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At least 127 records · Page 7Linked to original sources
PubMed2005

Genetic modification of hematopoietic cells using retroviral and lentiviral vectors: safety considerations for vector design and delivery into target cells.

The recent development of leukemia in three patients following retroviral vector gene transfer in hematopoietic stem cells, resulting in the death of one patient, has raised safety concerns for the use of integrating gene transfer vectors for human gene therapy. This review discusses these serious adverse events from the perspective of whether restrictions on vector design and vector-modified target cells are warranted at this time. A case is made against presently establishing specific restrictions for vector design and transduced cells; rather, their safety should be ascertained by empiric evaluation in appropriate preclinical models on a case-by-case basis. Such preclinical data, coupled with proper informed patient consent and a risk-benefit ratio analysis, provide the best available prospective evaluation of gene transfer vectors prior to their translation into the clinic.

Genetic Therapy↗
PubMed2005

Vector solution of the diffraction task using the Hertz vector.

A large class of diffraction problems can be solved on the basis of the Huygens principle. However, methods of solving diffraction problems based on this principle exhibit narrow boundaries of applicability. The goal of the present work is to offer a relatively simple physically based and mathematically strict "dipole wave" vector theory of non-paraxial diffraction of electromagnetic radiation which allows analytical solutions of typical diffraction problems. The suggested theory logically retains the wave approach used in the Kirchhoff method and does not exhibit strict limitations to applicability inherent in the Kirchhoff integral. The diffraction problem is solved by using the Hertz vector in the Kirchhoff integral instead of the field vector. The method efficiency is illustrated in several examples. Analytical solutions of diffraction base problems have been obtained for linearly polarized radiation on an infinite slit and on various-shaped holes at an arbitrary angle of incidence and polarization. It was shown the possibility of vector addition particular solutions to obtain diffraction patterns from several holes. The diffraction of radiation with azimuthal and radial directions of polarization on a ring slit is also considered. The main qualitative feature of the obtained solutions is the presence of "poles" one or two points of zero field in the diffraction pattern which are superimposed on the common system of light and dark fringes. The poles are located along electrical field vector directions. The vector analytical formulas describing the propagation of some laser beams in the free space have been obtained too. The solutions of the diffractive problems satisfy the Maxwell equations and the reciprocity principle.

Journal Article↗
PubMed2002

Germ-line transformation of the South American malaria vector, Anopheles albimanus, with a piggyBac/EGFP transposon vector is routine and highly efficient.

Stable and efficient germ-line transformation was achieved in the South American malaria vector, Anopheles albimanus, using a piggyBac vector marked with an enhanced green fluorescent protein gene regulated by the Drosophila melanogaster polyubiquitin promoter. Transgenic mosquitoes were identified from four independent experiments at frequencies ranging from 20 to 43% per fertile G0. Fluorescence was observable throughout the body of larvae and pupae, and abdominal segments of adults. Transgenic lines analysed by Southern hybridization had one to six germ-line integrations, with most lines having three or more integrations. Hybridized transposon vector fragments and insertion site sequences were consistent with precise piggyBac-mediated integrations, although this was not verified for all lines. The piggyBac/PUbnlsEGFP vector appears to be a robust transformation system for this anopheline species, in contrast to the use of a piggyBac vector in An. gambiae. Further tests are needed to determine if differences in anopheline transformation efficiency are due to the marker systems or to organismal or cellular factors specific to the species.

Animals↗
PubMed2004

New-generation multicistronic expression platform: pTRIDENT vectors containing size-optimized IRES elements enable homing endonuclease-based cistron swapping into lentiviral expression vectors.

Capitalizing on a proven multicistronic expression vector platform we have designed novel pTRIDENT vectors which (1). enable coordinated expression of three desired transgenes, (2). are size-optimized, (3). take advantage of small highly efficient internal ribosome entry sites of the GTX or Rbm3 type, (4). harbor various sites specific for homing endonucleases facilitating promoter/multicistronic expression unit/polyadenylation site swapping as well as (5). straightforward integration into human HIV-l-based lentiviral expression vectors tailored to contain compatible homing endonucleases. Multicistronic expression profiles of novel pTRIDENT vectors engineered for different tricistronic expression configurations encoding human low-molecular-weight urokinase-type plasminogen activator (u-PA(LMW)) or Bacillus stearothermophilus-derived alpha-amylase (SAMY), human vascular endothelial growth factor (hVEGF), and human placental secreted alkaline phosphatase (SEAP) have been quantified in Chinese hamster ovary cells (CHO-K1), mouse fibroblasts (NIH/3T3), and/or human fibrosarcoma (HT-1080) cells. In addition, a pTRIDENT-derived SAMY-VEGF-SEAP expression cassette transferred into a compatible lentiviral expression vector enabled simultaneous high-level transgene expression following transduction of transgenic lentiviral particles into primary human chondrocytes.

Cloning, Molecular↗
PubMed1998

Adenovirus vectors as transcomplementing templates for the production of replication defective retroviral vectors.

We have generated an adenovirus containing a retroviral vector sequence encoding the neomycin phosphotransferase (neo) gene (AV-LXSN). AV-LXSN transduction of retroviral packaging cell lines led to production of LXSN retroviral vector with alternative viral envelopes; exposure of target cells to retroviral containing supernatants confirmed envelope specific tropism. Retroviral titers (G418 cfu/ml) were comparable to those produced by standard techniques. Retrovirus could be detected in supernatants within 24 hours of AV-LXSN transduction and persisted as long as 120 hours. Southern blot analysis of DNA purified from populations of G418 cells showed the presence of a single neo containing restriction fragment of the appropriate size that could only be generated by reverse transcription of LXSN to produce LXSN provirus. This adeno-retroviral chimeric vector system could simplify the generation and testing of different retroviral vectors, particularly where assessment of vectors with alternative envelopes carrying novel targeting ligands is required.

Adenoviridae↗
PubMed1999

A versatile bait vector allowing rapid isolation of prey vectors in Gal4-dependent yeast two-hybrid screens.

Two-hybrid screens have been widely used in recent years for identifying and isolating new interaction partners to known proteins. Current Gal4-dependent two-hybrid screens employ mostly bait and library vectors, which both have the ampicillin gene as a selection marker in bacteria. The isolation of the library plasmids takes several days, because library and bait plasmid cannot be separated easily. We have replaced the ampicillin gene by a kanamycin gene in a Gal4 DNA binding domain bait vector. This vector reduces four- to fivefold the time period required for the isolation of library plasmid DNA. In addition we have changed the multicloning site in the modified vector for easy cloning of cDNA inserts. This vector is advantageous not only in standard two-hybrid screens, but also in mass screens that require multiple screening rounds in order to characterize networks of protein-protein interactions.

Base Sequence↗
PubMed2001

Promoter strength in adenovirus transducing vectors: down-regulation of the adenovirus E1A promoter in 293 cells facilitates vector construction.

Most adenovirus transducing vectors have the cytomegalovirus major immediate-early (CMV) or the Rous sarcoma virus long terminal repeat (RSV) promoter driving expression of the transgene. Both of these promoters are highly active in transfection and transduction assays in 293 cells, in which transducing vectors are constructed and grown, and in HeLa cells. The CMV promoter exhibits rapid activation while the RSV promoter exhibits a lag prior to the onset of viral DNA replication in transduction assays. While the use of very strong promoters facilitates expression of the transgene, high-level expression of certain gene products hinders virus construction and growth. For such genes, the use of the adenovirus type 5 E1A promoter offers advantages. The E1A promoter exhibits modest activity in HeLa cells after transfection or transduction, but very little activity in 293 cells, suggesting that the E1A promoter would permit construction and growth of vectors encoding deleterious gene products that could not be constructed with the CMV and RSV promoters. This idea was tested through attempts to construct viruses encoding the immunoglobulin loop 6 and transmembrane regions of the prostaglandin F2alpha receptor regulatory protein (FPRP), a product that inhibits adenovirus vector construction for reasons that are not clear. Only the E1A promoter permitted construction and growth of the transducing vector encoding the fragment of FPRP.

Adenovirus E1A Proteins↗
PubMed1991

Vectors for inserting selectable markers in vector arms and human DNA inserts of yeast artificial chromosomes (YACs).

To facilitate studies of gene expression and homologous recombination, plasmids have been developed which permit the insertion of neomycin resistance-encoding gene (NmR) into either the human DNA insert or the vector arm of a yeast artificial chromosome (YAC). To integrate into the YAC arm, the plasmid pRV1 contains a LYS2 (encoding alpha-aminoadipate reductase) gene for selection in the yeast host, and a NmR gene for subsequent selection after transfection of mammalian cells. These two sequences are bracketed by fragments of the URA3 gene (encoding orotidine-5'-phosphate decarboxylase) that can disrupt the URA3 gene in the YAC arm by homologous recombination in yeast. To integrate a selectable marker into the insert, the plasmid pRV2 contains a NmR gene and an intact copy of the URA3 gene, bracketed by segments of an L1 (LINEs) repetitive element. In this case, the vector has been designed for use with YACs that have already been fitted in the vector arm with a different marker (i.e., TK) that has disrupted the URA3 gene in the vector arm. Selection is for the restoration of URA3 gene activity attendant on recombination into an L1 element in the YAC insert. Use of the vectors is illustrated with a YAC clone containing ribosomal DNA.

Aldehyde Oxidoreductases↗
PubMed1993

Polycos vectors: a system for packaging filamentous phage and phagemid vectors using lambda phage packaging extracts.

A new class of hybrid vectors, 'polycos' vectors, incorporate a phage lambda cos site and filamentous phage origin to allow high-efficiency cloning via in vitro lambda packaging extracts. The head-filling mechanism of cos site recognition by lambda packaging proteins permits concatemers of several of these small cos-containing vectors to be packaged per phage particle. Excision of vector monomers after infection is accomplished via a lambda ZAP-like M13 excision process. This system has the advantage of adapting high-efficiency lambda packaging extracts to M13 and phagemid cloning vectors.

Bacteriophage M13↗
PubMed1995

Plasmid insertion vectors that facilitate construction of herpes simplex virus gene delivery vectors.

Plasmid insertion vectors were designed for the expression of foreign genes in recombinant herpes simplex virus (HSV) vectors. One vector, pGal9, was designed for the insertion of foreign genes with their own promoter; a second vector, pGal10, was designed for the insertion of coding sequences downstream from the HSV immediate-early 110K promoter. The 110K promoter directed efficient expression of foreign genes, particularly in replication-incompetent virus recombinants, as shown by the expression of the lacZ and IFN alpha genes. These vectors should be useful for the characterization of various promoters for gene delivery, and for the efficient expression of foreign genes in a variety of cell types.

Animals↗
PubMed2006

Retroviral vectors for vaccine development: induction of HIV-1-specific humoral and cellular immune responses in rhesus macaques using a novel MLV(HIV-1) pseudotype vector.

Retroviral vectors have yet not been tested for their potential as vaccines despite their frequent utilization in gene therapy allowing for highly efficient gene transfer into a number of cell types and their suitability for large-scale production in biotechnology. To investigate MLV-based vectors suitability for inducing immune response against HIV-1-antigens, we generated a MLV(HIV-1) pseudotype vector enabling CD4-specific transduction of HIV-1 genes env, vpu, tat and rev originating from the pathogenic SHIV-89.6P. Functional expression of the lentiviral genes in packaging cells, human and rhesus CD4+ target cells was demonstrated by various assays. Following highly efficient ex vivo transduction, up to 3.4x10(7) autologous, transfer vector-positive rhesus peripheral blood mononuclear cells (rhPBMCs) were re-inoculated into a rhesus macaque. Five weeks after the initial inoculation HIV-1 Env-specific antibodies were detected using ELISA. ELIspot-assay revealed the induction of a HIV-1 Rev and Env-specific CTL-response 7.5 weeks after immunization. Thus, these novel MLV(HIV-1) vectors facilitate efficient transduction and subsequent expression of HIV-1-genes in CD4-positive host cells. Induction of both humoral and cellular HIV-1-specific immune responses in vivo confirmed their potential as an effective HIV-1 vaccine to be further studied in SHIV/rhesus macaque model of lentivirus infection.

AIDS Vaccines↗
PubMed2005

Recombinant adenovirus type 5 vectors that target DC-SIGN, ChemR23 and alpha(v)beta3 integrin efficiently transduce human dendritic cells and enhance presentation of vectored antigens.

Recombinant adenoviruses (rAds) represent a promising system for vaccine delivery but transduce dendritic cells (DC) relatively poorly. To address this concern, we used a biotin-avidin linkage to conjugate rAd vectors to ligands which bind with high affinity to selected receptors on DC (ChemR23, alpha(v)beta3 integrin, and DC-SIGN). The targeted vectors had an enhanced ability to transduce human monocyte-derived DC compared to untargeted virus. In addition, DC transduced with targeted rAd vectors were more efficient at stimulating cytokine production by autologous memory CD8+ T cells, against a vector-encoded antigen. These results expand the range of cell surface receptors that can be used to target rAd5 vectors to DC, and may facilitate future development of rAd-based vaccines.

Adenoviridae↗
PubMedDate not supplied

The use of lentiviral vectors in gene therapy of leukemia: combinatorial gene delivery of immunomodulators into leukemia cells by state-of-the-art vectors.

Our goal is to develop cell vaccines against leukemia cells, genetically modified to express molecules with potent immune-stimulatory capacities. Pre-clinical evaluation of this approach in murine models has demonstrated efficient anti-leukemic responses with the expression of immunomodulators, in particular GM-CSF and CD80, in irradiated cell vaccines. We have previously shown efficient insertion of GM-CSF and CD80 genes into primary human leukemia cells with the use of second and third generation self-inactivating (SIN) lentiviral vectors (Blood 96 (2000), 1317; Leukemia 16 (2002), 1645). The advantages of lentiviral vectors for development of autologous leukemia cell vaccines include: (1) efficient and consistent gene delivery; (2) high levels of transgene expression; (3) persistent expression of the transduced gene; (4) no viral proteins, as only the transduced gene is expressed; (5) no undesirable cytotoxic effects, and; (6) simplicity of use [leukemia cells are exposed to vector(s) only once]. In this work, we evaluated the insertion of the central polypurine tract and the central termination sequence into a SIN lentiviral vector encoding for GM-CSF and CD80, which significantly enhanced the transduction efficiency of primary leukemia cells and provided higher levels of GM-CSF and CD80 co-expression. We also demonstrate a methodology to deliver simultaneously a combination of immunomodulatory molecules (GM-CSF, CD80, IL-4, and CD40L) to activate different pathways of immune stimulation. Therefore, lentiviral vectors offer a simple, versatile, and reliable approach for engineering leukemic cells for use as cell vaccines.

Adjuvants, Immunologic↗
PubMed1997

Gene transfer into enteric neurons of the rat small intestine in organ culture using a replication defective recombinant herpes simplex virus type 1 (HSV1) vector, but not recombinant adenovirus vectors.

We have designed a system in which to test gene transfer into gut neurons consisting of an organ culture of neonatal rat small intestine. The tissue was exposed to herpes simplex- and adenovirus-derived vectors: (1) a temperature-sensitive herpes simplex virus-1 (HSV1) vector (tsK-beta gal) containing the lacZ gene encoding beta-galactosidase (beta-gal), under the transcriptional control of the HSV1 immediate-early 3 (IE3) promoter; (2) RAd35, an E1-/E3- replication-deficient adenovirus expressing lacZ under the control of a truncated HCMV major IE promoter; and (3) RAd122, an E1-/E3- replication-deficient adenovirus expressing the lacZ under the control of the RSV LTR. Forty-eight hours after the vector was added to the organ culture, we detected beta-gal using immunohistochemistry or X-gal histochemistry in tissue sections examined by light microscopy. We encountered a distinctive staining of cells arranged in two concentric circles corresponding in location to the myenteric and submucosal plexuses. Cells in these areas were of similar size and morphology to neonatal enteric neurons, as visualized by NADPH-diaphorase histochemistry and immunocytochemical staining with antibodies to the neuronally expressed proteins PGP 9.5, or neurofilaments. Double labelling with antibodies recognizing neurofilaments and beta-galactosidase revealed that most cells infected by tsK were neurons, while the RAd35 and 122 vectors only infected non-neuronal cells. We thus demonstrate that both HSV1- and adenovirus-derived vectors can be used to transfer genes to the gut in vitro, but they transduce different populations of target cells.

Adenoviridae↗
PubMed2005

PEGylated helper-dependent adenoviral vectors: highly efficient vectors with an enhanced safety profile.

Transgene expression from helper-dependent adenoviral (HD-Ad) vectors is effective and long lasting, but not permanent. Their use is also limited by the host response against capsid proteins that precludes successful gene expression upon readministration. In this report, we test the hypothesis that PEGylation of HD-Ad reduces its toxicity and promotes transgene expression upon readministration. PEGylation did not compromise transduction efficiency in vitro and in vivo and reduced peak serum IL-6 levels two-fold. IL-12 and TNF-alpha levels were reduced three- and seven-fold, respectively. Thrombocytopenia was not detected in mice treated with the PEGylated vector. Serum transaminases were not significantly elevated in mice treated with either vector. Mice immunized with 1 x 10(11) particles of unmodified HD-Ad expressing human alpha-1 antitrypsin (hA1AT) were rechallenged 28 days later with 8 x 10(10) particles of unmodified or PEG-conjugated vector expressing beta-galactosidase. Trace levels of beta-galactosidase (52.23+/-19.2 pg/mg protein) were detected in liver homogenates of mice that received two doses of unmodified HD-Ad. Mice rechallenged with PEGylated HD-Ad produced significant levels of beta-galactosidase (5.1+/-0.4 x 10(5) pg/mg protein, P=0.0001). This suggests that PEGylation of HD-Ad vectors may be appropriate for their safe and efficient use in the clinic.

Adenoviridae↗
PubMed2006

Mosaic vectors comprised of modified AAV1 capsid proteins for efficient vector purification and targeting to vascular endothelial cells.

Vascular-targeted gene therapies have the potential to treat many of the leading causes of mortality in the western world. Unfortunately, these therapies have been ineffective due to poor vascular gene transfer. The use of alternative virus serotypes and the incorporation of vascular targeting ligands into vectors has resulted in only modest increases in vascular gene transfer. Adeno-associated virus (AAV) 1 has shown the most promise among the AAV vectors for the transduction of vascular endothelial cells. However, no straightforward small-scale purification strategy exists for AAV1 as it does for AAV2 making it difficult to quickly produce AAV1 vector for analysis. Here we have combined two AAV1 capsid protein modifications to enhance vascular gene transfer and allow easy purification of vector particles. Mosaic vector particles have been produced comprised of capsid proteins containing the well-characterized RGD4C modification to target integrins present on the vasculature, and capsid proteins containing a modification that permits metabolic biotinylation and efficient purification of mosaic particles by avidin affinity chromatography. We show that the RGD modification results in a 50-100-fold enhancement in endothelial cell gene transfer that is maintained in biotinylated mosaic AAV1 particles. These results suggest that mosaic virions hold significant promise for targeted gene delivery to the vasculature.

Cell Line↗
PubMed1999

HSV-1-based vectors for gene therapy of neurological diseases and brain tumors: part II. Vector systems and applications.

Many properties of HSV-1 are especially suitable for using this virus as a vector to treat diseases affecting the central nervous system (CNS), such as Parkinson's disease or malignant gliomas. These advantageous properties include natural neurotropism, high transduction efficiency, large transgene capacity, and the ability of entering a latent state in neurons. Selective oncolysis in combination with modulation of the immune response mediated by replication-conditional HSV-1 vectors appears to be a highly promising approach in the battle against malignant glioma. Helper virus-free HSV/AAV hybrid amplicon vectors have great promise in mediating long-term gene expression in the PNS and CNS for the treatment of various neurodegenerative disorders or chronic pain. Current research focuses on the design of HSV-1-derived vectors which are targeted to certain cell types and support transcriptionally regulatable transgene expression. Here, we review the recent developments on HSV-1-based vector systems and their applications in experimental and clinical gene therapy protocols.

Brain Neoplasms↗