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PubMed2002

Enhanced antitumor effect and reduced vector dissemination with fiber-modified adenovirus vectors expressing herpes simplex virus thymidine kinase.

There are at least two hurdles confronting the use of the adenovirus (Ad)-mediated herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) system for the treatment of cancer. One is inefficient Ad vector-mediated gene transfer into tumor cells lacking the primary receptor, i.e., the coxsackievirus and adenovirus receptor (CAR). The other is hepatotoxicity due to unwanted vector spread into the liver, even when Ad vectors are injected intratumorally. Herein, we present an attractive strategy for overcoming such limitations based on use of a fiber-modified Ad vector containing an RGD peptide motif in the fiber knob. HSVtk-expressing Ad vectors containing mutant fiber (AdRGD-tk) or wild-type fiber (Ad-tk) were injected intratumorally into CAR-negative B16 melanoma cells inoculated into mice, after which GCV was injected intraperitoneally for 10 days. AdRGD-tk showed approximately 25 times more antitumor activity than Ad-tk. Histopathological studies suggested that liver damage in mice injected with AdRGD-tk was significantly lower than that in mice injected with Ad-tk. Intratumoral administration of luciferase-expressing Ad vectors containing the mutant fiber (AdRGD-L2) resulted in nearly 40 times more luciferase production in the tumor, but 8 times less production in the liver than the conventional Ad vectors (Ad-L2). These results indicate that combination of fiber-modified vectors and a HSVtk/GCV system is a potentially useful and safe approach for the treatment of tumors lacking CAR expression, and that fiber-modified vectors could be of great utility for gene therapy and gene transfer experiments.

Adenoviridae↗

PubMed1999

Use of helper-dependent adenoviral vectors of alternative serotypes permits repeat vector administration.

We have developed a new helper adenovirus (Ad) based on serotype 2, Ad2LC8cCARP, for use in the Cre/loxP system (Parks et al. Proc Natl Acad Sci USA, 1996; 93: 13565-13570) to generate Ad vectors deleted of all protein coding sequences (helper-dependent Ad vectors (hdAd)). A comparison of Ad2LC8cCARP and our original helper virus (based on serotype 5, Ad5LC8cluc) showed that the two helper viruses amplified hdAd with a similar efficiency, and resulted in a similar yield and purity after large-scale preparation of vector. In vitro, the resulting hdAd2 had a similar transduction efficiency and expression kinetics of transgene (beta-gal) as the hdAd5. An important feature of the helper-dependent system is that all virion components, except the virion DNA, derive from the helper virus. Consequently, vectors produced with help from Ad2LC8cCARP were not neutralized by antibodies against Ad5, and vectors produced with Ad5 helper were resistant to neutralizing antibodies against Ad2. Analysis of transgene expression in mouse liver after intravenous injection of the Ad2-based hdAd showed that the vector could efficiently transduce the liver, and produce high levels of a foreign transgene, similar to those expressed by the hdAd generated with the Ad5 helper virus. Mice immunized with hdAd2 produced Ad2-neutralizing antibodies, which did not cross-react with hdAd5. To determine if successful repeat Ad vector administration could be achieved by sequential use of alternative Ad serotypes, we injected mice with hdAd2 (hSEAP) followed 3 months later by a lacZ-expressing hdAd of either the same or different serotype. Repeated administration of hdAd2 resulted in a 30- to 100-fold reduction in transgene expression compared with naive animals. In contrast, no decrease in transgene expression was observed when the second vector was of a different serotype. These results demonstrate that effective vector readministration can be achieved by the sequential use of hdAds based on alternative serotypes.

Adenoviridae↗

PubMed2004

Transient foamy virus vector production by adenovirus vectors.

The genome of the prototype foamy virus (PFV) has been introduced into an adenoviral/PFV hybrid vector and tested for stable in vitro gene transfer. Three different adenoviruses are used to encode: (i) the PFV structural genes gag and pol (Ad-GagPolDeltaPacI); (ii) the PFV structural gene env (Ad-Env); and (iii) the PFV vector genome (Ad-MD9) encoding the transgene (the enhanced green fluorescent protein (eGFP) gene). Following cotransduction by the three adenoviruses, the target cells become transient PFV vector-producing cells, resulting in the in situ release of recombinant PFV at a titre of up to 10(3) vector particles/ml, which can then infect surrounding cells, leading to stable integration of the expression cassette. Stable eGFP expression, observed for up to 60 days (11 passages) in cells transduced with all three adenoviral vectors, was shown by PCR to be the result of PFV integration. In contrast, cells transduced with only the adenovirus encoding the PFV vector genome showed a marked decrease in eGFP expression by passage 2 (16 days post-transduction) and did not contain integrated PFV vector. In short, this paper describes the production of a hybrid vector capable of high in vitro transduction and stable transgene expression using adenovirus and PFV vectors.

Adenoviridae↗

PubMed1997

Ds excision from extrachromosomal geminivirus vector DNA is coupled to vector DNA replication in maize.

Analysis of transposition products generated after Activator (Ac) excision from the P locus in maize suggest that Ac excises either during or after replication of the P locus. The frequency of excision of the non-autonomous Ac derivative, Dissociation (Ds), from extrachromosomal replicating and nonreplicating vector DNAs in transfected black mexican sweet maize protoplasts was compared to assess directly a role of extrachromosomal vector DNA replication in Ds excision. Replicating (rep+) and nonreplicating (rep-) vector DNAs comprised a Ds element that harbored a geminivirus, wheat dwarf virus (WDV), origin of replication and WDV genes required for viral DNA replication (rep+) or mutant, inactive derivatives of these genes (rep-). Excision of Ds was detected only in those cell nuclei co-transfected with the replicating Ds-vector DNA and a transposase expression vector. Quantitative reconstruction experiments showed that Ds excised at least 3 x 10(5)-fold more frequently from replicating vector DNA as compared with nonreplicating vector DNA. Therefore, these results provide direct evidence for a coupling of Ds excision from extrachromosomal vector DNA to vector DNA replication in maize.

Cell Nucleus↗

PubMed2003

A triad of costimulatory molecules synergize to amplify T-cell activation in both vector-based and vector-infected dendritic cell vaccines.

The activation of a T cell has been shown to require two signals via molecules present on professional antigen presenting cells: signal 1, via a peptide/MHC complex, and signal 2, via a costimulatory molecule. Here, the role of three costimulatory molecules in the activation of T cells was examined. Poxvirus (vaccinia and avipox) vectors were employed because of their ability to efficiently express multiple genes. Murine cells provided with signal 1 and infected with either recombinant vaccinia or avipox vectors containing a TRIad of COstimulatory Molecules (B7-1/ICAM-1/LFA-3, designated TRICOM) induced the activation of T cells to a far greater extent than cells infected with vectors expressing any one or two costimulatory molecules. Despite this T-cell "hyperstimulation" using TRICOM vectors, no evidence of apoptosis above that seen using the B7-1 vector was observed. Results employing the TRICOM vectors were most dramatic under conditions of either low levels of first signal or low stimulator cell to T-cell ratios. Experiments employing a four-gene construct also showed that TRICOM recombinants could enhance antigen-specific T-cell responses in vivo. These studies thus demonstrate the ability of vectors to introduce three costimulatory molecules into cells, thereby activating both CD4+ and CD8+ T-cell populations to levels greater than those achieved with the use of only one or two costimulatory molecules. This new threshold of T-cell activation has broad implications in vaccine design and development. Dendritic cells infected with TRICOM vectors were found to greatly enhance naïve T-cell activation, and peptide-specific T-cell stimulation. In vivo, peptide-pulsed DCs infected with TRICOM vectors induced cytotoxic T lymphocyte activity markedly and significantly greater than peptide-pulsed DCs.

Adoptive Transfer↗

PubMed1997

Immune responses to reporter proteins and high viral dose limit duration of expression with adenoviral vectors: comparison of E2a wild type and E2a deleted vectors.

Experiments designed to evaluate the effect of deletion of E2a on duration of expression using adenoviral vectors led to a series of observations regarding host responses to adenoviral vectors and reporter proteins. In studies using human alpha1-antitrypsin (hAAT) as a reporter gene, we found that the duration of expression is very brief for C3H/J and CBA/J mice but is prolonged for C57BL/6J mice, that disappearance of hAAT from the blood is correlated with the appearance of antibodies, and that immunization against hAAT can prevent appearance of the protein in the blood after administration of an adenoviral vector. Deletion of E2a in hAAT vectors did not prolong expession in C3H/J or CBA/J mice and did not shorten duration of expression in C57BL/6J mice. Using similar vectors expressing Escherichia coli beta-galactosidase (beta-Gal) in immunocompetent mice, short duration of expression with a beta-Gal reporter was remarkably different from the long expression with an identical vector expressing hAAT in C57BL/6J. In the case of vectors expressing hAAT, adenoviral sequences persisted in the liver, and inflammatory responses were minimal compared to vectors expressing beta-Gal, where adenoviral sequences disappeared from the liver concomitant with a prominent inflammatory response. The duration of expression of beta-Gal in hepatocytes was increased in transgenic mice expressing the reporter in keratinocytes, indicating that host immune responses to the reporter can limit duration of expression. Dosage studies indicated that persistence of expression of hAAT can be markedly decreased by administration of high doses of vector in a manner consistent with a nonimmune-mediated toxicity following injection. These experiments indicate that host responses to reporter genes rather than host responses to adenoviral proteins can be the primary determinant of duration of expression under many experimental conditions.

Adenoviridae↗

PubMed1997

Co-packaging of non-vector RNAs generates replication-defective retroviral vector particles: a novel approach for blocking retrovirus replication.

A Moloney murine leukemia virus (MoMuLV)-derived packaging retroviral vector, pUCMoTN-PR3, was previously developed in which the packaging (psi) signal was cloned within the 5'-long terminal repeat (LTR) U3-r and U5 sequences. The MoTN-PR3 vector particles released from a transfected packaging cell line contain RNAs with r-psi-U5 sequences at the 5'-end and U3-r sequences at the 3'-end. Upon infection, these vector particles can efficiently transduce the neomycin phosphotransferase (neo) gene to the target cells. The structure of the proviral DNA synthesized in these cells was shown to contain modified 5'- and 3'-LTRs with U3-r-psi-U5 sequences, indicating that this vector can undergo reverse transcription and integration. Analysis of psi signal-containing RNAs revealed that in addition to vector RNA transcribed from the MoMuLV 5'-LTR promoter, readthrough neo RNA transcribed from the internal herpes simplex virus (HSV) thymidine kinase (tk) promoter and cellular RNAs transcribed from the MoMuLV 3'-LTR promoter are produced. Of these, the downstream cellular RNAs are also packaged within the vector particles. These vector particles containing the vector and non-vector RNAs carrying the MoMuLV psi signal are non-infectious. It is proposed that intracellular expression of packageable non-viral RNAs may represent an effective strategy for inhibiting animal and plant virus replication.

3T3 Cells↗

PubMed1995

E- vectors: development of novel self-inactivating and self-activating retroviral vectors for safer gene therapy.

We have developed novel self-inactivating and self-activating retroviral vectors based on the previously observed high-frequency deletion of direct repeats. We constructed spleen necrosis virus (SNV)-based viral vectors that contained large direct repeats flanking the viral encapsidation sequence (E). A large proportion of the proviruses in the target cells had E and one copy of the direct repeat deleted. Direct repeats of 1,333 and 788 bp were deleted at frequencies of 93 and 85%, respectively. To achieve a 100% deletion efficiency in target cells after ex vivo infection and drug selection, we constructed a self-activating vector that simultaneously deleted E and reconstituted the neomycin phosphotransferase gene. Selection of the target cells for resistance to G418 (a neomycin analog) ensured that all integrated proviruses had E deleted. The proviruses with E deleted were mobilized by a replication-competent virus 267,000-fold less efficiently than proviruses with E. We named these self-inactivating vectors E- (E-minus) vectors. These vectors should increase the safety of retroviral vector-mediated gene therapy by preventing the spread of vector sequences to nontarget cells in the event of coinfection with helper virus. We propose that direct-repeat deletions occur during RNA-dependent DNA synthesis and suggest that template switches occur without a requirement for RNA breaks. The minimum template dissociation frequency was estimated as 8%/100 bp per replication cycle. These vectors demonstrate that large direct repeats and template-switching properties of reverse transcriptase can be utilized to delete any sequence or reconstitute genes during retroviral replication.

Animals↗

PubMed1996

Pseudotransduction of hepatocytes by using concentrated pseudotyped vesicular stomatitis virus G glycoprotein (VSV-G)-Moloney murine leukemia virus-derived retrovirus vectors: comparison of VSV-G and amphotropic vectors for hepatic gene transfer.

Recombinant retrovirus vectors are widely used for gene transfer studies. The recent development of a pseudotyped Moloney murine leukemia virus vector that contains the G envelope protein from the vesicular stomatitis virus allows for efficient concentration of vector and offers hope for potential use of these vectors for gene expression in vivo. A standard amphotropic vector expressing a serum marker protein, human alpha 1-antitrypsin, was infused into regenerating mouse liver and was 10-fold more efficient at achieving stable gene expression than was an equivalent pseudotyped vector. Discrepant results were obtained with cultured hepatocytes infected with an Escherichia coli beta-galactosidase-producing pseudotype and amphotropic vector. High rates of beta-galactosidase-positive cells were detected with the vesicular stomatitis virus G glycoprotein vector under culture conditions known to be relatively nonpermissive for retrovirus-mediated gene transfer. Subsequent studies demonstrated that beta-galactosidase protein was concentrated and copurified during pseudotype vector preparation, resulting in high rates of protein transfer rather than stable gene transfer, a process referred to as pseudotransduction. The cotransfer of protein with concentrated pseudotyped retroviruses indicates that caution must be used when interpreting gene transduction efficiencies in gene therapy experiments.

3T3 Cells↗

PubMed2005

A single-cycle vaccine vector based on vesicular stomatitis virus can induce immune responses comparable to those generated by a replication-competent vector.

Live attenuated vaccine vectors based on recombinant vesicular stomatitis virus (VSV) are effective in several viral disease models. In this study, we asked if a VSV vector capable of only a single cycle of replication might be an effective alternative to replication-competent VSV vectors. We compared the cellular immune responses to human immunodeficiency virus (HIV) envelope protein (Env) expressed by replication-competent and single-cycle VSV vectors and also examined the antibody response to Env. The single-cycle vector was grown by complementation with VSV G protein and then tested initially for immunogenicity when given by four different routes. When given by the intramuscular route in mice, we found that the single-cycle vector was equivalent to the replication-competent VSV vector in generating high-level primary and memory CD8 T-cell responses as well as antibody responses to Env. Cellular responses were analyzed using major histocompatibility complex class I tetramers and direct measurement of cytotoxic T-lymphocyte activity in vivo. We also found that the recall responses after boosting were equivalent in animals vaccinated with replication-competent or single-cycle vectors. Additionally, we observed recall and heightened memory responses after boosting animals with a single-cycle vector complemented with G protein from a different vesiculovirus. Because expression of HIV Env by G-deleted VSV might allow replication in human cells expressing CD4, we generated a single-cycle VSV recombinant expressing a secreted form of the HIV Env protein. This virus was just as effective as the recombinant expressing the membrane-anchored Env protein at producing CD8 T cells and antibody responses.

AIDS Vaccines↗

PubMed1994

Adeno-associated virus vector gene expression occurs in nondividing cells in the absence of vector DNA integration.

Adeno-associated virus type 2 (AAV2)-based vectors are capable of stable expression in the airway epithelium and may be useful for gene therapy for human diseases, such as cystic fibrosis. Certain virus vectors, such as retroviruses, require active cell division for integration and expression, but this has not been formally evaluated in the case of AAV2. The cystic fibrosis bronchial epithelial cell line, IB3-1, which can be transduced by AAV2 vectors, was shown to undergo a decrease in DNA synthesis to undetectable levels when grown to confluence. Cultures in which < 0.1% of cells were dividing could still be efficiently transduced with AAV-lacZ or AAV-neo vectors, with a linear dose response, up to 91% with a multiplicity of 3,000 vector particles per cell. The fate of vector DNA in nondividing target cells was investigated by Southern blotting of both low molecular weight, nonintegrated DNA and high molecular weight, genomic DNA fractions. Detectable levels of vector DNA were only seen in the nonintegrated state. These results indicate that AAV2-based vectors, unlike retrovirus vectors, do not require active cell division or integration for expression to occur and thus possess a unique profile of biologic properties.

Bronchi↗

PubMed2002

Lentiviral vector transduction of NOD/SCID repopulating cells results in multiple vector integrations per transduced cell: risk of insertional mutagenesis.

Efficient vector transduction of hematopoietic stem cells is a requirement for successful gene therapy of hematologic disorders. We asked whether human umbilical cord blood CD34(+)CD38(lo) nonobese diabetic/severe combined immunodeficiency (NOD/SCID) repopulating cells (SRCs) could be efficiently transduced using lentiviral vectors, with a particular focus on the average number of vector copies integrating into these primitive progenitor cells. Mouse bone marrow was analyzed by fluorescence-activated cell-sorter scanner and by semiquantitative polymerase chain reaction (PCR) to determine the transduction efficiency into SRCs. Lentiviral vector transduction resulted in an average of 22% (range, 3%-90%) of the human cells expressing green fluorescent protein (GFP), however, multiple vector copies were present in human hematopoietic cells, with an average of 5.6 +/- 3.3 (n = 12) copies per transduced cell. To confirm the ability of lentiviral vectors to integrate multiple vector copies into SRCs, linear amplification mediated (LAM)-PCR was used to analyze the integration site profile of a selected mouse showing low-level engraftment and virtually all human cells expressing GFP. Individually picked granulocyte macrophage colony-forming unit colonies derived from the bone marrow of this mouse were analyzed and shown to have the same 5 vector integrants within each colony. Interestingly, one integration site of the 5 that were sequenced in this mouse was located in a known tumor-suppressor gene, BRCA1. Therefore, these findings demonstrate the ability of lentiviral vectors to transduce multiple copies into a subset of NOD/SCID repopulating cells. While this is efficient in terms of transduction and transgene expression, it may increase the risk of insertional mutagenesis.

ADP-ribosyl Cyclase↗

PubMed2003

From virus evolution to vector revolution: use of naturally occurring serotypes of adeno-associated virus (AAV) as novel vectors for human gene therapy.

Gene transfer vectors based on the human adeno-associated virus serotype 2 (AAV-2) have been developed and tested in pre-clinical studies for almost 20 years, and are currently being evaluated in clinical trials. So far, all these studies have provided evidence that AAV-2 vectors possess many properties making them very attractive for therapeutic gene delivery to humans, such as a lack of pathogenicity or toxicity, and the ability to confer long-term gene expression. However, there is concern that two restrictions of AAV-2 vectors might limit their clinical use in humans. First, these vectors are rather inefficient at transducing some cells of therapeutic interest, such as liver and muscle cells. Second, gene transfer might be hampered by neutralizing anti-AAV-2 antibodies, which are highly prevalent in the human population. In efforts to overcome both limitations, an increasing number of researchers are now focusing on the seven other naturally occurring serotypes of AAV (AAV-1 and AAV-3 to -8), which are structurally and functionally different from AAV-2. To this end, several strategies have been devised to cross-package an AAV-2 vector genome into the capsids of the other AAV serotypes, resulting in a new generation of "pseudotyped" AAV vectors. In vitro and in vivo, these novel vectors were shown to have a host range different from AAV-2, and to escape the anti-AAV-2 immune response, thus underscoring the great potential of this approach. Here the biology of the eight AAV serotypes is summarized, existing technology for pseudotyped AAV vector production is described, initial results from pre-clinical evaluation of the vectors are reviewed, and finally, the prospects of these promising novel tools for human gene therapy are discussed.

Animals↗

PubMed1999

In vivo marking of rhesus monkey lymphocytes by adeno-associated viral vectors: direct comparison with retroviral vectors.

We have compared adeno-associated virus (AAV)-based and retrovirus-based vectors for their ability to transduce primary T lymphocytes in vitro and then tracked the persistence of these genetically marked lymphocytes in vivo, using the rhesus monkey model. To avoid the complication of immune rejection of lymphocytes transduced with xenogeneic genes in tracking studies primarily designed to investigate transduction efficiency and in vivo kinetics, the vectors were designed without expressed genes. All vectors contained identically mutated beta-galactosidase gene (beta-gal) and neomycin resistance gene (neo) DNA sequences separated by different length polylinkers, allowing simple differentiation by polymerase chain reaction (PCR). Each of 2 aliquots of peripheral blood lymphocytes from 4 rhesus monkeys were transduced with either AAV or retroviral vectors. The in vitro transduction efficiency (mean vector copy number/cell) after the ex vivo culture was estimated by PCR at 0.015 to 3.0 for AAV, varying depending on the multiplicity of infection (MOI) used for transduction, and 0.13 to 0.19 for the retroviral transductions. Seven days after transduction, Southern blot analysis of AAV-transduced lymphocytes showed double-stranded and head-to-tail concatemer forms but failed to show integration of the AAV vector. AAV and retroviral aliquots were reinfused concurrently into each animal. Although the retrovirally marked lymphocytes could be detected for much longer after infusion, AAV transduction resulted in higher short-term in vivo marking efficiency compared with retroviral vectors, suggesting possible clinical applications of AAV vectors in lymphocyte gene therapy when long-term vector persistence is not required or desired.

Animals↗

PubMed2005

Adeno-associated virus as a gene therapy vector: vector development, production and clinical applications.

Adeno-associated virus (AAV) has emerged as an attractive vector for gene therapy. AAV vectors have successfully been utilized to promote sustained gene expression in a variety of tissues such as muscle, eye, brain, liver, and lung. As the significance of AAV as a gene therapy vector has been realized over the past years, recent developments in recombinant AAV (rAAV) production and purification have revolutionized the AAV field. It is now possible to produce high yields of vector (10(12)-10(13) genome-containing particles per mL) that are free of contaminating cellular and helper virus proteins. Such vectors have been successfully used in preclinical applications in animal models such as those of hemophilia, lysosomal storage diseases and vision deficiency, all of which have shown therapeutic benefits from rAAV treatment. Clinical trials using rAAV2 for the treatment of hemophilia B, cystic fibrosis, alpha-1-antitrypsin deficiency, and Canavan disease have begun, and reports from these phase I trials support the safety seen in preclinical trials. Eventually, tissue-specific vectors that can potentially evade the immune system will be required to optimize success in gene therapy. In recent years, this has led to the development of retargeted rAAV2 vectors and the identification and characterization of new serotypes from human and nonhuman primates that could potentially achieve these goals. AAV virologists and gene therapists alike have just begun to scratch the surface in terms of the utility of this small virus in a clinical setting. In this chapter, we will provide a comprehensive overview of the recent advances in rAAV vector production and purification, vector development, and clinical applications.

Animals↗

PubMed2004

Functional applications of novel Semliki Forest virus vectors are limited by vector toxicity in cultures of primary neurons in vitro and in the substantia nigra in vivo.

The Semliki Forest virus (SFV) system has been shown to be highly efficient in transduction of cell lines and primary cells. We employed a novel "noncytotoxic" SFV(PD) vector for transduction of primary ventral midbrain floor cultures in vitro and rat substantia nigra in vivo. Rapid protein expression was noted with preferential transduction of neuronal cells including the dopaminergic subpopulation. To examine the suitability of the SFV vector system for functional gene expression, SFV(PD) vectors encoding for antiapoptotic proteins Bcl-X(L) and XIAP were designed. Despite effective transgene expression, SFV(PD) vectors were unable to rescue dopaminergic neurons from MPP+-induced apoptosis. In vivo, virus injection into substantia nigra resulted in fast onset of transgene expression, but elicited an activation of microglia and an inflammation response. We conclude that the use of novel SFV(PD) vectors is currently limited by persistent neurotoxicity of the vector system. Although SFV(PD) vectors may be useful for protein localization studies in dopaminergic neurons, functional applications will require the development of even less cytopathic vector systems.

1-Methyl-4-phenylpyridinium↗

PubMedDate not supplied

Approaches to vector control: new and trusted. 3. Prospects for genetic manipulation of insect vectors.

Insect vector control has proved an effective method of reducing the transmission of disease-causing organisms to human populations in many tropical countries. A variety of methods has been employed for suppressing vector populations, including the application of biological control agents and the elimination of breeding sites, with a continuing and heavy reliance on the use of chemical insecticides. However, the development of insecticide resistance by vector insects, the cost of developing and registering new insecticidal compounds, and the increase in legislation to combat the detrimental effects of insecticidal residues on the environment, have emphasized the need to assess a variety of alternatives to vector control. What is required is a completely novel approach either to suppress vector populations, or to alter their ability to transmit disease-causing organisms in such a way as to have a profound and long-lasting effect on disease transmission. Genetic manipulation of insect vectors may provide just such an approach. The major requirements for being able to manipulate the genomes of insects are reviewed together with the progress which has been made to create transgenic vector insects. The potential applications of this technology are then explored, emphasizing that its most immediate use will be as an analytical tool. Finally, the feasibility of creating refractory vector strains by genetic manipulation and releasing them into the environment is assessed in relation to its future use as a disease control strategy.

Animals↗

PubMed2004

Spliced spleen necrosis virus vector RNA is not encapsidated: implications for retroviral replication and vector design.

RNA splicing is a complex event in the retroviral life cycle and can involve multiple steps, as well as cis-acting sequences, to maintain a proper balance of spliced and unspliced viral RNA for translation and encapsidation. The retroviral RNA can be processed by cellular machinery and enables the removal of intronic sequences. We aimed to utilize the removal of a synthetic intron for targeted gene expression. To analyze intron removal and gene expression, we have constructed a novel self-inactivating gene-activating (SIGA) vector for potential universal gene therapy. New vectors for gene therapy are necessary for safe and effective gene delivery in humans. The SIGA vector is derived from spleen necrosis virus (SNV), which is an avian reticuloendotheliosis virus. The vector was designed so that expression of a therapeutic gene is blocked in helper cell lines due to an intervening sequence containing various blocks in transcription and translation. However, after one round of retroviral replication, the intervening sequence should be removed by the cellular machinery and the therapeutic gene will be selectively expressed in target cells. Our studies show that the intervening sequence in SIGA vector RNA is partially spliced. However, spliced vector RNA was not transduced to target cells. Previous studies showed that an infectious SNV vector enabled transduction of spliced RNA. However, yet-undefined differences in infectious and replication-deficient retroviral replication may have an effect on the transduction of spliced RNA. The results of this study present key information on spliced RNA and its encapsidation, as well as data for the construction of a new generation of SNV-derived retroviral vectors.

Animals↗