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At least 307 records · Page 17Linked to original sources
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↗
PubMed1997

HSV/AAV hybrid amplicon vectors extend transgene expression in human glioma cells.

Novel hybrid vectors, which incorporate critical elements of both herpes simplex virus type 1 (HSV-1) amplicon vectors and adeno-associated virus (AAV) vectors, are able to sustain transgene expression in dividing glioma cells for over 2 weeks. These vectors combine the high infectibility and large transgene capacity of HSV-1 vectors with the potential for episomal amplification and chromosomal integration of AAV vectors. The hybrid vectors contain the HSV-1 origin of DNA replication, oriS, and the DNA cleavage/packaging signal, pac, which allow amplicon replication and packaging in HSV-1 virions. The lacZ reporter gene under control of the CMV IE1 promoter is flanked by AAV inverted terminal repeat (ITR) sequences, which facilitate replication and genomic integration of this cassette in the host cell nucleus. Constructs were generated with or without the AAV rep gene (rep+ and rep-) to assess its importance in extending transgene expression. Expression of Rep proteins was confirmed by Western blot analysis. An HSV-1 amplicon construct containing the reporter gene, but no AAV sequences, was used as a control. Constructs were packaged into HSV-1 virions with or without helper virus and these vector stocks were used to infect human U87 glioma cells in culture. The hybrid vectors supported transgene retention and expression for over 2 weeks, whereas the control amplicon vector lost the transgene after 10 days. Expression was somewhat longer for the rep+ as compared to the rep- hybrid vectors. Toxicity due to the HSV-1 helper virus was eliminated using helper virus-free amplicon vector stocks. Transgene constructs could also be packaged in AAV virions, using AAV and adenovirus or HSV-1 helper functions. These HSV/AAV hybrid vectors should allow long-term, nontoxic gene delivery of DNA constructs to both dividing and nondividing cells.

Animals↗
PubMed1999

The molecular basis of multiple vector insertion by gene targeting in mammalian cells.

Gene targeting using sequence insertion vectors generally results in integration of one copy of the targeting vector generating a tandem duplication of the cognate chromosomal region of homology. However, occasionally the target locus is found to contain >1 copy of the integrated vector. The mechanism by which the latter recombinants arise is not known. In the present study, we investigated the molecular basis by which multiple vectors become integrated at the chromosomal immunoglobulin mu locus in a murine hybridoma. To accomplish this, specially designed insertion vectors were constructed that included six diagnostic restriction enzyme markers in the Cmu region of homology to the target chromosomal mu locus. This enabled contributions by the vector-borne and chromosomal Cmu sequences at the recombinant locus to be ascertained. Targeted recombinants were isolated and analyzed to determine the number of vector copies integrated at the chromosomal immunoglobulin mu locus. Targeted recombinants identified as bearing >1 copy of the integrated vector resulted from a Cmu triplication formed by two vector copies in tandem. Examination of the fate of the Cmu region markers suggested that this class of recombinant was generated predominantly, if not exclusively, by two targeted vector integration events, each involving insertion of a single copy of the vector. Both vector insertion events into the chromosomal mu locus were consistent with the double-strand-break repair mechanism of homologous recombination. We interpret our results, taken together, to mean that a proportion of recipient cells is in a predetermined state that is amenable to targeted but not random vector integration.

Animals↗
PubMed1992

Unusually high frequency of reconstitution of long terminal repeats in U3-minus retrovirus vectors by DNA recombination or gene conversion.

Recently, we described a retrovirus vector system with which to study formation of cDNA genes (R. Dornburg and H. M. Temin, Mol. Cell. Biol. 6:2328-2334, 1988; Mol. Cell. Biol. 8:64-72, 1990; J. Virol. 64:886-889, 1990). For these studies, retrovirus vectors were constructed in which the U3 region of the 3' long terminal repeat (LTR) was deleted. After one round of retrovirus replication, such vectors formed a provirus with two U3-minus LTRs. However, the insertion of some additional sequences into such vectors promoted vector rearrangements with an efficiency greater than 95%. Such rearranged vectors behaved like vectors with two wild-type LTRs. Proviruses derived from such vectors were investigated by Southern blot analysis, polymerase chain reaction, and DNA sequencing. We found that the U3 region was reconstituted, resulting in vectors with LTRs like wild-type virus. The sequences that reconstituted the U3 region of the vector LTR were derived from LTR sequences present in the helper cell. Since no retroviral protein coding sequences were detected in infected target cells, recombination of vector sequences with coencapsidated helper cell sequences during reverse transcription seems very unlikely. Thus, it appears that the recombination (or gene conversion) events leading to a vector with reconstituted LTRs occurred at the DNA level. The high frequency of this recombination (or gene conversion) was dependent on internal vector sequences.

Animals↗
PubMed1995

Adeno-associated virus vectors transduce primary cells much less efficiently than immortalized cells.

Immortalized cell lines have been used to study infection and replication of adeno-associated virus (AAV) in culture, but primary cells presumably provide a better model for AAV behavior in animals. Here, we have evaluated the ability of AAV vectors to transduce primary and immortalized strains of human epithelial cells and fibroblasts. Two AAV vectors were used, one that transduced an alkaline phosphatase gene (AAV-LAPSN), and one that transduced a beta-galactosidase/neomycin phosphotransferase fusion gene (AAV-L beta geo). The transduction efficiency of the AAV-LAPSN vector, quantitated by measurement of alkaline phosphatase-positive cell foci following infection, was 10 to 60 times greater in immortalized human cells than in primary cells, and total alkaline phosphatase activity in cell lysates was 40 to 50 times greater in immortalized cells. The AAV-L beta geo vector gave similar results. In contrast, the transduction efficiency of a retrovirus vector encoding alkaline phosphatase was equivalent in primary and immortalized cells. Analysis of the quantity and state of the AAV vector genomes in cells showed that primary and immortalized cells contained comparable numbers of vector copies per cell and that the vast majority of vector DNA was not integrated into the cell genome. Additionally, the level of AAV vector-derived message paralleled the transduction efficiency. These results indicate that the block to functional transduction in primary cells occurred after virus entry and limited the abundance of vector-derived message. Data from AAV transduction in cultures of human cells containing immortalizing genes suggest that cellular changes secondary to the introduction of immortalizing genes increased permissiveness for transduction by AAV vectors. In summary, our data demonstrate that AAV vectors transduce primary human cells much less efficiently than immortalized cells and indicate the importance of using primary cells to evaluate AAV vectors for gene therapy applications.

Cells, Cultured↗
PubMed1996

Molecular characterization of replication-competent variants of adenovirus vectors and genome modifications to prevent their occurrence.

Adenovirus (Ad) vectors for gene therapy are made replication defective by deletion of E1 region genes. For isolation, propagation, and large-scale production of such vectors, E1 functions are supplied in trans from a stable cell line. Virtually all Ad vectors used for clinical studies are produced in the 293 cell, a human embryonic kidney cell line expressing E1 functions from an integrated segment of the left end of the Ad type 5 (Ad5) genome. Replication-competent vector variants that have regained E1 sequences have been observed within populations of Ad vectors grown on 293 cells. These replication-competent variants presumably result from recombination between vector and 293 cell Ad5 sequences. We have developed Ad2-based vectors and have characterized at the molecular level examples of replication-competent variants. All such variants analyzed are Ad2-Ad5 chimeras in which the 293 cell Ad5 E1 sequences have become incorporated into the viral genome by legitimate recombination events. A map of Ad5 sequences within the 293 cell genome developed in parallel is consistent with the proposed recombination events. To provide a convenient vector production system that circumvents the generation of replication-competent variants, we have modified the Ad2 vector backbone by deleting or rearranging the protein IX coding region normally present downstream from the E1 region such that the frequency of recombination between vector and 293 cell Ad5 sequences is greatly reduced. Twelve serial passages of an Ad2 vector lacking the protein IX gene were carried out without generating replication-competent variants. In the course of producing and testing more than 30 large-scale preparations of vectors lacking the protein IX gene or having a rearranged protein IX gene, only three examples of replication-competent variants were observed. Use of these genome modifications allows use of conventional 293 cells for production of large-scale preparations of Ad-based vectors lacking replication-competent variants.

Adenovirus E1 Proteins↗
PubMed1997

Persistence of recombinant adenovirus in vivo is not dependent on vector DNA replication.

Recombinant adenovirus vectors represent an efficient means of transferring genes into many different organs. The first-generation E1-deleted vector genome remains episomal and, in the absence of host immunity, persists long-term in quiescent tissues such as the liver. The mechanism(s) which allows for persistence has not been established; however, vector DNA replication may be important because replication has been shown to occur in tissue culture systems. We have utilized a site-specific methylation strategy to monitor the replicative fate of E1-deleted adenovirus vectors in vitro and in vivo. Methylation-marked adenovirus vectors were produced by the addition of a methyl group onto the N6 position of the adenine base of XhoI sites, CTCGAG, by propagation of vectors in 293 cells expressing the XhoI isoschizomer PaeR7 methyltransferase. The methylation did not affect vector production or transgene expression but did prevent cleavage by XhoI. Loss of methylation through viral replication restores XhoI cleavage and was observed by Southern analysis in a wide variety of, but not all, cell culture systems studied, including hepatoma and mouse and macaque primary hepatocyte cultures. In contrast, following liver-directed gene transfer of methylated vector in C57BL/6 mice, adenovirus vector DNA was not cleaved by XhoI and therefore did not replicate, even after a period of 3 weeks. Although replication may occur in some tissues, these results show that stabilization of the vector within the target tissue prior to clearance by host immunity is not dependent upon replication of the vector, demonstrating that the input transduced DNA genomes were the persistent molecules. This information will be useful for the design of optimal adenovirus vectors and perhaps nonviral episomal vectors for clinical gene therapy.

Adenoviridae↗
PubMed1998

Production and characterization of improved adenovirus vectors with the E1, E2b, and E3 genes deleted.

Adenovirus (Ad)-based vectors have great potential for use in the gene therapy of multiple diseases, both genetic and nongenetic. While capable of transducing both dividing and quiescent cells efficiently, Ad vectors have been limited by a number of problems. Most Ad vectors are engineered such that a transgene replaces the Ad E1a, E1b, and E3 genes; subsequently the replication-defective vector can be propagated only in human 293 cells that supply the deleted E1 gene functions in trans. Unfortunately, the use of high titers of E1-deleted vectors has been repeatedly demonstrated to result in low-level expression of viral genes still resident in the vector. In addition, the generation of replication-competent Ad (RCA) by recombination events with the E1 sequences residing in 293 cells further limits the usefulness of E1-deleted Ad vectors. We addressed these problems by isolating new Ad vectors deleted for the E1, E3, and the E2b gene functions. The new vectors can be readily grown to high titers and have several improvements, including an increased carrying capacity and a theoretically decreased risk for generating RCA. We have also demonstrated that the further block to Ad vector replication afforded by the deletion of both the E1 and E2b genes significantly diminished Ad late gene expression in comparison to a conventional E1-deleted vector, without destabilization of the modified vector genome. The results suggested that these modified vectors may be very useful both for in vitro and in vivo gene therapy applications.

Adenoviridae↗
PubMed2000

Development of minimal lentivirus vectors derived from simian immunodeficiency virus (SIVmac251) and their use for gene transfer into human dendritic cells.

Lentivirus-derived vectors are very promising gene delivery systems since they are able to transduce nonproliferating differentiated cells, while murine leukemia virus-based vectors can only transduce cycling cells. Here we report the construction and characterization of highly efficient minimal vectors derived from simian immunodeficiency virus (SIVmac251). High-fidelity PCR amplification of DNA fragments was used to generate a minimal SIV vector formed from a 5' cytomegalovirus early promoter, the 5' viral sequences up to the 5' end of gag required for reverse transcription and packaging, the Rev-responsive element, a gene-expressing cassette, and the 3' long terminal repeat (LTR). Production of SIV vector particles was achieved by transfecting 293T cells with the vector DNA and helper constructs coding for the viral genes and the vesicular stomatitis virus glycoprotein G envelope. These SIV vectors were found to have transducing titers reaching 10(7) transducing units/ml on HeLa cells and to deliver a gene without transfer of helper functions to target cells. The central polypurine tract can be included in the minimal vector, resulting in a two- to threefold increase in the transduction titers on dividing or growth-arrested cells. Based on this minimal SIV vector, a sin vector was designed by deleting 151 nucleotides in the 3' LTR U3 region, and this SIV sin vector retained high transduction titers. Furthermore, the minimal SIV vector was efficient at transducing terminally differentiated human CD34(+) cell-derived or monocyte-derived dendritic cells (DCs). Results show that up to 40% of human primary DCs can be transduced by the SIV vectors. This opens a new perspective in the field of immunotherapy.

Cell Line↗
PubMed2000

Human beta interferon scaffold attachment region inhibits de novo methylation and confers long-term, copy number-dependent expression to a retroviral vector.

Moloney murine leukemia virus-based retroviral vector expression is gradually lost during prolonged in vitro culture of CEMSS T cells. However, when the human beta interferon scaffold attachment region (IFN-SAR) was inserted into the vector immediately upstream of the 3' long terminal repeat (LTR), expression was maintained for the length of the study (4 months). Clonal analysis of the retrovirus vector-infected CEMSS cells showed that SAR-containing retroviral vector expression levels were positively correlated with the proviral copy numbers (P < 0.0001), while there was no correlation between the proviral copy numbers and expression levels in control vector-infected clones. Thirty-three percent of the CEMSS cell clones infected with the control vector showed evidence of partial or complete methylation in the 5' LTR region. In sharp contrast, we detected no methylation in the clones infected with the SAR-containing vector. To demonstrate a direct inhibitory effect of methylation on retroviral vector expression, we have transfected 293 cells with in vitro-methylated proviral DNA. In transiently transfected cells, expression of methylated LTR was reduced but not completely inhibited, irrespective of the presence of the IFN-SAR sequence. In stably transfected cells, however, methylation completely abolished expression of the control vector but not of the SAR-containing vector. Furthermore, the expression of the SAR-containing vector was stable over time, indicating the ability of the SAR sequence to alleviate methylation-mediated transcriptional repression of a vector. This study extends our understanding of the mechanisms of retroviral vector inactivation by methylation and provides insight into a functional role for the SAR elements.

Animals↗
PubMed2000

DNA methylation of helper virus increases genetic instability of retroviral vector producer cells.

Retroviral vector producer cells (VPC) have been considered genetically stable. A clonal cell population exhibiting a uniform vector integration pattern is used for sustained vector production. Here, we observed that the vector copy number is increased and varied in a population of established LTKOSN.2 VPC. Among five subclones of LTKOSN.2 VPC, the vector copy number ranged from 1 to approximately 29 copies per cell. A vector superinfection experiment and Northern blot analysis demonstrated that suppression of helper virus gene expression decreased Env-receptor interference and allowed increased superinfection. The titer production was tightly associated with helper virus gene expression and varied between 0 and 2.2 x 10(5) CFU/ml in these subclones. In one analyzed subclone, the number of integrated vectors increased from one copy per cell to nine copies per cell during a 31-day period. Vector titer was reduced from 1.5 x 10(5) CFU to an undetectable level. To understand the mechanism involved, helper virus and vectors were examined for DNA methylation status by methylation-sensitive restriction enzyme digestion. We demonstrated that DNA methylation of helper virus 5' long terminal repeat occurred in approximately 2% of the VPC population per day and correlated closely with inactivation of helper virus gene expression. In contrast, retroviral vectors did not exhibit significant methylation and maintained consistent transcription activity. Treatment with 5-azacytidine, a methylation inhibitor, partially reversed the helper virus DNA methylation and restored a portion of vector production. The preference for methylation of helper virus sequences over vector sequences may have important implications for host-virus interaction. Designing a helper virus to overcome cellular DNA methylation may therefore improve vector production. The maintenance of increased viral envelope-receptor interference might also prevent replication-competent retrovirus formation.

Cell Line↗
PubMed2001

Chromosomal integration pattern of a helper-dependent minimal adenovirus vector with a selectable marker inserted into a 27.4-kilobase genomic stuffer.

Helper-dependent minimal adenovirus vectors are promising tools for gene transfer and therapy because of their high capacity and the absence of immunostimulatory or cytotoxic viral genes. In order to characterize this new vector system with respect to its integrative properties, the integration pattern of a minimal adenovirus vector with a neo(r) gene inserted centrally into a noncoding 27.4-kb genomic stuffer element derived from the human X chromosome after infection of a sex chromosome aneuploid (X0) human glioblastoma cell line was studied. Our results indicate that even extensive homologies and abundant chromosomal repeat elements present in the vector did not lead to integration of the vector via homologous or homology-mediated mechanisms. Instead, integration occurred primarily by insertion of a monomer with no or little loss of sequences at the vector ends, apparently at random sites, which is very similar to E1 deletion adenovirus vectors. It is therefore unlikely that the incorporation of stuffer elements derived from human genomic DNA, which were shown to allow long-term transgene expression in vivo in a number of studies, leads to an enhanced risk of insertional mutagenesis. Furthermore, our findings indicate that the potential of minimal adenovirus vectors as tools for targeted insertion and gene targeting is limited despite the possibility of incorporating long stretches of homologous sequences. However, we found an enhanced efficiency of stable neo(r) transduction of the minimal adenovirus vector compared to an E1 deletion adenovirus vector, possibly caused by the absence of potential growth-inhibitory viral genes. Complete integration of the vector and tolerance of the integrated vector sequences by the cell might indicate a potential use of these vectors as tools for stable transfer of (large) genes.

Adenoviruses, Human↗
PubMed2002

RNA 3' readthrough of oncoretrovirus and lentivirus: implications for vector safety and efficacy.

The expression of reporter genes driven by the same human elongation factor 1alpha (EF1alpha) promoter in murine leukemia virus (MLV)- and human immunodeficiency virus type 1 (HIV-1)-based vectors was studied in either transfected or virally transduced cells. The HIV-1 vectors consistently expressed 3 to 10 times higher activity than the MLV vectors at both the RNA and protein levels. The difference was not attributable to transcriptional interference, alternative enhancer/silencer, or differential EF1alpha intron splicing. Based on nuclear run-on assays, both vectors exhibited similar EF1alpha transcriptional activity. The reduced RNA levels of MLV vectors could not be explained by the decrease in RNA half-lives. Southern analysis of proviral DNA indicated that both HIV-1 and MLV vectors efficiently propagated the EF1alpha intron in the transduced cells. To decipher the discrepancy in transgene expression between MLV and HIV-1 vectors, the role of RNA 3'-end processing was examined using a sensitive Cre/lox reporter assay. The results showed that MLV vectors, but not HIV-1 vectors, displayed high frequencies of readthrough of the 3' polyadenylation signal. Interestingly, the polyadenylation signal of a self-inactivating (SIN) HIV-1 vector was as leaky as that of the MLV vectors, suggesting a potential risk of oncogene activation by the lentiviral SIN vectors. Together, our results suggest that an efficient polyadenylation signal would improve both the efficacy and the safety of these vectors.

Animals↗