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The goal of correcting such genetic lung diseases as cystic fibrosis could be achieved by vector-mediated gene transfer to airway epithelia. The principle of using viral vectors to complement genetic defects is well documented in cell culture and in animal models. Moreover, strategies for the successful implementation of gene transfer-based therapies for patients are beginning to emerge (1). However, the airway epithelium has evolved many defensive barriers against microbial invasion (2,3). Indeed, these same barriers likely impede gene transfer efficiency with viral and nonviral vectors (3). [ABSTRACT FROM AUTHOR]

Direct in vivo gene transfer to the central nervous system (CNS) using recombinant lentiviral vectors (rLV) has emerged as a powerful technique to overexpress various genes of interest in different neuronal populations. This interest is exemplified by the increasing number of studies using rLV vectors to evaluate therapeutic proteins to correct disorders of the nervous system (1-3) or to explore a protein's involvement in normal function or pathological processes (4). Compared to conventional trangenic techniques for overexpression, rLVs have several attractive features. For example, the level of transgene expression in cells transduced with a rLV is typically higher than obtained with knock-in techniques and is present already at 3-4 d after injection in vivo (5). Second, the expression may be directed to specific regions or cell populations depending on the anatomical location of the rLV injections as well as the design of the vector system. This may be advantageous for the study design since it allows unilateral overexpression, thereby creating an internal control in functional and morphological studies. Finally, it is considerably less time consuming than creating transgenic mouse lines. [ABSTRACT FROM AUTHOR]

Lentivirus vectors are promising tools for gene transfer (1,2). Retroviral vectors in general offer the unique advantage of stably integrating into the genome of the host cell, thus providing the basis for sustained gene expression. In contrast to the classical oncoretrovirus-derived vectors, lentivirus vectors are highly efficient at infection of nondividing cells. This unique ability is owing to the presence of nuclear localization signals on several virion associated proteins, which include matrix (MA), viral protein R (VPR), and integrase (IN) (3) and a cis-acting element termed the central polypurine tract (cPPT) (4,5). [ABSTRACT FROM AUTHOR]

The recovery of vectors that are suitable for an in vivo gene delivery has been a recurrent theme in gene therapy research over the last decade. Several challenging hurdles need to be overcome to reach such a goal. First, is a need for methods that allow the preparation of vectors at high titers and in culture systems with potential for large scale-up need to be optimized. Second, the gene transfer vectors should not be recognized by the host immune system in order to avoid inactivation. Upon delivery into gene therapy recipients, vectors should also be able to circumvent the numerous biological barriers that are likely to limit their diffusion and biodistribution in the target organism. They should, therefore, be able to recognize specifically and to penetrate cells of the gene therapy target tissue. Third, they should be able to replicate and to express a transgene in cells that are either not or only slowly proliferating, a predominant situation in vivo. Last, but not least, they should be accepted by both ethical and regulatory authorities. In this respect, the development of vectors derived from viruses that are not pathogenic to human may be preferred. [ABSTRACT FROM AUTHOR]

Lentiviral vectors combine two useful vector properties: permanent integration and transduction of nondividing cells. Replication-defective lentiviral vectors were originally derived entirely from the human lentivirus human immunodeficiency virus type 1 (HIV-1) (1,2). Application of pseudotyping with broadly tropic, physically stable envelope glycoproteins (3) permitted efficient transduction of nonlymphocytes by HIV-1 vectors in vitro and in vivo (4,5). The first nonprimate lentivirus-based vector was derived from feline immunodeficiency virus (FIV) (6). Subsequently, substantial improvements have been made in the design and capabilities of FIV vectors, and recently identified FIV elements (central polypurine tract, central termination sequence, and packaging signal) have been incorporated (7,8). This chapter will briefly present an overview of FIV vectors, describe recent improvements, and then explain practical methods for production and use. [ABSTRACT FROM AUTHOR]

Lentivirus vectors are expected to become excellent tools for gene transfer in gene therapy and functional genomics and for engineering transgenic animals. When fully developed, their use will cover a whole gamut of endeavors, from discovery to therapeutics. Because they integrate into the host cell genome and become a permanent member of the genetic makeup of the recipient cell, retroviruses are well suited for stable expression of the transgenes. This property is shared by all retroviruses, including lentiviruses (1). Apart from the poor suitability of the conventional retroviral vectors for human gene therapy, what brought the prospects of lentiviral vectors to the forefront was their ability to cause gene transfer into nondividing, quiescent cells as well as dividing cells (2). This attribute will become increasingly important as more knowledge is gained about the biology of stem cells and a more optimized technology is developed for transplantation of genetically modified cells. [ABSTRACT FROM AUTHOR]

A mammalian cornea can be maintained in vitro for days (1,2), allowing ex vivo transduction of this tissue prior to transplantation (3,4). Ex vivo transduction of the cornea is useful for studying the efficacy of gene products on inhibition of corneal neovascularization (4), amelioration of corneal inflammatory disease, or promotion of corneal wound healing in animal models. Ex vivo transduction can also be used for transfer of genes into corneal epithelial stem cells (5), thereby allowing genetically modified stem cells to be reimplanted into the limbal area (6), providing a means for long-term gene-replacement (e.g., βig-h3 gene) therapy for various inherited corneal diseases (7). [ABSTRACT FROM AUTHOR]

Mutations within genes that are specifically expressed within the retina and/or the retinal pigment epithelium (RPE) are associated with the majority of inherited retinal degenerations (1). Retinal degenerative disorders are highly heterogeneous, both genetically and phenotypically (2). Different mutations within a single gene (such as rhodopsin) can not only result in different modes of inheritance (autosomal dominant as well as recessive) of the same disease (retinitis pigmentosa), but can also result in different retinal disease (congenital stationary night blindness (CSNB) (3). Numerous mutations within the rhodopsin gene and mutations within other genes in the phototransduction pathway have been detected in patients with retinitis pigmentosa (RP), a progressive degenerative disease that leads to death of rod photoreceptors and results in loss of vision. To date, 41 forms of RP, autosomal dominant, recessive, or X-linked, have been mapped, and 21 genes have so far been identified that carry mutations responsible for the disease process (4,5). Many retinal degenerative diseases are caused by mutations within genes that are involved in the phototransduction pathway or are key structural components of the outer segments of photoreceptors and are thereby expressed exclusively in photoreceptor cells. However, when altered, genes for RPE65, RPE-retinal G protein-coupled receptor (RGR), cellular retinaldehyde-binding protein (CRALBP), and Best macular dystrophy (VMD2), which are expressed in the RPE and not in rod photoreceptors, can also cause similar retinal dysfunction. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) is a common inherited disorder affecting a variety of epithelial tissues. The disease is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) that lead to abnormal secretions, recurrent infection and inflammation, bronchiectasis, and premature death. Because airways disease is the major cause of morbidity and mortality in cystic fibrosis, gene therapy efforts have focused on luminal delivery of vector to the airways of CF patients. Retroviruses are attractive as a gene transfer vector system since integration of the wild-type CFTR cDNA into the host genome may lead to long-term expression and perhaps, a cure. However, simple retroviruses are limited as vectors for airway gene transfer by the low rates of epithelial cell proliferation in human airways (∼0.1-0.2%) combined with the traditionally low titers. Advances in vector design and production have improved titers, and the development of human and animal lentiviruses may help overcome the requirement for cell proliferation. These developments have raised hopes for retroviral approaches for treatment of CF lung disease. [ABSTRACT FROM AUTHOR]

The ability of lentivirus vectors to stably transduce postmitotic cells by genomic integration and early evidence of a capacity for retrograde axoplasmic transport, depending on the envelope pseudotype, underpin the immense potential of this vector system for neuronal transduction (1-3). The bulk of studies performed to date have focused on central nervous system (CNS) neurons despite the relative merits of peripheral nervous system (PNS) neurons as targets for gene transfer (4-6). The neural circuitry of the PNS is relatively simple, accessible, and anatomically discrete. Dorsal root ganglion (DRG) sensory neurons exemplify these properties, are readily cultured ex vivo after dissociation or as intact DRG explants, and as a consequence, have been extensively exploited as a model system by neurobiologists (7). The demonstrated ability of human immunodeficiency virus type 1 (HIV-1)-derived lentivirus vectors to stably transduce DRG sensory neurons (8) extends the utility of this model system for both basic science investigations of neuronal structure and function and for the development and evaluation of gene therapy strategies targeting PNS neuropathology. [ABSTRACT FROM AUTHOR]

Conventional ways of introducing genes into cells, such as calcium phosphate transfections and liposome or lipofectamine-mediated methods, work poorly in cardiomyocytes. They can be useful for gene reporter studies, but are of little value when a gene needs to be introduced into a large number of cardiac cells. Therefore, viral vectors are used to achieve this goal. Type 5 adenoviruses work well in cardiomyocytes, but they are hard to produce, and moreover, their immunogenicity prevents their use in long-term in vivo experiments (1). The adeno-associated virus is suitable for in vivo myocardial gene transduction because of its low or absent immunogenic potential (1), but its use for in vitro studies is limited by the low gene expression achieved (less then 10% in cardiomyocytes) (2). The classical retroviral systems are of no practical value either for studies of growth-arrested cardiomyocytes owing to the requirement of M phase for integration with host genomic DNA. Lentiviruses, however, can enter the nucleus even without mitosis (3), and recently, a new variant of third generation lentivirus ("advanced" generation) has been described (4) in which sequences of the pol gene of human immunodeficiency virus type 1 (HIV-1), central polypurine tract (cPPT) and of the posttranscriptional regulatory element of woodchuck hepatitis virus (WPRE) have been inserted. The cPPT is a cis element that has been associated with increased gene expression in growth-arrested human hematopoietic progenitor cells (4). [ABSTRACT FROM AUTHOR]

It has proven notoriously difficult to express foreign genes in primary neuronal cells in vitro. Some success has been achieved with lipid-mediated plasmid DNA transfection (1), transduction via mechanical methods (e.g., gene gun) (2,3), and infection with a variety of neurotropic viral vectors: adeno-associated virus (AAV) (4), Semliki Forest virus (SFV) (5), measles virus (6), Sindbis virus (7), and lentivirus (8). Of these methods, lentiviruses show the longest survival of infected neurons (up to 6 wk), with the least associated cytopathology. However, expression is not seen for at least 48 h postinfection, and expression levels are much lower than those resulting from Sindbis, SFV, and adenovirus. This makes lentiviruses ideal vectors for experiments in which effects of low levels of foreign gene expression are studied over long-term and less desirable for protein production or massive overexpression. We have found lentivirus impractical for acute slice recordings, as the preparations do not live long enough to demonstrate visible expression of reporter genes (e.g., green fluorescent protein [GFP]); for these cultures, AAV or SFV is preferable. In organotypic and dissociated cultures, however, neurons may be infected at any age in vitro, with a high rate of success (70 to >90% for dissociated cultures) (Fig. 1). [ABSTRACT FROM AUTHOR]

There has been much effort in isolating hematopoietic stem cells (HSC) and some confusion within the literature on what even a HSC is, with some investigators using the term inappropriately (1). A HSC is a cell capable of both self-renewal and of deriving progeny of all hematopoietic cell lineages, that is, a single HSC can regenerate erythroid, myeloid, megakaryocytic, and lymphoid lineages in a myeloablated host. Progenitor cells, on the other hand, are cells committed to one or a few lineages having a limited capacity for self-renewal. Progenitors are the progeny of HSC. [ABSTRACT FROM AUTHOR]

Adult bone marrow contains both hematopoietic and nonhematopoietic stem cells, also termed mesenchymal stem cells (MSCs), or mesenchymal progenitor cells. MSCs are cells capable of differentiating into cells of the mesenchymal lineage (1-3). These cells were previously referred to as "marrow stromal cells" or "colony-forming unit-fibroblast" (CFU-F), reflecting their origin and morphology in culture (4,5). Bone marrow stroma is the main source of these multipotent nonhematopoietic mesenchymal stem cells (6,7). MSCs derived from bone marrow can be readily isolated and expanded in vitro. Due to their ability of self-renewal and their potential to differentiate into terminal osteocytes, chondrocytes, myocytes, tenocytes, adipocytes, and neural cells in vivo and in vitro (8-11), bone marrow-derived MSCs have attracted considerable attention as potential tools for therapeutic gene transfer. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) are a heterogeneous population of bone marrow-derived antigen-presenting cells (APCs) that populate various lymphoid and nonlymphoid tissues, including the skin (Langerhans cells), lymph nodes (interdigitating and follicular DCs), spleen, and thymus. DCs are considered professional APCs, with a remarkable ability for initiating T-cell activation and the consequent immune response, but also for inducing tolerance (1-3). The regulation of these two distinct functions is not completely understood to date. Extensive recent studies have improved our understanding of DC development, differentiation, activation, and function. DCs exist as distinct subsets that differ in their lineage affiliation, surface molecule expression, and biological function. Each of these types of DCs can present antigens in either an immunogenic or tolerogenic manner according to their maturation state and antigen capture properties. Hence, DCs either induce the appropriate immune response to pathogens or prevent autoimmune reactivity, and could thus be useful for manipulating the immune system by cellular immunotherapy. [ABSTRACT FROM AUTHOR]

Lentiviral vectors derived from human immunodeficiency virus (HIV) have demonstrated exceptional promise as tools for gene therapy applications (1,2), but have also raised safety concerns because of potential creation of replication-competent lentivirus (RCL) by uncontrolled recombination (3-6). In order to be safe for clinical use, vector preparations must be formally and extensively tested to show they are absolutely free of RCL. Various methods have been proposed to detect RCL, all of which report high specificity and sensitivity and all of which can discriminate between replication-defective virus and RCL. There is currently a compelling need for standardization and validation of this method. [ABSTRACT FROM AUTHOR]

The manipulation of gene expression in primary lymphocytes has been shown to have potential therapeutic applications for immunodeficiencies (1-3), and cancer immunotherapy (4,5). Efficient gene transfer is an absolute prerequisite for developing successful clinical gene therapy protocols. At present, viral gene transfer vectors based on murine leukemia virus (MLV) are used in most clinical trials and have been used successfully to transduce cells from various tissues, including cells of hematopoietic origin (6-8). However, transduction of primary lymphocytes has been limited primarily by the requirement that the cells be proliferating for stable retroviral integration (9). Although potent induction of cell proliferation in vitro has been used to overcome this problem (1,10-12), it has now become evident that activation stimuli such as antibodies and mitogens result in alteration of the CD4/CD8 ratios and T-cell receptor (TCR) repertoire composition of T lymphocytes and induce changes of their cytokine secretion profile, thus precluding preservation of the pool of naïve lymphocytes and their functional integrity (13,14). [ABSTRACT FROM AUTHOR]

Lentiviruses are members of a subgroup of enveloped Retroviridae. Lentivirus-based gene delivery vectors have gained popularity in gene therapy field because of their notable potentials in delivering and integrating transgenes into both mitotic active and inactive cells in vitro and in vivo, a characteristic that overcomes many of the barriers of therapeutic gene therapy application. In the past few years, different lentiviral vector systems have been developed based on primate and nonprimate lentiviruses, including human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), equine infectious anemia virus (EIAV), and feline immunodeficiency virus (FIV) (seerefs. 1-4). However, HIV-1-based lentiviral vector remains the best-studied and well-developed lentiviral vector system that has shown high transduction efficiencies targeting many human and experimental animal cell types, including stem cells (embryonic and adult) (5-10), terminally differentiated somatic cells (i.e., neurons) (7,11,12), muscles (7,13,14), skin (15), liver (13,16,17), islet (18,19), lung epithelium (20,21), retina (22,23), primary T lymphocytes (24,25), and fetal tissues (21,26) (Table 1). [ABSTRACT FROM AUTHOR]

Lentiviral vectors have traditionally been produced in human embryonic kidney 293T cells (1), involving transient transfection procedures that were originally established for the production of retroviral vectors based on Moloney murine leukemia (MoMLV) virus (2). Simian virus 40 (SV40)-transformed African green monkey kidney (COS-7) cells (3) and human TE671 rhabdomyosarcoma cells (4) have also been used to generate human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors (5-7), but titers obtained in 293T cells are generally higher than those observed in other cell lines. Page et al. (5) were the first to describe an HIV-1-based vector harboring a selectable transgene. The HIV-1 genome was rendered replication defective in this study by replacing the envelope (Env)-encoding gp160 sequence with a guanine-phosphoribosyl transferase (gpt) gene driven by the SV40 early promoter. Transient cotransfection of COS-7 cells with this env-deleted vector and a gp160 expression vector resulted in packaging of the defective HIV-gpt genome into infectious virions. Upon infection of susceptible cells, the gpt drug resistance gene was transmitted and expressed, allowing transduced cells to be selected in mycophenolic acid (5). Landau et al. (8) subsequently demonstrated that expression of heterologous Env proteins, including the MoMLV Env and the human T-cell leukemia virus type I (HTLV-I) Env, in cells transfected with the HIV-gpt vector resulted in the production of vector pseudotypes capable of infecting human as well as murine cells with titers reaching 105 colony-forming units (CFU) per mL. [ABSTRACT FROM AUTHOR]

The accurate quantification of functional vector particles within a given vector preparation or stock is of fundamental importance to virtually all potential gene transfer applications for any viral vector of interest. This accuracy is especially critical if anticipated clinical applications will require scale-up of vector production or if multiple different vector stocks will be required for serial transductions of target cell populations over the course of a series of experiments. Standard notations designating the quantity of functional particles in a vector preparation include infectious units (i.u.) per milliliter and transducing units (TU) per milliliter (1). These units are interchangeable and represent the number of functional vector particles per volume of vector containing supernatant. The quantification of infectious or transducing units per unit volume allows transductions to be conducted based on the number of vector particles added per target cell and will therefore be standardized yielding more reproducible results. The numerical relationship between vector particles utilized per single target cell is designated as the multiplicity of infection (MOI). Establishing an appropriate MOI for a given transduction will, in part, depend on the desired transduction efficiency or, to an extent, the approximate number of chromosomal integrants desired per cell. Accurate vector stock titers will allow for the establishment of controlled transduction conditions and will, therefore, reduce experimental variability and simplify the interpretation of results obtained. [ABSTRACT FROM AUTHOR]

Transducing lentiviral vectors that transfer the exogenous gene(s) into target cells are typically defective for viral replication because at least some of the trans-acting sequences encoding the viral proteins have been deleted. To propagate vector virus, the viral proteins are supplied either by transient transfection of plasmids expressing the viral proteins or by using packaging cell lines, which contain the viral expression plasmids stably integrated into their cell genome (1). Packaging cells provide a safety advantage for propagating retroviral vector virus because they reduce the chance of recombination that might result in the production of replication-competent (RC) virus. During development of the packaging cells, the expression plasmids can be introduced sequentially at different times so that they will be located at different places in the cell genome. In addition, avoiding cotransfection of all of the trans-acting components simultaneously, a recombinagenic step (2), further reduces the likelihood of recombination. [ABSTRACT FROM AUTHOR]

Although a member of the lentivirus group, the equine infectious anemia virus (EIAV) was the first nonplant virus discovered in the first decade of the 20th century (1), lentiviruses were considered as rather mysterious viruses until the isolation of the human immunodeficiency virus type 1 (HIV-1) occurred at the beginning of 1980s. Lentiviruses are enveloped viruses carrying two copies of single-strand positive (i.e., codifying) RNA and are considered the ethiologic agents of acquired immunodeficiency syndromes for a broad range of animal species, such as humans, primates, cats, horses, sheep, and goats. Such syndromes develop in multiorgan diseases and share a long period of incubation (with viral persistence despite a potent immunological response) and a fatal outcome. The name lentiviruses (from Latin, lenti, slow) originated from the uniquely prolonged incubation period (i.e., from months to years) needed for the infecting virus to induce the disease, a feature joining the most popular lentivirus, HIV-1, with a large number of nonprimates lentiviruses. Lentiviruses belong to the Lentiviridae subfamily of the Retroviridae family, which also includes the Oncoviridae, for the most part viruses inducing cell transformation, and the Spumaviridae, viruses establishing persistent as well as nonpathogenic infections (a deeper treatment of this topic can be found in ref. 2). [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus (HIV)-derived lentiviral vectors can integrate into the genome of dividing and nondividing cells, in vitro as well as in vivo (reviewed in refs. 1-3). Lentiviral vectors are particularly efficient in transducing multipotent stem cells, such as hematopoietic stem cells (HSC), without compromising their self-renewing and organ repopulation capacity upon transplantation in vivo (4-6). This is a crucial advantage over oncoretroviral vectors derived from the Moloney murine leukemia virus (MoMLV) since transplantation of genetically modified stem cells (hematopoietic, neural, or epidermal) is a potential therapy for a variety of genetic and acquired disorders, including diabetes, multiple sclerosis, cancer, and acquired immunodeficiency syndrome (AIDS). Several years of research have drastically improved both design and packaging of lentiviral vectors, minimized the use of HIV structural and regulatory sequences in both the transfer and the packaging constructs and have virtually abolished the safety concerns originally raised by the idea of transducing human cells with a derivative of HIV (reviewed in ref. 3). These vectors are now a very promising alternative to transduce transplantable stem cells ex vivo and such postmitotic tissues as the central nervous system (CNS) or liver in vivo. [ABSTRACT FROM AUTHOR]