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PubMed2005

Engineering chromosomes for delivery of therapeutic genes.

The ability to create fully functional human chromosome vectors represents a potentially exciting gene-delivery system for the correction of human genetic disorders with several advantages over viral delivery systems. However, for the full potential of chromosome-based gene-delivery vectors to be realized, several key obstacles must be overcome. Methods must be developed to insert therapeutic genes reliably and efficiently and to enable the stable transfer of the resulting chromosomal vectors to different therapeutic cell types. Research to achieve these outcomes continues to encounter major challenges; however recent developments have reiterated the potential of chromosome-based vectors for therapeutic gene delivery. Here we review the different strategies under development and discuss the advantages and problems associated with each.

Animals↗
PubMed2003

aadA confers streptomycin resistance in Borrelia burgdorferi.

To enhance genetic manipulation of the Lyme disease spirochete Borrelia burgdorferi, we assayed the aadA gene for the ability to confer resistance to the antibiotics spectinomycin and streptomycin. Using the previously described pBSV2 as a backbone, a shuttle vector, termed pKFSS1, which carries the aadA open reading frame fused to the B. burgdorferi flgB promoter was constructed. The hybrid flgB promoter-aadA cassette confers resistance to spectinomycin and streptomycin in both B. burgdorferi and Escherichia coli. pKFSS1 has a replication origin derived from the 9-kb circular plasmid and can be comaintained in B. burgdorferi with extant shuttle vector pCE320, which has a replication origin derived from a 32-kb circular plasmid, or pBSV2, despite the fact that pKFSS1 and pBSV2 have the same replication origin. Our results demonstrate the availability of a new selectable marker and shuttle vector for genetically dissecting B. burgdorferi at the molecular level.

Anti-Bacterial Agents↗
PubMed2002

Heterologous expression of nonribosomal peptide synthetases in B. subtilis: construction of a bi-functional B subtilis/E coli shuttle vector system.

A major obstacle in investigating the biosynthesis of pharmacologically important peptide antibiotics is the heterologous expression of the giant biosynthetic genes. Recently, the genetically engineered strain Bacillus subtilis KE30 has been reported as an excellent surrogate host for the heterologous expression of an entire nonribosomal peptide synthetase (NRPS) gene cluster. In this study, we expand the applicability of this strain, by the development of four Escherichia coli/B. subtilis shuttle expression vectors. Comparative overproduction of hybrid NRPS proteins derived from both organisms revealed a significant beneficial effect of overproducing proteins in B. subtilis KE30 as underlined by the production of stable nondegradative proteins, as well as the formation of active phosphopantetheinylated holo-proteins.

Anti-Bacterial Agents↗
PubMed1996

Positive selection vectors for allelic exchange.

We describe here the development and use of two new allelic exchange vectors, pKAS32 and pKAS46. These vectors can be used for allelic exchange in a wide variety of bacterial species because their R6K origin of replication functions only in bacteria engineered to produce the replication protein pi. In addition, these vectors express the Escherichia coli rpsL gene, encoding ribosomal protein S12, which provides a positive selection for bacteria that have exchanged cloned plasmid sequences with the corresponding chromosomal sequences. In this report, we show that these vectors can be used to efficiently introduce point mutations and deletions into the chromosome of Vibrio cholerae.

Alleles↗
PubMed2003

The alphavbeta5 integrin of hematopoietic and nonhematopoietic cells is a transduction receptor of RGD-4C fiber-modified adenoviruses.

Epithelial and endothelial cells expressing the primary Coxsackie virus B adenovirus (Ad) receptor (CAR) and integrin coreceptors are natural targets of human Ad infections. The fiber knob of species A, C, D, E and F Ad serotypes binds CAR by mimicking the CAR-homodimer interface, and the penton base containing arginine-glycine-aspartate (RGD) motifs binds with low affinity to alphav integrins inducing cell activation. Here, we generated seven different genetically modified Ad vectors with RGD sequences inserted into the HI loop of fiber knob. All mutants bound and infected CAR and alphav integrin-positive epithelial cells with equal efficiencies. However, the Ads containing two additional cysteines, both N and C terminals of the RGD sequence (RGD-4C), were uniquely capable of transducing CAR-less hematopoietic and nonhematopoietic human tumor cell lines and primary melanoma cells. Both binding and transduction of RGD-4C Ad were blocked by soluble RGD peptides. Flow cytometry of cell surface integrins and virus binding to CAR-less cells in the presence of function-blocking anti-integrin antibodies indicated that the alphavbeta5 integrin, but not alphavbeta3, alphaIIbbeta3 or beta1,alpha5 or alpha6-containing integrins served as a functional transduction receptor of the RGD-4C Ads. However, in cells with low levels of alphavbeta5 integrin, the function-blocking anti-alphavbeta5 antibodies were not effective, unlike soluble RGD peptides. Collectively, our data demonstrate that the alphavbeta5 integrin is a functional transduction receptor of RGD-4C Ads in the absence of CAR, and that additional RGD receptors are targets of these viruses. The RGD-4C vectors further extend the tropism of Ads towards potential human therapies.

Adenoviridae↗
PubMed2005

AdEasy-based cloning system to generate tropism expanded replicating adenoviruses expressing transgenes late in the viral life cycle.

Replicating adenoviral vectors (RAds) hold great promise for the treatment of cancer. Significant therapeutic effects of these vectors do not only rely on tumor targeting but also on efficient release of viral progeny from host cells. Cytotoxic genes expressed late in the adenoviral life cycle can significantly enhance viral release and spreading. Therefore, an adenoviral cloning system that allows easy integration of established tumor targeting techniques together with late expression of transgenes can be a valuable tool for the development of RAds. We expanded the features of the widely used AdEasy adenoviral cloning system toward the production of tropism modified replicating adenoviral vectors that express transgenes late in the viral life cycle. Three vectors (pIRES, pFIBER and pAdEasy-Sce) that facilitate easy manipulation of the adenoviral fiber region were established. Unique BstBI and I-Sce-1 restriction sites facilitate the introduction of retargeting peptides in the fiber HI-loop and of genes of interest in the fiber transcription unit. We validated the system by constructing an E1-positive adenovirus with an RGD motif in the fiber HI-loop and green fluorescent protein (GFP) expressed from the fiber transcription unit (AdDelta24Fiber-rgd-GFP). Additionally, assessment of E1-negative replication-deficient vectors confirmed strict dependence upon E1 expression for the expression of transgenes inserted into the fiber transcription unit. This flexible cloning system allows for straightforward construction of tropism expanded replicating adenoviral vectors that express transgenes late in the adenoviral life cycle.

Adenoviridae↗
PubMed2004

Lentiviral vectors efficiently transduce human gonadotroph and somatotroph adenomas in vitro. Targeted expression of transgene by pituitary hormone promoters.

Despite important advances in human therapeutics, no specific treatment for both non-functioning gonadotroph and resistant somatotroph adenomas is available. Gene transfer by viral vectors can be considered as a promising way to achieve a specific and efficient treatment. Here we show the possibility of efficient gene transfer in human pituitary adenoma cells in vitro using a human immunodeficiency virus (HIV)-type 1-derived vector. Using enhanced green fluorescent protein (eGFP) gene as a marker placed under the phosphoglycerate kinase (PGK) promoter, gonadotroph and somatotroph adenomas were transduced even with moderate viral loads. The expression started at day 2, reached a peak at day 5, and it was still present at day 90. For targeting somatotroph and gonadotroph adenomas, human growth hormone (GH) promoter (GH -481, +54 bp) and two fragments of the human glycoprotein hormone alpha-subunit promoter (alpha-subunit 1 -520, +33 bp, and alpha-subunit 2 -907, +33 bp) were tested. In gonadotroph adenomas, the percentage of identified fluorescent cells and the fluorescence intensity analyzed by fluorescence-activated cell sorting indicated that the strength of the alpha-subunit 1 and alpha-subunit 2 promoters were comparable to that of the PGK promoter. Primary cultures of rat pituitary cells showed that alpha-subunit 1 is more selective to thyreotroph and gonadotroph phenotypes than alpha-subunit 2. GH promoter activity appeared weak in somatotroph adenomas. The human GH enhancer did not increase the GH promoter activity at all but the human prolactin promoter (-250 bp) allowed 4-fold more fluorescent cells to be obtained than the GH promoter. Several cell lines appeared too permissive to test cell-specificity of pituitary promoters. However, on human non-pituitary cell cultures, the tested pituitary promoters seemed clearly selective to target endocrine pituitary phenotypes. This study gives a starting point for a gene-therapy program using lentiviral vectors to transfer therapeutic genes in human pituitary adenomas.

Adenoma↗
PubMed2003

Receptor targeting of adeno-associated virus vectors.

Adeno-associated virus (AAV) is a promising vector for human somatic gene therapy. However, its broad host range is a disadvantage for in vivo gene therapy, because it does not allow the selective tissue- or organ-restricted transduction required to enhance the safety and efficiency of the gene transfer. Therefore, increasing efforts are being made to target AAV-2-based vectors to specific receptors. The studies summarized in this review show that it is possible to target AAV-2 to a specific cell. So far, the most promising approach is the genetic modification of the viral capsid. However, the currently available AAV-2 targeting vectors need to be improved with regard to the elimination of the wild-type AAV-2 tropism and the improvement of infectious titers. The creation of highly efficient AAV-2 targeting vectors will also require a better understanding of the transmembrane and intracellular processing of this virus.

Animals↗
PubMed1998

Site-specific gene delivery in vivo through engineered Sendai viral envelopes.

Inspite of several stimulating developments in gene therapy, the formulation of a targeted gene delivery "vector" is still far from ideal. We have demonstrated the potential of reconstituted Sendai viral envelopes containing only the fusion glycoprotein (F-virosomes) in targeted delivery of reporter genes to liver cells of BALB/c mouse in vivo. The membrane fusion-mediated high efficiency of gene transfer to liver cells was ascertained following a critical evaluation of the level of the DNA, mRNA, and relevant proteins. Furthermore, the involvement of viral glycoprotein both as a unique natural ligand and as a membrane fusogen could lead to preferential transfection of parenchymal cell types of liver. The integration of transgenes in the mouse chromosomal DNA and its stable expression up to 4 mo after single i.v. administration of this gene carrier has bolstered its efficiency and novelty. Moreover, the F-virosomes did not elicit significant humoral immune response against the fusion protein in the injected animal. The findings reported here open up the possibility for considering "F-virosomes" as a promising "vehicle" for site-specific DNA delivery in gene therapy.

Animals↗
PubMed2002

Adenovirus binding to the coxsackievirus and adenovirus receptor or integrins is not required to elicit brain inflammation but is necessary to transduce specific neural cell types.

Intracranial administration of adenovirus vectors elicits rapid, capsid-mediated dose-dependent brain inflammation. The mechanisms through which adenovirus capsids trigger inflammation in the brain remain unknown. We determined whether adenovirus interaction with the primary and secondary cell surface receptors for infection (CAR and alphav integrins) was necessary to trigger acute adenovirus-mediated brain inflammation, and, furthermore, whether capsid mutations that abrogated CAR and integrin binding altered vector tropism in the brain. Vectors ablated for CAR binding, but retaining integrin binding function, transduced equivalent areas of brain compared to vectors with wild-type capsids; however, vector tropsim was dramatically altered. Vectors with wild-type capsids predominantly transduced oligodendrocytes, whereas mutation of the fiber protein to ablate CAR binding resulted in a loss of oligodendrocyte transduction and a consequent redirection of transduction to neurons and other types of glial cells. Combined mutations of fiber and penton base that ablate both CAR and integrin binding almost abolished brain transduction. Thus, doubly-ablated capsids engineered to express new ligands should allow complete vector retargeting in the central nervous system. Although transduction by the doubly-ablated vectors was reduced by greater than 95%, inflammation was not reduced compared to wild-type vectors, demonstrating that brain inflammation occurs independently of adenovirus binding and infection of cells via CAR and integrin receptors.

Adenoviridae↗
PubMed2004

Construction of retroviral vectors with enhanced efficiency of transgene expression.

Retroviral vectors have been widely used in gene therapy due to their simple genomic structure and high transduction efficiency. We report a construction of Moloney murine sarcoma virus (MoMSV) and Moloney murine leukemia virus (MoMLV) hybrid-based retroviral vectors with significantly improved efficiency of transgene expression after stable incorporation into the host genome. In these vectors, the residual gag gene coding sequence located in the extended region of packaging signal was removed. These vectors, therefore, contain no coding sequence for the gag, pol, or env gene that can be used for homologous recombination with sequences introduced in the packaging system for a recombinant competent retrovirus (RCR) generation. A strong splice acceptor site obtained from the exon/intron junction of either the chimpanzee EF1-alpha gene or the human CMV major immediate early gene was placed downstream of the MoMSV packaging signal (Psi), significantly improving the efficiency of transgene expression. The 5' LTR U3 sequence was replaced with an extended human CMV major immediate early gene enhancer/promoter for a strong expression of full-length messages from the viral backbone, helping to maintain high levels of viral titer. These newly developed retroviral vectors should facilitate RCR-free gene transfer with significantly improved efficacy in clinical gene therapy trials.

Animals↗
PubMed2006

Use of herpes simplex virus type 1-based amplicon vector for delivery of small interfering RNA.

Silencing of gene expression by small interfering RNAs (siRNAs) is rapidly becoming a powerful tool for genetic analysis of mammalian cells. The use of DNA-based plasmid vectors to achieve transient and stable expression of siRNA has been developed to avoid the problems of double-stranded oligonucleotides transfection. These vectors direct the transcription of small hairpin RNAs (shRNAs) from a polymerase-III (H1 or U6)-RNA gene promoter. However, numerous disadvantages remain, including low transfection efficiency and difficulty in transfecting primary cells. To overcome some of these problems, the use of viral vectors for siRNA delivery has been described. Retroviral, adenoviral, adeno-associated and herpes viral shRNAs delivery systems have been successfully used to silence genes, in vitro and in vivo. The use of a herpes simplex virus type 1 (HSV-1)-based amplicon vector for siRNA delivery into mammalian cells, using human polyomavirus BK (BKV)-transformed cells as a model system is described. The results demonstrate the ability of amplicon vectors to inhibit the expression of BKV T-Ag and tumorigenicity of BKV-transformed cells. We show that the use of the amplicon vector is highly efficient for the delivery of siRNA molecules. The unique ability of these vectors to deliver multiple copies of siRNA may provide a useful tool in the development of novel anticancer therapy.

Animals↗
PubMed2000

Development and optimization of herpes simplex virus vectors for multiple long-term gene delivery to the peripheral nervous system.

Herpes simplex virus (HSV) has often been suggested as a suitable vector for gene delivery to the peripheral nervous system as it naturally infects sensory nerve terminals before retrograde transport to the cell body in the spinal ganglia where latency is established. HSV vectors might therefore be particularly appropriate for the study and treatment of chronic pain following vector administration by relatively noninvasive peripheral routes. However parameters allowing safe and efficient gene delivery to spinal ganglia following peripheral vector inoculation, or the long-term expression of delivered genes, have not been comprehensively studied. We have identified combinations of deletions from the HSV genome which allow highly efficient gene delivery to spinal dorsal root ganglia (DRGs) following either footpad or sciatic nerve injection. These vectors have ICP34.5 deleted and have inactivating mutations in vmw65. We also report that peripheral replication is probably necessary for the efficient establishment of latency in vivo, as fully replication-incompetent HSV vectors allow efficient gene expression in DRGs only after peripheral inoculation at a high virus dose. Very low transduction efficiencies are otherwise achieved. In parallel, promoters have been developed that allow the long-term expression of individual or pairs of genes in DRGs by using elements from the latently active region of the virus to confer a long-term activity onto a number of promoters which otherwise function only in the short term. This work further defines elements and mechanisms within the latently active region that are necessary for long-term gene expression and for the first time allows multiple inserted genes to be expressed from HSV vectors during latency.

Animals↗
PubMed1992

Development and use of field application vectors to express nonadaptive foreign genes in competitive environments.

Many potential applications of genetically engineered microorganisms in environmental and agricultural biotechnology involve introducing genetic capabilities into nonsterile competitive environments in which they provide no advantage to the host. Field application vectors have been designed for the purpose of creating a temporary niche for the host in such environments. This technique involves the addition to the target environment of a selective substrate readily utilizable by the host microorganism but unavailable to most indigenous species. Thirteen nonionic and anionic detergents, representing a wide range of structural complexities and molecular weights, were screened as potential selective substrates. Competition experiments in soil, using Warburg respirometry, indicated that isolates from six different detergent enrichment cultures were more active on their corresponding detergents than the indigenous microorganisms. Detergents of intermediate structural complexities and molecular weights were most effective for use as selective substrates. A field application vector that utilizes 1.0% Igepal CO-720 (detergent) as the selective substrate and Pseudomonas paucimobilis 1IGP4 as the host was tested for its ability to increase the presence of nonadaptive tetracycline resistance marker genes in soil. In soil amended with the selective substrate, strain 1IGP4 plate counts increased by three orders of magnitude and tetracycline-resistant transformant (pRK293) counts increased from 1.8 x 10/g of soil to 4.3 x 10/g in 2 days. Inoculation in the absence of substrate amendment or amendment with a nonselective substrate did not result in growth of strain 1IGP4. These results demonstrate the effectiveness of field application vectors for increasing the concentration of nonadaptive genes in competitive environments.

Journal Article↗
PubMed1997

Anaerobic bacteria as a gene delivery system that is controlled by the tumor microenvironment.

A fundamental obstacle in gene therapy for cancer treatment is the specific delivery of an anticancer gene product to a solid tumor. Although several strategies exist to control gene expression once a vector is directly introduced into a tumor, as yet no systemic delivery system exists that specifically targets solid tumors. Nonpathogenic, obligate anaerobic bacteria of the genus Clostridium have been used experimentally as anticancer agents because of their selective growth in the hypoxic regions of solid tumors after systemic application. In this report we further describe a novel approach to cancer gene therapy in which genetically engineered clostridia are used as tumor-specific vectors for the delivery of antitumor genes. We have introduced into a strain of C. beijerinckii the gene for an E. coli nitroreductase known to activate the nontoxic prodrug CB 1954 to a toxic anticancer drug. Nitroreductase produced by these clostridia enhanced the killing of tumor cells in vitro by CB 1954, by a factor of 22. To demonstrate the specificity of this approach for tumor targeting, we intravenously injected the inactive spore form of C. beijerinckii, which upon transition to a reproductive state will express the E. coli nitroreductase gene. Nitroreductase activity was detectable in 10 of 10 tumors during the first 5 days after intravenous injection of inactive clostridial spores, indicating a rapid transition from spore to reproductive state. Tumors harboring clostridial spores which did not possess the E. coli nitroreductase gene were devoid of nitroreductase activity. Most importantly, E. coli nitroreductase protein was not found in a large survey of normal mouse tissues following intravenous injection of nitroreductase containing clostridia, strongly suggesting that obligate anaerobic bacteria such as clostridia can be utilized as highly specific gene delivery vectors for cancer therapy.

Animals↗
PubMed1998

Genetic engineering of an industrial strain of Saccharopolyspora erythraea for stable expression of the Vitreoscilla haemoglobin gene (vhb).

Several Actinomycetes/Streptomycetes expression vectors are described for expression of the Vitreoscilla haemoglobin gene (vhb) in an industrial erythromycin-producing strain of Saccharopolyspora erythraea. Cloning of vhb under the control of either the thiostrepton-inducible PtipA promoter or the constitutive PermE* promoter led to the production of chemically active haemoglobin (VHb) in Streptomyces lividans TK24 transformed with these constructs. However, theplasmids could not be transformed into Sac. erythraea. Transformants of Sac. erythraea and/or exconjugants were obtained using a novel Escherichia coli/Streptomyces shuttle vector comprised of vhb under the control of the PermE* promoter, the Streptomyces plasmid pIJ350 origin of replication, the thiostrepton-resistance gene (tsr) for selection, and the oriT region which is necessary for conjugal transfer. Increased plasmid stability in Sac. erythraea was obtained by construction of a vector for chromosomal integration. This vector contained the Streptomyces phage phi C31 attachment site for chromosomal integration and vhb expressed under the PmerR promoter and was stably maintained in the chromosome of Sac. erythraea. Shake-flask cultivations of the transformed Sac. erythraea strain with the chromosomally integrated vhb gene show that vhb is expressed in an active form. The corresponding amount of erythromycin produced in the vhb-expressing strain was approximately 60% higher relative to the original VHb-negative strain.

Bacterial Proteins↗
PubMed1985

High-level expression of RNAs and proteins: the use of oligonucleotides for the precise fusion of coding-to-regulatory sequences.

We show that the fusion between regulatory sequences present on expression vectors and coding sequences can be efficiently achieved by oligonucleotide-directed mutagenesis. We have constructed single-stranded (ss) expression vectors that facilitate this process. These plasmids derive from vectors that have been used for the synthesis of quantities of proteins in Escherichia coli or RNAs in vitro. By inserting the origin of replication of the ss phage f1 into these plasmids it became possible to package their ss DNA into phage rods. Deletion of unwanted sequences or simple base changes can then be obtained by oligonucleotide-directed mutagenesis using the vector ss DNA as a template. We discuss the results of several experiments where this technique was applied to our expression vectors and we demonstrate the construction of a plasmid which efficiently synthesizes in vitro a regulatory RNA molecule that is involved in the control of plasmid copy number.

Chromosome Deletion↗
PubMed2006

Generation of variable and fixed length siRNA from a novel siRNA expression vector.

Small interfering RNA (siRNA) expression vectors using a Pol III promoter fall into two categories, vectors with a single Pol III promoter that express small hairpin RNA (shRNA) and vectors with two head-to-head (convergent) Pol III promoters that express siRNA. There are technical difficulties in preparing convergent siRNA vectors from cDNA. Here, we report construction of a novel convergent siRNA expression vector, pTHUB. Two XcmI sites were inserted between opposing Pol III promoters. After linearization with XcmI, pTHUB has a single 3' A overhang at each end that allows direct cloning of partially DNase I digested cDNA fragments (20-30 bp) tailed with ddT. A derivative method for generating 19 bp siRNA in pTHUB is also described. The suppression efficiency of the pTHUB vector is comparable to those of conventional shRNA vectors. We have made a siRNA library from a single cDNA. The same approach can be used to construct whole-genome siRNA libraries from cellular cDNA.

Base Sequence↗