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PubMed1995

Retroviruses as vectors.

Recombinant retroviruses have long been used to deliver heterologous genes to mammalian cells. Convenient packaging cell lines and vector plasmids have been distributed widely and 'home-made' retroviral vectors have now become a useful research tool in many laboratories. Compared to more traditional methods of gene transfer, retroviral vectors are extraordinarily efficient gene delivery vehicles which cause no detectable harm as they enter their target cells. In the nucleus the retroviral necleic acid becomes integrated into chromosomal DNA, ensuring its long-term persistence and stable transmission to all future progeny of the transduced cell. Up to 8 kilobases of foreign gene sequence can be packaged in a retroviral vector and this is more than enough for most gene therapy applications. Retroviral vectors can also be manufactured in large quantities to meet very stringent safety specifications. They have therefore been selected as the vectors of choice in 80% of the clinical gene therapy trials that have been approved to date. So far there have been no reported short- or long-term toxicity problems associated with their use in human gene therapy trials, now dating back to 1989. However, despite this impressive record, there is still great scope (and need) for the development of new, improved retroviral vectors and packaging systems to fuel further advances in the field of human gene therapy. In the following discussion, existing retroviral vectors are reviewed and current areas of technological development are emphasised.

Gene Expression Regulation, Viral↗
PubMed1987

Stable expression of recombinant factor VIII molecules using a bovine papillomavirus vector.

The bleeding disorder in hemophilia A results from a deficiency or abnormality of Factor VIII (FVIII), a member of the coagulation cascade. FVIII is a large glycoprotein (approximately 350,000 daltons) that is activated by a series of proteolytic cleavages. During activation, a large internal domain (B domain) is removed, resulting in an active complex comprised of the amino and carboxyl subunits of the parental molecule. Using a bovine papillomavirus expression vector system, we have established stable, genetically engineered cell lines harboring either full-length FVIII cDNA or variant FVIII cDNA (delta FVIII), the latter containing an extensive deletion in the region encoding the B domain. We demonstrate that the two recombinant FVIII molecules manifest the biological attributes of native FVIII. Relative to full-length FVIII transformants, cells harboring delta FVIII cDNA are five to eight times more efficient in expressing coagulant activity. This difference is due to a post-transcriptional event.

Bovine papillomavirus 1↗
PubMedDate not supplied

Silencing of gene expression: implications for design of retrovirus vectors.

Transcriptional silencing of retroviruses poses a major obstacle to their use as gene therapy vectors. Silencing is most pronounced in stem cells which are desirable targets for therapeutic gene delivery. Many vector designs combat silencing through cis-modifications of retroviral vector sequences. These designs include mutations of known retroviral silencer elements, addition of positive regulatory elements and insulator elements to protect the transgene from negative position effects. Similar strategies are being applied to lentiviral vectors that readily infect non-dividing quiescent stem cells. Collectively these cis-modifications have significantly improved vector design but optimal expression may require additional intervention to escape completely the trans-factors that scan for foreign DNA, establish silencing in stem cells and maintain silencing in their progeny. Cytosine methylation of CpG sites was proposed to cause retroviral silencing over 20 years ago. However, several studies provide evidence that retrovirus silencing acts through methylase-independent mechanisms. We propose an alternative silencing mechanism initiated by a speculative stem cell-specific "somno-complex". Further understanding of retroviral silencing mechanisms will facilitate better gene therapy vector design and raise new strategies to block transcriptional silencing in transduced stem cells.

Chromatin↗
PubMed1997

Sustainable cutaneous gene delivery.

Durable gene delivery to human skin is necessary for lasting correction of human genetic skin disease. Current cutaneous gene-delivery strategies, however, have achieved only transient gene expression, often only within a small percentage of tissue cells. The recent inability to sustain phenotypic correction of human genetic skin disease due to loss of therapeutic gene expression in regenerated epidermal tissue has highlighted this current limitation. In an effort to surmount this problem, we have generated gene delivery vectors that produce more durable gene delivery in human skin tissue in vivo.

Animals↗
PubMed2000

Cotransduction of nondividing cells using lentiviral vectors.

Diseases such as AIDS and cancers may require the introduction of multiple genes into either stem cells or nondividing cells, among others, for therapeutic purposes. Such genes may act at different points of the disease pathway, or may constitute a regulatory loop to bypass or rectify the defective gene or pathway underpinning the disease. Ideally, the therapeutic genes must be transduced together in diverse combinations, and the introduction should occur without constraints. Since lentiviral vectors can transduce both dividing and nondividing cells, they are ideal vehicles to investigate combinatorial gene transfer into diverse cells. In this study, we demonstrate that by using two independent lentiviral vectors, pseudotyped with the protein g of vesicular stomatitis virus, up to four genes can be introduced simultaneously into single dividing and nondividing cells. Up to 45% and 73% of dividing and nondividing cells, respectively, could be transduced with two lentiviral vectors. The efficiency of cotransducing a single cell was the product of the individual transduction efficiencies and suggested the absence of viral interference. Multiple and combinatorial gene transduction using lentiviral vectors may prove useful in gene therapy.

Actins↗
PubMed2007

Construction and characterization of efficient, stable and safe replication-deficient foamy virus vectors.

As serious side effects affected recent virus-mediated gene transfer studies, novel vectors with improved safety profiles are urgently needed. In the present study, replication-deficient retroviral vectors based on feline foamy virus (FFV) were constructed and analyzed. The novel FFV vectors are devoid of almost the complete env gene plus the internal promoter - accessory bel gene cassette including the gene for the viral transcriptional transactivator Bel1/Tas. In these Bel1/Tas-independent vectors, expression of the lacZ (beta-galactosidase) marker gene is directed by the heterologous, constitutively active human ubiquitin C promoter (ubi). Env-transcomplemented vectors have un-concentrated titers of more than 10(5) transducing units/ml. The vectors allow efficient transduction of a broad array of diverse target cells, which can be increased by repeated vector exposure. However, the number of lacZ marker gene expressing cells decreased slightly upon serial passages of the transduced cells. Vectors carrying a self-inactivating (SIN) deletion of the TATA box and most parts of the viral promoter were not rescued by wt FFV whereas those with the intact or a partially deleted promoter were readily reactivated. This finding indicates that the viral promoters are in fact non-functional, pointing to a highly advantageous safety profile of these new FFV-ubi-lacZ-SIN vectors.

Animals↗
PubMed2006

System of centromeric, episomal, and integrative vectors based on drug resistance markers for Saccharomyces cerevisiae.

Integrative, centromeric, and episomal plasmids are essential for easy, fast, and reliable genetic manipulation of yeast. We constructed a system of shuttle vectors based on the widely used plasmids of the pRS series. We used genes conferring resistance to Geneticin (kanMX4), nourseothricin (natNT2), and hygromycin B (hphNT1) as markers. The centromeric and episomal plasmids that we constructed can be used the same way as the traditional auxotrophic marker-based shuttle vectors (pRS41x and pRS42x series). Additionally, we created a set of nine yeast integrative vectors with the three dominant markers. These plasmids allow for direct integration in the LEU2, URA3, and HIS3 locus of any yeast strain and the concomitant partial deletion of the gene. This prevents multiple integrations and allows for the rapid identification of correct integrants. The set of new vectors considerably enhances the flexibility of genetic manipulations and gene expression in yeast. Most notably, the new vectors allow one to work with natural yeast isolates, which do not contain auxotrophic markers.

Anti-Bacterial Agents↗
PubMed1987

Expression in Escherichia coli of BCY1, the regulatory subunit of cyclic AMP-dependent protein kinase from Saccharomyces cerevisiae. Purification and characterization.

The regulatory (R) subunit of cAMP-dependent protein kinase from the yeast Saccharomyces cerevisiae was expressed in Escherichia coli by engineering the gene for yeast R, BCY1, into an E. coli expression vector that contained a promoter from phage T7. Oligonucleotide-directed mutagenesis was used to create an NdeI restriction site at the natural ATG of the yeast R. This facilitated construction of the T7 expression vector so that the sequence of the protein produced was identical to the natural R subunit. Yeast R was highly expressed in a soluble form. 20 mg of purified yeast R was obtained from 4 liters of E. coli. N-terminal amino acid sequencing revealed that the expressed protein began with the natural sequence. 60% of the molecules contained an N-terminal methionine, and 40% initiated with valine, the second amino acid of yeast R. The protein produced in E. coli migrated on a sodium dodecyl sulfate-polyacrylamide gel with an Mr of 52,000. The yeast R bound 2 mol of cAMP/mol of R monomer with a Kd of 76 nM. The protein was treated with urea to remove bound cAMP. Sedimentation values before and after the urea treatment were identical (s20,w = 5.1). Addition of purified R subunit to a preparation of yeast C subunit (TPK1) rendered catalytic activity cAMP-dependent with an activity ratio of 4.6. The yeast R was autophosphorylated by yeast C to a level of 0.8 mol of phosphate/mol of R monomer. By these criteria, the R subunit produced in E. coli was structurally and functionally identical to the natural yeast R subunit and similar to mammalian type II R subunits.

Base Sequence↗
PubMed2006

Engineering mutations: deconstructing the mouse gene by gene.

Over the past years new vectors and methodologies have been developed to carry out large-scale genome-wide insertional mutagenesis screens in the mouse. Gene trapping, the most commonly used technique, is based on the insertion of a retroviral- or plasmid-based vector into a gene, resulting in a loss-of-function mutation, while simultaneously reporting its expression pattern and providing a molecular tag to facilitate cloning. The discovery of vertebrate DNA transposons in the mouse and recent improvements has also led to their increased use in insertional mutagenesis screens. Several public resources have been set-up recently by the academic community to distribute information and materials generated from these large-scale screens. These new resources should accelerate the study and understanding of biological and developmental processes.

Animals↗
PubMed2003

Recent developments in ocular gene therapy.

The past two to three years have witnessed a remarkable increase in the number of gene therapy studies to treat almost every disease of the eye. All types of delivery systems, viral and non-viral, have been used. Experiments have begun to move from the use of reporters, to genes with potential therapeutic value. In this paper, rather than giving an overview from the beginning of ocular gene therapy, I have chosen to review its most recent advances. Although numerous issues remain to be solved, the emerging picture is encouraging. Within the experimental setting, conditions in the anterior and posterior segments have been improved by the administration of genes encoding beneficial proteins. In one case, vision has been restored in a congenitally blind animal. Limitations do exit, however a greater understanding of the molecular biology of eye tissues coupled with the development of low immunogenicity vectors will continue edging the way for a future use of gene therapy in the clinical setting.

Animals↗
PubMed2006

Ectopic expression of glucagon-like peptide 1 for gene therapy of type II diabetes.

Glucagon-like peptide 1 (GLP-1) is a promising candidate for the treatment of type II diabetes. However, the short in vivo half-life of GLP-1 has made peptide-based treatments challenging. Gene therapy aimed at achieving continuous GLP-1 expression presents one way to circumvent the rapid turnover of GLP-1. We have created a GLP-1 minigene that can direct the secretion of active GLP-1 (amino acids 7-37). Plasmid and adenoviral expression vectors encoding the 31-amino-acid peptide linked to leader sequences required for secretion of GLP-1 yielded sustained levels of active GLP-1 that were significantly greater than endogenous levels. Systemic administration of expression vectors to animals using two diabetic rodent models, db/db mice and Zucker Diabetic Fatty (ZDF) rats, yielded elevated GLP-1 levels that lowered both the fasting and random-fed hyperglycemia present in these animals. Because the insulinotropic actions of GLP-1 are glucose dependent, no evidence of hypoglycemia was observed. Improved glucose homeostasis was demonstrated by improvements in %HbA1c (glycated hemoglobin) and in glucose tolerance tests. GLP-1-treated animals had higher circulating insulin levels and increased insulin immunostaining of pancreatic sections. GLP-1-treated ZDF rats showed diminished food intake and, in the first few weeks following vector administration, a diminished weight gain. These results demonstrate the feasibility of gene therapy for type II diabetes using GLP-1 expression vectors.

Adenoviridae↗
PubMed2001

Alphavirus vectors: development and potential therapeutic applications.

Alphaviruses are RNA enveloped viruses that are proving their value as expression vectors. They are particularly well-suited for this role as they are easily and quickly engineered and can be used to produce high levels of proteins of interest. A promising and important use is as vaccines against disease-causing agents such as HIV. The three alphaviruses now serving as vectors are Sindbis virus, Semliki Forest virus (SFV) and Venezuelan equine encephalitis (VEE) virus. Sindbis virus and SFV are well-known models for studies in molecular and cell biology; VEE virus is a human pathogen and had received some previous notoriety as a potential biological weapon. It is now becoming a potentially valuable vaccine vector. All three viruses are being tested as vaccines but, at present, only Sindbis virus and SFV have been considered for other uses. Sindbis virus vectors have been developed to screen libraries for the identification of new proteins and to devise sensitive assays to detect viruses more difficult to grow in culture. Both Sindbis virus and SFV vectors are serving as tools for fundamental studies in biology, examples include development in insects and analysis of protein functions in neuronal cells. In this article the replication strategy of alphaviruses and the different ways they can be engineered to serve as expression vectors is described. This provides an introduction to the ways these vectors have been used and illustrates the promise these vectors offer.

Alphavirus↗
PubMed2001

Using HSV-thymidine kinase for safety in an allogeneic salivary graft cell line.

Extreme salivary hypofunction is a result of tissue damage caused by irradiation therapy for cancer in the head and neck region. Unfortunately, there is no currently satisfactory treatment for this condition that affects up to 40,000 people in the United States every year. As a novel approach to managing this problem, we are attempting to develop an orally implantable, fluid-secreting device (an artificial salivary gland). We are using the well-studied HSG salivary cell line as a potential allogeneic graft cell for this device. One drawback of using a cell line is the potential for malignant transformation. If such an untoward response occurred, the device could be removed. However, in the event that any HSG cells escaped, we wished to provide additional patient protection. Accordingly, we have engineered HSG cells with a hybrid adeno-retroviral vector, AdLTR.CMV-tk, to express the herpes simplex virus thymidine kinase (HSV-tk) suicide gene as a novel safety factor. Cells were grown on plastic plates or on poly-L-lactic acid disks and then transduced with different multiplicities of infection (MOIs) of the hybrid vector. Thereafter, various concentrations of ganciclovir (GCV) were added, and cell viability was tested. Transduced HSG cells expressed HSV-tk and were sensitive to GCV treatment. Maximal effects were seen at a MOI of 10 with 50 microM of GCV, achieving 95% cell killing on the poly-L-lactic acid substrate. These results suggest that engineering the expression of a suicide gene in an allogeneic graft cell may provide additional safety for use in an artificial salivary gland device.

Artificial Organs↗
PubMed1995

The biotechnology and applications of antibody engineering.

The exquisite specificity of monoclonal antibodies (MAb) has long provided the potential for creating new reagents for the in vivo delivery of therapeutic drugs or toxins to defined cellular target sites or improved methods of diagnosis. However, many difficulties associated with their production, affinity, specificity, and use in vivo have largely confined their application to research or in vitro diagnostics. This situation is beginning to change with the recent developments in the applied molecular techniques that allow the engineering of the genes that encode antibodies rather than the manipulation of the intact antibodies themselves. Techniques, such as the polymerase chain reaction, have provided essential methods with which to generate and modify the genetic constituents of antibodies, allow their conjugation to toxins or drugs, provide ways of humanizing murine antibodies, and allow discrete modular antigen binding components to be produced. More recent developments of in vitro expression systems and powerful phage surface display technologies will without doubt play a major role in future antibody engineering and in the successful development of new diagnostic and therapeutic antibody-based reagents.

Animals↗
PubMed2000

Gene transfer into stimulated and unstimulated T lymphocytes by HIV-1-derived lentiviral vectors.

Genetic modification of T lymphocytes holds great potential for treatments of cancer, T cell disorders and AIDS. While in the past recombinant murine retroviruses were the vectors of choice for gene delivery to T cells, vectors based on lentiviruses can provide additional benefits. Here, we show that VSV-G pseudotyped HIV 1 vector particles delivering the enhanced green fluorescent protein (EGFP) efficiently transduce human T lymphocytes. Transduction efficiency was optimal when infection included centrifugation of cells with concentrated vector supernatant in the presence of Polybrene. In contrast to previous reports describing murine retrovirus-mediated gene transfer to T lymphocytes, fibronectin did not improve the transduction efficiency of the VSVG-pseudotyped HIV-1 particles. Similar gene transfer efficiencies were observed following stimulation of cells with PHA/IL-2 or anti-CD3i/CD28i antibodies, although greater transgene expression was observed in the latter case. Interestingly, production of vectors in the absence of the accessory proteins Vif, Vpr, Vpu and Nef was accompanied by a 50% decrease in transduction efficiency in activated T cells. Transduction of T cells that were not stimulated before infection was achieved. No transduction of non-prestimulated cells was observed with a GAL V-pseudotyped murine retroviral vector. The requirement for accessory proteins in non-prestimulated cells was more pronounced. Our results have implications for lentiviral vector targeting of other cells of the hematopoietic system including stem cells.

Cell Line↗
PubMed2006

Viral-based modelling and correction of neurodegenerative diseases by RNA interference.

Experimental recapitulation of recessive human genetic neurodegenerative disease in rodents can be classically addressed through genetic disruption of the related gene. Although very informative, this specific gene targeting is restricted to mice and precludes a species scale-up towards non-human primates. Concomitantly, this requirement to silence a specific gene in a broad range of animal models is important in the design of therapeutic approaches to dominantly inherited neurodegenerative diseases. The emergence of RNA interference (RNAi), a highly specific mechanism of post-translational gene silencing, has opened a plethora of biological application ranging from reverse genetic analysis to therapeutic schemes. Recombinant viral vectors, by promoting a long-lasting delivery of genetic instructions in a broad range of cellular types of different species origins, represent potential platforms mandating silencing of specific gene products through RNAi. This review aims at providing an overview of the different viral systems engineered so far for efficient in vitro and in vivo delivery of RNAi instructions. Additionally, the potential of RNAi for functional analysis and therapy for polyglutamine disorders or amyotrophic lateral sclerosis is discussed.

Animals↗
PubMed2004

Vaccinia viral/retroviral chimeric vectors.

Retroviral vectors have become important tools in gene therapy due to a number of desirable properties, including efficient gene delivery and stable genomic integration. Some shortcomings, however, still remain to be solved. Retroviral vectors cannot be grown to as high titers as for example adenoviruses or vaccinia viruses, 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 trying to combine the broadly estimated features of retroviral vectors with advantageous properties of a second viral vector system. Chimeric systems with retroviruses and adeno-alphavirus and herpesviruses have been reported. This review is dedicated to vaccinia virus, a widely used vector in molecular and cell biology, as a chimeric carrier for retroviral vector units. In the first poxviral/retroviral constructs, retroviral vector units integrated into a defective vaccinia vector, gave rise to transduction competent particles. Due to the high insertion capacity of the vaccinia system, also the packaging components could be inserted into the carrier virus resulting in a system that is independent of retroviral packaging cell lines. Moreover, since vaccinia is a cytoplasmic virus that does not recognize nuclear transcription and processing signals, retroviral vectors with introns and internal transcription stops could be constructed that transduce complex gene cassettes. The topic of this review is the vaccinia viral / retroviral vector system and possible applications to gene therapy.

Animals↗