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PubMed1983

Continuous vectorcardiography in acute myocardial infarction. Natural course of ST and QRS vectors.

We made continuous recordings of the X, Y and Z Frank leads in 43 patients with their first myocardial infarction admitted within 4 hours of the onset of pain. Sequential hourly analysis of the ST and QRS vector changes during the first 24 hours was performed. In short-term survivors (n = 38) mean serial changes of ST vectors showed an initial rapid decline until the 8th-10th hour, whereas QRS vector changes lasted longer and were completed within 13.5 +/- 3.6 hours (inferior infarction) and 10.2 +/- 2.4 hours (anterior infarction) respectively. The initial ST vector magnitude was significantly correlated to the subsequent cumulative QRS vector change (r = 0.82). The individual ST vector changes showed a rapid decline in 33 of 38 patients (87%), whereas in 5 patients the ST vector magnitude increased to reach its maximum after 4-6 hours. After the initial decline new increases of the ST vector magnitude was noted in 16 patients on 20 occasions. In 13 instances this was associated with recurrent pain. The spatial change of ST vector direction with reference to the initial direction was significantly greater when recurrent ST rises were accompanied by additional QRS vector changes, compared to those without associated QRS changes (P less than 0.025). The individual QRS vector slopes could be characterized as (1) monophasic (n = 21, 55%), (2) polyphasic (n = 11, 29%) and (3) irregular (n = 6, 16%). We conclude that continuous vectorcardiography is a suitable method for following ST and QRS vector changes that accompany acute myocardial infarction and that ST vector changes can be used to predict subsequent QRS vector changes.

Adult↗
PubMed1981

Dynamic electrocardiography. III. The "imaginary cardiac vector" hypothesis: theoretical basis.

The "imaginary cardiac vector" hypothesis states that the "cardiac vector" is not a "real" vector entity. The hypothesis arises from the fact that voltage is not a vector but a scalar quantity. Nevertheless, in classical electrocardiographic analysis and teaching, the ECG voltage is treated as a vector. These voltages are used as "vector" components--first, to calculate a "resultant manifest potential difference", represented by the "cardiac vector" arrow in electrocardiography, and second, to draw the "cardiac vector" loop in vectorcardiography. The resultant "cardiac vector" is usually considered to be a "real" vector quantity describing the electrical activity of the heart. It is also widely believed that this "vector" was first described by Einthoven in 1913. To evaluate the hypothesis, we have utilised the actual values and methods presented by Einthoven, and demonstrated that the theoretical basis of the "cardiac vector" is false. It appears that Einthoven followed scalar and not vector procedures and that he did not describe a vector.

Electrocardiography↗
PubMed2005

Upscaling of lentiviral vector production by tangential flow filtration.

BACKGROUND: HIV-1-derived vectors are promising tools for gene transfer into the brain. Application of these vectors for gene therapy or for the creation of animal models for neurodegenerative diseases requires standardization and upscaling of lentiviral vector production methods. METHODS: In this study, serum-free HIV-1 vector production was efficiently upscaled by use of cell factories and the introduction of tangential flow filtration (TFF) prior to centrifugation. RESULTS: Vector titers (TU/ml) and p24 values (pg p24/ml) for a serum-free HIV-1 vector produced in cell factories and using TFF prior to centrifugation were comparable to those of small-scale productions. TFF allowed a 66-fold concentration of the vectors with complete vector recovery. Further concentration of the vector (30-fold) was achieved either by low-speed centrifugation or by ultracentrifugation. Combination of TFF and ultracentrifugation resulted in a vector recovery of 90-100% and titers that increased 1800-fold and 900-fold for transducing units and p24 concentration, respectively. CONCLUSIONS: With this new standardized method for lentiviral vector production and concentration, 1 ml of concentrated vector is routinely produced with titers of 10(9)-10(10) TU/ml starting from 2 l of cell-culture medium. Moreover, stereotactic injection of this vector in mouse striatum resulted in a large transduced brain volume in the absence of any immune response.

Animals↗
PubMed2002

Systemic interleukin-6 responses following administration of adenovirus gene transfer vectors to humans by different routes.

Administration of adenovirus (Ad) vectors to animals induces innate immune responses, typified by elevated interleukin-6 (IL-6). To assess innate responses to Ad vectors in humans, we evaluated serum IL-6 following administration of E1(-) E3(-) Ad vectors to different human hosts and the relationship among peak IL-6 and peak anti-Ad neutralizing antibodies. We administered: 1) Ad(GV)CFTR.10, a vector carrying the normal human CFTR cDNA (3 x 10(7) to 2 x 10(10) particle units (pu)) to airways of individuals with cystic fibrosis (CF); 2) Ad(GV)VEGF121.10, a vector carrying the normal human vascular endothelial growth factor (VEGF)121 cDNA, to the myocardium (4 x 10(8) to 4 x 10(10) pu) of individuals with coronary artery disease (CAD) and to lower extremity muscles (4 x 10(8) to 4 x 10(9.5) pu) of individuals with peripheral vascular disease (PVD); and 3) Ad(GV)CD.10, a vector carrying the Escherichia coli cytosine deaminase gene to skin (7 x 10(7) to 7 x 10(9) pu) and airways (7 x 10(8) to 7 x 10(10) pu) of normal individuals and to liver metastasis (4 x 10(8) to 4 x 10(9) pu) of individuals with colon carcinoma. IL-6 increased mildly (up to 220 pg/ml) following vector administration to skin and lung airways of normal individuals and of individuals with CF, and to muscle and liver metastasis of individuals with PVD and colon cancer, respectively. IL-6 responses were higher (up to 1100 pg/ml) following myocardial administration. Control individuals who had chest surgery and bronchoscopy, but no vector administration, had comparable IL-6 increases. Thus, both administration of Ad vectors of humans up to 10(10) pu and the procedures used to administer the vectors elicit systemic IL-6 responses. There was no correlation among peak IL-6 and peak anti-Ad antibodies. These observations indicate that the innate host responses following administration of Ad vectors to humans may result from the procedures used to administer the vector, and from the vector per se.

Adenoviridae↗
PubMed1996

Immediate-early baculovirus vectors for foreign gene expression in transformed or infected insect cells.

Baculovirus expression vectors are used routinely for foreign gene expression and are under intense development as improved biological pesticides. Conventional baculovirus expression vectors are recombinant viruses that can express a foreign gene in insect cells under the control of the polyhedrin promoter, which provides high-level transcription during the very late phase of infection. For some applications, including foreign glycoprotein production and insect pest control, it might be advantageous to have baculovirus vectors that could express foreign gene products in uninfected cells or earlier after infection. To fulfill this need, we have constructed a new set of plasmids that can be used to clone and express foreign genes under the control of a baculovirus ie1 promoter, which is active in uninfected insect cells and throughout infection. We used a subset of these new plasmids to isolate recombinant baculoviruses containing various foreign genes and compared expression of these genes by the resulting immediate-early baculovirus vectors and by conventional baculovirus vectors. As expected, the immediate-early vectors began to express each foreign gene earlier in infection but, by 36-48 h postinfection, the conventional vectors had produced more of each foreign protein. Conventional baculovirus vectors also produced more enzymatic activity from two different procaryotic genes than the immediate-early baculovirus vectors. However, immediate-early vectors produced as much or more enzymatic activity from two different eucaryotic genes encoding secretory pathway proteins than the conventional vectors, even at 48 h postinfection. Hence, this report describes a new set of plasmids that can be used to clone and express foreign genes under the control of the baculovirus ie1 promoter and suggests that immediate-early baculovirus vectors might be as useful as conventional baculovirus expression vectors for producing biologically active eucaryotic secretory pathway proteins.

Animals↗
PubMed2001

Generation of replication-defective helper-free vectors based on simian immunodeficiency virus.

A systematic study on generating simian immunodeficiency virus (SIV)-based vectors was carried out. The goal was to generate helper-free, replication-defective SIVmac-based vectors at high titers. The general approach was to cotransfect into human 293T cells a plasmid carrying the vector construct along with two helper plasmids that together expressed the SIVmac virion proteins. Initial vectors carried the bacterial beta-galactosidase gene (beta-gal). These vectors had a technical difficulty: "pseudotransduction" of beta-gal protein produced during the 293T cell transfections. As a result, infection of cultures with these vector stocks also resulted in passive transfer into, and X-gal staining of, cells that had not actually been infected by the vector. A second generation of vectors expressing the enhanced jellyfish green fluorescence protein (EGFP) was not subject to this artifact. A systematic study of the SIVmac-based EGFP vectors was carried out. Helper-free vector stocks were obtained when helper plasmids lacking the SIVmac packaging signals were used. By employing envelope helper plasmids derived from different SIVmac isolates, it was possible to generate SIVmac-based vectors pseudotyped with envelope proteins of different cell tropism. Optimization of vector and helper plasmid structures, transfection conditions, and infection procedures ultimately yielded vector titers in excess of 10(6)/ml.

Animals↗
PubMed2005

Comparison of HIV- and EIAV-based vectors on their efficiency in transducing murine and human hematopoietic repopulating cells.

The use of lentiviral vectors for gene transfer into hematopoietic stem cells has raised considerable interest as these vectors can permanently integrate their genome into quiescent cells. Vectors based on alternative lentiviruses would theoretically be safer than HIV-1-based vectors and could also be used in HIV-positive patients, minimizing the risk of generating replication-competent virus. Here we report the use of third-generation equine infectious anemia virus (EIAV)- and HIV-1-based vectors with minimal viral sequences and absence of accessory proteins. We have compared their efficiency in transducing mouse and human hematopoietic stem cells both in vitro and in vivo to that of a previously documented second-generation HIV-1 vector. The third-generation EIAV- and HIV-based vectors gave comparable levels of transduction and transgene expression in both mouse and human NOD/SCID repopulating cells but were less efficient than the second-generation HIV-1 vector in human HSCs. For the EIAV vector this is possibly a reflection of the lower protein expression levels achieved in human cells, as vector copy number analysis revealed that this vector exhibited a trend to integrate equally efficiently compared to the third-generation HIV-1 vector in both mouse and human HSCs. Interestingly, the presence or absence of Tat in viral preparations did not influence the transduction efficiency of HIV-1 vectors in human HSCs.

Animals↗
PubMed1997

Development and characterization of a binary gene expression system based on bacteriophage T7 components in adenovirus vectors.

To explore the utility of the bacteriophage T7 binary system in adenovirus (Ad) vectors we constructed three Ad5-based vectors containing the T7 RNA polymerase (T7pol) gene in either early region 1 (E1) or E3. The recombinant Ad vectors were either deficient (AdT7pol1, AdT7pol2) or competent (AdT7pol3) for replication in human cells other than Ad5 transformed (293) cells. To test the ability of the T7 polymerase produced by these vectors to drive gene expression, a reporter vector was constructed with an E1 substitution comprising the bacterial beta-galactosidase (betaGal) (lacZ) gene under the control of the T7 gene 10 promoter (T7pro) and linked to the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) (AdBHG10T7betaGal). Coinfections were performed with the various AdT7pol vectors and the reporter vector, and expression was analysed in three different human cell lines: 293, A549 and MRC-5. Depending on the AdT7pol vector used, different levels of expression were obtained from the reporter gene. In 293 cells, expression was detected following infection at very low multiplicities of infection (moi) with all of the T7pol vectors when coinfected with the reporter vector AdBHG10T7betaGal. In A549 and MRC-5 cells very little expression was detected using AdT7pol1 or pol2 and efficient expression was only obtained when relatively high moi values of the replication-competent vector were used in the coinfections. We also constructed a single vector containing both elements of the T7 system (T7pol in E3 and T7 promoter driving expression of the chloramphenicol acetyl transferase (cat) gene in E1). This vector proved difficult to rescue but was stable once isolated. Finally, experiments performed to evaluate the 'leakiness' of the Ad-T7 system detected very little expression from the T7pro in the absence of T7 polymerase suggesting this system may be useful for the cloning and expression of genes encoding cytotoxic proteins.

Adenoviridae↗
PubMed2003

Characterization of in vitro and in vivo gene transfer properties of adenovirus serotype 35 vector.

We have recently developed a replication-defective, recombinant adenovirus (Ad) vector composed of the whole Ad serotype 35 (Ad35), a member of subgroup B. We describe herein the in vitro and in vivo gene transfer properties of Ad35 vector in comparison with Ad serotype 5 (Ad5) and the Ad5F35 vector, which is a fiber-substituted Ad5 vector containing Ad35 fiber proteins. In vitro, Ad35 vector efficiently transduced not only human CAR-positive cells but also CAR-negative cells. Following intravenous administration into mice, both Ad5 and Ad35 vectors were rapidly cleared from the bloodstream with a half-life of approximately 3 min. Ad5 vector-mediated transgene expression predominantly occurred in liver parenchymal cells, although the Ad5 vector was delivered to both liver parenchymal and nonparenchymal cells. In contrast, Ad35 vector was efficiently taken up by liver nonparenchymal cells and mediated transduction efficiency in the liver on a level 4 log orders lower than the Ad5 vector. These findings demonstrate that Ad35 vector is an attractive vehicle for gene transfer into human cells, while the biodistribution profile of Ad35 vector in mice is much different from that of the Ad5 vector.

Adenoviridae↗
PubMed1980

Weather, host and vector--their interplay in the spread of insect-borne animal virus diseases.

The spread of insect-borne animal virus diseases is influenced by a number of factors. Hosts migrate, move or are conveyed over long distances: vectors are carried on the wind for varying distances in search of hosts and breeding sites; weather and climate affect hosts and vectors through temperature, moisture and wind. As parasites of host and vector, viruses are carried by animals, birds and insects, and their spread can be correlated with the migration of hosts and the carriage of vectors on winds associated with the movements of the Intertropical Convergence Zone (ITCZ) and warm winds to the north and south of the limits of the ITCZ. The virus is often transmitted from a local cycle to a migratory cycle and back again.Examples of insect-borne virus diseases and their spread are analysed. Japanese, Murray Valley, Western equine, Eastern equine and St Louis encephalitis represent viruses transmitted by mosquito-bird or pig cycles.THE AREAS EXPERIENCING INFECTION WITH THESE VIRUSES CAN BE DIVIDED INTO A NUMBER OF ZONES: A, B, C, D, E and F. In zone A there is a continuous cycle of virus in host and vector throughout the year; in zone B, there is an upsurge in the cycle during the wet season, but the cycle continues during the dry season; there is movement of infected vectors between and within zones A and B on the ITCZ and the virus is introduced to zone C by infected vectors on warm winds; persistence may occur in zone C if conditions are right. In zone D, virus is introduced each year by infected vectors on warm winds and the arrival of the virus coincides with the presence of susceptible nestling birds and susceptible piglets. The disappearance of virus occurs at the time when migrating mosquitoes and birds are returning to warmer climates. The virus is introduced to zone E only on occasions every 5-10 years when conditions are suitable. Infected hosts introduced to zone F do not lead to circulation of virus, since the climate is unsuitable for vectors. Zones A, B and C correspond to endemic and zones D and E to epidemic conditions.Similar zones can be recognized for African horse sickness, bluetongue, Ibaraki disease and bovine ephemeral fever - examples of diseases transmitted in a midge-mammal cycle. In zones A and B viruses are transported by infected midges carried on the wind in association with the movement of ITCZ and undergo cycles in young animals. In these zones and in zone C there is a continual movement of midges on the warm wind between one area and another, colonizing new sites or reinforcing populations of midges already present. Virus is introduced at times into fringe areas (zones D and E) and, as there is little resistance in the host, gives rise to clinical signs of disease. In some areas there is persistence during adverse conditions; in others, the virus is carried back to the endemic zones by infected midges or vectors.Examples of viruses maintained in a mosquito/biting fly-mammal cycle are Venezuelan equine encephalitis and vesicular stomatitis. These viruses enter a migratory cycle from a local cycle and the vectors in the migratory cycle are carried over long distances on the wind. Further examples of virus spread by movement of vectors include West Nile, Rift Valley fever, yellow fever, epizootic haemorrhagic disease of deer and Akabane viruses.In devising means of control it is essential to decide the relationship of host, vector and virus and the nature of the zone in which the area to be controlled lies. Because of the continual risk of reintroduction of infected vectors, it is preferable to protect the host by dipping, spraying or by vaccination rather than attempting to eliminate the local population of insects.

African Horse Sickness↗
PubMed2001

FIV vector systems.

Why is feline immunodeficiency virus (FIV) such an appealing candidate for gene therapy vector development? Phylogenetic analysis suggests FIV is only distantly related to the primate lentiviruses, and despite repeated exposure, neither seroconversion nor other detectable evidence of human infection occurs. FIV naturally infects diverse Felidae worldwide, including the domestic cat. Here, the disease progression parallels the immunodeficiency caused by HIV, and for that reason, FIV and the cat provide an excellent model for anti-virals and AIDS vaccine research. Simple genome organization also facilitates vector development and analysis: FIV has only three accessory/regulatory proteins. To overcome FIV's cat-specific tropism, feline vectors are equipped with hybrid LTRs, since the FIV LTR shows low activity in human cells. Recombinant FIV vectors generate titers comparable to other lentiviral systems, are capable of incorporating heterologous envelopes and efficiently transduce dividing and nondividing cells in the presence and absence of the accessory proteins in vitro. Compared to HIV vectors, FIV vector development is still in its infancy, but initial in vivo data in various species and tissues indicate long-term gene expression at therapeutic levels, and thus FIV vectors hold great promise. Future efficacy studies in animal models and primates will determine the FIV vectors' suitability for gene therapy. The design of recombinant FIV vectors incorporates safety features described for primate lentiviral vectors with the benefit that biosafety testing of FIV vectors can occur in the natural host. Currently, FIV vectors are generated in a transient fashion, but the availability of a stable producer system amenable to better characterization and scale-up will considerably increase the potential for use of FIV vectors in the clinic.

Animals↗
PubMed2000

Ovine adenovirus vectors mediate efficient gene transfer to skeletal muscle.

Ovine adenovirus (OAV) vectors represent a promising tool for human gene therapy since these vectors overcome the problem of pre-existing immunity against human adenovirus vectors. In this report we investigated the in vivo characteristics of this novel vector system with respect to its potential for gene transfer into skeletal muscle. We found that moderate doses of an OAV-derived vector expressing the human alpha1-antitrypsin gene (OAVhaat) infected skeletal muscle in mice very efficiently resulting in high serum hAAT levels. The infection was restricted to skeletal muscle, but gene expression was transient and vector DNA was rapidly cleared. Vector clearance was also observed with a vector that lacked the transgene. The loss of vector DNA was accompanied by a cellular immune response in the infected muscle but was not connected with detectable expression of early or late genes of the viral backbone as analyzed by RT-PCR. A very low dose of OAVhaat (3x 10(7) infectious particles) was sufficient to produce reasonable amounts (>100 ng/ml) of serum hAAT, and this was accompanied by a weak immune response to the vector. Under these conditions, a second intramuscular injection of the same recombinant OAV vector was successful. Our study expands the known tissue tropism of OAV-derived vectors in vivo and points to the possible utility of the vector for muscle gene transfer and vaccination.

Animals↗
PubMed2003

Efficient gene transfer into human CD34+ cells by an adenovirus type 35 vector.

Efficient gene transfer into human hematopoietic stem cells (HSCs) is the most important requirement for gene therapy of hematopoietic disorders and for study of the hematopoietic system. An adenovirus (Ad) vector based on the Ad serotype 5 (Ad5) is known to transduce HSCs, including CD34(+) cells, with very low efficiency because of low-level expression of its primary receptor, coxsackievirus and adenovirus receptor (CAR). In the present study, we developed a recombinant Ad vector composed of the whole Ad serotype 35 (Ad35), which recognizes an unidentified receptor different from CAR for its infection. A transduction study showed that the Ad35-based vectors exhibit a higher transduction efficiency in human CD34(+) cells than the conventional Ad5 vectors and the Ad5F35 vectors, which are fiber-substituted Ad5 vectors containing Ad35 fiber proteins. The mean of fluorescence intensity in the CD34(+) cells transduced with the Ad35 vectors was 12-76 and 1.4-3 times higher than that in the cells transduced with the Ad5 and Ad5F35 vectors, respectively. The percentages of green fluorescent protein (GFP)-positive CD34(+) cells by transduction with Ad35, Ad5, and Ad5F35 vectors expressing GFP at 300 PFU/cell were 53%, 5%, and 52%, respectively, suggesting that Ad35 vectors mediate a more efficient gene transfer into human CD34(+) cells than Ad5 and Ad5F35 vectors, although the percentage of transduced cells was similar between Ad35 and Ad5F35 vectors. The Ad vector based on Ad35 could be very useful in gene therapy for blood disorders and gene transfer experiments using HSCs.

Adenoviridae↗
PubMed2003

Effective transduction and stable transgene expression in human blood cells by a third-generation lentiviral vector.

Difficulty in gene transduction of human blood cells, including hematopoietic stem cells, has hampered the development of gene therapy applications for hematological disorders, encouraging the development and use of new gene delivery systems. In this study, we used a third-generation self-inactivating (SIN) lentiviral vector system based on human immunodeficiency virus type 1 (HIV-1) to improve transduction efficiency and prevent vector-related toxicity. The transduction efficiency of the HIV-1-based vector was compared directly with the Moloney murine leukemia virus (MLV) SIN vector in human leukemia cell lines. Initial transduction efficiencies were almost 100% for the HIV and less than 50% for the MLV vectors. Similar results were observed in 11 types of primary cells obtained from leukemia or myeloma patients. Transgene expression persisted for 8 weeks in cells transduced with the HIV vector, but declined with the MLV vector. In addition, resting peripheral blood lymphocytes and CD34(+) hematopoietic cells were transduced successfully with the HIV vector, but not with the MLV vector. Finally, we confirmed vector gene integration in almost all colony-forming cells transduced with the HIV vector, but not with the MLV vector. In conclusion, this lentiviral vector is an excellent gene transduction system for human blood cells because of its high gene transduction and host chromosome integration efficiency.

Cells, Cultured↗
PubMed2003

Gene therapy progress and prospects: adenoviral vectors.

In September 1999, the perceptions of the use of adenoviral (Ad) vectors for gene therapy were altered when a patient exposed via the hepatic artery to a high dose of adenoviral vector succumbed to the toxicity related to vector administration. Appropriately, concerns were raised about continued use of the Ad vector system and, importantly, there were increased efforts to more fully understand the toxicity. Today it is recognized that there is no ideal vector system, and that while Ad vectors are not suitable for all applications, the significant advantages over other vector systems including efficient transduction of a variety of cell types, both quiescent and dividing, make it optimal for certain applications. These include protocols where high levels of short-term expression are sufficient to provide a therapeutic benefit. Potential target applications include therapeutic angiogenesis, administration into immune-privileged sites such as the CNS, or treatments where the adjuvant effect of adenovirus can be of benefit such as cancer vaccines. Broader applicability of Ad vectors will require resolution of toxicity issues. This review will therefore focus on studies conducted over the last 2 years that have advanced our understanding of the toxicity associated with Ad vectors, studies that have employed methods to reduce toxicity and improvements in Ad vectors themselves that will reduce toxicity by one of several mechanisms. These mechanisms include retargeting vector to the tissue of interest, minimizing or eliminating viral gene expression that is thought to result in loss of transduced cells, or by methods that seek to reduce the vector dose required for therapeutic benefit. An area where there remains significant room for improvement is when readministration of vector is required because transgene expression has decreased to background levels.

Adenoviridae↗
PubMed1999

Stable transduction of quiescent CD34(+)CD38(-) human hematopoietic cells by HIV-1-based lentiviral vectors.

We compared the efficiency of transduction by an HIV-1-based lentiviral vector to that by a Moloney murine leukemia virus (MLV) retroviral vector, using stringent in vitro assays of primitive, quiescent human hematopoietic progenitor cells. Each construct contained the enhanced green fluorescent protein (GFP) as a reporter gene. The lentiviral vector, but not the MLV vector, expressed GFP in nondivided CD34(+) cells (45.5% GFP+) and in CD34(+)CD38(-) cells in G0 (12.4% GFP+), 48 hr after transduction. However, GFP could also be detected short-term in CD34(+) cells transduced with a lentiviral vector that contained a mutated integrase gene. The level of stable transduction from integrated vector was determined after extended long-term bone marrow culture. Both MLV vectors and lentiviral vectors efficiently transduced cytokine-stimulated CD34(+) cells. The MLV vector did not transduce more primitive, quiescent CD34(+)CD38(-) cells (n = 8). In contrast, stable transduction of CD34(+)CD38(-) cells by the lentiviral vector was seen for over 15 weeks of extended long-term culture (9.2 +/- 5.2%, n = 7). GFP expression in clones from single CD34(+)CD38(-) cells confirmed efficient, stable lentiviral transduction in 29% of early and late-proliferating cells. In the absence of growth factors during transduction, only the lentiviral vector was able to transduce CD34(+) and CD34(+)CD38(-) cells (13.5 +/- 2.5%, n = 11 and 12.2 +/- 9.7%, n = 4, respectively). The lentiviral vector is clearly superior to the MLV vector for transduction of quiescent, primitive human hematopoietic progenitor cells and may provide therapeutically useful levels of gene transfer into human hematopoietic stem cells.

ADP-ribosyl Cyclase↗
PubMed2002

Superior tissue-specific expression from tyrosinase and prostate-specific antigen promoters/enhancers in helper-dependent compared with first-generation adenoviral vectors.

The ability to target specific tissues is important in many applications of gene therapy. In this respect, a disadvantage of adenoviral vectors is the relative lack of specificity with which they transduce cells. One approach to overcome this is to express the therapeutic gene under the control of a tissue-specific promoter. However, the specificity and activity of these promoters may be altered by adenoviral sequences in the vector backbone. In contrast, helper-dependent adenoviral (HDAd) vectors [Parks, R.J., Chen, L., Anton, M., Sankar, U., Rudnicki, M.A., and Graham, F.L. (1996). Proc. Natl. Acad. Sci. U.S.A. 93, 13565-13570] are almost completely devoid of adenovirus sequences, and this may preserve the specificity of these heterologous promoters. We have compared HDAd and first-generation adenoviral (FGAd) vectors with respect to tissue-specific expression from prostate-specific antigen (PSA) or tyrosinase promoters/enhancers. A PSA-positive cell line (LNCaP) and a panel of PSA-negative cell lines were infected with HDAd vectors expressing luciferase under the control of three different kinds of PSA promoter/enhancer constructs. The results showed that these PSA promoter/enhancer cassettes in HDAd vectors maintained strict tissue-specific expression, but lost specificity when expressed from FGAd vectors. Similar results were observed with tyrosinase promoter-carrying vectors, except that the tyrosinase promoter retained a small degree of tissue specificity in FGAd vectors. Insertion of a murine cytomegalovirus immediate-early gene promoter-beta-galactosidase (MCMV-lacZ)-expressing cassette into a second site in the HDAd vector backbone significantly impaired the tissue specificity of the PSA and tyrosinase promoters. These results indicate that HDAd vectors are superior to FGAd vectors in their ability to maintain high levels of tissue-specific expression from PSA and tyrosinase promoters/enhancers. They also suggest that tissue-specific expression can be influenced not only by Ad sequences, but also by other viral and/or strong constitutive promoter/enhancers (such as the MCMV promoter) in the vector backbone.

Adenoviridae↗
PubMed1994

Improved methods of retroviral vector transduction and production for gene therapy.

To facilitate clinical applications of retroviral-mediated human gene transfer, retroviral vectors must be of high titer and free of detectable replication-competent retroviruses. The purpose of this study was to optimize methods of retroviral vector production and transduction. Studies were conducted using 22 retroviral vector producer cell lines. Inactivation of retroviral vectors was greater at 37 degrees C than at 32 degrees C. A 5- to 15-fold increase of vectors was produced at 32 degrees C compared to 37 degrees C; the vector increase at 34 degrees C was intermediate. For example, PA317/G1Na.40 grew to a titer of 1.8 x 10(7) cfu/ml at 32 degrees C, compared to 5.0 x 10(5) cfu/ml at 37 degrees C. The production of retroviral vectors was scalable achieving similar results in flasks, roller bottles, or a CellCube Bioreactor. Retroviral vectors were concentrated 15-24 times with vector recovery ranging from 91 to 96% in a Pellicon tangential flow filtration system. Retroviral supernatants were successfully lyophilized. The combination of glucose or sorbitol with gelatin resulted in recovery rates of 64-83%. In studies on transduction by retroviral vectors, centrifugation of vector supernatants onto target cells significantly increased transduction efficiency as measured by vector titration for G418 resistance, fluorescence-activated cell sorting (FACS), and polymerase chain reaction (PCR) analyses. The combination of the above methods has significantly increased the growth and transduction by this vector system.

3T3 Cells↗