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At least 271 records · Page 15Linked to original sources
PubMed2003

Cytotoxic effect of replication-competent adenoviral vectors carrying L-plastin promoter regulated E1A and cytosine deaminase genes in cancers of the breast, ovary and colon.

Prodrug activating transcription unit gene therapy is one of several promising approaches to cancer gene therapy. Combining that approach with conditionally replication-competent viral vectors that are truly tumor specific has been an important objective of recent work. In this study, we report the construction of a new conditionally replication-competent bicistronic adenoviral vector in which the cytosine deaminase (CD) gene and the E1a gene are driven by the L-plastin tumor-specific promoter (AdLpCDIRESE1a). A similar vector driven by the CMV promoter has also been constructed (AdCMVCDIRESE1a) as a control. We have carried out in vitro cytotoxicity in carcinomas of the breast, ovary and colon, and in vivo efficacy studies with these vectors in an animal model of colon cancer. While the addition of the AdLpCDIRESE1a vector to established cancer cell lines showed significant cytotoxicity in tumor cells derived from carcinomas of the breast (MCF-7), colon (HTB-38) and ovary (Ovcar 5), no significant toxicity was seen in explant cultures of normal human mammary epithelial cells (HMEC) exposed to this vector. The addition of 5-fluorocytosine (5FC) significantly increased the cytotoxicity in an additive fashion of both the AdLpCDIRESE1a and AdCMVCDIRESE1a vectors as well as that of the AdLpCD replication incompetent vector to established tumor cell lines. However, no significant cytotoxicity was observed with the addition of 5FC to explant cultures of normal human mammary epithelial cells that had been exposed to the L-plastin-driven vectors. Studies with mixtures of infected and uninfected tumor cell lines showed that the established cancer cell lines infected with the AdLpCDIRESE1a vector generated significant toxicity to surrounding uninfected cells (the "bystander effect") even at a ratio of 0.25 of infected cells to infected + uninfected cells in the presence of 5FC. The injection of the AdLpCDIRESE1a vector into subcutaneous deposits of human tumor nodules in the nude mice was potentiated by administering 5-FC by intraperitoneal injection. This treatment resulted in a decreased tumor size and a decreased tumor cell growth rate. The mice treated with a combination of the AdLpCDIRESE1a vector intratumoral injection and intraperitoneal 5FC injections lived much longer than the other experimental groups exposed to the viral vector alone or to the combination of the intratumoral AdLpCD replication incompetent vector injections plus intraperitoneal 5-FC injections. These encouraging results with our newly constructed AdLpCDIRESE1a vector suggest a need for further study of its utility in a preclinical model of intracavitary therapy of pleural or peritoneal carcinomatosis.

Adenoviridae↗
PubMed2002

Titering lentiviral vectors: comparison of DNA, RNA and marker expression methods.

To better characterize lentiviral vector supernatants, we compared three methods of titer assessment. These titer methods include assessment of vector RNA sequences in supernatants, DNA sequences in transduced cells, and vector expression in transduced cells (using a vector which expressed the green fluorescence protein, GFP). For analysis of RNA and DNA, we developed a real-time PCR method for detecting the lentiviral packaging sequence and used this methodology to quantitate the number of vector sequences. Vector expression was assessed by flow cytometric analysis for GFP. As functional titers (DNA and GFP expression titers) are dependent on transduction efficiency, we calculated the titer of a lentiviral vector, RRL-CMV-GFP, after transduction of 293, HeLa, or Mus dunni cells. Genomic DNA was extracted at 4 and 14 days after transduction and the number of vector DNA molecules was determined against a plasmid standard. Of the three cell lines tested, 293 cells provided the highest rate of transduction (PCR estimated DNA titer for RRL-CMV-GFP vector was 2.52 +/- 0.25 x 10(6) molecules/ml at 14 days, and 2.31 +/- 0.15 x 10(6) molecules/ml at 4 days). When titer was calculated based on GFP expression, the highest titer was also obtained on 293 cells (0.26 +/- 0.04 x 10(6) TU/ml at 14 days, and 0.24 +/- 0.03 +/- 10(6) TU/ml at 4 days). The titers obtained by GFP expression assay were approximately one log lower than those obtained by DNA analysis suggesting that variability in vector expression may underestimate titer. Measurement of RNA titers directly from vector supernatants against a plasmid standard indicated that the RNA titers are substantially higher than the DNA (approximately 10(3)-fold) and GFP titers (approximately 10(4)-fold). To show that the lentiviral probe and primers could be used for titering a variety of lentiviral vectors, we have also used the real-time PCR method to determine the DNA titers of two other HIV1 derived vectors, RRL-PGK-GFP (6.1 +/- 1.4 x 10(5) molecules/ml), and SMPU-RRE-BN (1.26 +/- 0.2 x 10(6) molecules/ml). We conclude that of the three methods tested, titers assessed by DNA analysis of transduced cells provide the most reliable estimate of functional titers as these are least likely to be influenced by factors, such as defective interfering particles and vector expression levels. The real-time PCR method described offers a reproducible method for lentiviral titering and can be applied to a wide variety of vectors, regardless of transgene.

Animals↗
PubMed2003

Retroviral vectors for high-level transgene expression in T lymphocytes.

Efficient expression of genes transferred by retroviral vectors is a prerequisite for gene therapy, especially when the biological effect depends on the amount of transgene product. High-level gene expression is desirable for several gene therapy approaches involving T lymphocytes. We evaluated standard retroviral vectors with cis-regulatory control elements of the Moloney murine leukemia virus (Mo-MLV) with or without the human T cell-specific CD2 enhancer. For comparison, vectors containing the long terminal repeat (LTR) of myeloproliferative sarcoma virus (MPSV) and an improved 5' untranslated region were used (MP71 vectors), with or without the woodchuck hepatitis virus posttranscriptional regulatory element (PRE). All vectors expressed the enhanced green fluorescent protein (GFP) to measure transgene expression. In mouse T cells MP71 vectors with and without the PRE yielded an up to 10-fold higher expression level compared with the Mo-MLV-based vectors currently used for gene transfer into T lymphocytes. A high multiplicity of infection (MOI) of standard Mo-MLV vectors could not reach expression levels obtained with a low MOI of MP71 vector. Ex vivo-transduced mouse T lymphocytes maintained the vector-dependent differences in level of transgene expression in Rag-1-deficient mice when adoptively transferred. In four human T cell lines and human primary T lymphocytes MP71 vectors yielded an up to 75-fold higher GFP expression level in comparison with the standard Mo-MLV vector. In contrast to mouse T cells, the integration of the PRE into MP71 vectors induced in human T cells a further significant increase in transgene expression level. Southern blot analysis of CEM T cells revealed that the superior performance of MP71 vectors was not due to a higher rate of viral integration. In summary, MP71 vectors are useful tools for stable, high-level gene expression in T lymphocytes, for example, in the expression of T cell receptor genes.

3T3 Cells↗
PubMed1997

LacZ gene transfer to skeletal muscle using a replication-defective herpes simplex virus type 1 mutant vector.

Herpes simplex virus type 1 (HSV-1) represents a promising new viral vector capable of efficient transduction of myofibers in vivo. Here we report on the use of a replication-defective HSV-1 mutant vector (DZ) deleted for the essential immediate early (IE) gene ICP4 for studies of reporter gene transfer and expression following direct inoculation of mouse skeletal muscle. The recombinant vector was engineered to contain the Escherichia coli lacZ gene under transcriptional control of the strong human cytomegalovirus (HCMV) IE promoter. The effect of vector cytotoxicity on the durability of transgene expression following infection of muscle cells in culture and myofibers in vivo revealed that this first-generation HSV vector was cytopathic, limiting the persistence of vector-transduced cells. UV irradiation of vector preparations reduced viral cytotoxicity for myoblasts in culture without reducing significantly beta-galactosidase production. Moreover, muscle cell viability and the durability of transgene expression was enhanced by several days following UV inactivated-vector infection in vivo. Nevertheless, the viral DNA was subsequently lost from vector-inoculated muscle tissue within 2 weeks. This observation indicated that vector toxicity alone did not account for the lack of persistent transgene expression. Longer-term vector transduction and transgene expression was observed, however, following inoculation of immunodeficient SCID mice, indicating that host immunocompetence played an important role in determining the duration of transgene expression in animals. To support this hypothesis, cells expressing CD4 and CD8 antigens have been found in the HSV-1 injected muscle of immunocompetent mice. These data demonstrated that both vector toxicity and vector-induced immunity are significant obstacles to the use of HSV-1 vectors for muscle gene transfer. These impediments must be overcome to further develop HSV vectors for muscle gene therapy applications.

Animals↗
PubMed1997

Adeno-associated virus vector integration junctions.

Vectors derived from adeno-associated virus (AAV) have the potential to stably transduce mammalian cells by integrating into host chromosomes. Despite active research on the use of AAV vectors for gene therapy, the structure of integrated vector proviruses has not previously been analyzed at the DNA sequence level. Studies on the integration of wild-type AAV have identified a common site-specific integration locus on human chromosome 19; however, most AAV vectors do not appear to integrate at this locus. To improve our understanding of AAV vector integration, we analyzed the DNA sequences of several integrated vector proviruses. HeLa cells were transduced with an AAV shuttle vector, and integrated proviruses containing flanking human DNA were recovered as bacterial plasmids for further analysis. We found that AAV vectors integrated as single-copy proviruses at random chromosomal locations and that the flanking HeLa DNA at integration sites was not homologous to AAV or the site-specific integration locus of wild-type AAV. Recombination junctions were scattered throughout the vector terminal repeats with no apparent site specificity. None of the integrated vectors were fully intact. Vector proviruses with nearly intact terminal repeats were excised and amplified after infection with wild-type AAV and adenovirus. Our results suggest that AAV vectors integrate by nonhomologous recombination after partial degradation of entering vector genomes. These findings have important implications for the mechanism of AAV vector integration and the use of these vectors in human gene therapy.

Base Sequence↗
PubMed1997

Activation of transgene expression by early region 4 is responsible for a high level of persistent transgene expression from adenovirus vectors in vivo.

The persistence of transgene expression has become a hallmark for adenovirus vector evaluation in vivo. Although not all therapeutic benefit in gene therapy is reliant on long-term transgene expression, it is assumed that the treatment of chronic diseases will require significant persistence of expression. To understand the mechanisms involved in transgene persistence, a number of adenovirus vectors were evaluated in vivo in different strains of mice. Interestingly, the rate of vector genome clearance was not altered by the complete deletion of early region 4 (E4) in our vectors. The GV11 (E1- E4-) vector genome cleared with a similar kinetic profile as the GV10 (E1-) vector genome in immunocompetent and immunocompromised mice. These results suggest that the majority of adenovirus vector genomes are eliminated from transduced tissue via a mechanism(s) independent of T-cell, B-cell, and NK cell immune mechanisms. While the levels of persistence of transgene expression in liver or lung transduced with GV10 and GV11 vectors expressing beta-galactosidase, cystic fibrosis transmembrane conductance regulator, or secretory alkaline phosphatase were similar in immunocompetent mice, a marked difference was observed in immunocompromised animals. Levels of transgene expression initially from both GV10 and GV11 vectors were the same. However, GV11 transgene expression correlated with loss of vector genome, while GV10 transgene expression persisted at a high level. Coadministration and readministration of GV10 vectors showed that E4 provided in trans could activate transgene expression from the GV11 vector genome. While transgene expression activity per genome from the GV10 vector is clearly activated, expression from a cytomegalovirus promoter expression cassette in a GV11 vector appeared to be further inactivated as a function of time. Understanding the molecular mechanisms underlying these expression effects will be important for developing persistent adenovirus vectors for chronic applications.

Adenoviridae↗
PubMed2000

Viral vectors for gene transfer: a review of their use in the treatment of human diseases.

The efficient delivery of therapeutic genes and appropriate gene expression are the crucial issues for clinically relevant gene therapy. Viruses are naturally evolved vehicles which efficiently transfer their genes into host cells. This ability made them desirable for engineering virus vector systems for the delivery of therapeutic genes. The viral vectors recently in laboratory and clinical use are based on RNA and DNA viruses processing very different genomic structures and host ranges. Particular viruses have been selected as gene delivery vehicles because of their capacities to carry foreign genes and their ability to efficiently deliver these genes associated with efficient gene expression. These are the major reasons why viral vectors derived from retroviruses, adenovirus, adeno-associated virus, herpesvirus and poxvirus are employed in more than 70% of clinical gene therapy trials worldwide. Among these vector systems, retrovirus vectors represent the most prominent delivery system, since these vectors have high gene transfer efficiency and mediate high expression of therapeutic genes. Members of the DNA virus family such as adenovirus-, adeno-associated virus or herpesvirus have also become attractive for efficient gene delivery as reflected by the fast growing number of clinical trials using these vectors. The first clinical trials were designed to test the feasibility and safety of viral vectors. Numerous viral vector systems have been developed for ex vivo and in vivo applications. More recently, increasing efforts have been made to improve infectivity, viral targeting, cell type specific expression and the duration of expression. These features are essential for higher efficacy and safety of RNA- and DNA-virus vectors. From the beginning of development and utilisation of viral vectors it was apparent that they harbour risks such as toxicities, immunoresponses towards viral antigens or potential viral recombination, which limit their clinical use. However, many achievements have been made in vector safety, the retargeting of virus vectors and improving the expression properties by refining vector design and virus production. This review addresses important issues of the current status of viral vector design and discusses their key features as delivery systems in gene therapy of human inherited and acquired diseases at the level of laboratory developments and of clinical applications.

Adenoviridae↗
PubMed2003

HIV-2 derived lentiviral vectors: gene transfer in Parkinson's and Fabry disease models in vitro.

Lentiviral vectors are prime candidate vectors for gene transfer into dividing and non-dividing cells, including neuronal cells and stem cells. For safety, HIV-2 lentiviral vectors may be better suited for gene transfer in humans than HIV-1 lentiviral vectors. HIV-2 vectors cross-packaged in HIV-1 cores may be even safer. Demonstration of the efficacy of these vectors in disease models will validate their usefulness. Parkinson's disease and Fabry disease provide excellent models for validation. Parkinson's disease is a focal degeneration of dopaminergic neurons in the brain with progressive loss of ability to produce the neurotransmitter dopamine. Current treatment entails administration of increasing doses of L-dopa, with attendant toxicity. We explore here the hypothesis that gene transfer of aromatic acid decarboxylase (AADC), a key enzyme in the pathway, will make neuronal cells more efficiently convert L-dopa into dopamine. Fabry disease on the other hand is a monogenic inherited disease, characterized by alpha-galactosidase A (AGA) deficiency, resulting in glycolipid accumulation in several cell types, including fibroblasts. Animal models for preclinical investigations of both of these diseases are available. We have designed monocistronic HIV-1 and HIV-2 vectors with the AADC transgene and monocistronic and bicistronic HIV-2 vectors with the AGA and puromycin resistance transgenes. They were packaged with either HIV-2 cores or HIV-1 cores (hybrid vectors). Gene transfer of AADC gene in neuronal cells imparted the ability on the transduced cells to efficiently convert L-dopa into dopamine. Similarly, the AGA vectors induced Fabry fibroblasts to produce high levels of AGA enzyme and caused rapid clearance of the glycolipids from the cells. Both monocistronic and bicistronic vectors were effective. Thus, the insertion of a second gene downstream in the bicistronic vector was not deleterious. In addition, both the self-packaged vectors and the cross-packaged hybrid vectors were effective in gene transfer.

Aromatic-L-Amino-Acid Decarboxylases↗
PubMed1993

Targeting vector configuration and method of gene transfer influence targeted correction of the APRT gene in Chinese hamster ovary cells.

A 21-bp deletion in the third exon of the APRT gene in Chinese hamster ovary (CHO) cells was corrected by transfection with a plasmid containing hamster APRT sequences. Targeted correction frequencies in the range of 0.3-3.0 x 10(-6) were obtained with a vector containing 3.2 kb of APRT sequence homology. To examine the influence of vector configuration on targeted gene correction, a double-strand break was introduced at one of two positions in the vector prior to transfection by calcium phosphate-DNA coprecipitation or electroporation. A double-strand break in the region of APRT homology contained in the vector produced an insertion-type vector, while placement of the break just outside the region of homology produced a replacement-type vector. Gene targeting with both linear vector configurations yielded equivalent ratios of targeted recombinants to nontargeted vector integrants; however, targeting with the two different vector configurations resulted in different distributions of targeted recombination products. Analysis of 66 independent APRT+ recombinant clones by Southern hybridization showed that targeting with the vector in a replacement-type configuration yielded fewer targeted integrants and more target gene convertants than did the integration vector configuration. Targeted recombination was about fivefold more efficient with electroporation than with calcium phosphate-DNA coprecipitation; however, both gene transfer methods produced similar distributions of targeted recombinants, which depended only on targeting vector configuration. Our results demonstrate that insertion-type and replacement-type gene targeting vectors produce similar overall targeting frequencies in gene correction experiments, but that vector configuration can significantly influence the yield of particular recombinant types.

Adenine Phosphoribosyltransferase↗
PubMed2003

Gene transfer into rabbit keratocytes using AAV and lipid-mediated plasmid DNA vectors with a lamellar flap for stromal access.

Development of gene transfer methods that can precisely deliver therapeutic genes to the localized or targeted tissue(s) would be highly beneficial in developing new gene therapy approaches and may also extend animal models for studying in vivo gene function and regulation at molecular levels in the selected tissues. We investigated lipid- and AAV-mediated gene transfer in rabbit cornea using a lamellar flap-technique. The goals of this study were to (1) analyze methods for in situ gene transfer into keratocytes, (2) identify efficient and suitable vectors for gene transfer into keratocytes, and (3) characterize times of first detectable expression, localization and duration of transgene expression in keratocytes with different vectors. A lamellar flap was produced in the rabbit cornea with a microkeratome. Recombinant adeno-associated viral vector (rAAV) expressing either beta-galactosidase (rAAV-beta-gal) or chloramphenicol acetyltransferase (rAAV-CAT) reporter genes, or plasmid-cationic lipid complexes expressing CAT (pMP6-CAT) or beta-galactosidase (pTR-beta-gal) were applied beneath the lamellar flap for two minutes. The flap was repositioned and eyelids sutured overnight. Corneas were removed at 4hr, 12hr, 36hr, 3 days, 7 days, or 10 days after application and either fixed in 2% formaldehyde, cryosectioned and stained for beta-galactosidase activity or homogenized and measured for CAT levels by ELISA. Corneas infected with rAAV-beta-gal vector showed positive beta-gal staining in the center and periphery of the flap interface in whole corneas and corneal beds at 3, 7, and 10 days, but not at earlier time points. Corneas treated with pTR-beta-gal plasmid vector showed positive beta-gal expression at the interface at 4, 12 and 36hr, but not at 3 or 7 days. The posterior surface of the lamellar interface where the vector was applied showed more expression than the overlying anterior surface with both plasmid and viral vectors. The level of gene expression was less with plasmid vector than viral vector monitored using beta-gal staining. CAT-ELISA confirmed expression of the CAT reporter gene with either the plasmid or rAAV vector. These results demonstrate that foreign genes can be introduced into keratocytes with plasmid or viral vectors using a lamellar flap to gain access to the stroma. The expression profile of the reporter genes depended on the vector. Transfection of keratocytes with plasmid vectors produced rapid expression of the reporter genes, but for a short duration. Reporter gene expression following transduction by rAAV vector was delayed several days, but was at higher levels and for a longer duration. This is the first report to demonstrate selective gene transfer into keratocytes and would be highly useful in studying function and regulation of genes in vivo and may eventually furnish a tool for the treatment of corneal dystrophies.

Adenoviridae↗
PubMed1999

Variables affecting the transduction efficiency of adenovirus vectors in bovine aortic endothelial cells.

Features and kinetics of Adenovirus (Ad)-mediated gene transfer to endothelial cells (EC) are not ultimately determined. We tested variables pertinent to the efficiency of Ad-mediated gene transfer to bovine aortic endothelial cells (BAEC) including: (1) Ad-vectors with different promoters, (2) kinetics of transduction efficiency of LacZ gene to BAEC, (3) the concentration and volume of vector-containing medium, (4) the period of incubation time of Ad vectors with BAEC, (5) the duration of transgene expression. An Ad5-LacZ vector with a cytomegalovirus (CMV) promoter transduced the LacZ gene to the cells more efficiently than vectors with the Rous sarcoma virus (RSV) promoter. However, both vectors exhibited a dose-dependent relationship between the vector multiplicity of infection (moi) and the percentage of LacZ-expressing cells. The higher moi of both vectors achieved nearly 100% of transduction efficiency in cultured BAEC. Although the Ad-CMV-LacZ vector better transduced the LacZ gene to BAEC than Ad-RSV-LacZ, a long period of vector exposure to BAEC could overcome the slightly difference in transduction efficiency between the two vectors. These results indicate that both Ad vectors are efficient for gene transfer to endothelial cells, and higher moi of vectors or a longer period exposure of vectors to EC can facilitate efficient transduction of foreign gene into EC in culture.

Adenoviridae↗
PubMed2002

Gene therapy for prostate cancer: toxicological profile of four HSV-tk transducing adenoviral vectors regulated by different promoters.

Adenoviral vector delivery of the Herpes simplex virus thymidine kinase (HSV-tk) gene in combination with the prodrug ganciclovir (GCV) has been tested in phase I clinical trials for prostate cancer and found to exhibit a satisfactory toxicity profile. We have developed additional adenoviral vectors with differing promoters to optimize the expression profile and in the present study evaluate the potential systemic toxicity of these vectors. Four recombinant adenoviral vectors that express the HSV-tk gene were generated using three different promoters: CMV (leftward orientation); RSV (both rightward and leftward orientation); and the mouse caveolin-1 (cav-1) promoter (leftward orientation). Efficacy was determined in vitro by cytotoxicity assays in a mouse prostate cancer cell line, RM-9, and in vivo by treating orthotopic tumors. Potential toxicity was evaluated from liver histology and apoptotic cell counts and enzyme levels in the serum following intravenous adenoviral vector injection. Although there were differences in HSV-tk expression at the protein level among the four vectors there were no significant differences in in-vitro cytotoxicity studies with GCV or in vivo in tumor growth suppression of an orthotopic mouse prostate cancer model in GCV treated mice. Intravenous delivery of high doses of all adenoviral vectors lead to abnormalities in liver function as measured by specific serum markers and histological evaluation of liver tissue and increased levels of apoptosis in the liver. These abnormalities were most prevalent with the vector containing the CMV promoter and the rightward oriented RSV promoter. They were least prevalent in the vector regulated by the cav-1 promoter. Upregulation of specific chemokines, MIP-2 and MIP-1beta was correlated with apoptotic counts. Our results demonstrate that comprehensive toxicological analysis of adenoviral vectors provides internally consistent information that can differentiate vectors with comparable efficacy based on toxicity. In these studies vectors with the cav-1 promoter-driven and leftward RSV-driven HSV-tk gene demonstrated minimal toxicities with cytotoxic effectiveness comparable to more toxic vectors. Our studies further suggest that promoter selection can influence the toxic effects of an adenoviral gene therapy vector.

Adenocarcinoma↗
PubMed1998

Increased gene transfer into human CD34+ progenitor cells using retroviral vectors produced by a canine packaging cell line.

Using retroviral supernatants derived from the amphotropic murine packaging cell line PA317 and the amphotropic canine packaging cell line (DA), cord blood and mobilized peripheral blood CD34+ cells were transduced with the vector LN (neomycin resistance) and the vector L-TR/TAT neo (neomycin resistance in conjunction with a double-hammerhead ribozyme conferring anti-HIV activity). Different multiplicities of infection (MOI) were applied in the setup according to vector titrations on NIH-3T3 cells. PA317-based supernatants were tested at MOI of 10 and 30. Purified concentrated DA-derived vector preparations were tested at MOI of 10, 30, 100, and 300. Immediately after transduction, CD34+ cells were plated into colony assays in the presence and absence of G418 to evaluate the amount of gene transfer and potential toxic effects of the vectors on colony growth. The remaining cells were subjected to G418 selection in liquid culture for 12 days and subsequently challenged with HIV-1JR-FL to test for efficacy of the anti-HIV gene in macrophages derived from transduced CD34+ cells. Transduction by the PA317-packaged vectors was maximal at the lowest MOI used and did not increase with increasing MOI. In contrast, transduction by the DA-packaged vectors could be progressively increased using increased MOI. The net transduction efficiency per unit of reverse transcriptase activity in the DA vector preparations was 8.7-fold higher than in the PA317 vector supernatants. HIV-1 challenge of the cells transduced by the ribozyme vector derived from the PA317 packaging cells resulted in a 1.5 log inhibition of p24 output compared with the control cells containing neomycin resistance only. A 2.5 log inhibition of p24 output could be observed in the cell population transduced with DA-packaged vector supernatants. Compared with retroviral supernatants from PA317 packaging cell lines, DA packaging line-derived vector preparations demonstrated higher transduction efficiency into CD34+ cells, particularly at higher MOI, and increased efficacy of the transferred anti-HIV gene when challenged with HIV-1JR-FL. The increase in transduction efficiency may be due to a higher ratio of intact vs. defective vector particles in the DA-derived vector preparations.

Animals↗
PubMed1999

Effect of scaffold attachment region on transgene expression in retrovirus vector-transduced primary T cells and macrophages.

The scaffold attachment region of the human interferon beta gene (IFN-SAR) inserted into a retroviral vector improved transgene expression in human primary CD4+ and CD8+ T cells, and in primary monocytemacrophages. In T cells, expression of the Maloney murine leukemia virus (Mo-MuLV)-based retroviral vectors was high in activated cells but low in resting cells. Addition of the IFN-SAR sequence enhanced vector expression 2- to 10-fold, and the effect was particularly pronounced in resting T cells. In CD33+CD14+CD4+ monocyte-macrophages derived from transduced hematopoietic stem/progenitor cells (HSPCs) in vitro, the IFN-SAR enhanced vector expression three- to sixfold. We have used the IFN-SAR-containing vectors to express the RevM10 gene, a trans-dominant mutant of the human immunodeficiency virus type 1 (HIV-1) rev gene. Compared with a standard retroviral vector, the IFN-SAR-containing vector was significantly (p < 0.01) more potent at inhibiting HIV-1 replication in infected CD4+ peripheral blood lymphocytes. In monocytes, however, addition of the IFN-SAR did not significantly improve antiviral efficacy. To understand better the reason for the strong effect of the SAR on antiviral efficacy in T cells we have studied the expression of HIV, Mo-MuLV, and Mo-MuLV + SAR vectors in resting and activated cells. While the expression of all three vectors was lower in resting compared with activated cells, the kinetics of the decrease in expression were fastest for the Mo-MuLV vector, followed by the HIV vector and then the Mo-MuLV + SAR vector. Thus, higher level expression of the Mo-MuLV + SAR vector relative to wild-type HIV at all stages of T cell activation is the most likely explanation for the strong antiviral efficacy. Overall, this study demonstrates the utility of the IFN-SAR sequence for achieving high-level retroviral vector expression in lymphoid and myeloid hematopoietic cells.

Animals↗
PubMed1994

Retroviral vectors containing chimeric promoter/enhancer elements exhibit cell-type-specific gene expression.

Retroviral vectors were constructed in which the U3 promoter/enhancer of Moloney murine leukemia (Mo-MLV) was replaced by the corresponding region from five related murine retroviruses--AKR murine leukemia virus (AKV), Harvey murine sarcoma virus (HaMSV), myeloproliferative sarcoma virus (MPSV), SL3-3, and the NZB-xenotropic virus (Xeno). In these vectors the chimeric long terminal repeat (chLTR) drives the expression of the chloramphenicol acetyl transferase (CAT) reporter gene that is followed by an internal SV40 virus early region promoter linked to the neomycin phosphotransferase II (NEO) gene. As an initial measure of the relative promoter/enhancer strength of the chLTR vectors, the murine NIH-3T3 cell line and the human JURKAT cell lines were transfected and assayed for CAT reporter activity. Relative to the MoMLV vector, the HaMSV construct was the most active in NIH-3T3 cells whereas the SL3-3 vector displayed the greatest activity in JURKAT cells. Retroviral vector producer cell populations and cell clones were established for each chLTR vector, and all were capable of yielding high vector titers (> 10(5) G418R cfu/ml on NIH-3T3). Supernatant from these cells was used to transduce both mouse and human cell lines and primary cells. In NIH-3T3 cells and two murine fibrosarcoma cell lines, the HaMSV chLTR vector was slightly more active than the MoMLV chLTR vector. In the human HepG2 and HeLa cell lines, the MPSV chLTR vector was the most active. Data from the human JURKAT T-cell line and a T cell line derived from an ADA-deficient severe combined immunodeficiency (SCID) patient demonstrate that the SL3-3 chLTR is the most active in these lymphoid cell lines. The greatest difference in the comparison of the different chLTR vectors was observed in primary human umbilical vein endothelial cells, where the MoMLV vector produced up to 100 times more CAT activity than the SL3-3 vector. These data suggest that the use of specific promoter/enhancer elements may lead to higher levels of gene expression following retroviral-mediated gene transfer into specific cell types and these observations may be useful in the design of human gene therapy experiments.

Animals↗
PubMed1999

Integrating adenovirus-adeno-associated virus hybrid vectors devoid of all viral genes.

Recently, we demonstrated that inverted repeat sequences inserted into first-generation adenovirus (Ad) vector genomes mediate precise genomic rearrangements resulting in vector genomes devoid of all viral genes that are efficiently packaged into functional Ad capsids. As a specific application of this finding, we generated adenovirus-adeno-associated virus (AAV) hybrid vectors, first-generation Ad vectors containing AAV inverted terminal repeat sequences (ITRs) flanking a reporter gene cassette inserted into the E1 region. We hypothesized that the AAV ITRs present within the hybrid vector genome could mediate the formation of rearranged vector genomes (DeltaAd.AAV) and stimulate transgene integration. We demonstrate here that DeltaAd.AAV vectors are efficiently generated as by-products of first-generation adenovirus-AAV vector amplification. DeltaAd.AAV genomes contain only the transgene flanked by AAV ITRs, Ad packaging signals, and Ad ITRs. DeltaAd.AAV vectors can be produced at a high titer and purity. In vitro transduction properties of these deleted hybrid vectors were evaluated in direct comparison with first-generation Ad and recombinant AAV vectors (rAAVs). The DeltaAd.AAV hybrid vector stably transduced cultured cells with efficiencies comparable to rAAV. Since cells transduced with DeltaAd.AAV did not express cytotoxic viral proteins, hybrid viruses could be applied at very high multiplicities of infection to increase transduction rates. Southern analysis and pulsed-field gel electrophoresis suggested that DeltaAd.AAV integrated randomly as head-to-tail tandems into the host cell genome. The presence of two intact AAV ITRs was crucial for the production of hybrid vectors and for transgene integration. DeltaAd.AAV vectors, which are straightforward in their production, represent a promising tool for stable gene transfer in vitro and in vivo.

Adenoviridae↗
PubMed1999

Minimum requirements for efficient transduction of dividing and nondividing cells by feline immunodeficiency virus vectors.

The development of gene delivery vectors based on feline immunodeficiency virus (FIV) is an attractive alternative to vectors based on primate sources for the delivery of genes into humans. To investigate the requirements for efficient transduction of dividing and nondividing cells by vector particles based on FIV, a series of packaging and vector constructs was generated for which viral gene expression was minimized and from which unnecessary cis-acting sequences were deleted. Pseudotyped vector particles produced in 293T cells were used to transduce various target cells, including contact-inhibited human skin fibroblasts and growth-arrested HT1080 cells. FIV vectors in which the U3 promoter was replaced with the cytomegalovirus promoter gave rise to over 50-fold-higher titers than FIV vectors containing the complete FIV 5' long terminal repeat (LTR). Comparison of the transduction efficiencies of vectors containing different portions of the FIV Gag coding region indicates that at least a functional part of the FIV packaging signal (Psi) is located within an area which includes the 5' LTR and the first 350 bp of gag. Transduction efficiencies of vectors prepared without FIV vif and orf2 accessory gene expression did not differ substantially from those of vectors prepared with accessory gene expression in either dividing or nondividing cells. The requirement for FIV rev-RRE was, however, demonstrated by the inefficient production of vector particles in the absence of rev expression. Together, these results demonstrate the efficient transduction of nondividing cells in vitro by a multiply attenuated FIV vector and contribute to an understanding of the minimum requirements for efficient vector production and infectivity. In addition, we describe the ability of an FIV vector to deliver genes in vivo into hamster muscle tissue.

Animals↗
PubMed1999

The resistance of retroviral vectors produced from human cells to serum inactivation in vivo and in vitro is primate species dependent.

The ability to deliver genes as therapeutics requires an understanding of the vector pharmacokinetics similar to that required for conventional drugs. A first question is the half-life of the vector in the bloodstream. Retroviral vectors produced in certain human cell lines differ from vectors produced in nonhuman cell lines in being substantially resistant to inactivation in vitro by human serum complement (F. L. Cosset, Y. Takeuchi, J. L. Battini, R. A. Weiss, and M. K. Collins, J. Virol. 69:7430-7436, 1995). Thus, use of human packaging cell lines (PCL) may produce vectors with longer half-lives, resulting in more-efficacious in vivo gene therapy. However, survival of human PCL-produced vectors in vivo following systemic administration has not been explored. In this investigation, the half-lives of retroviral vectors packaged by either canine D17 or human HT1080 PCL were measured in the bloodstreams of macaques and chimpanzees. Human PCL-produced vectors exhibited significantly higher concentrations of circulating biologically active vector at the earliest time points measured (>1, 000-fold in chimpanzees), as well as substantially extended half-lives, compared to canine PCL-produced vectors. In addition, the circulation half-life of human PCL-produced vector was longer in chimpanzees than in macaques. This was consistent with in vitro findings which demonstrated that primate serum inactivation of vector produced from human PCL increased with increasing phylogenetic distance from humans. These results establish that in vivo retroviral vector half-life correlates with in vitro resistance to complement. Furthermore, these findings should influence the choice of animal models used to evaluate retroviral-vector-based therapies.

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