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Retrograde intrabiliary infusion (RII) has recently been characterized as a safe and effective administration route for liver-targeted gene delivery. Efficient transgene expression in the liver has been achieved by infusing a variety of gene vectors including adenovirus, retrovirus, lipoplexes, polyplexes, and naked DNA through the common bile duct. Here, we describe the RII technique and key infusion parameters for delivering plasmid DNA and DNA nanoparticles to the rat liver. After RII of plasmid DNA, the level of transgene expression in rat liver is comparable to that achieved by hydrodynamic injection of plasmid DNA, which is considered to be "gold standard" for liver-targeted gene delivery. RII has also been shown to significantly enhance the gene delivery efficiency by polymer/DNA nanoparticles in comparison with intravenous and intraportal infusions. This method induces minimal level of cytotoxicity and damage to the liver and bile duct. Due to these advantages, RII has the potential to be used for delivering various gene vectors in clinical setting through the endoscopic retrograde cholangiopancreatography procedure.

Electroporation has been used successfully to introduce macromolecules such as DNA into the chick embryo for at least 15 years. Purified plasmid DNA is microinjected into embryo and then a series of low voltage electrical pulses are applied to the embryo which allows naked DNA to enter cells. Following entrance into the cytoplasm, the DNA is transported to the nucleus where it is transiently expressed. This powerful technique is useful for studies involving overexpression, misexpression, and knockdown of genes of interest at a variety of developmental timepoints.

Eukaryotic genomes are wrapped in nucleosomes. These nucleosomes could be a barrier or could help facilitate the binding of transcription or replication factors. To understand what biological role nucleosomes play, an accurate and reliable method for measuring not only the position of a nucleosome but the fraction of the population that is bound by a nucleosome is needed. Here is described a method for determining nucleosome occupancy that takes advantage of the difference in the rate of digestion of DNA by micrococcal nuclease when naked DNA is compared to the same DNA bound by a nucleosome. Curve fitting to a function that describes the amount of DNA remaining following a series of digestions over a broad range of micrococcal nuclease allows the calculation of nucleosome occupancy anywhere in the genome under many different conditions in vivo.

Current transgenic technologies for gene transfer into the germline of mammals cause a random integration of exogenous naked DNA into the host genome that can generate undesirable position effects as well as insertional mutations. The vectors used to generate transgenic animals are limited by the amount of foreign DNA they can carry. Mammalian artificial chromosomes have large DNA-carrying capacity and ability to replicate in parallel with, but without integration into, the host genome. Hence they are attractive vectors for transgenesis, cellular protein production, and gene therapy applications as well. ES cells mediated chromosome transfer by conventional blastocyst injection has a limitation in unpredictable germline transmission. The demonstrated protocol of laser-assisted microinjection of artificial chromosome containing ES cells into eight-cell mouse embryos protocol described here can solve the problem for faster production of germline transchromosomic mice.

Bifunctional DNA damaging agents continue to be the mainstay in various chemotherapeutic regimens used in the clinic. DNA interstrand crosslinks are considered to be the critical cytotoxic lesions for the biological activity of such agents. Gel-based electrophoretic assays can efficiently separate denatured single-stranded DNA from double-stranded, covalently-linked DNA resulting from the presence of an interstrand crosslink. The methods described here offer a simple way for the assessment of crosslinking efficiencies of bifunctional agents in both long fragments of DNA (e.g. 1-5 kb) and short oligonucleotide DNA duplexes. As the repair of interstrand crosslinks is a key determinant of cellular and clinical chemosensitivity, these methods can be useful for the characterization and isolation of site-directed adducted substrates for use in subsequent biochemical analysis of cellular recognition and DNA repair processes.

Genetic modification of peripheral blood T lymphocytes (PBL) or hematopoietic stem cells (HSC) has been shown to be promising in the treatment of cancer (Nat Rev Cancer 3:35-45, 2003), transplant complications (Curr Opin Hematol 5:478-482, 1998), viral infections (Science 285:546-551, 1999), and immunodeficiencies (Nat Rev Immunol 2:615-621, 2002). There are also significant implications for the study of T cell biology (J Exp Med 191:2031-2037, 2000). Currently, there are three types of vectors that are commonly used for introducing genes into human primary T cells: oncoretroviral vectors, lentiviral vectors, and naked DNA. Oncoretroviral vectors transduce and integrate only in dividing cells. However, it has been shown that extended ex vivo culture, required by oncoretroviral-mediated gene transfer, may alter the biologic properties of T cells (Nat Med 4:775-780, 1998; Int Immunol 9:1073- 1083, 1997; Hum Gene Ther 11:1151-1164, 2001; Blood 15:1165-1173, 2002; Proc Natl Acad Sci U S A, 1994). HIV-1-derived lentiviral vectors have been shown to transduce a variety of slowly dividing or nondividing cells, including unstimulated T lymphocytes (Blood 96:1309-1316, 2000; Gene Ther 7:596-604, 2000; Blood 101:2167-2174, 2002; Hum Gene Ther 14:1089-1105, 2003). However, achieving effective gene transfer and expression using lentivirus vectors can be complex, and there is at least a perceived risk associated with clinical application of a vector based on a human pathogen (i.e., HIV-1). Recently it has been found that oncoretroviral and lentiviral vectors show a preference for integration into regulatory sequences and active genes, respectively (Cell 110:521-529, 2002; Science 300:1749-1751, 2003). Additionally, insertional mutagenesis has become a serious concern, after several patients treated with an oncoretroviral vector for X-linked SCID developed a leukemia-like syndrome associated with activation of the LMO2 oncogene (Science 302:415-419, 2003). Naked DNA-based genetic engineering of human T lymphocytes also requires T cells to be activated prior to gene transfer (Mol Ther 1:49-55, 2000; Blood 101:1637-1644, 2003; Blood 107:2643-2652, 2006). In addition, random integration by electroporation is of low efficiency. We have recently reported that the Sleeping Beauty transposon system can efficiently mediate stable transgene expression in human primary T cells without prior T cell activation (Blood 107:483-491, 2006). This chapter describes methodology for the introduction of SB transposons into human T cell cultures with subsequent integration and stable long-term expression at noticeably high efficiency for a nonviral gene transfer system.

The main challenges for application of gene therapy to patients are poor selectivity in vector targeting, insufficient gene transfer, and great difficulties in systemic treatment in association with safety concerns for particular vector systems. For success in gene therapy, safe, applicable, and efficient transfer technologies are required. Because of the complex nature of targeted vector delivery to the tumor, our strategy for gene therapy is focused on the development of local nonviral gene transfer. This approach of local interference with tumor growth and progression could contribute to better control of the disease. Transfer of naked DNA is an important alternative to liposomal or viral systems. Different physical procedures are used for improved delivery of naked DNA into the target cells or tissues in vitro and in vivo. Among the various nonviral gene delivery technologies, jet-injection is gaining increased attractiveness, because this technique allows gene transfer into different tissues with deep penetration of naked DNA by circumventing the disadvantages associated with, e.g., viral vectors. The jet-injection technology is based on jets of high velocity for penetration of the skin and underlaying tissues, associated with efficient transfection of the affected area. The jet-injection technology has been successfully applied for in vivo gene transfer in different tumor models. More importantly, the efficacy and safety of jet-injection gene transfer have recently been investigated in a phase I clinical trial.

The importance of DNA formulation in safe and efficient electrogene transfer is increasingly recognized as electroporation technology enters into clinical development. A phenomenal increase in naked DNA delivery by electroporation offers new opportunities for nonviral gene therapies previously considered difficult because of insufficient delivery. However, significant tissue damage related to harsh electroporation conditions raises serious safety concerns with the use of electroporation in healthy tissues, which limits its current applications to only nonhealthy tissues such as tumors. DNA formulations designed to minimize tissue damage or enhance expression at weaker electric pulses have been examined to address these concerns. These include formulations fortified with the addition of transfection reagent(s), membrane-permeating agents, tissue matrix modifiers, targeted ligands, or agents modifying electrical conductivity or membrane stability to enhance delivery efficiency or reduce tissue damage. These advancements in DNA formulation could prove to be useful in improving the safety of electroporation protocols for human applications.

The concept of using gene therapy for cancer treatment was initially met with enthusiasm. The possibility of replacing or altering damaged genes, the introduction of suicide genes into cancer cells, and the alteration of cell function as a consequence of exogenous gene expression were advocated. Therapeutic genes can be transferred to patients through a variety of vehicles. These include retroviruses, herpes viruses, adenoviruses, adeno-associated viruses, lentiviruses, baculoviruses, liposomes, bacterial hosts, naked DNA, DNA precipitates, and protein-DNA conjugates (Table 1) (1,2). However, the practical application of gene therapy to treat cancer has been somewhat disappointing so far. Major obstacles remain, including the inability to target appropriate tissues and deliver therapeutic genes to a sufficient number of target cells, the inability to monitor the level of expression of the therapeutic gene, the loss of therapeutic gene expression over time, and the inability to correlate the level and duration of gene expression with therapeutic outcome.

Gene therapy requires efficient vectors for delivering therapeutic genes. Advances in developments of nonviral vectors have been established for improving the efficiency of gene delivery. This chapter describes different nonviral methods as well as their applications. Some new directions in developing nonviral vectors are also discussed.

Many of the problems related with mammalian gene expression, such as low translation efficiency and mRNA halflife, can be solved by means of a rational gene design, based on modern bioinformatics, followed by the de novo generation of a synthetic gene. Moreover, high expression rates and prolonged mRNA stability are not only crucial for heterologous mammalian expression, but, in particular, are important for the generation of effective DNA vaccines. In this chapter we show that an optimized synthetic gene encoding the HIV-1 Pr55gag outperforms wild-type gene driven expression by several orders of magnitude. RNA analysis revealed that this positive effect was mostly due to increased mRNA stability of the optimized transcripts. Moreover, mice vaccinated with the optimized gag gene elicited a much stronger immune response against Pr55gag than the control groups immunized with the respective wild-type gene.

The ability to deliver genes to cells and tissues in vivo offers the potential to develop potent vaccines and treat many hereditary diseases that are currently considered incurable, e.g., cancer, cystic fibrosis (CF), severe combined immunodeficiency (SCID), and acquired immune deficiency syndrome (AIDS) (1-6). Considering the tremendous promise of DNA-delivery technology, in addition to the extensive genetic information now available from the Human Genome Project, it is not surprising that gene therapy is being touted as the next revolution in medicine. Although a strict definition of “gene therapy” would be limited to therapeutic approaches that aim to use polynucleotides as a template for the in vivo production of proteins, the term is often used to refer to a wide variety of strategies that employ nucleotide-based molecules (e.g., vaccines, antisense, ribozymes, siRNA). To date, 70 clinical protocols have been approved for the delivery of naked DNA, comprising approx 11% of the total number of gene-therapy clinical protocols (http://www.wiley.co.uk/genetherapy/clinical). Slightly more studies (~12%) have employed nonviral, lipid-based vectors to facilitate DNA delivery. In comparison, the large majority of clinical gene therapy trials (>70%) utilizes viruses to deliver therapeutic genes because viruses are more efficient than contemporary synthetic gene-delivery systems. The higher efficiency of viruses should not be surprising if we recognize that these organisms have been evolving their gene-delivery machinery for billions of years. In contrast, the development of nonviral systems for therapeutic gene delivery can be traced back a mere 15 years (7). Although more efficient nonviral gene-delivery systems continue to be developed, synthetic systems have yet to replicate the efficiency of viruses. One significant drawback of viral delivery is the immunogenicity of viruses, which causes significant inflammation in vivo (8), and eliminates the potential for multiple dosing (anyone who has ever had a cold is familiar with the fever and inflammation associated with an immune response to viruses). In fact, the adverse reactions associated with viral delivery have been implicated as the cause of death in clinical trials (9,10). Also, a clinical trial involving liver infusion of an adenoassociated virus (AAV) for the treatment of hemophilia B was halted because of the presence of the viral vector in patient semen, and the concern that the genetic alteration could be passed to offspring (11). More recently, two patients treated with ex vivo genetherapy for the treatment of SCID have developed leukemia owing to insertional mutagenesis caused by the retroviral vector used in the study (12,13). Considering the potential safety risk involved in employing viruses as a therapeutic moiety, there is renewed interest in developing safe, efficient, nonviral gene-delivery systems.

The aim of gene therapy is to treat inherited or acquired genetic deficiencies (e.g., cystic fibrosis) or viral diseases (e.g., hepatitis B, HIV) by introduction of DNA encoding a therapeutic protein or a specific virus antigen, respectively, into the nucleus of the target cell. Because naked DNA will barely pass cellular membranes, a carrier system is required for transfection (1-4). Cationic polymers, which condense DNA by ionic interaction, form a promising class of nonviral transfection agents. Well-known examples of these polymers are DEAE dextran, poly(L-lysine), poly(ethylenimine), and poly(2- [dimethylamino]ethyl methacrylate) (pDMAEMA) (5-10). In order to achieve transfection, aplasmid must be delivered into the nucleus, which requires cellular uptake of polymerDNA complexes, generally referred to as "polyplexes" (11), which most likely occurs via endocytosis, followed by endosomal escape and transport to the nucleus. The polyplex must dissociate, either in the cytosol or in the nucleus, which may be a critical step in the transfection process.

Intramuscular injection of naked plasmid DNA is known (1-3) to elicit humoral and cell-mediated immune responses against the encoded antigen. It is thought (2,3) that immunity follows DNA uptake by muscle cells, leading to the expression and extracellular release of the antigen which is then taken up by antigen presenting cells (APC). In addition, it is feasible that some of the injected DNA is taken up directly by APC. Disadvantages (1-3) of naked DNA vaccination include: uptake of DNA by only a minor fraction of muscle cells, exposure of DNA to deoxyribonuclease in the interstitial fluid thus necessitating the use of relatively large quantities of DNA, and, in some cases, injection into regenerating muscle in order to enhance immunity. We have recently proposed (1,4) that DNA immunization via liposomes (phospholipid vesicles) could circumvent the need of muscle involvement and instead facilitate (5) uptake of DNA by APC infiltrating the site of injection or in the lymphatics, at the same time protecting DNA from nuclease attack (6). Moreover, transfection of APC with liposomal DNA could be promoted by the judicial choice of vesicle surface charge, size and lipid composition, or by the co-entrapment, together with DNA, of plasmids expressing appropriate cytokines (e.g., interleukin 2), or immunostimulatory sequences.

The ability of naked DNA to induce immune responses against encoded antigen has been clearly demonstrated for infectious diseases (1). In many cases, the induced immunity is able to protect against infection, and can approach the efficacy of exogenous antigen (2).

The potential applications of using plasmid DNA for immunization and other gene therapy approaches have been discussed in an increasing number of publications in the past few years. Injection of mouse muscle with naked DNA (plasmid DNA in saline) resulted in significant episomal expression from a number of encoded reporter genes such as firefly luciferase, chloramphenicol acetyltransferase, and β-galactosidase (1). DNA vaccination has been shown to induce neutralizing antibodies against the gene product, helper T-cell responses of the Th1 phenotype, and cytotoxic T lymphocyte responses (2). Vaccination with plasmid DNA stimulates immunogenicity and provides protection against various infectious diseases in pre-clinical animal models. Examples include hepatitis B in chimpanzees (3), bovine herpes virus in mice (4), influenza A virus in ferrets (5), human immunodeficiency virus in rhesus monkeys (6), Mycobacterium tuberculosis in mice (7,8), malaria in mice (9,10), and genital herpes simplex virus in guinea pigs (11). Recently, DNA vaccines for the protection against influenza (Merck Research Laboratories, Rahway, NJ), malaria (Vical Inc., San Diego, CA), and HIV (Apollon Inc., Philadelphia, PA), have entered phase I human clinical trials. Rapid progress has been made in the areas of adjuvants for DNA vaccines (12), route of immunization (13), industrial scale fermentation and pharmaceutical grade purification (14). One major interest in the commercial development of DNA vaccines, especially for developing countries, is to increase DNA vaccine stability at room temperature, to reduce the requirement for costly cold storage, and to extend product shelf-life.

Plasmid DNA immunization has emerged as a promising vaccine strategy against infectious agents, as well as a potential intervention for the treatment of cancer, autoimmunity, and allergy (1). Until recently, however, the cellular events by which injected plasmid DNA elicits potent antibody and cytotoxic T-lymphocyte (CTL) responses were largely unknown. Upon intramuscular (i.m.) injection of naked DNA, predominant expression of transfected DNA occurs in the myofibers (2), but no direct transfection of antigen presenting cells (APC) has been reported. There are essentially three different mechanisms by which CTLs can be primed by the injected DNA (3). The first possibility is that the transfected muscle cells directly activate CTLs by presenting the antigenic peptide on their MHC class I molecules. Alternatively, the priming of CTLs may be mediated by professional APC taking up antigen released from muscle cells. Finally, CTL priming may involve direct transfection of APC occurring, albeit at low level, and that the CTLs are activated by the transfected APC.

Inoculation of plasmid DNA vectors encoding immunogenic proteins induce humoral as well as cell-mediated immune responses. Protection against challenge with pathogens has provided protective immunity in several instances in animal models. (1,2). DNA vaccines allow the simultaneous expression of antigens and immune-stimulatory cytokines via an internal ribosomal entry site (IRES). Here we describe the construction of a DNA vaccine against malignant melanomas using: (i) the tumor-associated antigen gp100 (or pmel17), known to be over-expressed in many malignant melanomas (3,4), and (ii) the granulocyte macrophage stimulating factor (GM-CSF) which has been shown to have a stimulatory effect on humoral and cellular immune responses (5).

Direct injection of naked DNA into tissues as a gene-delivery method has been extensively studied for genetic immunization and gene therapy (1). When naked plasmid DNA was used as a negative control while cationic liposomes were studied, it was surprisingly found that direct injection of naked DNA into skeletal muscle resulted in higher gene expression than injection of cationic liposomes (2). Naked DNA was reported as more efficient than retroviral and adenoviral vectors following direct injection into adult mouse skeletal muscle (3). Subsequently, cardiac muscles, thyroid, joint, liver, lung, brain, kidney, as well as solid tumors were found capable of taking up and expressing naked DNA (4-10). In some tumors, gene expression following intratumoral injection of naked DNA was higher than that of DNA associated with cationic liposomes Fig. 1) (7). This is probably because of the faster dessimination rate of naked plasmid DNA in the tumor than that of DNA complexed with cationic liposomes (111). Fig. 1. CAT reporter gene expression in murine BL6 melanoma. pUCCMVCAT plasmid DNA was injected into the tumor. Mice were sacrificed 2 d later and tumor protein extracts were assayed for CAT activity. Lanes 16 and 17: 0.005 and 0.01 units of purified CAT standard enzyme, respectively. Lane 18: extracts from tumors injected with 5% glucose only [with the permission from (7)].

Drug-DNA Interaction Protocols, Second Edition Editor: Keith R. Fox, PhD Contents Preface Contributors 1. Quantitative Analysis of Small Molecule- Nucleic Acid Interactions with a Biosensor Surface and Surface Plasmon Resonance Detection Yang Liu and W. David Wilson 2. Thermal Melting Studies of Ligand DNA Interactions Aurore Guedin, Laurent Lacroix, and Jean-Louis Mergny 3. Circular and Linear Dichroism of Drug-DNA Systems Alison Rodger 4. Drug Binding to DNA*RNA Hybrid Structures Richard T. Wheelhouse and Jonathan B. Chaires 5. Quantification of Binding Data Using Capillary Electrophoresis Fitsumbirhan Araya, Graham G. Skellern, and Roger D. Waigh 6. Determination of Equilibrium Association Constants of Ligand-DNA Complexes by Electrospray Mass Spectrometry Valerie Gabelica 7. Detection of Adriamycin-DNA Adducts by Accelerator Mass Spectrometry Kate Coldwell, Suzanne M. Cutts, Ted J. Ognibene, Paul T. Henderson, and Don R. Phillips 8. Molecular Modelling Methods to Quantitate Drug-DNA Interactions Hao Wang and Charles A. Laughton 9. Application of Anomalous Diffraction Methods to the Study of DNA and DNA-Complexes Derrick Watkins, Tinoush Moulaei, Seiji Komeda, and Loren Dean Williams 10. DNase I Footprinting. Antonia S. Cardew and Keith R. Fox 11. Methods to Characterize the Effect of DNA-Modifying Compounds on Nucleosomal DNA Vidya Subramanian, Robert M. Williams, Dale L. Boger, and Karolin Luger 12. REPSA: Combinatorial Approach for Identifying Preferred Drug-DNA Binding Sequences Michael W. Van Dyke 13. In Vitro Transcription Assay for Resolution of Drug-DNA Interactions at Defined DNA Sequences Benny J. Evison, Don R. Phillips, and Suzanne M.Cutts 14. In Vitro Footprinting of Promoter Regions within Supercoiled Plasmid DNA Daekyu Sun 15. Topoisomerase I-Mediated DNA Relaxation as a Tool to Study Intercalation of Small Molecules into Supercoiled DNA Paul Peixoto, Christian Bailly, and Marie-Helene David-Cordonnier 16. A High-Throughput Assay for DNA Topoisomerases and Other Enzymes, Based on DNA Triplex Formation. Matthew R. Burrell, Nicolas P. Burton, and Anthony Maxwell 17. Measurement of DNA Interstrand Crosslinking in Individual Cells Using the Single Cell Gel Electrophoresis (Comet) Assay Victoria J. Spanswick, Janet M. Hartley, and John A. Hartley 18. Measurement of DNA Interstrand Crosslinking in Naked DNA Using Gel-Based Methods Konstantinos Kiakos, Janet M. Hartley, and John A. Hartley 19. An Evaluation Cascade for G-Quadruplex Telomere Targeting Agents in Human Cancer Cells Mekala Gunaratnam and Stephen Neidle