the cell cycle [75]. It is known that the nuclear envelope is dismantled during late mitosis, hence perhaps these findings can be explained by the length of time required for transfection complexes to proceed from endocytosis to the outer nuclear membrane being roughly equal to the amount of time needed for the cell to progress from G1 or S phase to late mitosis. Other work indicates the barrier to transfection presented by the nuclear envelope through results that reveal very low transfection efficiencies in nondividing cells when cationic lipid-mediated vectors were used [76].

Aside from entering the nucleus by virtue of location during mitotic nuclear membrane disruption, genes and gene delivery complexes can be delivered to the nucleus via import. Possible mechanisms for nuclear import include interactions between the complexes and other proteins bound for nuclear import and interactions between the complexes and nuclear import receptors themselves. Proteins in the cytoplasm that are bound for nuclear import typically have a conserved peptide sequence that is recognized by the cell as a nuclear localization signal (NLS). Such signals can be manufactured in the laboratory for inclusion as an integral part of the gene delivery complexes [77,78]. Many factors govern the success of NLS-mediated nuclear import: the type of NLS, the manner in which the NLS is incorporated, DNA form, and the proper definition of the complexes [79].

14.5 Cell and Tissue Targeting

Each of the physical delivery methods listed in Section 14.3.3 is an excellent way to deliver genes to a specific region. Certainly, microinjection is a very straightforward way to introduce genes into a single cell of interest. However, physical delivery methods can be technically difficult, insubstantial in the number of cells that are transfected, or impossible for delivery to remote areas of the body. To provide feasible alternatives when such challenges exist, molecular targeting methods have been developed, yielding encouraging results.

The use of cell-specific ligands or receptors to target extracellular attributes is a good method for gene delivery to specific cell types or classes. Neuronal cells have been targeted for transduction via a neuron-specific viral envelope in conjunction with specific glycoproteins, leaving nonneuronal cells unaffected [80]. Other examples of ligands that have been used include LOX-1 to potentially target endothelial cells [81], the human hepatitis B virus preS1 peptide to target hepatocytes [82], plus transferrin [83], folate [84], anti-CD34 [85], and galactose [86], among others. The list of potential and realized target ligands is very large, which suggests the utility of this targeting technique.

Using cell-specific transcription regulation sequences (binding elements such as promoters or enhancers) is another way to target cells without modification of the gene delivery vehicle, which allows for cell targeting with known delivery and release kinetics without having to repeat the same work for modified

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vectors. This type of targeting, known as promoter or expression-targeting, works on the principle that many cell types could take up the delivered genes, but only the cells of interest will transcribe them because the transcriptional machinery that binds to the utilized promoters is present only in the target cells. This type of targeting has been used to target megakaryocytes via the promoter for integrin αIIβ (for a possible treatment for platelet disorders) [87]. Targeting liver cells has been achieved by using the promoter for liver-type pyruvate kinase and SV40 enhancer [88]. Expression-targeting has also been used to target an entire class of cells (Cox-2 overexpressing cells), which could be an important technique in the treatment of carcinomas or inflammation [89]. The use of two specific binding elements, a promoter/enhancer combination, has been employed to deliver the suicide gene for thymidine kinase to prostate tumors [90]. Expression-targeted gene delivery could prove to be a valuable tool in clinical gene therapy, alone or in conjunction with ligand-targeted systems.

14.6 Applications

14.6.1 In Vitro

In 1977, a chemically created somatostatin gene was fused to an Escherichia coli β-galactosidase gene and introduced into a bacterium resulting in expression of the gene [91]. The alteration of both cells and cellular functions has expanded greatly since then to produce many new experimental applications. In vitro modeling is valuable in learning more about living systems without the involvement of animals, while potentially leading to in vivo applications. For example, liposomal delivery of the gene for adenomatous polyposis coli (APC), delivered to produce a potential anticancer agent, has produced excellent results in diminishing a human duodenal cancer in the presence of bile in vitro [92].

The proliferation of cells in vitro not only serves as a model for in vivo studies but also serves as a valuable component of most ex vivo applications. However, it is often the case that cell behavior differs between in vitro and in vivo environments. Hematopoietic stem cells show this characteristic, displaying substantial proliferation in vivo but dividing at a more conservative rate in vitro. Building on the retroviral overexpression of the homeobox B4 gene, a 40-fold increase in in vitro growth has been observed in mouse bone marrow cells [93]. However, the difference between cell behavior inside and outside the living organism along with the involvement of a wider array of cell types and cellular proteins continue to be significant barriers to scientific research, and are reasons why in vitro investigations are not enough to produce treatments ready for the clinic. In vitro research is still important because it allows greater control over experimental conditions for molecular studies and reduces the need for research animals.

In vitro gene therapy also has applications in the creation of regulation vehicles for the complexes needed in vivo. The delivery of l-dopa and dopamine to the nervous system by genetically altered cells can be regulated by embedding the cells in hydrogels, created and currently being tested in vitro, and implanting these systems into the affected area [94]. Another regulation device has been created from freeze-dried PEI/DNA complexes along with sucrose and PLGA to create a porous medium filled with encapsulated transfection complexes [95]. In vitro, the use of this PLGA sponge has caused expression of a reporter gene in adherent fibroblasts for 15 days.

Besides delivery regulation mechanisms there are in vitro transfection applications that treat cells as microfactories to create pharmaceutical products. An example of using gene delivery to utilize cells as manufacturing plants is the injection of engineered plasmids into silkworm eggs for the production of type III procollagen [96]. This application is a useful way of producing protein-based polymers with high sequence specificity and low polydispersity. This technology could be used to create scaffolds for cell-seeding in tissue engineering applications.

14.6.2 Ex Vivo

Ex vivo gene therapy involves the methods of in vitro cell therapy coupled with the reintroduction of the altered cells into an organism. Likely candidates for this approach are bone marrow progenitor cells of

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the adult (mesenchymal and hematopoetic) embryonic and adult stem cells. Delivery of the transfecting (or transducing) gene can be accomplished via any of the methods mentioned previously in this chapter. There has been much success with this application of gene therapy, which has progressed to the clinical trial stage. Transplantation of nerve growth factor (NGF)-producing grafts of neural progenitor cells into the septum and nucleus basalis of the rat cerebrum has been shown to diminish the atrophy of the brain associated with aging [97]. Several more ex vivo investigations are outlined in Section 14.7 of this chapter.

14.6.3 In Vivo

To transport gene delivery complexes into living organisms, there exist several strategies that do not entail surgery. The first method that might come to mind is injection via syringe. Injections can be very simple, entailing introduction of complexes into a vein [98], into the peritoneum [99], or directly into a tissue such as muscle [98] or tumor [100]. Even the blood–brain barrier can be circumvented via direct injection [101] or injection into the jugular vein using the brain-specific promoter GFAP [102]. Additional approaches for gene delivery to the central nervous system include injection into the cranial nerves for retrograde transport of PEI/DNA complexes into the brainstem [103], and intraveneous administration of lipoplexes conjugated with transferrin to effectively cross the blood–brain barrier via its transferrin receptors [104]. Permanent transfection via nonrandom integration has even been achieved via high-pressure tail vein injection, in which plasmids coding for bacteriophage φC31 integrase were co-injected with plasmids containing attB and a gene for human factor IX to yield site-specific genomic integration of the factor IX gene in rat hepatocytes at native pseudo attP sites [105]. This concept could be applied to achieve permanent transfection in many nonviral applications, which lend themselves to IV administration.

Another method of complex administration in vivo is through inhalation. Inhalation of aerosols is an intuitive choice for gene delivery to the respiratory system. In mice, aerosol administration of a liposome– DNA complex has yielded transfection results that persisted for 21 days [106]. More recently, aerosol delivery of adeno-associated viral vectors through the nasal cavity produced positive transfection that was apparent in the bloodstream in addition to that in the lungs [107]. Aerosol delivery is a logical method of delivering transfection complexes to the lungs, while intravenous delivery might be better suited for organs such as the spleen or liver, as demonstrated by results from a recent study that compared the two delivery methods for PEI/DNA transfection complexes [108].

Mucosal administration of gene delivery complexes by oral or rectal routes is another option for in vivo gene therapy. Mice that were fed chitosan-coated plasmid particles showed expression of the delivered gene in the intestinal epithelium [109]. Another delivery method involves particle-mediated gene delivery directly into the oral mucosa [110]. In the cited study, several marker and cancer-targeting genes were delivered and shown to be expressed in the oral cavities of canines. Transrectal complex administration (enema) has also been used to deliver genes to canines, where a recombinant adenovirus vector carrying a marker gene (β-galactosidase) was used to demonstrate successful transduction in the colon [111].

14.7 Clinical Applications

To date, over 900 reported gene therapy clinical trials are underway worldwide; approximately 2% of these are phase III investigations [112]. Well over half of the current trials utilize viruses as the gene delivery vehicle. Physical and chemical (especially liposomal) delivery methods are also represented. Over half of the current trials are cancer investigations [112]. Following is an overview of some of the more visible trials.

Severe combined immunodeficiency (SCID) was the first clinical application of gene therapy to go into human trials, commencing in 1990. The treatment involved removing bone marrow stem cells, altering them by exposure to healthy T-cells in culture, and then reintroducing the differentiated marrowderived cells to the host organism [113]. Another malady involving immunocompromise is the HIV infection, a retrovirally caused disease that affects T-helper cells and can eventually lead to acquired immunodeficiency syndrome (AIDS). Phase I, II, and III clinical trials are currently underway for several applications of HIV-related gene therapy. Ex vivo manipulation of T lymphocytes with ribozymes, followed

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by reintroduction of the cells into the host, shows promise in that the T-cells have a greater survival time and are clinically safe for the subject [114]. A separate study conducted on twins was completed in 2002, and showed the safety and efficacy of ex vivo lymphocyte manipulation for expression of an anti-HIV gene (revTD) [115].

Clinical investigations for tissueor organ-related maladies are also underway. Ischemic heart disease is the result of poor circulatory perfusion of cardiac muscle, commonly from coronary artery blockage. The delivery of vascular endothelial growth factor (VEGF), first described as a tumor-produced vascular permeability factor, has been shown to stimulate angiogenesis in several tissues including cardiac muscle [116]. Phase I studies have indicated that direct injection of naked [117] or adenovirally delivered [118] plasmids encoding VEGF into cardiac tissue yields both safe and effective transfection/transduction.

Cystic fibrosis is another tissue/organ malady. The disease is the result of a mutation in a gene encoding a cellular cAMP-mediated chloride channel, known as the cystic fibrosis transmembrane conductance regulator (CFTR). With inadequate chloride transport capabilities, affected cells will develop an osmotic gradient that is relieved by internalization of water from the extracellular environment. In cells that are bathed in mucus, such as lung airway epithelia, the result is a loss of water from the coating mucus, raising mucus viscosity and lowering the body’s ability to transport it via ciliary movement. Bacterial colonization is often the result of such stasis. Clinical trials for the treatment of cystic fibrosis have been conducted using different target administrations. A trial of liposome-mediated transfection for cystic fibrosis patients showed the efficacy and safety of delivery of the CFTR gene to nasal epithelium cells [119]. Aerosolized adenoviral particles carrying CFTR cDNA have also been used clinically for transduction in the lungs [120].

Neurologic disorders have also been the target of clinical gene therapy trials. A phase I study of Alzheimer’s disease involved the removal of primary fibroblasts from subjects, effectively transducing the harvested cells with nerve growth factor and reintroducing the cells into the brain [121] (also reviewed in Reference 122). The use of nerve growth factor to target the cholinergic neurons was employed with the aim of preventing neural degradation and elevating levels of acetylcholine transferase.

Gene therapy applications for cancer treatment are very well represented in the group of clinical trial investigations. Phase I and II trials directed at malignant gliomas are using a modified herpes virus to target glioma cells in order to cause an immune response to diminish the number of cancerous cells [123]. The referenced study is currently focused on drug dose amount in subjects with recurrent glioblastoma. A phase II study of ovarian cancer commenced in the year 2000 to determine the safety and efficacy of genetically altered herpes simplex thymidine kinase-producing cells (HSV-TK) delivered into canceraffected ovaries [124]. The amount of cancer research that is underway is too large to allow a proper presentation of the area in sufficient detail here.

14.8 Summary

Gene therapy is a very diverse and rapidly growing field. Researchers in many areas, including biology, chemistry, physics, engineering, and medicine can find new applications for their creations and discoveries in gene therapeutics. Since molecular biology and genetic recombination laid the foundation for controlled genetic alteration, the field of gene therapy has expanded dramatically. The ability to alter cellular function through the introduction of exogenous genetic material has drawn considerable hope that this technology can be used to discover new disease treatments as well as explicate some of the mysteries of life at the level of basic science. As laboratory and clinical trials proceed and knowledge of the area becomes more rational and objective within the general public, gene therapy should prove to deliver beneficial and lasting advances from the world of biomolecular science.

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Gene Therapy

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