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Retroviral vectors based on Moloney Murine Leukemia Virus (MLV), described more than 15 years ago (1), first entered clinical trials in 1990 (2). Since then, a greater understanding of the basic retrovirus biology and how it relates to the production of recombinant retroviral vectors has helped overcome some of the initial hurdles, including low titer, outbreak of replication-competent retrovirus (RCR), and complement inactivation of vector (3-6). Subsequently, retroviral vectors have become a very safe and powerful tool for efficient in vitro, ex vivo, and in vivo gene transfer for proliferating cells. Several refinements in recombinant MLV vector technology were necessary, particularly for clinical trials using systemic administration of very high vector doses (>1010 transducing units), thus necessitating efficient large-scale manufacture of safe and high-titer retroviral vector preparations. [ABSTRACT FROM AUTHOR]

Original standard protocols for the generation of recombinant adeno-associated viruses (rAAVs) have generally involved the cotransfection of 293 cells with a rAAV plasmid vector (pAAV) and a helper/packaging plasmid, followed by over-infection with a helper virus, normally an adenovirus (Ad) at low multiplicity of infection (MOI) (1). The pAAV vector contains the transgene of interest flanked by the AAV inverted terminal repeats (ITRs) and the helper/packaging plasmid contains the rep and cap genes of wild-type AAV. The drawback with this procedure is the presence of contaminating Ad helper, together with wild-type-like AAV particles in the derived virus vector stocks. This is because of recombination between the rAAV plasmid and rep and cap genes on the packaging plasmid resulting in the restoration of a wild-type virus. In addition, Ad infection can result in lysis of more than 50% of cells prematurely, releasing rAAV particles into the supernatant and therefore reducing virus particle yields from cellular extracts (2). [ABSTRACT FROM AUTHOR]

The integration of foreign DNA into the genomes of host cells is of fundamental importance for viral oncology, evolution, transgenic organisms, and gene therapy applications. Expression of foreign genes in eukaryotic cells is highly dependent upon the efficiency of integration events and the site of insertion. Integration can sometimes have detrimental effects on the host cell, such as insertional mutagenesis or activation of proto-oncogenes. For gene therapy, it would therefore be attractive to target insertion to innocuous chromosomal sites. Understanding the mechanisms for integration of foreign DNA and target site selection is crucial for approaches where long-term expression from delivered transgenes is required. [ABSTRACT FROM AUTHOR]

Retroviral vectors have become important tools in gene therapy as a result of a number of highly desirable properties, including stable genomic integration, which are rarely found with other gene delivery vectors. However, after two decades of experience with this technology, some shortcomings still remain to be solved. Retroviral vectors cannot be grown to as high titers, as for example, adenoviruses; they tend to be unstable and are sensitive to lysis by complement when transfused into patients. The search for more robust retroviral delivery systems has led to the development of hybrid viral vectors to combine the broadly estimated features of retroviral vectors with advantageous properties of another viral vector system. Chimeric systems with adeno- or alphavirus vectors have already been reported (1-3). Furthermore, herpesvirus amplicons expressing gag, pol, and env genes were shown to rescue defective lacZ retro virus in a cell line carrying a corresponding pro virus (4). This chapter is dedicated to vaccinia virus, one of the most widely used vectors in molecular and cell biology (5,6) as a chimeric carrier for small RNA viruses, such as retroviruses. The methods provided in this chapter disclose the necessary steps for the generation of such hybrid vectors and for their use to produce retroviral vector particles in packaging cells. However, the system offers a number of interesting opportunities that will require individual cloning concepts. To fully exploit the possibilities of the hybrid vector system, it is therefore advisable to consider the life cycle and the specific properties of the two vectors that are fused in this chimera. [ABSTRACT FROM AUTHOR]

Retroviral vectors developed from the murine leukemia virus (MLV) were the first and most widely used vectors for gene transfer experiments (1). Delineation of their replication strategy allowed extensive manipulation of their genome and the development of vector packaging cell lines; the latter provide a reliable tool for the production of high titer vector stocks and have been key to the popularity of MLV-type vectors (2). Because integration into the host cell genome is a sine qua non of the retroviral life cycle, MLV-type vectors were extensively used to transduce hematopoietic stem cells (HSCs) where stable genomic integration allows transmission of a gene to the differentiated progeny cells (3-5). However, a number of problems with these vectors have limited their use in the clinical setting; low transduction rates on primate and human HSCs, complete dependence on cell division for integration and silencing of the transferred gene are the major problems with the MLV-type vectors (6-8). In an effort to address these issues, two other retro viruses have been explored for their gene transfer potential; lentiviruses, like the human immunodeficiency virus (HIV) and, to a lesser extent, foamy (or spuma) viruses (9,10). Although many details of HIV biology have been unveiled owing to the extensive research on the HIV-related human diseases, relatively little is known about the foamy viruses. As a result, foamy vectors have not yet been as extensively developed as HIV and MLV-type vectors. [ABSTRACT FROM AUTHOR]

Recombinant protein expression is one of the basic elements of modern molecular biology. The completion of the sequencing of the human genome has, in fact, accelerated the need of rapid and efficient expression systems. Extensive efforts have been dedicated to the development of expression systems for bacterial, yeast, insect, and mammalian cells. The reason for using mammalian expression systems is obviously the correct posttranslational modification capacity and the most authentic environment for production of therapeutically interesting proteins. Today, it is also important that large quantities of recombinant proteins be produced and subjected to purification and structural studies to obtain high-resolution structures to aid rational drug design. [ABSTRACT FROM AUTHOR]

Semliki Forest virus (SFV), Sindbis virus, and several pathogenic encephalitis-producing viruses (e.g., Ross river virus, RRV) are members of the family Togaviridae and genus Alphavirus. A typical alphavirus is an enveloped virus with single-stranded, positive-polarity RNA genome of approx 12,000 nucleotides. The genome is capped at the 5′ end and polyadenylated at the 3′ terminus. The alphaviral genome functions directly as a messenger RNA, encoding a polycistronic protein that is cleaved into four nonstructural proteins (nsP1-4). These proteins associate with cellular factors and form so-called replicase complexes. These complexes mediate the replication of the plus-strand genome into full-length minus strands, which efficiently produce both new genomic RNAs and a second subgenomic RNA in an autocatalytic manner 1(see Fig. 1). Subgenomic RNA encodes the structural proteins that are building blocks for the alphavirus nucleocapsid. Alphavirus gene expression is transient by nature and takes place exclusively in the cytoplasm of the host cells. Replication of alphaviruses is extremely efficient with approximately 105 new virions per cell being produced with the aid of the host's own translational machinery. For review of alphaviruses, seeref. 1. [ABSTRACT FROM AUTHOR]

A number of inherited and acquired disorders can potentially be treated by gene-based therapies. To be successful, gene therapy requires efficient delivery and continued expression of the therapeutic gene in the target cell. Toward this goal, a variety of methods have been developed for delivering genes into various cell types and tissues (for reviews, seerefs. 1 and 2). Common viralbased methods utilize vectors derived from oncoretroviruses, adenovirus type 5, adenoassociated virus type 2, herpes simplex virus type 1 (HSV-1), and, most recently, lentiviruses. Among these, oncoretroviral vectors (primarily those based on Moloney murine leukemia virus) have been the most widely used to date in gene therapy applications, mainly because of their capacity to stably integrate into cellular DNA in the absence of wild-type virus (3-5). However, a major limitation of oncoretroviral vectors is their inability to transduce nondividing cells (6-9). [ABSTRACT FROM AUTHOR]

Retroviruses use homologous recombination to reverse transcribe a doublestranded copy of DNA from their RNA genome. Safety advancements in retroviral vector design include the development of packaging systems that provide all of the retroviral proteins in trans to a replication-defective gene transfer vector. The packaging component expresses viral structural proteins that are required for assembly of the virus particle and is stripped of cis-acting sequences necessary for efficient transfer and replication of the viral genome (1). These cis- acting sequences are retained within the gene transfer vector where they facilitate its encapsidation, reverse transcription, and integration. Nonetheless, this split design does not completely exclude encapsidation of other viral and cellular RNA, including that derived from the viral packaging construct. Copackaging of packaging and vector RNA genomes enables genetic recombination to occur during reverse transcription and thus, the rejoining of viral structural protein-coding sequences and cis-acting sequences of the vector. If recombinants are formed that contain replicative functions it is possible they may propagate and spread to infect other cells. Thus, the inadvertent production of replication-competent retrovirus (RCR) (2-5) constitutes the principal safety concern for the use of retroviral vectors in human clinical protocols (6-8). [ABSTRACT FROM AUTHOR]

Lentiviral vectors are promising tools for gene transfer (1-4). Like oncoret-roviral vectors, they 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, lentiviral vectors are highly efficient at infection of nondividing cells because of the presence of nuclear localization signals on several virion associated proteins, which include matrix (MA), viral protein R (VPR), and integrase (IN) in the case of human immunodeficiency virus type-one (HIV-1) (5). [ABSTRACT FROM AUTHOR]

Feline immunodeficiency virus (FIV) is an appealing candidate for viral vector development because recombinant FIV vectors efficiently transduce dividing and nondividing cells and thus provides an alternative to primate lentiviral vectors derived from simian immunodeficiency virus (SIV) or human immunodeficiency virus (HIV). FIV naturally infects diverse Felidae worldwide and feline gene therapy vectors are equipped with human-tropic envelopes and hybrid long terminal repeats (LTRs), to overcome FIV's cat-specific tropism and low activity of the FIV LTR in human cells, respectively. The simple genome organization of FIV with only three accessory/regulatory proteins facilitates vector development. Recombinant FIV vectors generate titers comparable to other lentiviral systems, and accessory proteins are not needed for high titer or efficient transduction. Compared to HIV vectors, FIV vector development is still in its infancy, but initial in vivo data in various species and tissues indicate long-term gene expression at therapeutic levels, and, therefore, FIV vectors hold great promise for gene therapy applications. Before we describe the generation of high-titer, replication-incompetent feline gene delivery systems, we will briefly discuss FIV biology and genome organization to highlight the questions that needed to be addressed during vector development. [ABSTRACT FROM AUTHOR]

Recombinant viral vectors have been used to study a variety of fundamental issues in developmental neurobiology, as well as pathogenesis and treatments for various neurodegenerative diseases. Lentiviral vectors are valuable tools for neurobiology research, because of their ability to transduce nondividing cells such as neurons, to introduce new genes into central nervous system (CNS) cells in vivo and in vitro. In this chapter, we provide a variety of detailed methods for evaluating lentiviral transduction of the CNS in the mucopolysaccharidosis type VII (MPS VII) mouse model. [ABSTRACT FROM AUTHOR]