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PubMed2002

Specific packaging of spliced retroviral vector transcripts lacking the Psi-region.

The effect of a cryptic splice acceptor (cSA) site located at the end of the extended packaging signal in murine leukemia virus (MLV)-based vectors was investigated. Although this cSA is also present in wild type MLV, it was found to result in a smaller transcript in which the packaging signal (Psi) had been removed by splicing only in MLV-derived vectors. Splicing occurs both in packaging cells producing the MLV-vectors as well as in the infected target cells. Transcripts lacking the Psi-sequence (Psi(-)) are packaged relatively efficiently into virus particles, even in the presence of wild type Psi(+)-vector transcripts. The Psi(-)-viral RNA is reverse transcribed in vector transduced cells as is any other retroviral genome. The titer obtained from the Psi(-)-vector was only 1000-fold reduced in comparison to the same Psi(+)-vector. These results suggest that Psi(-)-transcripts may be packaged more frequently than previously supposed and that splicing patterns should be carefully analysed on an individual basis for retroviral vectors used in gene therapy.

Animals↗
PubMed2000

Pharmacodynamic approach to study the gene transfer process employing non-viral vectors.

In the present work we set out to apply pharmacodynamic concepts derived from dose-response curves (Potency and Efficacy) to characterize the gene transfer efficiency of a vector:DNA complex. We employed two widely used vectors, the cationic lipid DOTAP (N,N, N-trimethyl 1-2-3-bis (1-oxo-9-octa-decenyl)oxy-(Z, Z)-1-propanaminium methyl sulfate) and the cationic polymer PEI (polyethylenimine, 800 kDa) to transfect several constructions of the green fluorescent protein cDNA. The analysis of dose-response curves indicated that in all cases the goodness-of-fit was > 0.99. Potency is a measure that provides information on gene activity per amount of DNA. Efficacy is a measure of maximum gene expression achievable using a specific vector:DNA complex, and depends on both the intrinsic efficacy of the gene (evaluated using different vectors to transfer the same gene construct) and on vector efficacy in DNA delivery (evaluated using a single vector to deliver different gene constructs). The results suggest that Potency and Efficacy are objective parameters for describing and comparing the goodness of vectors, as well as the intrinsic efficacy of a given gene construct. Furthermore, they are useful tools that may contribute to a better understanding of the mechanistic gene transfer process of each vector.

Animals↗
PubMed2003

A herpesvirus vector can transduce axotomized brain neurons.

If gene therapy is to be used to promote axon regeneration after spinal cord injury, a suitable vector for transgene delivery must be obtained. Replication-defective herpes simplex virus (HSV) vectors are promising candidates. We have examined whether they can express a LacZ transgene in injured neurons of adult rat brain. We transected the medial forebrain bundle, injected replication-defective HSV/LacZ vectors close to the lesion site, and looked for transgene expression at 2-14 days after the lesion. The vectors carried the LacZ transgene controlled either by the cytomegalovirus immediate-early promoter (vector CS5) or the HSV latency-associated promoter (vector CS1). CS5 transfected many cells near the lesion at 2 days, but did not give persistent expression at 5 days. CS1, in contrast, labeled many neurons in midbrain regions remote from the injection site at 5 days, and much of this expression remained at 12-14 days. The neurons of most interest were in the substantia nigra pars compacta and parabrachial nuclei, which were axotomized by the lesion. Vector-driven beta-galactosidase expression was detected in neurons in both regions. These were confirmed as axotomized by double immunofluorescence for c-Jun. By 12-14 days, many substantia nigra neurons had disappeared but some transduced neurons remained; there was no net loss of transduced neurons from the parabrachial nuclei. These results show that an HSV vector is capable of transducing axotomized cells in the central nervous system and producing transgene expression in them for at least 2 weeks after injection.

Animals↗
PubMed2002

Genetic markers for study of the anopheline vectors of human malaria.

Human malaria is truly a disease of global proportions and is one of the most broadly distributed vector-borne infections. Anopheline mosquitoes are the exclusive vectors of human malaria. A handful of species predominate as the most notorious malaria vectors, but the species and forms involved in the transmission of human malaria world-wide are incredibly diverse. Many of the anophelines that vector malaria exist as members of species complexes that often contain vector and non-vector species. Additionally, single anopheline species often exhibit significant heterogeneity across the species' range. This phenotypic and genotypic plasticity exacerbates the difficulties in identification of vector populations and implementation of effective surveillance and control strategies. Polytene chromosome investigations were among the first to provide researchers with tangible genetic markers that could be used to differentiate between what are now recognised as species and chromosomal forms of anopheline mosquitoes. The advent of the polymerase chain reaction gave access to the molecular genetics of genomes and the techniques that followed have facilitated investigation of the genetics of individual specimens or population size samples. The variety and number of genetic markers available for the study of malaria vectors has literally exploded in the last 10 years. Markers have expanded from the 'traditional tools' to include a vast array of molecular markers. Contemporary markers range from what are now referred to as 'classical genetic markers' to methods used to detect and identify single nucleotide polymorphisms and finally to highly polymorphic markers. One of the greatest advantages of this wide variety of genetic markers is that researchers may choose to utilise any combination of markers or techniques to address multifaceted questions relating to malaria transmission. These molecular markers have proven useful in a wide variety of applications including molecular taxonomy, evolutionary systematics, population genetics, genetic mapping, and investigation of defined phenotypes.

Animals↗
PubMed1997

Versatile vectors for transient and stable expression of recombinant antibody molecules in mammalian cells.

We have developed new cassette expression vectors for the cloning of any intact V-region gene followed by any C-region gene. Both the heavy-and light chain vectors harbor a strong hCMV promoter, restriction site cassettes for cloning of both V- and C-region genes, transcription termination signals, fl-ori for single stranded DNA (ssDNA) synthesis, selection marker for Neomycin and SV40 ori for transient expression. The vectors accept VH and VL chain genes obtained by RT-PCR. Reamplification of the V genes is then performed with a new set of primers which are designed specifically for each individual V gene. Cloning into the vectors is aided by restriction sites located just outside the V-gene coding region, thus keeping the V-genes intact. The vectors also contain cloning sites for the exchange of genomic C-genes so that the resulting Ig genes may code for complete antibodies, antibody fragments or fusion proteins. A simple subcloning step permits the expression of both heavy and light chain genes from one single vector, thus avoiding co-transfection of the two vectors. The usefulness of the vectors was confirmed by construction of mouse-human chimeric antibodies. The V-genes were derived from a hybridoma cell line, TP-3, and was combined with human C kappa, C gamma 3 and C gamma 1 genes as well as with CH1 gamma 3. High yields of recombinant antibody products in NSO cells were obtained. Transient expression was also demonstrated.

Animals↗
PubMed1999

Viral vectors for veterinary vaccines.

Whatever strategy is adopted for the development of viral vectors for delivery of veterinary vaccines there are several key points to consider: (1) Will the vectored vaccine give a delivery advantage compared to what's already available? (2) Will the vectored vaccine give a manufacturing advantage compared to what's already available? (3) Will the vectored vaccine provide improved safety compared to what's already available? (5) Will the vectored vaccine increase the duration of immunity compared to what's already available? (6) Will the vectored vaccine be more convenient to store compared to what's already available? (7) Is the vectored vaccine compatible with other vaccines? If there is no other alternative available then the answer to these questions is easy. However, if there are alternative vaccines available then the answers to these questions become very important because the answers will determine whether a vectored vaccine is merely a good laboratory idea or a successful vaccine.

Animals↗
PubMed2005

Lentiviral vector delivery of recombinant small interfering RNA expression cassettes.

Lentiviral vectors are able to transduce nondividing cells and maintain sustained long-term expression of transgenes. Many cells types, including brain, liver, muscle and hematopoietic stem cells, have been successfully transduced with lentiviral vectors carrying a variety of genes. These properties make lentiviral vectors attractive vehicles for delivering small interfering RNA (siRNA) genes into mammalian cells. RNA polymerase III (pol III) promoters are most commonly used for expressing siRNAs from lentiviral vectors. Pol III promoters are relatively small, have high activity, and use simple termination signals of short stretches of Us. It is possible to include several pol III expression cassettes in a single lentiviral vector backbone to express different siRNAs or to combine siRNAs with other transgenes. This chapter describes the delivery of pol III-promoted siRNAs by human immunodeficiency virus (HIV)-based lentiviral vectors and covers vector design, production, and verification of siRNA expression and function. This chapter should be useful for establishing a lentiviral vector-based delivery of siRNAs in experiments that require long-term gene knockdown or developing siRNA-based approaches for gene therapy applications.

Base Sequence↗
PubMed1994

Generation of high-titer pseudotyped retroviral vectors with very broad host range.

Encapsidation of the VSV G protein into the virions of MoMLV-derived retroviral vectors in the absence of other VSV-encoded proteins is shown to be an efficient process, although the exact mechanism for this process is currently unclear. Unlike the conventional retroviral vectors bearing the amphotropic envelope protein, the pseudotyped virus has the ability to withstand the shearing forces encountered during ultracentrifugation. This property of the pseudotyped virus enables the generation of high-titer retroviral vector stocks and has potential application for in vivo gene therapy studies. We have found as many as four copies of a pseudotyped vector to integrate into the genome of a single cell when a high multiplicity of infection was used to infect the cells. Multiple integration events were not observed with amphotropic retroviral vectors, probably because of their low virus titers. In addition, when retroviral vectors are pseudotyped with the VSV G protein, they acquire the host range of VSV and are able to infect nonmammalian cells derived from fish, Xenopus, mosquito, and Lepidoptera. Since techniques for efficient gene transfer in some of these nonmammalian systems are not currently available, retrovirus-mediated gene transfer described here should be useful for transgenic and other genetic studies in lower vertebrate species. The inability to establish a stable cell line expressing the VSV G protein, however, limits large-scale production of the pseudotyped retroviral vectors. Generation of stable packaging cell lines for the pseudotyped retroviral vectors is a major challenge for the future.

3T3 Cells↗
PubMed1997

Defective viral vectors as agents for gene transfer in the nervous system.

Viral vectors have attracted great interest as vehicles for gene therapy. Due to concerns regarding continued viral gene expression in several systems, new approaches have been sought for gene transfer in the nervous system. This article reviews the general concepts and basic biology of defective viral vectors. These are vectors which can package into a viral coat but contain no viral genes, thereby allowing efficient gene transfer in the absence of viral gene expression in target cells. The defective herpes simplex virus (HSV) vector has been applied to numerous interesting questions in neurobiology. The inability to completely eliminate helper viruses has raised concern regarding the application of this vector to human disease. The adeno-associated virus (AAV) vector has recently been introduced into the nervous system. This vector harbors no viral genes, however helper viruses can also be completely eliminated from the system. Although the smaller size may limit the range of applications for this vector, it has received great interest as a potential agent for gene therapy in the nervous system. Potential future directions are discussed as well.

Animals↗
PubMed2002

Specific transduction of HIV-1 envelope expressing cells by retroviral vectors pseudotyped with hybrid CD4/CXCR4 receptors.

Infection of a target cell by HIV is initiated by the interaction of the envelope glycoprotein with the CD4 receptor molecule on the surface of the target cell. This is followed by binding of a coreceptor of the chemokine receptor family and subsequently fusion of viral and cellular membranes. Membrane fusion is independent of whether the viral envelope protein is on the viral or on the cellular membrane. Accordingly, targeting of HIV infected cells by retroviral vectors has been previously achieved both by coincorporation of CD4 and coreceptors into murine leukemia virus (MLV) and lentivirus based vector particles. It was, therefore, tested whether hybrid genes of CD4 and CXCR4 are also able to yield 'receptor' vectors. A construct containing the four extracellular loops of CD4 fused to CXCR4 (CD4-D4-X4) allowed gene transfer into HIV-1 envelope expressing cells by vectors based on either MLV or lentiviruses. The CD4-D2-X4 hybrid receptor, containing the first two extracellular CD4 domains, allowed gene transfer only by lentiviral vectors. Attempts to increase vector titres by deletion of the intracellular part of CXCR4 failed. Vector titres obtained by hybrid receptors were slightly lower than published titres obtained by separate expression of CD4 and CXCR4. Thus, CD4-D4-CXCR4 hybrids are useful for the generation of retroviral and lentiviral vectors with specificity for HIV-1 envelope expressing cells.

CD4 Antigens↗
PubMed1998

Optimization of packaging of adeno-associated virus gene therapy vectors using plasmid transfections.

Adeno-associated Virus (AAV) is attracting wide attention as a potential human gene therapy vector. The advantages of this vector system are that it is naturally defective, it readily integrates into the target cell's genome and is considered to be nonpathogenic. AAV infects a wide variety of cell and tissue types. The major disadvantages of the vector are its small size and the labor-intensive procedures required to prepare large amounts of the vector for clinical studies. In this manuscript we have systematically tested a number of variables in the packaging procedure to determine the optimal conditions for successful vector preparation. Using an AAV vector that expresses the green fluorescent protein and the most commonly used packaging plasmid, pAAV/Ad, we determined the optimal conditions for; lysis of the transfected cultures, ratio of packaging to vector plasmids, day of harvest after transfection, storage conditions, multiplicity of infection of helper adenovirus, and the time of adenovirus infection. These results have important relevance for investigators that are using AAV vectors for heterologous gene transfer studies.

Cytopathogenic Effect, Viral↗
PubMed1999

Stable expression of the ecotropic retrovirus receptor in amphotropic packaging cells facilitates the transfer of recombinant vectors and enhances the yield of retroviral particles.

Retroviral vectors are used widely as gene transfer vehicles. Vector particles are generated by packaging cell lines, which supply the structural proteins gag, pol and env needed to package the retroviral vector RNA. The most efficient way to introduce the vector genome into the packaging cell line is cross-infection with a retroviral vector. Since the infection of a packaging cell line by the produced virus is blocked due to the down regulation of the retrovirus receptor by the envelope glycoprotein, the vector genome should be introduced by a virus with a host tropism different from the one of the packaging cell line. The murine ecotropic retrovirus receptor was expressed in the human amphotropic packaging cell line FLYA13 to generate a cell line which can be infected by murine ecotropic retroviruses. Vector transfer can now be facilitated by cross-infection with the appropriate ecotropic retroviral vectors and provides a simple and efficient method for the generation of amphotropic packaging lines.

3T3 Cells↗
PubMed2000

Use of recombinatory PCR to insert subtle genetic markers into Moloney murine leukemia virus-based retroviral vectors.

As tools to examine template switches and recombination events during the process of reverse transcription, two nearly identical Moloney murine leukemia virus-based (MoMLV) retroviral vectors were constructed using the technique of recombinatory polymerase chain reaction (PCR). The experimental vectors designed for this study were based on the well-characterized LN series vectors. The protein coding regions normally present in the retroviral genome have been replaced by the coding regions for two drug resistance markers, neomycin phosphotransferase (Neo) and hygromycin phosphotransferase (Hyg). With only one functional drug resistance gene in each vector, the individual vectors as well as recombination events between them can be followed by phenotypic selection. Utilization of recombinatory PCR allowed the insertion of very subtle nucleotide changes resulting in a series of restriction site polymorphisms in the two retroviral vectors. The ability to create these subtle mutations in specific locations of these retroviral vectors allowed the utilization of naturally occurring areas of variability in the vectors and avoid regions important for replication.

Animals↗
PubMed1999

Development of improved pUB110-based vectors for expression and secretion studies in Bacillus subtilis.

pUb110-based vectors are commonly used for expression and secretion studies in Bacillus subtilis. Two of these plasmids, pUB18P43 and pWB705, have been applies to produce several proteins of interest. To offer greater flexibility and compatibility in this system, the authors have also constructed a pE194-based plasmid vector (pE18). To determine whether the pUB110-based or the pE194-based vector serves as a better expression system, three structural genes encoding cytoplasmic BirA, extracytoplasmic PrsA and extracellular staphylokinase, respectively, were used as models. Production of these products using pUB110-based vectors was consistently 2--3-fold lower than that using the pE194-based vectors. The observed difference in the protein yield did not result from either the rearrangement of the plasmid or the difference in the plasmid copy number. By using three different approaches, the lower production yield from the pUB110-based vectors was found to be due to the transcription interference from the plasmid encoded genes. These findings illustrate that the orientation of the inserted gene in pUB110-based vector can greatly affect gene expression. Two new expression vectors, pUB19P43 and pWB980, were constructed to allow better gene expression.

Bacillus subtilis↗
PubMed2003

The effects of HHV-8 vMIP-II on SIVmac251 infection and replication competent and incompetent SIVmac239Delta3 vectors.

Human herpesvirus type 8 vMIP-II has one of the broadest ranges of chemokine receptor binding and therefore a multiplicity of biologic effects, both immunologic and antiviral. These properties make vMIP-II an attractive effector gene to be expressed from gene therapy vectors. The present studies were concerned with both therapeutic approaches: (1) an anti-simian immunodeficiency virus (SIV) biologic, and (2) an effector gene in SIV-based vectors. Regarding its antiviral properties, vMIP-II expressed from bacteria and SIV-based vectors bound the surface of CEMx174 cells and specifically suppressed SIVmac251 infection. A CCR3 monoclonal antibody partially inhibited vMIP-II binding, suggesting that both SIVmac251 and vMIP-II utilize a similar CCR3-like receptor for CEMx174 cell binding. Replication competent SIV-based vectors containing forward and reverse vMIP-II produced neither identifiable vMIP-II nor virions for the first 21 days. Virus replication occurred after this period. Significant sequence alterations in the forward vMIP-II containing replication competent vector transcripts were responsible for the failure of vMIP-II expression. The genetic basis for the initial failure to replicate virus and its later restoration was not determined but appeared in the II-PIMv containing vectors to coincide with deletions and compensatory rearrangements in nef 3' of the polypurine tract. Cells transfected with SIVmac239Delta3DeltaLTR-vMIP-II vectors expressed biologically active vMIP-II that bound CEMx174 cells and suppressed SIVmac251 infection. These data suggest that replication defective SIV vectors expressing immunobiolgic genes such as vMIP-II may prove useful in gene therapies, particularly in augmenting immune responses in chronically infected individuals.

Anti-HIV Agents↗
PubMed2001

Enhanced reporter gene expression in the rat brain from helper virus-free HSV-1 vectors packaged in the presence of specific mutated HSV-1 proteins that affect the virion.

Herpes simplex virus (HSV-1) gene expression is hypothesized to shut off promoters in HSV-1 vectors, but in a helper virus-free HSV-1 vector system, a number of promoters support only short-term expression. Thus, recombinant gene expression remains short-term in the absence of approximately 99% of the HSV-1 genome. To resolve this paradox, we hypothesized that specific HSV-1 proteins that affect the virion can shut off recombinant gene expression. This study evaluated expression from HSV-1 vectors, containing neuronal-specific promoters, that were packaged in the presence of specific mutated HSV-1 proteins that affect the virion. The mutated HSV-1 proteins that were examined included two protein kinases (U(L)13 and U(S)3), the virion host shut-off factor (vhs), the transactivator of immediate early promoters (VP16), and a virion protein that affects RNA metabolism (U(S)11). Helper virus-free packaging could occur in the presence of each mutated protein alone or specific combinations of two or three mutated proteins. In BHK and PC12 cells, vectors packaged in the presence of each mutated protein increased ( approximately 2-fold) the level of expression per cell, and vectors packaged in the presence of specific combinations of mutated proteins supported larger (4-7-fold) increases. In the rat striatum, vectors packaged in the presence of a mutated U(S)3 displayed enhanced gene transfer (13-18-fold increases in the number of cells at 4 days), and vectors packaged in the presence of mutated U(L)13 or VP16 enhanced long-term expression (2 months). Vectors packaged in the presence of mutated vhs or U(S)11 displayed minimal changes in expression.

Animals↗
PubMed2001

Efficient gene transfer by fiber-mutant adenoviral vectors containing RGD peptide.

One of the hurdles to adenovirus (Ad)-mediated gene transfer is that Ad vectors mediate inefficient gene transfer into cells lacking in the primary receptors, Coxsackievirus and adenovirus receptor (CAR). We previously developed a fiber-mutant Ad vector containing the Arg-Gly-Asp (RGD)-containing peptide motif on the HI loop of the fiber knob, and showed that the mutant vector had enhanced gene transfer activity to human glioma cells, which showed little CAR expression, compared to the vector containing wild type fiber. In this study, the feasibility of the Ad vector containing RGD peptide on the fiber knob was examined in a wide variety of cell types: CAR-positive or -negative human tumor cells, mouse cells, and leukemia cells. The mutant vector infected the cells, which lacked CAR expression but showed alpha(v) integrin expression, about 10-1000 times more efficiently than the vector containing wild type fiber via an RGD-integrin (alpha(v)beta3 and alpha(v)beta5)-dependent, CAR-independent cell entry pathway. The results of this study indicate that Ad vector containing RGD peptide on the fiber knob could be of great utility for gene therapy and gene transfer experiments.

Adenoviridae↗
PubMed2002

Adenovirus vectors containing chimeric type 5 and type 35 fiber proteins exhibit altered and expanded tropism and increase the size limit of foreign genes.

Adenovirus (Ad) fiber proteins are responsible for the initial attachment of the virion to the cell membrane. Most Ad vectors currently in use are based on the Ad type 5 (Ad5), which belong to subgroup C, and use the coxsackievirus and adenovirus receptors (CAR) as the initial receptor. Ad35, which belongs to subgroup B, recognizes unknown receptor(s) other than CAR. In this study, the feasibility of the Ad vector containing Ad5/35 chimeric fiber protein was examined in a wide variety of cell types, such as CAR-positive or -negative human tumor cells, rodent cells, and blood cells (a total of 20 cell types), and in mice in vivo. Transduction data suggested that the Ad vectors containing the Ad5/35 chimeric fiber protein exhibited altered and expanded tropism when compared with the Ad5-based vector. The chimeric vector also allows the packaging of larger foreign DNAs than the conventional Ad5-based vector, which can package approximately 8.1-8.2 kb of foreign DNA. The chimeric vector containing approximately 8.8 kb of foreign DNA was generated without affecting the viral growth rate and titer. These results suggested that inclusion of the Ad35 fiber protein into the Ad5-based vector could lead to an improved efficiency in gene therapy and in gene transfer experiments, especially for the cells lacking in sufficient CAR expression.

Adenoviridae↗