Showing papers in "New Comprehensive Biochemistry in 2003"

Locus control regions

Abstract: Publisher Summary Locus control regions (LCRs) are regulatory elements which are operationally defined by their ability to enhance the expression of linked genes in a tissue-specific and copy number-dependent manner at ectopic chromatin sites. The LCRs are composed of a series of DNase I-hypersensitive sites (HS) usually located near the genes they control. Each HS consists of a core element and flanking sequences. The core elements usually are approximately 200 nucleotides long and are composed of arrays of multiple ubiquitous and lineage-specific transcription factor binding sites. In this chapter, the β-globin locus LCRs of several species have been characterized and compared to the human LCR. The general organization and spatial array of the individual HSs has been conserved. The goat and human LCR share 6.5 kb that are roughly 60% conserved. The homology is 80–90% within and adjacent to the individual HSs. The mouse and human LCRs have an identical organization, although insertion of repetitive sequences within this region during evolution has altered the distances between the HSs. There are extended regions of significant homology, with the highest conservation within and adjacent to the individual HSs. The properties of LCRs given in the chapter are enhancer activity, copy number-dependent gene expression, timing and origin of DNA replication, and histone modification. The molecular mechanism by which the LCR functions over a long distance is still speculative. Three models have been proposed: looping, tracking, and linking. The mechanisms of globin–gene activation by the LCR are discussed in this chapter. Several LCR or LCR-like elements have been described in mammals including human, mouse, rat, chicken, rabbit, sheep, and goat. All of these LCRs are comprised of varying numbers of tissue-specific DNase I-HS.

Use of scaffold/matrix-attachment regions for protein production

Abstract: MARs (Matrix Attachment Regions), also called SARs (Scaffold Attachment Regions), are 300–3000 bp long DNA elements proposed to play a role in nuclear and chromosomal architecture. They were proposed to attach chromatin to proteins of the nuclear matrix and thereby partition the eukaryotic genomes into independent chromatin loops. Because of their co-localization with transcription units and regulatory elements in genomes, MARs have been implicated in the regulation of gene expression. For instance, MARs were shown to control gene expression by facilitating interactions between DNA activating complexes and genes, and by controlling chromatin accessibility. The ability of MARs to protect transgenes against transcriptional silencing effects has been used to augment expression of heterologous genes. This review provides a possible explanation on the function of MARs and presents recent data on their use to increase protein production.

Architecture and utilization of highly expressed genomic sites

Abstract: Publisher Summary A major reoccurring problem in molecular biology, bioengineering, and gene therapy is the variable expression of transgenes. One solution to this problem is sought in the use of nonviral episomal (extrachromosomal) vectors that replicate and segregate in synchrony with the host cell. Although progress has recently been reported, the development of this promising system is still in its infancy. If a stable long-term expression is desired, then integrating systems remain standard. According to conventional transfection protocols, integration occurs into random chromosomal loci and at variable copy numbers, which together cause unpredictable gene expression characteristics because of position-dependent inactivation and repeat-induced silencing processes. To circumvent problems of this kind, methodologies have been developed whereby the gene of interest (GOI) can be inserted into a pre-characterized chromosomal site at the location of a unique genomic tag. Retroviral vectors are considered particularly useful vehicles for a GOI because the retroviral integration machinery is well suited to introduce a single intact copy into a subclass of apparently favorable genomic sites. The chapter discusses the architecture of loci enabling stable and high-level expression, the methods for their characterization, and the principles which can be applied to guide a transgene into a characterized site with known properties.

Gene transfer and gene amplification in mammalian cells

Abstract: Publisher Summary This chapter discusses some aspects of gene transfer and amplification in dihydrofolate reductase-deficient ( dhfr -deficient) Chinese Hamster Ovary (CHO) cells, one of the most popular mammalian cell hosts for recombinant protein production. The observations made and conclusion drawn have value also for work with other mammalian cell hosts for recombinant protein production. Expression vectors containing the target gene are used in combination with a functional dhfr expression cassette either on the same or on a separate vector. Few firmly established insights have been gained about the integration of transfected DNA into the genome of the host cell, but it appears that mitotic activity of the host cell population during the transfection procedure is an important parameter. Selection and identification of recombinant cells is performed using the DHFR phenotype. Integration into a chromosomal site within the nucleus is random and rare, usually guaranteeing a singular event. Recombinant cell clones might vary with respect to the stability within the primary locus of integration. Profound effects on transcription rates and on the eventual fate of transfected DNA are exerted by the genomic DNA environment at the integration site. Identification of highly productive cell lines requires screening of candidate clonal cell populations. In spite of many unanswered questions, CHO cells have been extremely successful as a reliable source of large quantities of high-value proteins. No other cell line is available that has been studied to the same extent as CHO cells with respect to a multitude of parameters and characteristics. Though many details remain to be deciphered about gene transfer and amplification in mammalian cells, further study of CHO cells surely will lead to an improved understanding of these complex mechanisms.

Vectors for gene expression in mammalian cells

Abstract: Publisher Summary The achievement of robust and regulated protein production in mammalian cells is a complex process that requires careful consideration of many factors, including transcriptional and translational control elements, RNA processing, gene copy number, mRNA stability, the chromosomal site of gene integration, potential toxicity of recombinant proteins to the host cell, and the genetic properties of the host. Gene transfer into mammalian cells may be effected either by infection with virus that carries the recombinant gene of interest, or by direct transfer of plasmid DNA. This chapter discusses the molecular architecture of non-viral vectors for high-level protein production. Virus-based vectors for gene therapy, protein production, vaccine development and other applications are summarized in a table and described.

Metabolic engineering of mammalian cells for higher protein yield

Abstract: Publisher Summary The advent of recombinant DNA technology enabled heterologous gene expression in living cells and there large-scale production of protein therapeutics. Many protein pharmaceuticals are produced using bacterial and yeast cells because of their rapid growth and high expression levels. However, many of the most promising protein therapeutics require post-translational modifications such as glycosylation for full therapeutic efficacy. Therefore, mammalian cells are rapidly becoming the standard for industrial production of pharmaceutical proteins. Initial metabolic engineering strategies to improve mammalian production cell lines and reduce production costs focused on adaptation of mammalian cells for growth in suspension and serum,or even protein-free media. However, this strategy is time-consuming, unpredictable and difficult to standardize. Most cell lines currently used for biopharmaceutical manufacturing contain a constitutive expression unit stably integrated in the target chromosome. However, the transcriptional activity varies depending on the cellular levels of the relevant transcription factors and on the chromatin structure at the integration site. The dominance of illegitimate recombination (IR) in immortalized cell lines results in rare transgene integrations randomly distributed on the target chromosome. In biopharmaceutical manufacturing a high proliferation rate of production cell lines was long considered desirable since improved process yields often resulted from an increase in cell number. However, current research interests are focused on studying production under proliferation-controlled conditions. Cells from higher organisms are capable of committing suicide by initiating a highly conserved molecular program known as “apoptosis.” This chapter discusses the processes employed in counteracting suicidal tendencies; i.e., preventing cell death in mammalian cell cultures. Third generation metabolic engineering—multiregulated multigene metabolic engineering— is also described in the chapter.

Protein production by large-scale mammalian cell culture

Abstract: Publisher Summary In the past two decades, more than a dozen recombinant proteins have been approved and successfully commercialized as therapeutic medicines. Currently, over a hundred recombinant protein molecules, including humanized monoclonal anti-bodies, cytokines, and growth hormones, are in clinical trials, targeting a variety of diseases such as cancer, psoriasis, asthma, and viral infections. These therapeutic proteins offer patients new treatment options that are more effective, safer, and/or more convenient than traditional treatments. The typical dose for therapeutic proteins falls in the range of 20–500 mg. In combination with chronic usage and/or a large patient population, this translates into production requirements that can exceed 100 kg/annum in some cases. The current demand based on rapid and broad acceptance in the medical community exceeds the manufacturer's existing production capacity, and a large mammalian cell culture facility with several bioreactors at a scale greater than 10,000 liters is being constructed. Development of such a large-scale process, construction of the facility, and factory startup are no small tasks, which may take more than five years and need to be conducted according to regulations by agencies such as the US Food and Drug Administration (FDA). Large-scale mammalian cell culture technology is a comprehensive piece of art that only a handful of companies have been able to practice in routine manufacturing. With more than one hundred protein molecules in various stages of clinical development, the need for large-scale mammalian cell culture will certainly increase dramatically in the years to come. In this chapter, various process aspects of state-of-the-art large-scale mammalian cell culture technology for protein production have been briefly reviewed.

Virus-based vectors for gene expression in mammalian cells: Coronavirus

Abstract: Publisher Summary The coronavirus and the torovirus genera form the Coronaviridae family, which is closely related to the Arteriviridae family. Both families are included in the Nidovirales order. Recently, a new group of invertebrate viruses, the Roniviridae, with a genetic structure and replication strategy similar to those of coronaviruses, has been described. This new virus family has been included within the Nidovirales. Coronaviruses have several advantages as vectors over other viral expression systems: (1) coronaviruses are single-stranded RNA viruses that replicate within the cytoplasm without a DNA intermediary, making integration of the virus genome into the host cell chromosome unlikely, (2) these viruses have the largest RNA virus genome and, in principle, have room for the insertion of large foreign genes, (3) a pleiotropic secretory immune response is best induced by the stimulation of gut-associated lymphoid tissues, (4) the tropism of coronaviruses may be modified by manipulation of the spike (S) protein allowing engineering of the tropism of the vector, (5) non-pathogenic coronavirus strains infecting most species of interest (human, porcine, bovine, canine, feline, and avian) are available to develop expression systems, and (6) infectious coronavirus cDNA clones are available to design expression systems. Within the coronavirus two types of expression vectors have been developed: one requires two components (helper–dependent expression system) and the other a single genome that is modified either by targeted recombination or by engineering a cDNA encoding an infectious RNA. This chapter focuses on the advantages and limitations of these coronavirus expression systems, the attempts to increase their expression levels by studying the transcription-regulating sequences (TRSs), and the proven possibility of modifying their tissue and species-specificity.

Chromatin insulators and position effects

Abstract: Publisher Summary Most mechanisms of stable gene transfer in mammalian cells, including methods of physical transfection and virus transduction, require chromosomal integration This allows for the stable and faithful transmission of the transferred gene to all of the progeny of the targeted cell However, once integrated, expression of the transferred gene is subject to the influence of surrounding chromatin, a phenomenon known as “position effects” Because chromosomal integration is generally random and the majority of the mammalian genome is often in the form of silent and condensed heterochromatin, position effects generally manifest as the partial or complete loss of expression There are several potential approaches to overcoming the negative influence of position effects on the expression of randomly integrated genes Such effects can be mitigated through the use of appropriate transcriptional promoters or other cis -regulatory elements such as enhancers, matrix-attachment regions, and locus control regions (LCRs) Another approach involves the use of homologous recombination to integrate the transferred gene into a specific site where the surrounding chromatin can support the desired pattern of expression A growing body of literature has emerged on the use of an alternative class of cis -regulatory elements, known as “chromatin insulators,” as a means of overcoming the influence of position effects on the expression of genes transferred in mammalian cells These are naturally occurring DNA elements found in a diverse range of species which function as boundary elements to separate differentially regulated chromosomal loci This chapter discusses the problems of position effects as they apply to gene transfer and expression in mammalian cells, the properties of chromatin insulators which make them useful for abrogating these position effects, and several examples where chromatin insulators have been used successfully for this and other purposes

Pathways and functions of mammalian protein glycosylation

Abstract: Publisher Summary Protein glycosylation is a prevalent post-translational modification in eukaryotic cells, occurring on more than half of all proteins. The study of protein-linked glycans is a crucial adjunct to current efforts in proteomics and functional genomics. There are three families of protein-linked glycans categorized on the basis of the type of amino –acid side chains through which protein attachment occurs: those linked through asparagine side chains (the ‘‘N-linked’’ oligosaccharides), those linked through hydroxylated amino acid side chains (for example, the ‘‘O-linked’’ oligosaccharides linked through the side chains of serine or threonine), and finally those linked through the C-terminal amino acid of a protein (the glycosylphosphatidyl-inositols (GPIs)). A considerable investment of cellular resources is ultimately responsible for supporting these elaborate pathways of protein glycosylation. This investment includes not only the enzymes that process the oligosaccharides of nascent peptide chains but also the cellular systems responsible forthe synthesis and transport of donor substrates (nucleotide sugars and lipid-linked monosaccharides) or information-decoding lectins to specific subcellular locales. Thus, the glycosylation apparatus of the cell extends well beyond the set glycosyltransferases and hydrolases that execute biosynthesis. This chapter covers the biosynthetic pathways for each of the three major types of protein-linked glycans and reviews the factors that specify what glycan structures are ultimately attached to a given protein. It then concludes with a brief discussion of the emerging functional roles for protein-linked glycans that ultimately justify the large cellular investment in protein glycosylation.

Reporter genes for monitoring gene expression in mammalian cells

Abstract: Publisher Summary The advent of reporter gene technology has greatly facilitated the knowledge of the mechanisms of many cellular processes Reporter genes encode proteins with phenotypic properties that are both distinct (from the system being studied) and conveniently monitored. Linkage of the cellular activity being assayed to the phenotypic expression of the reporter is accomplished by the fusion of appropriate DNA sequences to the reporter gene. These sequences are either regulatory in nature, typically being responsive to the cellular event under examination, or structural, encoding proteins that mediate such an event. After introduction of the chimera into cells, the qualitative or quantitative activity of the cellular event is extrapolated from the expression of the linked reporter gene product. Application of reporter–gene technology to mammalian cells was introduced in 1982 with the development of plasmid vectors, encoding the bacterial enzymes chloramphenicol acetyltransferase (CAT) or β-galactosidase (β-gal), to study eukaryotic gene regulation. As in these initial studies, reporter genes have been traditionally used to characterize and dissect transcriptionally active regulatory regions. Although characterization of cis-elements remains a common usage, reporter gene technology has attained increasing levels of sophistication and is now used in numerous other applications: characterization of transcription factors and associated proteins such as co-activators, delineation of signal transduction pathways, identification of protein–protein interactions, determination of cell fates, visualization of cellular trafficking, high-throughput screening of chemicals for adverse or therapeutic effects, and optimization and monitoring of DNA delivery systems. Numerous genes, both of prokaryotic and eukaryotic origin, have been proposed for use as reporter genes in mammalian cells. Only a handful of these, however, are widely used in this capacity. Aside from the obvious characteristic that reporter gene products cannot be toxic, the commonly used reporters share two other features: (1) the gene products exhibit phenotypic characteristics that are unique (i.e., foreign to mammalian cells) or can be easily distinguished from any similar endogenous activity, and (2) methods for detection of the reporter are sensitive, generally quantitative, and exhibit a broad linear dynamic range; they are easy to perform, reproducible and reasonably cost-effective. In terms of the reporter genes themselves, the single most significant development since that time is the introduction and evolution of green fluorescent protein (GFP) as a powerful and multi-faceted reporter protein. The chapter discusses this system as well as some of the other reporter gene systems.

Methods for DNA introduction into mammalian cells

Abstract: Publisher Summary The development of methods for the introduction of DNA into cultured cells, especially mammalian cells, has proceeded in parallel with advances in molecular cloning techniques. The process of introducing DNA into vertebrate cells is generally referred to as “transfection,” although some authors have referred to the same process as “transformation,” by analogy with DNA transfer in prokaryotes. In this chapter, the term transfection is used to avoid confusion, as transformation can have a distinct meaning with regard to the growth and the morphologic state of mammalian cells. Ideally, a transfection efficiency of 100% (all target cells acquire and express the introduced DNA) associated with minimal toxicity (all cells survive the procedure) is desired. Although a number of transfection methods have been described, virtually all fall short of these ideals. Increased transfection efficiency often correlates with increased toxicity, necessitating a tradeoff. This chapter reviews the most commonly encountered methods, with consideration of their relative merits both in principle and in practice. The principal barriers to successful transfection are identified and discussed, as further research in this area will likely lead to improved methods in the future. Transfection methods can be grouped simplistically into three categories: physically mediated delivery, chemically mediated delivery and biological vector-mediated delivery of nucleic acid. Although mammalian cells have been transfected with nucleic acids from a variety of sources, including total genomic DNA and RNA, the vast majority of experiments involve DNA sequences that have been subcloned and propagated in Escherichia coli .

Virus-based vectors for gene expression in mammalian cells: Semliki Forest virus

Abstract: Publisher Summary Among alphaviruses the most popular members developed into expression vectors are Semliki Forest virus (SFV), Sindbis virus (SIN) and Venezuelan equine encephalitis virus (VEEV) The most commonly used SFV expression system is based on two plasmid vectors: the expression vector contains the SFV non-structural and the foreign genes and the helper vector harbors the SFV structural genes In vitro-transcribed RNA molecules from both vectors are co-transfected into BHK-21 cells, where immediate synthesis of non-structural SFV proteins occurs The third version of SFV vectors is the so called DNA-layered vector, where a cytomegalovirus (CMV) promoter or another eukaryotic RNA polymerase II type promoter is utilized to drive the transcription of self-amplifying SFV replicon vectors A fourth type of SFV expression system has been developed where both in the expression vector and the helper vector the SP6 RNA polymerase promoter was replaced by an RNA polymerase II-dependent promoter Co-transfection of SFV DNA expression and helper vectors generated titers of 106 particles/ml, which is approximately 100–1000 lower than using RNA-based SFV vectors This chapter discusses the use of replication-deficient vectors for gene expression both in vitro and in vivo, the expression studies on topologically different recombinant proteins, the use of various mammalian cell lines, and the scale-up for large-scale protein production Descriptions of applying SFV vectors for primary cells in cultures, for injection of organotypic hippocampal slices and in vivo expression, recent developments in using SFV vectors for gene therapy, SFV vectors used for retrovirus particle production and applications of both SFV particles as well as nucleic acid vectors (naked RNA and layered DNA vectors) are described the engineering of novel less-cytotoxic, temperature-sensitive and down-regulated SFV vectors as well as potential novel applications are discussed in the chapter

Protein synthesis, folding, modification, and secretion in mammalian cells

Abstract: Publisher Summary Protein synthesis in eukaryotic cells is a complex process dependent upon numerous mechanisms to ensure the successful production and targeting of proteins. Most protein synthesis begins in the cytoplasm, with the exception of a small number of mitochondrial-encoded proteins that are synthesized within the mitochondria. Proteins destined for the mitochondria, peroxisomes, and nucleus are fully translated in the cytosol and delivered post-translationally to their final destinations. Post-translational modifications can affect the half-life of a protein in the extracellular environment and may be required for recognition by receptors, cofactors, and/or substrates. Most of the pivotal processing events are localized to the ER, including signal peptide cleavage, asparagine-linked oligosaccharide addition and modification, and post-translational modification of specific amino–acid residues. These modifications promote the correct formation of final tertiary or quaternary structures before transit of proteins through the remainder of the secretory pathway. The machinery that directs post-translational modifications recognizes specific structural and/or sequence determinants within the polypeptide backbone. The efficiency of these reactions is determined by the host –cell enzymatic repertoire, the availability of cofactors, and the character of the polypeptide to be modified. Investigators have employed numerous techniques to evaluate the role of post-translational modification in protein function. This chapter discusses the mechanisms involved in the biosynthesis, post-translational modification, and transport of proteins through the secretory pathway.

Virus-based vectors for gene expression in mammalian cells: Baculovirus

Abstract: Publisher Summary The baculoviruses comprise a diverse group of lytic viruses with large double-stranded DNA genomes that infect arthropod species. Baculovirus vectors are, for the most part, derived from the Autographa californica nuclear polyhedrosis virus (AcMNPV), which infects cells from lepidopteran species. In the nearly 20 years since the baculovirus expression system was first introduced in 1983, these vectors have been used to express hundreds of recombinant proteins in insect cells. Insect cells carry out similar post-translational modifications to proteins as mammalian cells, providing a decided advantage over expression of proteins in bacterial systems. Since their introduction the vectors have been continuously engineered to make virus construction easier and more accessible to a wide spectrum of laboratories. In recent years, recombinant baculovirus vectors modified to contain mammalian cell-active promoters have been developed. These viruses can enter a wide variety of mammalian cells and direct the expression of recombinant proteins. The many advantages of this viral gene delivery system, combined with the inherent inability of the virus to replicate in mammalian target cells, make it a good choice for a wide variety of mammalian gene expression applications.

Virus-based vectors for gene expression in mammalian cells: Retrovirus

Abstract: Publisher Summary High-level expression of proteins in animal cells has been very informative in studies of protein and cellular function and in many cases has relied on virus-derived expression vectors. Many viruses have evolved to maximize expression of their proteins in host cells and are therefore a good starting point for construction of efficient expression vectors. Retroviruses are diploid, single-stranded, positive-sense RNA viruses. As an obligate step of the retrovirus life cycle, the RNA genome is converted into DNA and then integrated into the host-cell chromosome in the form of provirus. The provirus replicates as the host cell chromosome replicates and is transmitted to all progeny cells. This ability of retroviruses to stably introduce new genetic information into the target cells led to the development of retroviruses as vehicles for the stable transfer of genes. Retroviral vectors have been used for a variety of experimental applications, including insertional mutagenesis, cell lineage studies, the creation of transgenic animals and the expression of foreign genes into mammalian cells, both in vitro and in vivo as documented by the majority of gene therapy clinical trials. An ideal vector should guarantee not only high efficient gene transfer but also an appropriately regulated and stable gene expression from a safely integrated provirus. Currently, efforts are being devoted to achieve these goals. This chapter focuses on the recent progress on retroviral vector design and applications.

Virus-based vectors for gene expression in mammalian cells: Lentiviruses

Abstract: Publisher Summary Lentiviruses, represented by the human immunodeficiency virus type 1 (HIV-1) are retroviruses that possess complex genomes and a finely regulated mode of replication. These viruses share the common feature of being able to infect post-mitotic cells of the monocyte/macrophage lineage; a phenomenon closely related to their biology and induced pathologies. Gene therapy vectors based on lentiviruses enable the generation of a gene delivery system that combines features of vectors derived from murine oncoretroviruses (i.e., large coding capacity and stable integration of the transgene into host-cell genetic material) and the capability of transducing non-dividing cells. As such, vectors derived from HIV-1 have been designed and the proof of principle of stable transgene delivery in non-dividing cells has been established in a wide variety of systems. The major hurdle that prevents the progression of the HIV-1-based vector system to the clinic for evaluation of therapeutic potential is the association of the parental virus with an incurable and still largely fatal disease in humans. An alternative approach to the construction of HIV-1-derived vectors, is the use of viruses derived from less pathogenic human immunodeficiency virus type 2 (HIV-2) and from simian immunodeficiency virus (SIV). Non-primate lentiviral vector systems derived from feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), and visna/maedi virus (VMV) have also been described in the chapter. However, the impressive achievements accomplished with HIV-1-based vectors have largely overshadowed the characterization of these other lentiviral vector systems. Therefore, this chapter focuses on HIV-1 derived vectors to exemplify the basic design and improvements relevant to the development of a safe and efficient lentivirus-based gene delivery system.

Inducible gene expression in mammalian cells

Abstract: Publisher Summary Artificial control systems for adjusting transgene expression in mammalian cells, animals and eventually humans have generated tremendous impact on different areas of modern biomedical engineering ranging from basic gene-function analysis, drug discovery, drug testing in animals, the design of animal-based human disease models and biopharmaceutical manufacturing to gene therapy and tissue engineering strategies. Heterologous gene regulation systems have become an integral part of current spearhead therapeutic technologies focusing on production and delivery of therapeutic proteins where they are needed in the human body. Only a few of these systems have the assets for human therapeutic use including absence of any interference with endogenous regulatory networks, and graded as well as rapid response characteristics showing low basal and high maximum expression levels following administration of a clinically licensed drug (e.g., antibiotics, immunophilins and steroid hormones). The chapter focuses on these human-compatible systems that are currently competing in preclinical studies for optimal performance in adjusting expression of a single therapeutic (model) gene, (e.g., erythropoietin, insulin or human growth hormone). Recent initiatives to combine several compatible heterologous gene regulation systems have exemplified that complex artificial gene control configurations such as regulatory cascades and networks will develop in the next years from concept studies to a therapeutic reality. Such multiregulated multigene metabolic engineering will enable optimal integration of next-generation gene interventions in endogenous proliferation-, differentiation- and apoptosis-regulatory networks to achieve cell phenotypes designed to improve the understanding and therapy of currently untreatable human diseases. This chapter discusses different human-compatible heterologous gene regulation systems, their adaptation to specific expression configurations, and their potential to be integrated into higher order control systems to achieve next-generation gene therapy and tissue engineering strategies.

Intracellular targeting of antibodies in mammalian cells

Abstract: Publisher Summary Antibodies produced by the humoral immune system are so diverse that they are essentially able to bind target molecules of any nature whether they are proteins, nucleic acids, carbohydrates, or lipids. While diverse, each individual antibody produced maintains high specificity and affinity to its antigen. Based on their unique properties, antibodies have been widely used as powerful tools in diagnostic and therapeutic applications as well as in basic biomedical research. It is now possible to manipulate genes encoding antibodies into different forms utilizing recombinant DNA technology. Antibodies can be expressed not only as full-length intact forms but can also be expressed as antigen-binding domains such as Fab fragments, consisting of the entire light chain and partial heavy chain; as single-chain variable region fragments (sFv), consisting only of the heavy and light variable regions linked by a short interchain linker of usually 15 amino acids; or even as domain antibodies consisting of only the heavy chain variable region. When fused to well-characterized intracellular protein localization/trafficking signal peptides, antibodies can be expressed in different subcellular compartments depending on the trafficking signals that are used. Intracellular antibodies, termed “intrabodies,” represent a new family of molecules that can be expressed within the context of a cell to define or mediate function(s) of a particular gene product. Since its first applications a decade ago, intrabody technology has been utilized in a variety of research areas such as signal transduction, cancer, neurodegenerative disease as well as AIDS, and may hold great potential in functional genomics as well as in gene therapy applications. This chapter discusses the concept and principles of intrabody technology, and the critical parameters that have been identified that result in an effective intrabody molecule as well as strategies in the selection of functional intrabodies

Virus-based vectors for gene expression in mammalian cells: Adeno-associated virus

Abstract: Publisher Summary This chapter discusses the biology of adeno-associated virus (AAV). AAV is a non-pathogenic and replication-defective member of the parvoviridae family. AAV has no etiological association with any known diseases. Its propagation requires the co-infection of an unrelated virus, such as adenovirus or herpesvirus, to provide essential helper functions. Recombinant AAV vectors can be produced at very high titers (more than 10 13 viral particles/ml) by several different methods. All these methods share the same three essential components: (1) AAV vector component that contains the foreign transgene(s) flanked by the 145-bp AAV inverted terminal repeats (ITRs). (2) AAV replication gene (Rep) and AAV capsid gene (Cap) genes that provide non-structural and structural proteins for vector DNA replication and packaging. (3) Helper functions from adenovirus or herpesvirus that facilitate efficient AAV propagation. When the three components above are introduced either transiently or stably into a suitable host cell, such as human 293 or HeLa cells, the AAV vector DNA is replicated and packaged into viral particles. AAV vectors have been increasingly used for gene transfer and gene therapy, particularly in vivo , because of the safety, high efficiency and long-term gene transfer by the vector system. In vitro and in vivo gene transfer is discussed along with the immunological aspects of AAV vectors. Compared to other viral vector systems, such as those based on retrovirus and adenovirus, the AAV vector is relatively new with potential for further development.

Virus-based vectors for gene expression in mammalian cells: Adenovirus

Abstract: Publisher Summary Adenoviruses (Ad) were first described in the 1950s as causal agents of upper respiratory tract infections and were subsequently associated with only minor pathologies. There are more than 100 Ad serotypes currently identified both in mammals and birds. The Ad virion is a 70–100 nm icosahedral particle composed of 252 capsomers and contains a double-stranded DNA linear genome of 25–45 kb depending on the serotype. Their biology and molecular structure were more extensively characterized using human Ads from serotypes 2 and 5. As a result, the majority of adenoviral vectors (AdV) had been derived from these serotypes. AdVs have been extensively engineered in order to be adapted to several applications of gene transfer in mammalian cells and gene therapy in vivo. The chapter discusses AdV platforms currently developed along with the means to engineer, produce, and characterize them. The biological features of AdV have contributed to their popularity as tools for functional studies and gene therapy applications as demonstrated by the thousands of reports on AdV in recent years. Because efficacy and specificity are of paramount importance in gene therapy success, considerable efforts have been invested on their improvement. Although several challenges in targeting and reducing the host-immune response remain to be addressed, current progress has armed molecular biologists with a wealth of AdV adapted to numerous applications. New developments in the construction of Ad-retro, Ad-adeno-associated virus (AAV) or Ad-Epstein-Barr virus (EBV) hybrid viruses will further expand their use in therapy that requires long-term transgene expression. the recent development of Ad-based libraries will lead to new applications yet to be explored.

Virus-based vectors for gene expression in mammalian cells: Vaccinia virus

Abstract: Publisher Summary The chapter presents basic concepts and applications of poxviruses as recombinant expression vectors. Additional focused reviews on poxvirus molecular biology construction of recombinant poxviruses and their applications as vaccine vectors for infectious diseases and cancer immunotherapy Vaccinia virus (VV), along with smallpoxvirus, is a member of the orthopoxvirus genus, within the family Poxviridae . The Poxviridae also encompass the avipoxvirus genus, of which fowlpoxvirus (FPV) and canarypoxvirus (CPV) have been developed as candidate human vaccine vectors. This chapter focuses on the biology and the applications of recombinant vaccinia virus (rVV). Because of the conservation of the replication machinery within the Poxviridae, many of the concepts of recombinant vaccinia virus (rVV) can be applied to other poxviruses. The successful eradication of smallpox, in the late 1970s, was aided by several properties of VV: simple and inexpensive manufacture process, stability and antigenicity. VV vectors provide a degree of versatility that few other vector systems afford. The advantageous properties of the rVV system include (1) ease of use, (2) ability to accommodate over 25 kilobasepairs (kbp) of foreign DNA, (3) genomic stability, (4) broad host range, (5) cytoplasmic site of gene expression, and (6) ability to authentically process eukaryotic proteins. This vector has been used to express hundreds of recombinant genes for a variety of applications including: protein function analysis, antigen processing, reverse genetics of RNA viruses, and recombinant vaccine development.

Virus-based vectors for gene expression in mammalian cells: Herpes simplex virus

Abstract: Publisher Summary Herpes simplex virus (HSV)-based vectors are flexible and efficient vehicles for introducing experimental and therapeutic transgenes into a variety of tissues. The natural neurotropism and latency of the wild-type vector can be exploited to generate vectors with a particular utility for neuroscience and neurological applications. Gene delivery vectors can be generated from HSV by a series of genetic manipulations; these modifications remove pathogenic functions, while retaining features inherent to HSV biology that may enhance vector interactions with host tissues. Current vector development issues are centered on vector manufacture/production, cellular and tissue targeting, on further eliminating pathogenicity and viral gene expression, and regulating transgene expression. These vectors have proven useful in experimental gene delivery in a variety of settings, and the many successful applications to animal models of disease imply that these reagents might find clinical applications in the near future. This chapter discusses HSV biology and the ways of generating non-toxic vectors from the wild-type virus. The chapter concludes with a brief summary of published applications of these vectors, both within and outside the nervous system.

Virus-based vectors for gene expression in mammalian cells: Sindbis virus

Abstract: Publisher Summary Alphaviruses are relatively simple RNA-enveloped viruses that have been valuable models for learning about virus replication and transmission. Their wide host range, small genome length, simple gene organization, and high level of gene expression provided the original motivation to adapt them as gene-expression vectors. This chapter focuses mainly on Sindbis virus (SIN), the type species of the Alphavirus genus in the Togaviridae family. In nature, it is transmitted by a number of species of mosquitoes to vertebrates (mammals, birds, reptiles and amphibia), principally birds. Two other members of this genus, Semliki Forest virus (SFV) and Venezuelan equine encephalitis virus (VEEV) are also being developed as vectors. VEEV is a known human pathogen but a vaccine strain exists that is the basis for the VEEV vectors. The ways in which SIN vectors are being used cover a wide spectrum. In addition to the illustrations given in this chapter, there are many examples in which proteins were expressed to be able to study their cellular localization, modifications, and function. Alphaviruses enter cells through the endosomal pathway. SIN attaches to the cell surface using one of several potential receptors. It is then taken up into an endosome, that, when acidified, triggers the viral surface glycoproteins to fuse the virus membrane with the endosomal membrane, thus depositing the internal nucleocapsid into the cytoplasm. The genomic RNA is capped and polyadenylated, and is the mRNA for the viral nonstructural polyprotein. The polyprotein is proteolytically processed to produce the individual subunits, nsP1 through nsP4 by the nsP2 protease. The nonstructural proteins serve as the replicase-transcriptase that uses the genomic RNA as template to produce a full-length, negative-sense complement, which in turn is template for synthesis of additional genomic RNAs . The negative sense complement additionally contains a promoter that is used to produce a subgenomic mRNA for translation of the structural polyprotein . The capsid protein located at the amino-terminus is an autoprotease that cleaves itself from the nascent polypeptide, which is then inserted into cellular membranes and is cleaved by host cell proteases to produce the glycoproteins E1 and PE2 (precursor of the virus glycoprotein E2) and a small hydrophobic 6K protein. The capsid protein binds to genomic RNA, facilitated by a packaging signal in the nsP1-encoding sequences, to form nucleocapsids. (Packaging signals of other alphaviruses map to different parts of the nonstructural proteins coding region). The nucleocapsids interact with the cytoplasmic tail of the E2 glycoprotein, resulting in the budding and release of progeny virions through the host plasma membrane.

Lipid reagents for DNA transfer into mammalian cells

Abstract: Publisher Summary Synthetic DNA delivery agents are of great interest as alternatives to viral vectors because they display potentially fewer risks in terms of immunogenicity and propagation. Essentially two chemical alternatives have been developed: cationic polymers such as poly (ethylene imine) and lipid reagents. Lipid reagents are among the most frequently used chemical vectors for DNA transfer into mammalian cells: their association with DNA leads to supra-molecular entities, that promote DNA penetration into the cells. As early as 1978, it was discovered that DNA could be encapsulated in lipidic particles such as liposomes. Few years later, it was shown that cationic lipids could strongly interact with DNA and condense it in small ionic particles called “lipoplexes”. Since this discovery at the end of the 80s, a large variety of cationic lipids were developed to optimize in vitro and in vivo DNA transfer. These reagents are yet defined as the most promising systems for DNA transfer, especially because no limitation of the size of transferred DNA occurs with these systems. Many cationic lipids are commercially available and used in vitro because of their high efficiency. Lipoplexes present low efficiency in vivo as compared to other systems such as viral vectors. Most of the lipid systems used for DNA transfer contain at least 2 or 3 lipid reagents, which are complexed with DNA. The main lipid reagent is cationic and therefore permits DNA condensation; a neutral lipid is often used to promote particle formation and to increase its efficiency for gene transfer. In many cases, a polyethylene glycol PEGylated lipid is also added in order to stabilize and optimize bio-distribution of the particle. The resulting supra-molecular assemblies may allow a selective and efficient in vivo DNA transfer to specific cells or tissues, provided that targeting properties are added. A lot of work was devoted to optimize DNA encapsulation, and condensation of DNA is yet easily obtained from commercially available lipid reagents. Nevertheless, the targeting of specific tissues with lipid reagents has to be enhanced as well as the optimization of intracellular traffic to the nucleus: both extra- and intra-cellular DNA traffic must be improved to enhance DNA transfer into mammalian cells.

Why choose mammalian cells for protein production

Abstract: Publisher Summary There are many different types of hosts to use for production of natural or recombinant proteins: mammalian cells; bacteria, including Gram-negative, Gram-positive, and L-form; filamentous fungi and yeast, including Saccharomyces cerevisiae and Pichia pastoris; insects, including Drosophila melanogaster, Aedes albopictus, Spodoptera frugiperda, and Bombyx mori; Dictyostelium; Xenopus oocytes, and other types of cells, as well as plant tissue culture, transgenic animals and transgenic plants. Progress is continuing in the development of cell-free systems consisting of purified components. The choice of a suitable host cell or expression system for protein production depends on many considerations, such as cell growth characteristics, the ability to effect extracellular expression, post-translational modifications, folding and biological activity of the protein of interest, as well as regulatory and economic issues in the large-scale production of therapeutic proteins. The economics of the selection of a particular expression system requires a cost breakdown in terms of process, design, and other considerations. Key advantages of mammalian cells over other hosts are the ability to carry out proper protein folding, and complex N-linked and authentic O-linked glycosylation of mammalian proteins. Also, mammalian cells posses an extensive post-translational modification machinery, including the ability to produce mature proteins through proteolytic processing.

Co-transfer of multiple plasmids/viruses as an attractive method to introduce several genes in mammalian cells

Abstract: Publisher Summary Gene transfer into mammalian cells has become a standard technique with many applications. The transfer may be transient or may serve to establish stable cell lines using viral or non-viral methods, but the general goal in each case is to target the recombinant gene to the nucleus where it can be transcribed. The success rate of transient or stable introduction of genes into the nucleus is highly variable and is dependent on the choice of cell line and DNA transfer method. Despite tremendous progress in this technology, current methods do not allow strict control of the number of copies of the gene that get into the nucleus. This might appear to be a minor issue since success is measured as the amount of recombinant protein expressed, but the gene copy number in the nucleus is relevant to this output. Ignoring this variable may lead to a misinterpretation of experimental results This chapter describes phenomena that depend on the distribution of plasmids or viral genomes following their introduction into cells. Though this question relies heavily on statistical models and analyses, emphasis is not on calculations but on graphical presentations that attempt to describe DNA levels within the whole cell population. Understanding the principles of such distributions also helps to plan and optimize the co-transfer of multiple plasmids or viral vectors in one step. Although these two different approaches, non-viral and viral, are used for gene transfer, this chapter presents statistical models that can be applied to either approach.

Optimization of plasmid backbone for gene expression in mammalian cells

Abstract: Publisher Summary Two major groups of vectors are used for gene therapy: viral and non-viral. Viruses are considered to be very efficient vehicles for gene transfer. However, their use is limited by safety concerns, such as immune response, possible mutagenesis and carcinogenesis, and high production costs. Considering these limitations, the delivery of therapeutic genes to target cells upon direct in vivo administration of non- viral vectors, i.e., plasmids, is of great value for the development of gene therapy. However, the use of plasmids is plagued by poor transfer efficiency, intracellular penetration and nuclear localization, and low expression level. The success of non-viral gene therapy depends on the development of optimized plasmids. This chapter focuses on the importance of the plasmid backbone, which is often not considered in gene therapy experiments. Several features are considered in the chapter: bacterial DNA sequences, nuclear import, and safety. The chapter also describes two examples of vectors. .

Virus-based vectors for gene expression in mammalian cells: Epstein-Barr virus

Abstract: Publisher Summary Epstein-Barr virus (EBV) is a large DNA tumor virus identified as a causative agent for infectious mononucleosis and a risk factor in the development of Burkitt's lymphoma and nasopharyngeal carcinoma. EBV has been causally associated with B-cell lymphomas in immunocompromized patients, and with portions of Hodgkin's disease and gastric carcinoma. In the vast majority of humanity, however, lifelong infection with EBV is benign and free of any symptoms. EBV maintains its genome in cells extrachromosomally and this feature, even though EBV is a pathogen, has fostered interest in using vectors derived from it for gene therapy in a variety of diseases. This chapter discusses what is known of the mechanism, elements by which EBV replicates, and possible applications of its derived vectors. The understanding of how origin of plasmid replication ( oriP ) and Epstein-Barr nuclear antigen-1 (EBNA-1) contribute to replication of plasmid DNA has increased significantly over the past 10 years and promises to increase further in the next 5 years. As the knowledge of the mechanism by which EBV-derived plasmids replicate increases, the utility of such plasmids is likely to increase as well. EBV-derived plasmids have already proven to be useful tools in vitro with such applications as gene expression, cDNA library expression, recombinant shuttle vectors, and recombinant protein production being particularly successful. Vectors derived from EBV may hold promise for the treatment of both benign and malignant diseases by gene therapy. Their ability to replicate extrachromosomally using cellular replicative machinery with only two required viral elements makes them particularly attractive for future applications. Limitations of current studies arise from the lack of appropriate experimental models in which to test the EBV-derived vectors. However, data collected thus far using the available resources appear promising. A better understanding of the mechanism by which the EBV-elements, oriP and EBNA-1, support sustained plasmid replication should lead to the development of more efficient and safer vectors in the future.

Virus-based vectors for gene expression in mammalian cells: SV40

Abstract: Recombinant gene delivery vectors derived from Tag -deleted SV40 viruses (rSV40s) are potentially useful tools for transduction of many cell types, both in culture and in animals. Characteristically, these vectors: (i) can be made to very high titers (>10 12 infectious units/ml); (ii) infect almost all nucleated mammalian cell types, whether resting or dividing, with high efficiency such that selection is not necessary; (iii) integrate rapidly into the cellular genome and provide for permanent transgene expression without detectable diminution over time; (iv) lack immunogenicity, neither imparting antigenicity to transduced cells nor eliciting neutralizing immune responses against themselves and thus; (v) can be administered multiple times in vivo to normal, immunocompetent animals; (vi) can carry up to ≈5 kb of foreign DNA; and (vii) are safe. Recombinant SV40-derived vectors have been used with pol II promoters to deliver transgenes that encode intracellular, cell membrane, and secreted proteins. They have also been used with pol III promoters to deliver untranslated RNAs, including ribozymes, antisense, RNA decoys, and small inhibitory RNAs. Experimental therapeutic applications of these vectors have included inhibiting HIV in culture and in vivo ; delivering transgenes encoding important missing enzymes, in animal models of human diseases; protecting cells from free radical-induced damage; immunizing against lentiviral antigens; and manipulating the balance of neurotransmitters in the brain. These vectors are limited by their cloning capacity of 5 kb and by the fact that they do not easily express commonly used marker genes such as fluorescent proteins and β-galactosidase. Currently, human trials of rSV40 vectors are being planned.