*Corresponding Author:
P. Saraswat
Mahatma Gandhi Medical College and Hospital, RIICO Institutional Area, Sitapura, Jaipur-302 022, India
E-mail: saraswatmgmch@rediffmail.com
Date of Submission 08 February 2008
Date of Revision 25 August 2009
Date of Acceptance 12 September 2009
Indian J Pharm Sci, 2009, 71 (5): 488-498  


Human gene therapy is the introduction of new genetic material into the cells of an individual with the intention of producing a therapeutic benefit for the patient. Deoxyribonucleic acid and ribonucleic acid are used in gene therapy. Over time and with proper oversight, human gene therapy might become an effective weapon in modern medicine's arsenal to help fight diseases such as cancer, acquired immunodeficiency syndrome, diabetes, high blood pressure, coronary heart disease, peripheral vascular disease, neurodegenerative diseases, cystic fibrosis, hemophilia and other genetic disorders. Gene therapy trials in humans are of two types, somatic and germ line gene therapy. There are many ethical, social, and commercial issues raised by the prospects of treating patients whose consent is impossible to obtain. This review summarizes deoxyribonucleic acid-based therapeutics and gene transfer technologies for the diseases that are known to be genetic in origin. Deoxyribonucleic acid-based therapeutics includes plasmids, oligonucleotides for antisense and antigene applications, deoxyribonucleic acid aptamers and deoxyribonucleic acidzymes. This review also includes current status of gene therapy and recent developments in gene therapy research.


Gene therapy, nucleic acid therapeutics, antisense, gene transfer technology, gene therapy trials, DNA delivery systems, viral vectors, nonviral vectors, liposomes

Human gene therapy is defined as the introduction of new genetic material into the cells of an individual with the intention of producing a therapeutic benefit [1-3]. A number of human diseases are known to be genetic in origin (Huntington’s chorea and cystic fibrosis to name a few) and virtually all diseases, except for trauma, have a hereditary component [4]. Thus, the opportunity to treat such disorders by replacing the defective gene(s) with a normal healthy gene (gene therapy) offers a novel therapeutic approach for patients who suffer from such diseases. Now, gene therapy routinely is evoked to encompass the use of deoxyribonucleic acid (DNA) as a drug to alleviate the symptoms of a disease, even if the therapeutic genes are not strictly ‘corrective’ (in the sense of restoring a function known to be mutated in the affected cells). In its broadest terms, gene therapy represents an opportunity for the treatment of genetic disorders in adults and children by genetic modification of human body cells [5]. All of the gene therapy trials currently approved for use in human patients target somatic cells that will live only as long as the patient. This ensures that the genetic treatment will affect only one generation and will not alter the genetic makeup of any offspring of the patient, since there is no spread of the therapeutic gene(s) to the gametes. This is known as the somatic gene therapy, and its purpose is to alleviate disease in the treated individual alone. More than 300 clinical trials involving gene transfer in patients have been approved and the first nucleic acid drug, an antisense oligonucleotide, fomivirsen (marketed as Vitravene) has been approved by the United States Food and Drug Administration (USFDA) for the treatment of cytomegalovirus retinitis in immunocompromised patients [6]. In contrast, it is also possible to target directly the gametes (sperm and ova) in order to modify the genetic profile, not of the current, but of the subsequent generation of unborn ‘patients’. Gene transfer at an early stage of embryonic development also might have similar effects by achieving gene transfer to both somatic and germ line cells. This is known as the germ line gene therapy.

Apart from the inability to predict the long-term effects of altering the germ line by delivery of exogenous genetic material at the scientific level, there are many ethical, social, and commercial issues surround the technique. The social implications of such technology include the possibility that patients might suffer from depression as a result of being ‘genetically altered’ or might not be accepted by society in the way that they were before treatment. The commercial implications of such technology are that the insurance companies and other such institutions also would want to access the available information prior to them granting life insurance policies. So, it is obvious that a person shown to have a predisposition to a genetic disease could be severely penalized because of a mutation in their DNA, even though they might never develop the disease. The biggest setback for gene therapy occurred in 1999 when Jesse Gelsinger, an 18 year-old high-school graduate from Arizona, died as a result of a gene therapy experiment. Gelsinger developed a fever and blood clots throughout his body within hours of treatment to correct partial ornithine transcarbamylase (OTC) deficiency, a rare metabolic disease that can cause a dangerous build-up of ammonia in the body and died four days later [7]. Despite this attempt to preserve public confidence in gene therapy, the Washington post documented six unreported deaths on 3rd November 1999 that had occurred in trials conducted at the Cornell Medical Center, Manhattan and at the Tufts University, Boston [8].

Nucleic acids are one of the most important sources not only for the understanding of the fundamental basis of human life but also for the development of a novel group of therapeutics. One of the significant advantages of DNA-based drugs over currently available low molecular weight pharmaceuticals is their selective recognition of molecular targets and pathways, which imparts tremendous specificity of action. DNA-based therapeutics includes plasmids, oligonucleotides for antisense and antigene applications [9], DNA aptamers, and DNAzymes. In gene therapy, the gene transfer technologies are DNA delivery systems for nucleic acid based therapeutics.

Although most of the DNA and RNA based drugs are in early stages of clinical trials, these classes of compounds have emerged in recent years to yield extremely promising candidates for drug therapy for wide range of diseases, including cancer, AIDS, neurological disorders such as Parkinson’s disease and Alzheimer’s disease, and cardiovascular disorders [10,11]. Elucidation of the human genome has also provided a major impetus in identifying human genes implicated in diseases, which may eventually lead to the development of DNA and RNA based drugs for gene replacement or potential targets for gene ablation [12]. The Human Genome project will help determine genetic markers responsible for patient response to drug therapy, drug interactions, and potential side effects [13]. Developments in human genomics, transcriptomics, and proteomics will provide an additional impetus for the advancement of DNA based therapeutics by supplying novel targets for drug design, screening, and selection. In this review, we summarize DNA-based therapeutics, gene transfer technologies, current status of gene therapy and recent developments in gene therapy research.

Dna- Based Therapeutics


Plasmids are high molecular weight, double stranded DNA constructs containing transgenes, which encode specific proteins. On the molecular level, plasmid DNA molecules can be considered pro-drugs that upon cellular internalization employ the DNA transcription and translation apparatus in the cell to biosynthesize the therapeutic entity, the protein [14]. The mechanism of action of plasmid DNA requires that the plasmid molecules gain access into the nucleus after entering the cytoplasm. Nuclear access or lack thereof eventually controls the efficiency of gene expression. In addition to disease treatment, plasmids can be used as DNA vaccines for genetic immunization [15]. In the early stages of development, plasmid-based gene therapy was attempted to correct inheritable disorders resulting from a single gene defect. The first federally approved human gene therapy protocol was initiated in 1990 for the treatment of adenosine deaminase deficiency [16]. Since then, more than 500 gene therapy protocols have been approved or implemented [17]. In 2002, scientists reported the successful gene-therapybased cure for severe combined immunodeficiency (SCID) [18]. In 2003, the Chinese drug regulatory agency approved the first gene therapy product for head and neck squamous carcinoma under the trade name Gendicine [19]. Currently, diseases with complex etiologies such as cancer [20-21] and neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease [22] are being targeted. In addition, DNA vaccines for malaria, AIDS, and many other diseases are in development [23]. DNA vaccines have also been used to prevent allergic response [24].


Oligonucleotides are short single-stranded segments of DNA that upon cellular internalization can selectively inhibit the expression of a single protein. For antisense applications, oligonucleotides interact and form a duplex with the mRNA or the premRNA and inhibit their translation or processing, consequently inhibiting protein biosynthesis. For antigene applications, oligonucleotides must enter the cell nucleus; form a triplex with the double- stranded genomic DNA, and inhibits the translation as well as the transcription process of the protein. On the molecular level, numerous mechanisms have been proposed to explain the basis of oligonucleotide action [25-27]. For therapeutic purposes, oligonucleotides can be used to selectively block the expression of proteins that are implicated in diseases [28]. With successful antisense inhibition of proteins in animal models, the first antisense drug, fomivirsen sodium (Vitravene, Isis Pharmaceuticals, Carlsbad, CA) was approved for the treatment of cytomegalovirus retinitis in AIDS patients in 1998 [29]. Antisense oligonucleotides such as MG98 and ISIS 5132, designed to inhibit the biosynthesis of DNA methyltransferase and c-raf kinase, respectively, are in human clinical trials for cancer [30]. Synthetic antisense DNA oligonucleotides and oligonucleotide analogs [31], which inhibit the replication of several infectious agents such as hepatitis C virus [32], human cytomegalovirus [33], human immunodeficiency virus and papilloma virus [34-43], have also been designed.


DNA-Aptamers are double-stranded nucleic acid segments that can directly interact with proteins [10]. Aptamers interfere with the molecular functions of disease-implicated proteins or those that participate in the transcription or translation processes. Aptamers are preferred over antibodies in protein inhibition owing to their specificity, non-immunogenicity, and stability of pharmaceutical formulation [44]. DNA-aptamers that have demonstrated promise in intervention of pathogenic protein biosynthesis are HIV-1 integrase enzyme [45].


DNAzymes are analogs of ribozymes with greater biological stability [28]. The RNA backbone chemistry is replaced by the DNA motifs that confer improved biological stability. DNAzyme directed against the vascular endothelial growth factor receptor 2 was confirmed to be capable of tumor suppression by blocking angiogenesis upon intratumoral injections in mice [46].

Gene Transfer Technologies

Gene transfer technologies or DNA delivery methods can be classified into 3 general types; electrical techniques, mechanical transfection, and vector assisted delivery systems.

Mechanical and electrical techniques

Mechanical and electrical strategies of introducing naked DNA into cells include microinjection, particle bombardment, the use of pressure, and electroporation. Microinjection is highly efficient since one cell at a time is targeted for DNA transfer; however, this precision is achieved at the expense of time. Ballistic transfer of gold micro-particles can be achieved using particle bombardment equipment such as the gene gun. Electroporation uses high-voltage electrical current to facilitate DNA transfer. This technique results in high cell mortality and therefore is not suitable for clinical use [47-50].

Vector-assisted delivery systems

Vector-assisted DNA/gene delivery systems can be classified into 2 types based on their origin; biological viral DNA delivery systems and chemical nonviral delivery systems. In viral delivery systems, nonpathogenic attenuated viruses can be used as delivery systems for genes/DNA molecules; especially plasmids [51-53]. These viral DNA-delivery vectors include both RNA and DNA viruses. The viruses used as gene therapy vectors can be classified into 4 types; retroviruses [54], adenoviruses, adenoassociated viruses [55] and Herpes simplex viruses. Gene expression using viral vectors has been achieved with high transfection efficiencies in tissues such as kidney [56], heart muscle [57], eye [55], and ovary [58]. Viruses are currently used in more than 70% of human clinical gene therapy trials world-wide [59]. Gene therapy using viral systems has made considerable progress for the treatment of a wide range of diseases, such as muscular dystrophy [57], AIDS [60], and cancer [61]. The only approved gene therapy treatment (Gendicine) delivers the transgene using a recombinant adenoviral vector [20]. DNA delivery using viral vectors has been extensively reviewed [52,53,62]. The first-generation retroviral vectors were largely derived from oncoretroviruses, such as the Maloney murine leukemia virus (MMuLv), and were unable to transfer genes into non-dividing cells [63,64]. This limited the potential for their application as a delivery system in gene therapy. The utilization of the lentivirus family of retroviruses has overcome this shortcoming. Lentiviruses, which include Human immunodeficiency virus type1 (HIV-1), bovine immunodeficiency virus (BIV), feline immunodeficiency virus (FIV) and simian immunodeficiency virus (SIV), are able to transfer genes to non-dividing cells [64,65].

Retroviral vectors used in gene therapy are replication deficient, such that they are unable to replicate in the host cell and can infect only one cell [66,67]. This characteristic, although essential for the safety of viral vectors in gene therapy, imposes restrictions on the amounts of virus that can safely be administered [68,69]. Retroviral-mediated delivery of therapeutic DNA has been widely used in clinical gene therapy protocols, including the treatment of cancers, such as melanoma [70] and ovarian cancer [71], adenosine deaminase deficiency-severe combined immune deficiency (ADA-SCID) [72,73] and Goucher’s disease [74]. Retroviral vectors are capable of transfecting high populations (45-95%) of primary human endothelial and smooth muscle cells, a class of cells that are generally extremely difficult to transfer [75].

Adenoviruses have been used to deliver therapeutic DNA to patients suffering from metastatic breast, ovarian and melanoma cancers [76-78]. Indeed, the severe immune response of the host contributes to the limited survival of the adenoviral DNA in the targeted cells and results in a transient expression of the therapeutic gene since the adenoviral DNA is lost over time [79-83]. First- generation adenoviral vectors were able to accommodate the introduction of therapeutic genes over 7 Kb long (but rarely larger) into targeted cells [84]. However, the generation of gutless adenoviral vectors, which lack all viral genes, has facilitated adenoviral delivery of up to 30 Kb of a therapeutic DNA sequence [85-88] with decreased toxicity [89]. Adenoviral-mediated gene transfer in COS- 7 cells was significantly higher than that achieved by liposomal delivery systems [90].

The use of adeno-associated viral (AAV) vectors provides an alternative to adenoviral vectors for gene therapy and a means for long-term gene expression with a reduced risk of adverse reactions upon administration of the vector [91,92]. AAV viruses are linear, single stranded DNA parvoviruses that are not associated with any disease in humans [93]. In humans, the site of AAV viral DNA integration is on chromosome 19 [94,95]. In the engineering of AAV vectors, most of the AAV genome can be replaced with the therapeutic gene [96], which significantly reduces potential adverse responses of the host to viral infection. However, the size of the therapeutic gene is limited to approximately 5 Kb [97,98]. First generation adeno-associated viruses had a very small capacity of ~4.7 Kb for encapsulation of the plasmid DNA cargo. Recent reports demonstrate efficient production of second-generation AAV with higher encapsulating capabilities [99]. It has been demonstrated that adenoviruses in formulations may lose their potency after storage in commonly used pharmaceutical vials [100].

Herpes simplex virus (HSV) vector is a large and relatively complex enveloped, double-stranded DNA virus that has the capacity to encode large therapeutic genes and, like AAV, can remain latent in infected cells providing the potential for long term expression of the therapeutic gene [101]. Although, able to infect many cell types, HSV vectors currently are limited in their use by vector toxicity [102].

Non-viral delivery systems have the greatest advantage over viral delivery systems is the lack of immune response and ease of formulation and assembly. Commonly used non-viral vectors for delivery of DNA-based therapeutics can be classified into 3 major types; Naked DNA delivery systems, polymeric delivery systems, and liposomal delivery systems [30,103-105].

Naked DNA can be administered via two possible routes, either by ex vivo delivery or by in vivo delivery. The ex vivo method of naked DNA delivery has been used successfully for the introduction of DNA into endothelial and smooth muscle cells [106,107], its reliance on the culture of harvested cells renders it unsuitable for many cell types. In vivo delivery of naked DNA was first described in 1990 [108]. Efficiency of the delivery of naked DNA can be improved when administered in a pressure-mediated fashion [107,109]. Particle bombardment technology enables the localized delivery of DNA readily into skin or muscle [110]. Another technique for delivery of naked DNA directly into target cells is electroporation. The successful delivery of DNA by electroporation in vivo has been reported in tissues such as skin and muscle [111-114].

In polymeric delivery systems, cationic polymers are used in gene delivery because they can easily complex with the anionic DNA molecules [115]. The mechanism of action of these polycomplexes is based on the generation of a positively charged complex owing to electrostatic interaction of these cationic polymers with anionic DNA [48]. Commonly used polymers include polyethylenimine (PEI) [116], poly-L-lysine (PLL) [117], chitosans [118], and dendrimers [30]. Agents such as folates, transferin, antibodies, or sugars such as galactose and mannose can be incorporated for tissue targeting [30]. Synthetic polymers such as protective interactive non-condensing polymers (PINC), poly-L-lysine, cationic polymers and dendrimers offer an alternative to cationic lipids as a vehicle for DNA delivery into target cells [119-123]. Encapsulation of a DNA molecule or even a therapeutic viral vector within a biodegradable polymer has been demonstrated to permit the controlled release of the DNA in a targeted cell over a period of weeks or months [124,125]. The inclusion of proteins and peptides in the DNA complex that are recognized by receptors on targeted cells has led to an improvement in the efficiency of DNA uptake in several instances [126]. Some polymers have inherent potent pharmacological properties (such as hypercholesterolemia-induced by chitosans) that make them extremely unfavorable for human use [127].

Liposomes are one of the most versatile tools for the delivery of DNA therapeutics [28,103,104,128]. Liposome and drug/lipid complexes have been used for the delivery of the anticancer drugs doxorubicin and daunorubicin [129]. Liposomes can be used as DNA drug delivery systems either by entrapping the DNA-based therapeutics inside the aqueous core or complexing them to the phospholipids lamellae. Liposome can also be used for specialized gene delivery options that include long circulation half-life, sustained and targeted delivery [103].

Numerous studies have demonstrated the use of cationic liposomal formulations for the delivery of different plasmid constructs in a wide range of cells, both in vivo and in vitro [130]. The use of cationic lipids to transfer DNA into cells was first described as an in vitro method of DNA delivery [131]. Cationic liposomes have also been used in clinical trials to deliver therapeutic DNA [132-136]. Cationic liposomal formulations consist of mixtures of cationic and zwitterionic lipids [128,137,138]. Proprietary formulations of cationic lipids such as lipofectamine (Invitrogen, Carlsbad, CA), effectene (Qiagen, Valencia, CA), and tranfectam (Promega, Medison, WI) are commercially available [139], but most of the kits are useful only for in vitro experimentation. There are reports of improved efficiency of DNA delivery by cationic lipid via the coupling of specific receptor ligands or peptides to DNA/liposome complexes [126,140-143]. Cytotoxicity of cationic lipids has been established in numerous in vitro [144,145] and in vivo [146-148] studies. Low transfection efficiencies have been attributed to the heterogeneity and instability of cationic lipoplexes [149]. Another drawback in the use of cationic lipids is their rapid inactivation in the presence of serum [138,150]. Some in vivo studies have revealed that the gene transduction responses obtained by cationic lipoisomes were transient and short-lived [151,152].

As an alternative to cationic lipids, the potential of anionic lipids for DNA delivery has been investigated. The safety of anionic lipids has been demonstrated when administered to epithelial lung tissue. In recent years, a few studies, using anionic liposomal DNA delivery vectors have been reported. There have been attempts to incorporate anionic liposomes into polymeric delivery systems. However, these vectors have limited applications, mainly because of (1) inefficient entrapment of DNA molecules within anionic liposomes and (2) lack of toxicity data. Lack of further progress of these systems may be attributed, in part, to the poor association between DNA molecules and anionic lipids, caused by electrostatic repulsion between these negatively charged species [145,146,153-160].

Along with numerous cationic and anionic lipid derivatives, functionalized liposomal formulations serving specific therapeutic objectives have shown promise in gene therapy [103,161,162]. Specialized liposomal delivery platforms include pH-sensitive liposomes, immunoliposomes, and stealth liposomes. pH-Sensitive Liposomes can be generated by the inclusion of 1,2-dioleoyl-3-phosphoethanolamine (DOPE) into liposomes composed of acidic lipids such as cholesterylhemisuccinate or oleic acid.

At the neutral cellular pH 7, these lipids have the typical bilayer structure; however, upon endosomal compartmentalization they undergo protonation and collapse into a nonbilayer structure, thereby leading to the disruption and destabilization of the endosomal bilayer, which in turn helps in the rapid release of DNA into the cytoplasm [161]. Efficient gene delivery of the beta-galactosidase and luciferase reporter plasmids has been obtained using pH-sensitive liposomes in a variety of mammalian cell lines [163]. A chemical derivative of DOPE, Citraconyl-DOPE, has been used to deliver DNA-based therapeutics to cancer cells, thereby combining the targeting and the rapid endosome-releasing aspects of specialized liposomal delivery systems [164]. A phosphatidylcholine/ glycyrrhizin combination was also successful in pHsensitive gene delivery in mice [165]. Immunoliposomes are sophisticated gene delivery systems that can be used for cell targeting by the incorporation of functionalized antibodies attached to lipid bilayers [162]. Immunoliposomes containing an antibody fragment against the human transferring receptor were successfully used in targeted delivery of tumorsuppressing genes into tumors in vivo [166]. Tissuespecific gene delivery using immunoliposomes has been achieved in the brain [167], embryonic tissue [168], and breast cancer tissue [169]. Stealth liposomes are sterically stabilized liposomal formulations that include polyethylene glycol (PEG)-conjugated lipids [103].

Current status of gene therapy Research

Current gene therapy is experimental and has not proven very successful in clinical trials. Little progress has been made since the first gene therapy clinical trial began in 1990. In 1999, gene therapy suffered a major setback with the death of 18-year-old Jesse Gelsinger. Another major blow came in January 2003, when the FDA placed a temporary halt on all gene therapy trials using retroviral vectors in blood stem cells. FDA took this action after it learned that a second child treated in a French gene therapy trial had developed a leukemia-like condition. Both this child and another who had developed a similar condition in August 2002 had been successfully treated by gene therapy for X-linked severe combined immunodeficiency disease (X-SCID), also known as "bubble baby syndrome".

FDA’s Biological Response Modifiers Advisory Committee (BRMAC) met at the end of February 2003 to discuss possible measures that could allow a number of retroviral gene therapy trials for treatment of life-threatening diseases to proceed with appropriate safeguards. In April of 2003 the FDA eased the ban on gene therapy trials using retroviral vectors in blood stem cells.

Recent developments in gene Therapy

Nanotechnology and gene therapy yields treatment to torpedo cancer (March, 2009); The School of Pharmacy in London is testing a treatment in mice, which delivers genes wrapped in nanoparticles to cancer cells to target and destroy hard-to-reach cancer cells [170]. Results of world’s first gene therapy for inherited blindness show sight improvement (April, 2008); UK researchers from the UCL Institute of Ophthalmology and Moorefield’s Eye Hospital NIHR Biomedical Research Centre have announced results from the world’s first clinical trial to test a revolutionary gene therapy treatment for a type of inherited blindness. The results, published in the New England Journal of Medicine, show that the experimental treatment is safe and can improve sight. The findings are a landmark for gene therapy technology and could have a significant impact on future treatments for eye disease [171,172].

Previous information on this trial (May 1, 2007); A team of British doctors from Moorfields Eye Hospital and University College in London conduct first human gene therapy trials to treat Leber’s congenital amaurosis, a type of inherited childhood blindness caused by a single abnormal gene. The procedure has already been successful at restoring vision for dogs. This is the first trial to use gene therapy in an operation to treat blindness in humans [173].

A combination of two tumor suppressing genes delivered in lipid-based nanoparticles drastically reduces the number and size of human lung cancer tumors in mice during trials conducted in The University of Texas M. D. Anderson Cancer Center and the University of Texas Southwestern Medical Center [174]. Researchers at the National Cancer Institute (NCI), part of the National Institutes of Health, successfully reengineer immune cells, called lymphocytes, to target and attack cancer cells in patients with advanced metastatic melanoma. This is the first time that gene therapy is used to successfully treat cancer in humans [175].

Gene therapy is effectively used to treat two adult patients for a disease affecting nonlymphocytic white blood cells called myeloid cells. Myeloid disorders are common and include a variety of bone marrow failure syndromes, such as acute myeloid leukemia. The study is the first to show that gene therapy can cure diseases of the myeloid system (http://www. cincinnatichildrens.org/March 31, 2006). A research team at the University of California, Los Angeles is able to transport genes into the brain using liposomes coated with polyethylene glycol. The transfer of genes into the brain is a significant achievement because viral vectors are too big to get across the bloodbrain barrier. This method has potential for treating Parkinson’s disease [176].

RNA interference or gene silencing may be a new way to treat Huntington’s. Short pieces of doublestranded RNA (short, interfering RNAs or siRNAs) are used by cells to degrade RNA of a particular sequence. If a siRNA is designed to match the RNA copied from a faulty gene, then the abnormal protein product of that gene will not be produced [177]. New gene therapy approach repairs errors in messenger RNA derived from defective genes. Technique has potential to treat the blood disorder thalassaemia, cystic fibrosis, and some cancers [178]. Gene therapy for treating children with X-SCID (bubble boy) disease is stopped in France when the treatment resulted in leukemia in one of the patients [179]. Researchers at Case Western Reserve University and Copernicus Therapeutics are able to create tiny liposomes 25 nanometers across that can carry therapeutic DNA through pores in the nuclear membrane [180]. Sickle cell is successfully treated in mice [181].


  1. Anderson WF. Human gene therapy. Science 1992;256:808-13.
  2. Baltimore DP. Intracellular immunization. Nature 1988;335:395-7.
  3. Mizutani TN, Kato M, Hirota K, Sugiyama A, Murakami J, Shimotohno K. Inhibition of hepatitis C virus replication by antisense oligonucleotids in culture cells. Biochem Biophys Res Commun 1995;212:906-11.
  4. SoRelle R. Who owns your DNA? Who will own it? Circulation 2000;101:e67-8.
  5. Report of the United Kingdom Health Minister’s Gene Therapy Advisory Committee. Guidance on making proposals to conduct gene therapy research on human subjects. Hum Gene Ther 1995;6:335-46.
  6. Rosenberg SA, Blaese RM, Branner MK, Deisseroth AB, Ledley FD, Lotze MT, et al. Human gene marker/therapy protocols. Hum Gene Ther 2000;11:919-79.
  7. Lehrman S. Virus treatment questioned after gene therapy death. Nature 1999;401:517-8.
  8. Anon S. The increasing opacity of gene therapy. Nature 1999;402:107.
  9. Crooke ST. An overview of progress in antisense therapeutics. Antisense Nucleic Acid Drug Dev 1998;8:115-22.
  10. Stull RA, Szoka FC Jr. Antigene, ribozyme and aptamer nucleic acid drugs: Progress and prospects. Pharm Res 1995;12:463-5.
  11. Patil SD, Burgess DJ. DNA-based pharmaceuticals: therapeutics for the 21st century. AAPS Newsmagazine 2003;6:27.
  12. Baker BF. The role of antisense oligonucleotides in the wave of genomic information. Nucleosides Nucleotides Nucleic Acids 2001;20:397-9.
  13. Van Ommen GJ, Bakker E, Den Dunnen JT. The human genome project and the future of diagnostics, treatment, and prevention. Lancet 1999;354:5-10.
  14. Uherek C, Wels W. DNA-carrier proteins for targeted gene delivery. Adv Drug Deliv Rev 2000;44:153-66.
  15. Johnston SA, Talaat AM, McGuire MG. Genetic Immunization What’s in a name? Arch Med Res 2002;33:325-9.
  16. Anderson WF. Human gene therapy. Nature (London) 1998;392:25-30.
  17. Vorburger SA, Hunt KK. Adenoviral gene therapy. Oncologist 2002;7:46-59.
  18. Otsu M, Candotti F. Gene therapy in infants with severe combined immunodeÞciency. BioDugs 2002;16:229-39.
  19. Zhaohui P. China OKs Gene Therapy Drug. Genetic Engineering News 2003;6. [http://www. p53therapy.com/news/(November 19, 2003)].
  20. Galanis E, Russell S. Cancer gene therapy clinical trials: Lessons for the future. Br J Cancer 2001;85:1432-6.
  21. Mulherkar R. Gene therapy for cancer. Curr Sci 2001;81:555-60.
  22. Baekelandt V, De Strooper B, Nuttin B, Debysre Z. Gene therapeutic strategies for neurodegenerative diseases. Curr Opin Mol 2000;2:540-54.
  23. Bunnel BA, Morgan RA. Gene therapy for HIV infection. Dugs Today 1996;32:209-24.
  24. Horner AA, van Uden JH, Jubeldia JM, Broide D, Raz E. DNA-based immunothrapeutics for the treatment of allergic disease. Immunol Rev 2001;179:102-18.
  25. Crooke ST. Molecular mechanisms of antisense drugs. Biochem Biophys Acta 1999;1489:31-43.
  26. Crooke ST. Molecular mechanisms of antisense drugs: Human RNaseH. Antisense Nucleic Acid Drug Dev 1999;9:377-9.
  27. Speedie M. Antisense therapeutic agents. In: Williams DA, Lemke TL, editors. Foye’s Principles of Medicinal Chemistry. 5th ed. Philadelphia: Lippincott, Williams & Wilkins; 2005. p. 1053-4.
  28. Akhtar S, Hughes MD, Khan A. The delivery of antisense therapeutics. Adv Drug Deliv Rev 2000;44:3-21.
  29. Crooke ST. Vitravene another piece in the mosaic. Antisense Nucleic acid Drug Dev 1998;8:vii-v3.
  30. Mardan T, Kopecek J, Kissel T. Prospects for cationic polymers in gene and oligonucleotide therapy against cancer. Adv Drug Deliv Rev 2002;54:715-58.
  31. Agarawal S, Tang JY. GEM-91-an antisense oligonucleotide phosphorothioate as a therapeutic agent for AIDS. Antisense Res Dev 1992;2:261-6.
  32. Alt M, Ring R, Hofschneider PH, Paumgartner G, Caselmann WH. SpeciÞc inhibition of hepatitis C viral gene expression by antisense phosphorothioate oligodeoxynucleotides. Hepatology 1995;22:707-17.
  33. Azad RF, Driver VB, Tanaka K, Cooke RM, Anderson KP. Antiviral activity of a phosphorothioate oligonucleotide complementary to RNA of the human cytomegalovirus major immediate-early region. Antimicrob Agents Chemother 1993;37:1945-54.
  34. Bordier B, Helena C, Barr PJ, Litvak S, Sarih-Cottin L. In vitro effects of antisense oligonucleotides on human immunodeÞciency virus type 1 reverse transcription. Nucleic Acids Res 1992;20:1999-2006.
  35. Cowsert LM, Fox MC, Zon G, Mirabelli MK. In vitro evaluation of phosphorothioate oligonucleotides targeted to the E2 mRNA of papillomavirus: potential treatment for the genital warts. Antimicrob Agents Chemother 1993;37:171-7.
  36. Gerviax A, Li X, Kraus G, Wong-Staal F. Multigene antivirus vectors inhibit divers human immunodeÞciency virus type 1 clades. J Virol 1997;71:3048-53.
  37. Hanecak R, Brown-Driver V, Fox MC, Azad RF, Furusako S, Nozaki C, et al. Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes. J Virol 1996;70:5203-12.
  38. Kinchington D, Galpin S, Jeroszewski J, Ghosh K, Subashinghe C, Cohen JS. A comparision of gag, pol, and rev antisense oligodeoxynucleotides as inhibitors of HIV-1. Antiviral Res 1992;17:53-62.
  39. Lisziewicz J, Sun D, Klotman M, Agarwal S, Zamencik P, Gallo RC. SpeciÞc inhibition of human immunodeÞciency virus type 1 replication by antisense oligonuclotides: An in vitro model for treatment. Proc Natl Acad Sci USA 1992;89:11209-13.
  40. Lisziewicz J, Sun D, Metelev V, Zamencik P, Gallo RC, Agarwal S. Long term treatment of human immunodeÞciency virus infected cells with antisense oligonucleotide phosphorothioates. Proc Natl Acad Sci USA 1993;90:3860-4.
  41. Mirabelli CK, Bennet CF, Anderson K, Crooke ST. In vitro and in vivo pharmacologic activities of antisense oligonucleotides. AntiCancer Drug Design 1991;6:647-61.
  42. Morvan F, Porumb H, Degols G, Lefebvre I, Pampon A, Sproat BS, et al. Comparative evaluation of seven oligonucleotide analogues aspotential antisense agents. J Med Chem 1993;36:280-7.
  43. Tonkinson JL, Stein CA. Antisense nucleic acids-prospects for antiviral intervention. Antiviral Chem Chemother 1993;4:193-9.
  44. Jayasena SD. Aptamers: An emerging class of molecules that rival antibodies in diagnostics. Clin Chem 1999;45:1628-50.
  45. de Saultrait VR, Lozach PY, Altmeyer R, Tarrago-Litvak L, Litvak S, Andreola ML. DNA aptamers derived from HIV-1 RNaseH inhibitors are strong anti-integrase agents. J Mol Biol 2002;324:195-203.
  46. Zhang L, Gasper WA, Stass SA, Ioffe OB, Davis MA, Mixson AJ. Angiogenic inhibition mediated by a DNAzyme that target vascular endothelial growth factor receptor 2. Cancer Res 2002;62:5463-9.
  47. McAlister DV, Allen MG, Prausnitz MR. Microfabricated microneedles for gene and drug delivery. Annu Rev Biomed Eng 2000;2:289-313.
  48. Luo D, Saltzman WM. Synthetic DNA delivery systems. Nat Biotechnol 2000;18:33-7.
  49. Regnier V, Tahiri A, Andre N, Lemaitre M, Le Doan T, Preat V. Electroporation-mediated delivery of 3’-protected phosphodiester oligodeoxynucleotides to the skin. J Control Release 2000;67:337-46.
  50. Huang L, Viroonchatapan E. Introduction. In: Huang L, Huang M-C, Wagner E, editors. Nonviral vectors for gene therapy. San Diego, CA: Academic Press; 1999. p. 3-22.
  51. Kamiya H, Tsuchiya H, Yamazaki J, Harashima H. Intracellular trafÞcking and transgene expression of viral and non-viral gene vectors. Adv Drug Deliv Rev 2001;52:153-64.
  52. Mah C, Byrne BJ, Flotte TR. Virus-based gene delivery systems. Clin Pharmacokinet 2002;41:901-11.
  53. Lotze MT, Kost TA. Viruses as gene delivery vectors: Application to gene function, target validation, and assay development. Cancer Gene Ther 2002;9:692-9.
  54. McTaggart S, Al-Rubeai M. Retroviral vectors for human gene delivery. Biotechnol Adv 2002;20:1-31.
  55. Martin KR, Klein RL, Quigley HA. Gene delivery to the eye using adeno-associated viral vectors. Methods 2002;28:267-75.
  56. Lien Y-HH, Lie L-W. Gene therapy for renal disorders: What are the beneÞts for the elderly? Drugs Aging 2002;19:553-60.
  57. Chamberlain JS. Gene therapy of muscular dystrophy. Hum Mol Genet 2002;11:2355-62.
  58. Wolf JK, Jenkins AD. Gene therapy for ovarian cancer. Int J Oncol 2002;21:461-8.
  59. Walther W, Stein U. Viral vectors for gene transfer: A review of their use in the treatment of human diseases. Drugs 2000;60:249-71.
  60. Lever AML. Gene therapy in the Þght against AIDS. Expert Opin Ther Patents 1996;6:161-7.
  61. Zhao W, Kobayashi M, Hosokawa M, Seth P. Adenoviral vectors for cancer gene therapy. Curr Genomics 2002;3:163-80.
  62. Hale SJ, Green NK. Viral approaches to cancer gene therapy. Expert Opin Ther Patents 2002;12:369-78.
  63. Roe T, Reynolds TC, Yu G, Brown PO. Integration of murine leukemia virus DNA depends on mitosis. EMBO J 1993;12:2099-108.
  64. Lewis PF, Emerman M. Passage through mitosis is required for oncoretroviruses but not for the human immunodeÞciency virus. J Virol 1994;68:510-6.
  65. Naldini L, Blomer U, Gally P. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 1996;272:263-7.
  66. Mann R, Mulligan RC, Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell 1983;33:153-9.
  67. Cone RD, Mulligan RC. High-efÞciency gene transfer into mammalian cells: Generation of helper-free recombinant retrovirus with broad mammalian host range. Proc Natl Acad Sci USA 1984;81:6349-53.
  68. Kim SH, Yu SS, Park JS, Robbins PD, An CS, Kim S. Construction of retroviral vectors with improved safety, gene expression, and versatility. J Virol 1998;72:994-1004.
  69. Sheridan PL, Bodner M, Lynn A. Generation of retroviral packaging and producer cell lines for large-scale vector production and clinical application: Improved safety and high titer. Mol Ther 2000;2:262-75.
  70. Fujji S, Huang S, Fong TC. Induction of melanoma-associated antigen systemic immunity upon intratumoral delivery of interferon-gamma retroviral vector in melanoma patients. Cancer Gene Ther 2000;7:1220-30.
  71. Tait DL, Obermiller PS, Hatmaker AR. Ovarian cancer BRCA1 gene therapy: Phase I and II trial differences in immune response and vector stability. Clin Cancer Res 1999;5:1708-14.
  72. Bordignon C, Notarangilo LD, Nobili N. Gene therapy in peripheral blood lymphocytes and bone marrow for ADA-immunodeficient patients. Science 1995;270:470-5.
  73. Onodera M, Ariga T, Kawamura N. Successful peripheral T-lymphocyte-directed gene transfer for a patient with severe combined immunodeÞciency caused by adenosine deaminase deÞciency. Blood 1998;91:30-6.
  74. Dunbar CE, Kohn DB, Shiffman R. Retroviral transfer of the glucoceribrosidase gene into CD34+ cells from patients with Goucher disease: In vivo detection of transduced cells without myeloblation. Hum Gen Ther 1998;9:2629-40.
  75. Garton KJ, Ferri N, Raines EW. Efficient expression of exogenous genes in primary vascular cells using IRES-based retroviral vectors. Biotechniques 2002;32:830-43.
  76. Stewart AK, Lassam NJ, Quirt IC. Adenovector-mediated gene delivery of interleukin-2 in metastatic breast cancer and melanoma: Results of a phase 1 clinical trial. Gene Ther 1999;6:350-63.
  77. Alvarez RD, Barns MN, Gomez-Navarro J. A cancer gene therapy approach utilizing an anti-erbB-2 single-chain antibody-encoding adenovirus (AD21): A phase I trial. Clin Cancer Res 2000;6:3081-7.
  78. Alvarez RD, Gomez-Navarro J, Wang M. Adenoviral-mediated suicide gene therapy for ovarian cancer. Mol Ther 2000;2:524-30.
  79. Byrnes AP, Rusby JE, Wood MJ. Adenovirus gene transfer causes inßammation in the brain. Neuroscience 1995;66:1015-24.
  80. Yang Y, Jooss KU, Su Q. Immune responses to viral antigens versus transgene product in the elimination of recombinant adenovirus-infected hepatocytes in vivo. Gene Ther 1996;3:137-44.
  81. Yang Y, Su Q, Wilson JM. Role of viral antigens in destructive cellular immune responses to adenovirus vector-transduced cells in mouse lungs. J Virol 1996;70:7209-12.
  82. van Ginkel FW, McGhee JR, Liu C. Adenoviral gene delivery elicits distinct pulmonary-associated T helper cell responses to the vector and to its transgene. J Immunol 1997;159:685-93.
  83. Michou AI, Santoro L, Christ M. Adenovirus-mediated gene transfer: Inßuence of transgene, mouse strain and type of immune response on persistence of transgene expression. Gene Ther 1997;4:473-82.
  84. Bett AJ, Prevec L, Graham FL. Packaging capacity, and stability of human adenovirus type 5 vectors. J Virol 1993;67:5911-21.
  85. Clemens PR, Kochanek S, Sunada Y. In vivo muscle gene transfer of full-length dystrophin with an adenoviral vector that lacks all viral genes. Gene Ther 1996;3:965-72.
  86. Kochanek S, Clemens PR, Mitani K. A new adenoviral vector: replacement of viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and beta-galactosidase. Proc Natl Acad Sci USA 1996;93:5731-6.
  87. Parks RJ, Grahm FL. A helper-dependent system for adenovirus vector production helps deÞne a lower limit for efÞcient DNA packaging. J Virol 1997;71:3293-8.
  88. Morsy MA, Gu M, Motzel S. An adenoviral vector deleted for all viral coding sequences results in enhanced safety and extended expression of a leptin transgene. Proc Natl Acad Sci USA 1998;95:7866-71.
  89. Schiedner G, Morral N, Parks RJ. Genomic DNA transfer with a high-capacity adenovirus vector results in improved in vivo gene expression and decreased toxicity. Nat Genet 1998;18:180-3.
  90. Heider H, Verca HB, Rusconi S, Asmis R. Comparision of lipid-mediated, and adenoviral gene transfer in human monocyte-derived macrophages and COS-7 cells. Biotechniques 2000;28:260-70.
  91. Fisher KJ, Jooss K, Alston J. Recombinant adeno-associated virus for muscle directed gene therapy. Nat Med 1997;3:306-12.
  92. Jooss K, Yang Y, Fisher KJ. Transduction of dendritic cells by DNA viral vectors directs the immune response to transgene products in muscle Þbers. J Virol 1998;72:4212-23.
  93. Rose JA, Berns KI, Hoggan MD. Evidence for a single stranded adenovirus-associated virus genome: Formation of a DNA density hybrid on release of viral DNA. Proc Natl Acad Sci USA 1969;64: 863-9.
  94. Kotin RM, Siniscalco M, Samulski RJ. Site-specific integration by adeno-associated virus. Proc Natl Acad Sci USA 1990;87:2211-5.
  95. Samulski RJ, Zhu X, Xiao X. Targeted integration of adeno-associated virus (AAV) into human chromosome 19. EMBO J 1991;10:3941-50.
  96. Samulski RJ, Chang LS, Shenk T. Helper-stocks of recombinant adeno-associated viruses: Normal integration does not require viral gene expression. J Virol 1989;63:3822-8.
  97. Dong JY, Fang PD, Frizzel RA. Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Hum Gene Ther 1996;7:2101-12.
  98. Hermonat PL, Quirk JG, Bishop BM. The packaging capacity of adeno-associated virus (AAV) and the potential for wild-type-plus AAV gene therapy vectors. FEBS Lett 1997;407:78-84.
  99. Owens RA. Second generation adeno-associated virus type 2-based gene therapy systems with the potential for preferential integration into AAVS 1. Curr Gene Ther 2002;2:145-59.
  100. Nyberg-Hoffman C, Aguilar-Cardova E. Instability of adenoviral vectors during transport and its implication for clinical studies. Nat Med 1999;5:955-7.
  101. Carpenter DE, Stevens JG. Long term expression of a foreign gene from a unique position in the latent herpes simplex virus genome. Hum Gene Ther 1996;7:1447-54.
  102. Lowenstein PR, Morrison EE, Bain D. Use of recombinant vectors derived from herpes simplex virus 1 mutant tsK for short-tem expression of transgenes encoding cytoplasmic and membrane anchored proteins in post mitotic-polarized cortical neurons and glial cells in vitro. Neuroscience 1994;60:1059-77.
  103. Fattal E, Dellattre J, Dubernet C, Couvrur P. Liposomes for the delivery of nucleotides and oligonucleotides. S.T.P. Pharma Sciences 1999;9: 383-90.
  104. Fattal E, Dubernet C, Couvrur P. Liposome-based formulations for the delivery of oligonucleotids. S.T.P. Pharma Sciences 2001;11:31-44.
  105. Pedroso de Lima MC, Simoes S, Pires P, Faneca H, Duzguns N. Cationic lipid-DNA complexes in gene delivery: From biophysics to biological applications. Adv Drug Deliv Rev 2001;47:277-94.
  106. Nakamura M, Devila-Zavala P, Tokuda H. Uptake and gene expression of naked plasmid DNA in cultured brain microvessel endothelial cells. Biochem Biophys Res Commun 1998;245:235-39.
  107. Mann MJ, Gibbons GH, Hutchinson H. Pressure-mediated oligonucleotide transfection of rat and human cardiovascular tissues. Proc Natl Acad Sci USA 1999;96:6411-6.
  108. Wolff JA, Malone RW, Williams P. Direct gene transfer into mouse muscle in vivo. Science 1990;247:1465-8.
  109. Liu F, Song Y, Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther 1999;6:1258-66.
  110. Fynan EF, Webster RG, Fuller DH. DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations. Proc Natl Acad Sci USA 1993;90:11478-82.
  111. Wong TK, Neumann E. Electric Þeld mediated gene transfer. Biochem Biophys Res Commun 1982;107:584-7.
  112. Neumann E, Schaefer-Ridder M, Wang Y. Gene transfer into lyoma by electroporation in high electric Þelds. EMBO J 1982;1:841-5.
  113. Rols MP, Delteil C, Golzio M. In vivo electrically mediated protein and gene transfer in murine melanoma. Nat Biotechnol 1998;16:168-71.
  114. Rizzuto G, Cappelletti M, Maione D. Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation. Proc Natl Acad Sci USA 1999;96:6417-22.
  115. Hwang SJ, Davis ME. Cationic polymers for gene delivery: Designs for overcoming barriers to systemic administration. Curr Opin Mol Ther 2001;3:183-91.
  116. Lemkine GF, Demeneix BA. Polyethylenimins for in vivo gene delivery. Curr Opin Mol Ther 2001;3:178-82
  117. Lollo CP, Banaszczyk MG, Mullen PM. Poly-L-lysine based gene delivery systems: synthesis, puriÞcation, and application. Methods Mol Med 2002;69:1-13.
  118. Borchard G. Chitosans for gene delivery. Adv Drug Deliv Rev 2001;52:145-50.
  119. Boussif O, Lezoualc’h F, Zanta MA. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimin. Proc Natl Acad Sci USA 1995;92:7297-301.
  120. Wadhwa MS, Knoell DL, Young AP. Targeted gene delivery with a low molecular weight glycopeptide carrier. Bioconjug Chem 1995;6:283-91.
  121. Kukowska-Latello JF, Bielinska AU, Johnson J. EfÞcient transfer of genetic material into mammalian cells using Starbust polyamidoamine dendrimers. Proc Natl Acad Sci USA 1996;93:4897-902.
  122. Tang MX, Szoka FC. The influence of polymer structure on the interactions of cationic polymers with DNA and morphology of the resulting complexes. Gene Ther 1997;4:823-32.
  123. Mumper RJ, Wang J, Klakamp SL. Protective interactive noncondensing (PINC) polymers for enhanced plasmid distribution and expression in rat skeletal muscle. J Control Release 1998;52:191-203.
  124. Naughton BA, Dai Y, Sibanda B. Long-term expression of a retrovirally introduced beta-galactosidas gene in rodent cells implanted in vivo using biodegradable polymer meshes. Somat Cell Mol Genet 1992;18:451-62.
  125. Singh M, Briones M, Ott G. Cationic microparticles: A potent delivery system for DNA vaccines. Proc Natl Acad Sci USA 2000;97:811-16.
  126. Jenkins RG, Herrick SE, Meng QH. An integrin-targeted non-viral vector for pulmonary gene therapy. Gene Ther 2000;7:393-400.
  127. LeHoux JG, Grondin F. Some effects of chitosan on liver function in the rat. Endocrinology 1993;132:1078-84.
  128. Godbey WT, Mikos AG. Recent progress in gene delivery using non-viral transfer complexes. J Control Release 2001;72:115-25.
  129. Tejada-Berges T, Granai CO, Gordinier M, Gajewski W. Caelyx/Doxil for the treatment of metastatic ovarian and breast cancer. Expert Rev Anticancer Ther 2002;2:143-50.
  130. Marshal J, Yew NS, Eastman SJ, Jiang C, Sheule RK, Cheng SH. Cationic lipid-mediated gene delivery to the airways. In: Wagner E, editors. Nonviral vectors for gene therapy. San Diego, CA: Academic Press; 1999. p. 39-68.
  131. Felgner PL, Gadek TR, Holm M. Lipofection: A highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci USA 1987;84:7413-7.
  132. Caplen NJ, Alton EW, Middleton PG. Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic Þbrosis. Nat Med 1995;1:39-46.
  133. Hyde SC, Southern KW, Gileadi U. Repeate administration of DNA/ liposome to the nasal epithelium of patients with cystic Þbrosis. Gene Ther 2000;7:1156-65.
  134. Noone PG, Hohnker KW, Zhou Z. Safety and biological efÞcacy of a lipid-CFTR complex for gene transfer in the nasal epithelium of adult patients with cystic Þbrosis. Mol Ther 2000;1:105-14.
  135. Nabel GJ, Nabel EG, Yang ZY. Direct gene transfer with DNA-liposome complexes in melanoma: Expression, biological activity, and lack of toxicity in humans. Proc Natl Acad Sci USA 1993;90:11307-11.
  136. Nabel GJ, Gordon D, Bishop DK. Immune response in human melanoma after transfer of an allogenic class I major histocompatibility complex gene with DNA-liposome complexes. Proc Natl Acad Sci USA 1996;93:15388-93.
  137. Felgner JH, Kumar R, Sridhar CN. Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J Biol Chem 1994;269:2550-61.
  138. Hoßand HEJ, Shephard L, Sullivan SM. Formation of stable cationic lipid/DNA complexes for gene transfer. Proc Natl Acad Sci USA 1996;93:7305-9.
  139. Kang S-H, Zirbes EL, Kole R. Delivery of antisense oligonucleotides and plasmid DNA with various carrier agents. Antisense Nucleic Acid Drug Dev 1999;9:497-505.
  140. Ellison KE, Bisphoric NH, Webster KA. Fusigenic liposome-mediated DNA transfer into cardiac myocytes. J Mol Cell Cardiol 1996;28:1385-99.
  141. Simoes S, Slepushkin V, Gaspar R. Gene delivery by negatively charged ternary complexes of DNA, cationic liposomes and transferin or fusigenic peptides. Gene Ther 1998;5:955-64.
  142. Shinmura K, Morishita R, Aoki M. Catheter-delivered in vivo gene transfer into rat myocardium using the fusigenic liposomal mediated method. Jpn Heart J 2000;41:633-47.
  143. Compton SH, Mecklenbeck S, Mejia JE. Stable integration of large (>100kb) PAC constructs in HaCa T keratinocytes using an integrin-targeting peptide delivery system. Gene Ther 2000;7:1600-7.
  144. Lappalainen K, Jaaskelainen I, Syrjanen K, Urtti A, Syrjanen S. Comparison of cell proliferation and toxicity assays using two cationic liposomes. Pharm Res 1994;11:1127-31.
  145. Patil SD, Rhodes DG, Burgess DJ. Anionic liposomal delivery system for DNA transfection. The AAPS Journal 2004;6:E29.
  146. Dokka S, Toledo D, Shi X, Castranova V, Rojanasakul Y. Oxygen radical-mediated pulmonary toxicity induced by some cationic liposomes. Pharm Res 2000;17:521-5.
  147. Filion MC, Philips NC. Toxicity and immunomodulatory activity of liposomal vectors formulated with cationic lipids toward immune effector cells. Biochim Biophys Acta 1997;1329:345-56.
  148. Freimark BD, Blzinger HP, Florack VJ. Cationic lipids enhance cytokine and cell inßux levels in the lung following administration of plasmid: Cationic lipid complexes. J Immunol 1998;160:4580-6.
  149. Lee H, Williams SK, Allison SD, Anchordoquy TJ. Analysis of self-assembled cationic lipid-DNA gene carrier complexes using ßow Þeld-ßow fractionation and light scattering. Anal Chem 2001;73:837-43.
  150. Audouy S, Molema G, De Leij L, Hoekstra D. Serum as a modulator of lipoplex-mediated gene transfection: Dependence of amphiphile cell type and complex stability. J Gene Med 2000;2:465-76.
  151. Liu F, Qi H, Huang L, Liu D. Factors controlling the efficiency of cationic lipid-mediated transfection in vivo via intravenous administration. Gene Ther 1997;4:517-23.
  152. Wheeler CJ, Felgner PL, Tsai YJ. A novel cationic lipid greatly enhances plasmid DNA delivery and expression in mouse lung. Proc Natl Acad Sci USA 1996;93:11454-9.
  153. Patil SD, Rhodes DG. Inßuence of divalent cations on the conformation of phosphorothioate oligodeoxynucleotides: A circular dichroism study Nucleic Acids Res 2000;28:2439-45.
  154. Fillion P, Desjardins A, Sayasith K, Lagace J. encapsulation of DNA in negatively charged liposomes and inhibition of bacterial gene expression with ßuid-liposome encapsulated antisense oligonucleotides. Biochim Biophys Acta 2001;1515:44-54.
  155. Lakkaraju A, Dubinsky JM, Low WC, Rehman YE. Neurons are protected from excitotoxic death by p53 antisense oligonuclotides delivered in anionic liposomes. J Biol Chem 2001;276:32000-7.
  156. Patil SD, Rhodes DG. Conformation of oligodeoxynucleotides associated with anionic liposomes. Nucleic Acids Res 2000;28:4125-9.
  157. Patil SD, Rhodes DG, Burgess DJ. Biophysical characterization of anionic lipoplexes. Biochim Biophys Acta - Biomembranes 2005;7:E61-77.
  158. Lee RJ, Huang L. Lipidic vector systems for gene transfer. Crit Rev Ther Drug Carrier Syst 1997;14:173-206.
  159. Guo W, Gosselin MA, Lee RJ. Characterization of a novel diolein-based LPD II vector for gene delivery. J Control Release 2002;83:121-32.
  160. Perrie Y, Gregoriadis G. Liposome-entrapped plasmid DNA: Characterization studies. Biochim Biophys Acta 2000;1475:125-32.
  161. Venugopalan P, Jain S, Sankar S, Singh P, Rawat A, Vyas SP. pH-sensitive liposomes: Mechanism of triggered release to drug and gene delivery prospects. Pharmazai 2002;57:659-71.
  162. Maclean AL, Symonds G, Ward R. Immunoliposomes as targeted delivery vehicles for cancer therapeutics. Int J Oncol 1997;11:325-32.
  163. Legendre JY, Szoka FC Jr. Delivery of plasmid DNA into mammalian cell lines using pH-sensitive liposomes: Comparision with cationic liposomes. Pharm Res 1992;9:1235-42.
  164. Reddy JA, Low PS. Enhanced folate receptor mediated gene therapy using a novel pH-sensitive lipid formulation. J Control Release 2000;64:27-37.
  165. Sviridov YV, Zhadanov RI, Podobed OV, Tsvetkova TA, Konstantinov I, Bogdanenko EV. The LacZ gene transfer into L929 cells and [14C]-DNA tissue distribution following intraperitoneal administration of new pH-sensitive lipoplexes in mice. Cytobios 2001;106:7-14.
  166. Xu L, Huang CC, Huang W. Systemic tumor-targeted gene delivery by anti-transferrin receptor scFv-immunoliposomes. Mol Cancer Ther 2002;1:337-46.
  167. Shi N, Zhang Y, Zhu C, Boado RJ, Pardridge WM. Brain-specific expression of an exogenous gene after IV administration. Proc Natl Acad Sci USA 2001;98:12754-9.
  168. Khaw BA, da Silva J, Vural I, Narula J, Torchilin VP. Intracytoplasmic gene delivery for in vitro transfection with cytoskeleton-specific immunoliposomes. J Control Release 2001;75:199-210.
  169. Krauss WC, Park JW, Kirpotin DB, Hong K, Benz CC. Emerging antibody-based HER2 (ErbB- 2/new) therapeutics. Breast Disease 2000;11:113-24.
  170. Schatzlein A. Nano-treatment to torpedo cancer. Available from: http:// news.bbc.co.uk/2/hi/health/7935592.stm. [cited in 2009 Mar 10].
  171. James WB, Alexander JS, Susie SB, Scott R, Robert H, Kamaljit B, et al. Effect of gene therapy on visual function in Leber’s congenitalamaurosis. N Engl J Med 2008;358:2231-9.
  172. Albert MM, Francesca S, Aric AP, Edward NP, Federico M, Jeannette B, et al. Safety and efÞcacy of gene transfer for Leber’s congenital amaurosis. N Engl J Med 2008;358:2240-8.
  173. Ben H. Doctors test gene therapy to treat blindness. Available from: http://www.reuters.com/article/ scienceNews/idUSL016653620070501? pageNumber=1/. [cited in 2007 May 1].
  174. Scott M. Dual gene therapy suppresses lung cancer in preclinical test. Available from: http://www.newswise.com/p/articles/view/526526/. [cited in 2007 Jan 11].
  175. Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM, et al. Cancer regression in patients mediated by transfer of genetically engineered lymphocytes. Available from: http://www.cancer. gov/newscenter/pressreleases/MelanomaGeneTherapy/. [cited in 2006 Aug 31].
  176. Anil A. Undercover genes slip into the brain. Available from: http:// www.newscientist.com/section/science-news/. [cited in 2003 Mar 22].
  177. Bob H. Gene therapy may switch off Huntington’s. Available from: http://www.newscientist.com/section/ science-news/. [cited in 2003 Mar 13].
  178. Danny P. Subtle gene therapy tackles blood disorder. Available from: http://www.newscientist.com/section/ science-news/. [cited in 2002 Oct 11].
  179. Emma Y. ‘Miracle’ gene therapy trial halted. Available from: http:// www.newscientist.com/section/science-news/. [cited in 2002 Oct 3].
  180. Sylvia PW. DNA nanoballs boost gene therapy. Available from: http:// www.newscientist.com/section/science -news/. [cited in 2002 May 12].
  181. Jennifer FW. Murine Gene Therapy Corrects Symptoms of Sickle Cell Disease. The Scientist 2002;16:36. Available from: http://www.the-scientist.com. [cited in 2002 Mar 18].