Non-viral gene transfer therapy for cystic fibrosis

Non-viral methods of gene transfer are being investigated to treat cystic fibrosis (CF) and include naked DNA, lipid-DNA complexes and complexes of DNA with polycations such as poly-L-lysine (poly K) or polyethylenimine (PEI), all of which can carry the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most recent promising strategy is the use of polycation-DNA complexes, particularly those prepared with poly-K and substituted with polyethylene glycol. These complexes produced partial correction of the CF defect in a mouse model with minimal toxicity, and have advanced to clinical trial. Improvements in this and other non-viral methods are in process and include i). targeting the complexes to the desired cells using receptor ligands, ii). lessening toxicity by changing the mix of lipids or adding protective molecules to polycations, iii). modifying the plasmid DNA to reduce inflammatory CpG sequences and enhance intensity, duration and tissue specificity of expression, and iv). modification of the complexes to improve nuclear access.     

Non-viral gene transfer therapy for cystic fibrosis

Non-viral methods of gene transfer are being investigated to treat cystic fibrosis (CF) and include naked DNA, lipid–DNA complexes and complexes of DNA with polycations such as poly-L-lysine (poly K) or polyethylenimine (PEI), all of which can carry the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most recent promising strategy is the use of polycation–DNA complexes, particularly those prepared with poly-K and substituted with polyethylene glycol. These complexes produced partial correction of the CF defect in a mouse model with minimal toxicity, and have advanced to clinical trial. Improvements in this and other non-viral methods are in process and include i) targeting the complexes to the desired cells using receptor ligands, ii) lessening toxicity by changing the mix of lipids or adding protective molecules to polycations, iii) modifying the plasmid DNA to reduce inflammatory CpG sequences and enhance intensity, duration and tissue specificity of expression, and iv) modification of the complexes to improve nuclear access.     

Stem cell therapy and gene transfer for regeneration

The committed stem and progenitor cells have been recently isolated from various adult tissues, including hematopoietic stem cell, neural stem cell, mesenchymal stem cell and endothelial progenitor cell. These adult stem cells have several advantages as compared with embryonic stem cells as their practical therapeutic application for tissue regeneration. In this review, we discuss the promising gene therapy application of adult stem and progenitor cells in terms of modifying stem cell potency, altering organ property, accelerating regeneration and forming expressional organization.     

High-capacity adenoviral vectors for gene transfer and somatic gene therapy.

The availability of efficient and nontoxic gene delivery technologies is fundamental to the translation of therapeutic concepts into clinical practice by gene transfer. High-capacity adenoviral (HC-Ad) vectors are characterized by the ability to transduce cells in vitro and in vivo with more than 30 kb of nonviral DNA. This quality allows simultaneous gene transfer of several expression cassettes, large promoters, and some genes in their natural genomic context. Because all viral coding sequences are removed from these vectors, safety is considerably improved compared with previous-generation adenoviral vectors.     

mRNA-based gene transfer as a tool for gene and cell therapy

In the field of nonviral gene therapy, mRNA-based gene transfer has generated much interest over the last decade. The combination of RNA as a versatile protein delivery molecule and the dendritic cell as the most potent antigen-presenting cell is an attractive approach to induce cellular and potentially therapeutic immune responses in patients with cancer. The success of mRNA transfection stems from its superior cytoplasmic expression efficiency, simplicity over viral transduction protocols, and clinical safety profile (due to a strictly transient expression and inability to integrate into the host genome). Most researchers have exploited low-voltage electrical pulses (electroporation) as a means to introduce RNA into cells, but other methods with an even higher degree of in vivo applicability are under development. Recently, more insights into the immunological properties of RNA and optimized strategies to produce highly translatable mRNA have increased its efficacy in cellular vaccination. In addition, application of RNA gene transfer into areas other than immunotherapy are slowly emerging and underscore the potential of RNA transfection as a versatile gene therapy tool.     

Loss of transgene following ex vivo gene transfer is associated with a dominant Th2 response: implications for cutaneous gene therapy.

Host responses to therapeutic gene products are potentially serious complications in cutaneous gene therapy. Controlling immune responses to the therapeutic antigen may therefore be critical for an effective therapy. Both ex vivo and in vivo gene transfer to epidermal stem cells has been shown to induce transgene-specific immune responses; however, whether the mechanism of immune activation is the same is not clear. In this study, we have characterized transgene-specific immune responses in an ex vivo model of epidermal gene transfer using green fluorescent protein as a model antigen and retrovirus-mediated gene delivery. Contrary to T helper (Th)1-type responses induced following in vivo gene transfer to epidermis, rejection of ex vivo-transduced keratinocytes was associated with Th2/eosinophilc responses. These responses were characterized by interleukin (IL)-4 and IL-5 production by T cells, a predominance of anti-green fluorescent protein IgG1 in serum, the presence of numerous eosinophils within rejected skin, and a lack of class I-restricted cytotoxic T lymphocyte response. Pretreatment of mice receiving ex vivo transduced keratinocytes with neutralizing anti-IL-5 antibody prevented eosinophil infiltration and prolonged survival of transduced epidermis. These data indicate a role for the Th2/eosinophilic pathway in rejection of ex vivo-transduced keratinocytes, suggesting different requirements for achieving tolerance for ex vivo and in vivo approaches to gene therapy.     

Gene therapy progress and prospects: ultrasound for gene transfer

Ultrasound exposure (USE) in the presence of microbubbles (MCB) (e.g. contrast agents used to enhance ultrasound imaging) increases plasmid transfection efficiency in vitro by several orders of magnitude. Formation of short-lived pores in the plasma membrane ('sonoporation'), up to 100 nm in effective diameter lasting a few seconds, is implicated as the dominant mechanism, associated with acoustic cavitation. Ultrasound enhanced gene transfer (UEGT) has also been successfully achieved in vivo, with reports of spatially restricted and therapeutically relevant levels of transgene expression. Loading MCB with nucleic acids and/or disease-targeting ligands may further improve the efficiency and specificity of UEGT such that clinical testing becomes a realistic prospect.     

A new direction for gene therapy: intrathymic T cell-specific lentiviral gene transfer

Reports of neoplasia related to insertional activation of protooncogenes by retroviral vectors have raised serious safety concerns in the field of gene therapy. Modification of current approaches is urgently required to minimize the deleterious consequences of insertional mutagenesis. In this issue of the JCI, Adjali and colleagues report on their treatment of SCID mice lacking the 70-kDa protein tyrosine kinase, ZAP-70, with direct intrathymic injection of a ZAP-70-expressing T cell-specific lentiviral vector, which resulted in T cell reconstitution. Using lentiviral vectors and in situ gene transfer may represent a safer approach than using retroviral vectors for ex vivo gene transfer into HSCs, avoiding 3 factors potentially linked to leukemogenesis, namely HSC targets, ex vivo transduction and expansion, and standard Moloney leukemia virus-based retroviral vectors.     

Immune-mediated loss of transgene expression in skin: implications for cutaneous gene therapy

A clearer understanding of the immune-mediated loss of transgene from cutaneous epithelium is necessary for development of effective clinical gene therapy protocols for patients who carry null mutations in the target gene. We have used retrovirus-mediated transfer of lacZ to mouse skin as a model to investigate the mechanism of immune-mediated transgene loss in skin. Transduction of C57Bl/6 mouse skin resulted in elicitation of both humoral and cellular immune responses. Antibody responses did not play a major role in the loss of transgene. Infiltration of the transduced skin with CD4⁺ and CD8⁺ cells and induction of transgene-specific cytotoxic T lymphocytes implied a role for T-cell-mediated responses. Transduction of mice deficient in either major histocompatibility complex (MHC) class I or class II molecules resulted in transient transgene expression. Only in MHC^−/− mice lacking expression of both class I and class II MHC molecules was persistent transgene expression seen. These data indicate a primary role for T-cell-mediated responses in the immune-mediated loss of transgene expression. Furthermore, CD4 and CD8 T cells have overlapping roles and either population can effectively eliminate transduced cells. Therefore, long-term cutaneous gene therapy may require development of strategies to interfere with activation or function of both T cell populations.     

Applying horizontal gene transfer phenomena to enhance non-viral gene therapy

Horizontal gene transfer (HGT) is widespread amongst prokaryotes, but eukaryotes tend to be far less promiscuous with their genetic information. However, several examples of HGT from pathogens into eukaryotic cells have been discovered and mimicked to improve non-viral gene delivery techniques. For example, several viral proteins and DNA sequences have been used to significantly increase cytoplasmic and nuclear gene delivery. Plant genetic engineering is routinely performed with the pathogenic bacterium Agrobacterium tumefaciens and similar pathogens (e.g. Bartonella henselae) may also be able to transform human cells. Intracellular parasites like Trypanosoma cruzi may also provide new insights into overcoming cellular barriers to gene delivery. Finally, intercellular nucleic acid transfer between host cells will also be briefly discussed. This article will review the unique characteristics of several different viruses and microbes and discuss how their traits have been successfully applied to improve non-viral gene delivery techniques. Consequently, pathogenic traits that originally caused diseases may eventually be used to treat many genetic diseases.     

Bacteria-mediated DNA transfer in gene therapy and vaccination

The use of live attenuated bacterial vaccine strains allows the targeted delivery of macromolecules to mammalian cells and tissues via the mucosal route. Depending on their specific virulence mechanisms and inherent metabolic preferences, bacteria invade certain cell types and body niches where they consequently deliver their cargo. Recently, the ability of attenuated strains of Salmonella, Shigella and Yersinia spp., as well as Listeria monocytogenes and invasive Escherichia coli, to deliver eukaryotic expression plasmids into mammalian cells in vitro and in vivo has been discovered. The great potential of bacteria-mediated transfer of plasmid DNA encoding vaccine antigens and/or therapeutic molecules was demonstrated in experimental animal models of infectious diseases, tumours and gene deficiencies. The exact mechanism of DNA transfer from the bacterial vector into the mammalian host is not yet completely known. The understanding of molecular events during bacterial DNA transfer, however, will further the development of bacterial vector systems with great promise for various clinical applications.     

Recombinant AAV-mediated gene transfer to the retina: gene therapy perspectives

Retinal degenerative diseases such as retinal macular degeneration and retinitis pigmentosa constitute a broad group of diseases that all share one critical feature, the progressive apoptotic loss of cells in the retina. There is currently no effective treatment available by which the course of these disorders can be modified, and visual dysfunction often progresses to total blindness. Gene therapy represents an attractive approach to treating retinal degeneration because the eye is easily accessible and allows local application of therapeutic vectors with reduced risk of systemic effects. Furthermore, transgene expression within the retina and effects of treatments may be monitored by a variety of noninvasive examinations. An increasing number of strategies for molecular treatment of retinal disease rely on recombinant adeno-associated virus (rAAV) as a therapeutic gene delivery vector. Before rAAV-mediated gene therapy for retinal degeneration becomes a reality, there are a number of important requirements that include: (1) evaluation of different rAAV serotypes, (2) screening of vectors in large animals in order to ensure that they mediate safe and long-term gene expression, (3) appropriate regulation of therapeutic gene expression, (4) evaluation of vectors carrying a therapeutic gene in relevant animal models, (5) identification of suitable patients, and finally (6) manufacture of clinical grade vector. All these steps towards gene therapy are still being explored. Outcomes of these studies will be discussed in the order in which they occur, from vector studies to preclinical assessment of the therapeutic potential of rAAV in animal models of retinal degeneration.     

Lentiviral-mediated gene transfer to the sheep brain: implications for gene therapy in Batten disease

The neuronal ceroid lipofuscinoses (NCLs; Batten disease) are inherited neurodegenerative lysosomal storage diseases with common clinical features of blindness and seizures culminating in premature death. Gene-therapy strategies for these diseases depend on whether the missing activity is a secreted lysosomal protein taken up by neighboring cells, or an intramembrane protein that requires careful targeting. Therapies are best developed in animal models with large complex human-like brains. Lentiviral-mediated gene delivery to neural cell cultures from normal sheep and sheep affected with an NCL resulted in green fluorescent protein (GFP) expression in neurons and neuroblasts, more efficiently than in astrocytes. Similar transgene expression was obtained from two constitutive promoters, the viral MND promoter and the human EF1α promoter. In vivo studies showed stable and persistent GFP expression throughout the cell bodies, axons, and dendrites from intracortical injections and indicated ependymal and subependymal transduction. The sheep showed no ill effects from the injections. These data support continuing gene-therapy trials in the sheep models of Batten disease.     

Nonviral jet-injection gene transfer for efficient in vivo cytosine deaminase suicide gene therapy of colon carcinoma.

Jet-injection technology has developed into an efficient gene delivery system for nonviral in vivo gene transfer. In this study the jet-injector system was used for the intratumoral gene transfer of small volumes of naked DNA encoding the Escherichia coli cytosine deaminase (CD) suicide gene. In our in vivo studies human colon carcinoma (patient-derived tumor model Colo5734 and SW480 colon carcinoma)-bearing NMRI-nu/nu male mice received four jet injections (10 microl per injection) of the CD-gene-carrying plasmid, representing 40 microg plasmid DNA per animal. Forty-eight hours after jet-injection, treatment of tumors with 5-fluorocytosine (5-FC; 500 mg/kg ip) was started and during treatment tumor volumes were measured. Starting from day 5 of 5-FC treatment inhibition of tumor growth was seen in the CD-gene-transduced tumors compared to the respective control groups, which lasted for the entire observation time. Expression analysis at the mRNA and protein levels revealed efficient expression of the CD gene in the jet-injected tumors. Therefore, in this in vivo study jet-injection gene transfer of 40 microg CD-expressing naked plasmid DNA leads to a significant tumor growth inhibition. This study demonstrates the applicability of the jet-injection technology for in vivo gene transfer into tumors to achieve efficient tumor gene therapy.     

Intrapleural 'outside-in' gene therapy: therapeutics for organs of the chest via gene transfer to the pleura

The pleural space is an attractive site for using viral vectors to deliver gene products to the lung parenchyma, other thoracic structures and the systemic circulation. The advantages of intrapleural gene transfer using viral vectors include: (i) easy accessibility; (ii) large surface area; (iii) ability to provide high concentrations of secreted gene products to chest structures; (iv) low risk of detrimental effects of possible vector-induced inflammation compared with intravascular delivery; and (v) because it is local, lower vector doses can be used to deliver therapeutic genes to thoracic structures than less efficient systemic routes. Examples of pleural gene transfer include the use of adenovirus vectors to treat mesothelioma by transiently expressing genes that encode toxic proteins, immunomodulatory molecules or anti-angiogenesis factors. Intrapleural delivery of adeno-associated viral vectors represents an efficient strategy to treat alpha1-antitrypsin (alpha1AT) deficiency, achieving high lung and systemic therapeutic levels of alpha1AT. Intrapleural delivery of gene transfer vectors holds promise for the treatment of diseases requiring transient, localized gene expression, as well as sustained expression of genes to correct hereditary disorders requiring localized or systemic expression of the therapeutic protein.     

Progress and prospects: naked DNA gene transfer and therapy

Increases in efficiency have made naked DNA gene transfer a viable method for gene therapy. Intravascular delivery results in effective gene delivery to liver and muscle, and provides in vivo transfection methods for basic and applied gene therapy and antisense strategies with oligonucleotides and small interfering RNA (siRNA). Delivery via the tail vein in rodents provides an especially simple and effective means for in vivo gene transfer. Electroporation methods significantly enhance direct injection of naked DNA for genetic immunization. The availability of plasmid DNA expression vectors that enable sustained high level expression, allows for the development of gene therapies based on the delivery of naked plasmid DNA.