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PubMed2004

An assessment of relative transcriptional availability from nonviral vectors.

To design better delivery systems that enhance transfection efficiency of nonviral vectors, we need to improve our understanding of the mechanisms governing both the amounts of plasmid delivered to the nucleus and gene expression. What is needed is a measure of transcriptional availability (TA): the average level of gene expression per plasmid delivered to the nucleus over the course of an experiment. We describe a method to measure TA and demonstrate its application. The chloramphenicol acetyltransferase reporter gene was transfected into NIH/3T3 cells using either cationic liposomes (TFL-3; O,O'-ditetradecanoyl-N-(alpha-trimethylammonioacetyl) diethanolamine chloride (DC-6-14), dioleoylphosphatidylethanolamine (DOPE) and cholesterol, molar ratio 1/0.75/0.75) or cationic polymer (PEI; polyethylenimine). The time courses of both nuclear delivery of plasmids and reporter gene expression were measured for 4 h thereafter. For the conditions used, time courses of gene expression and plasmid nuclear delivery for the two vectors were different. To understand the origins of those differences, we applied a simple pharmacokinetic model, used the data to estimate the values of the model parameters, and interpret differences in estimated parameter values. The rate constant of delivery of plasmids into the nucleus for the TFL-3 vector was twice that of the PEI vector, whereas rate constant of elimination of plasmids in the nucleus for the PEI vector was four times that for the TFL-3 vector. The gene expression rate constant for the TFL-3 vector was estimated to be seven times larger than that of the PEI vector for the conditions used. The pharmacokinetically determined average exposure of a nucleus to plasmid was about 17 times larger for the TFL-3 vector, relative to the PEI vector. That greater exposure resulted in increased relative gene expression. Overall, the TA from the TFL-3 vector was about 13 times greater than from the PEI vector. The experimental design combined with the adoption of pharmacokinetic concepts and principles provide a method to measure TA along with detailed insights into the mechanisms governing gene delivery and expression.

Animals↗
PubMed2004

Porcine adenoviral vectors evade preexisting humoral immunity to adenoviruses and efficiently infect both human and murine cells in culture.

Preexisting immunity against human adenoviruses (HAd) limits the efficiency of transduction of HAd vectors in humans. In addition, development of a vector-specific immune response after the first inoculation with a HAd vector further lowers vector uptake following readministration. We investigated the usefulness of porcine adenovirus serotype 3 (PAd3)-based vectors as a supplement to HAd vectors. Here we demonstrate that preexisting HAd-specific neutralizing antibodies in humans do not cross-neutralize PAd3. In order to generate E1A-deleted PAd3 vectors, an E1-complementing cell line of porcine origin was produced. E1A-deleted PAd3 vector expressing green fluorescent protein; GFP (PAd-GFP) and E1-deleted HAd5 vector expressing GFP (HAd-GFP) transduced human cell lines with comparable efficiencies. Both of these vectors efficiently transduced murine MT1A2 breast cancer cell line, while PAd-GFP transduced murine NIH 3T3 fibroblast cell line significantly better (P < 0.05) than HAd-GFP. These results suggest that PAd3 vectors would be promising supplement to HAd vectors as a delivery vehicle for recombinant vaccines and gene therapy applications.

Adenovirus E1A Proteins↗
PubMed2004

Avidin-based targeting and purification of a protein IX-modified, metabolically biotinylated adenoviral vector.

While genetic modification of adenoviral vectors can produce vectors with modified tropism, incorporation of targeting peptides/proteins into the structural context of the virion can also result in destruction of ligand targeting or virion integrity. To combat this problem, we have developed a versatile targeting system using metabolically biotinylated adenoviral vectors bearing biotinylated fiber proteins. These vectors have been demonstrated to be useful as a platform for avidin-based ligand screening and vector targeting by conjugating biotinylated ligands to the virus using high-affinity tetrameric avidin (K(d) = 10(-15) M). The biotinylated vector could also be purified by biotin-reversible binding on monomeric avidin (K(d) = 10(-7) M). In this report, a second metabolically biotinylated adenovirus vector, Ad-IX-BAP, has been engineered by fusing a biotin acceptor peptide (BAP) to the C-terminus of the adenovirus pIX protein. This biotinylated vector displays twice as many biotins and was markedly superior for single-step affinity purification on monomeric avidin resin. However, unlike the fiber-biotinylated vector, Ad-IX-BAP failed to retarget to cells with biotinylated antibodies including anti-CD71 against the transferrin receptor. In contrast, Ad-IX-BAP was retargeted if transferrin, the cognate ligand for CD71, was used as a ligand rather than the anti-CD71. This work demonstrates the utility of metabolic biotinylation as a molecular screening tool to assess the utility of different viral capsid proteins for ligand display and the biology and compatibility of different ligands and receptors for vector targeting applications. These results also demonstrate the utility of the pIX-biotinylated vector as a platform for gentle single-step affinity purification of adenoviral vectors.

Adenoviridae↗
PubMed2005

Herpesvirus-based infectious titering of recombinant adeno-associated viral vectors.

Studies in animals and human clinical trials demonstrate the safety and persistence of recombinant adeno-associated viral (rAAV) serotype 2 vectors in a variety of tissues. rAAV vectors of other serotypes are also being developed for efficient gene transfer. To date, the literature describing these vectors has relied on physical or transducing titers to determine dose, but few, if any, infectious titers have been presented. This is due in large part to the lack of reagents and methods that would facilitate the infectious titering of vectors other than serotype 2. Here, we describe reagents and methods for infectious titering of AAV2 ITR-containing vectors pseudotyped with other AAV capsid serotypes and demonstrate their utility by titering pseudotyped rAAV1 or rAAV5 vectors. Cell lines are screened for optimal transduction using a vector of a particular serotype that expresses a marker transgene. Once a cell line and vector serotype are matched, a recombinant herpes simplex virus vector expressing AAV2 rep and cap genes provides helper functions that amplify the rAAV vector genome. The vector genomes are then detected and a titer is calculated. These methods generate reliable infectious titers for AAV vectors of different serotypes, thus enhancing product characterization and reducing risk in future clinical applications.

Animals↗
PubMed2000

Development of an adenoviral vector for intracerebral delivery of the dopamine D(2) receptor.

The age-related loss of striatal dopamine D(2) receptors (D(2)R) has been observed in numerous species, including rodents, monkeys, and man, and is partly responsible for impaired motor function in aged mammals. We have developed an adenoviral vector designed for intracerebral transfer of cDNA for D(2)R. Results of in vitro studies demonstrated that the vector produced abundant message for D(2)R and that the vector was membrane bound and capable of binding appropriate ligand. Results of in vivo studies provided clear evidence of D(2)R production when injected into the striatum of rats. The D(2)R produced were capable of binding appropriate ligand. In addition, evidence of functional receptors was produced by demonstrating apomorphine-induced rotational behavior in rats receiving a unilateral injection of the vector. Despite these successes, we have been unable to demonstrate improvement in the motor behavior of aged rats receiving bilateral injections of the vector. A major problem with this vector as with similar adenoviral vectors is the loss of expression beginning 3-5 days after injection to undetectable levels at 21 days. Because of the lack of motor functional effects in aged rats and the loss of expression of the vector, other strategies for development of the vector are being pursued. Regarding functional effects, we have examined the feasibility of manipulating hippocampal acetylcholine (ACh) release through D(2)R manipulation to improve memory performance. Using microdialysis, we have demonstrated in vivo in rats that treatment with a D(2)R agonist increases hippocampal ACh release while treatment with a D(2)R antagonist attentuates this effect as well as impairs performance in a complex maze task. In addition, a D(2)R null mutant mouse is being used to examine possible therapeutic effects of the vector. These mice show specific motor deficits. Recent studies using positron emission tomography have also demonstrated the feasibility of in vivo imaging of the vector. Thus, use of adenoviral vectors specific for neurotransmitter receptors can provide a highly useful research tool for examining age-related alterations in behavioral function and a possible strategy for therapeutic intervention.

Acetylcholine↗
PubMed2003

In vitro and in vivo activities of an oncolytic adenoviral vector designed to express GM-CSF.

Oncolytic adenoviruses are being tested as biological cancer therapeutics. Ar6pAE2fF (E2F vector) contains the E2F-1 promoter to regulate the expression of the E1a gene in cells with a disregulated retinoblastoma pathway. Ar6pAE2fmGmF (E2F-GM vector) includes the murine granulocyte-macrophage colony-stimulating factor (GM-CSF) transgene to enhance anti-tumor activity. Both vectors selectively killed human tumor cells in vitro. The E2F-GM vector expressed biologically active murine GM-CSF in vitro and GM-CSF was detected for several days in serum and tumor extracts following injections of established human xenograft tumors. In vivo, both vectors showed significant dose-dependent anti-tumor responses. The E2F-GM vector elicited greater efficacy compared to the E2F vector, demonstrating that GM-CSF enhanced the anti-tumor activity, even in immunodeficient nude mice. Histological analysis showed that both vectors induced necrosis and mononuclear cell infiltration, but only the E2F-GM vector resulted in eosinophil infiltration. Vector replication in vivo was demonstrated. The data showed that intratumoral injection of a GM-CSF-armed oncolytic vector induced potent anti-tumor responses in xenograft tumor models, likely as the result of both oncolytic vector activity and the induction of GM-CSF-mediated inflammation and innate immunity.

Adenoviridae↗
PubMed2003

HIV vector production mediated by Rev protein transduction.

HIV-1-based vectors are promising tools for gene therapy because of their ability to integrate into nondividing cells. Their safety in clinical applications remains a major concern. Recombination events occurring among plasmid constructs during vector production could potentially lead to the generation of replication-competent viruses. The safety of HIV-1-based vectors can be improved by removing all regions of the viral genome that are not absolutely required for vector production or function. In this study, we demonstrate that the HIV-1 rev gene is dispensable for the production of HIV-1-based vectors if the vector-producing cells are supplied with purified Rev protein. We compared the efficiency of vector production among Rev, TAT-Rev (Rev fused to the protein transduction domain of the HIV TAT protein), and Rev/Pep-1 (Rev complexed with the carrier peptide Pep-1). Our results showed that 293T cells efficiently internalized TAT-Rev and Rev/Pep-1 and high-titer vector preparations were obtained with this approach. Vectors generated by such an approach showed little difference in their efficiencies of transduction of established cell lines and primary cells compared with vectors generated by standard plasmid cotransfection. Eliminating the requirement for the HIV-1 rev gene during vector production should improve the safety of applying HIV vectors in human clinical trials.

Animals↗
PubMedDate not supplied

Production of retrovirus and adenovirus vectors for gene therapy: a comparative study using microcarrier and stationary cell culture.

In gene therapy, retrovirus and adenovirus vectors are extensively used as gene-delivery vehicles and further large-scale processing of these viral vectors will be increasingly important. This study examined stationary and microcarrier cell culture systems with respect to the production of a retrovirus vector (encoding a monounit hammerhead ribozyme gene with an intron) and an adenovirus vector (encoding a reporter lacZ gene). Cytodex 1 and Cytodex 3 solid microcarriers were found to be able to provide good cell growth and high-titer vector production in suspension cultures. Porous microcarriers such as Cytopore 2 gave slightly lower but still efficient growth but produced significantly lower titers of retrovirus and adenovirus vector from the producer cells. The specific retrovirus production was not proportionally related to the specific growth rate of the producer cells. High MOI infection was essential for high-titer production of adenovirus vector in 293 cells. Hydrodynamic shear forces on microcarrier-grown cells increased the production yield for retrovirus vector but decreased for adenovirus vector. The cellular productivity was much more efficient for adenovirus vector produced in 293 cells as compared to the retrovirus vector produced in PA317-RCM1 cells. These findings can provide further insight into the feasibility of applying microcarrier cell culture technology to produce gene-therapy virus vectors.

3T3 Cells↗
PubMed1999

Recombinant E1-deleted adenovirus vector induces apoptosis in two lung cancer cell lines.

Although replication-defective adenoviruses (Ads) are used as vectors for delivering therapeutic genes to cancer cells, various effects of the viruses on the proliferation of lung cancer cells have been reported. Experiments were carried out to determine whether or not E1-deleted Ad vectors (Ad5-CMV-lacZ) affected cell kinetics in two different types of lung cancer cell line in vitro. A dose-dependent relationship was measured between the vector multiplicity of infection (MOI) and the efficiency of lacZ gene transfer to lung cancer cells. The growth curves of vector-infected cells were shifted to the right compared with those of vehicle-exposed cells in a vector MOI-dependent fashion. The slowed cell proliferation resulted from both increased cell death and slower cell cycle progression of the vector-infected cells. The morphology of vector-exposed cells revealed apoptotic features including nuclear condensation and fragmented nuclei. These results indicate that using a higher vector MOI causes a higher gene transfer rate, but may induce apoptosis of infected cells. Although vector-induced apoptosis may be advantageous in inhibiting tumour growth, apoptosis of vector-infected cells may also reduce transgene expression in cancer cells. Minimization of the induction of apoptosis of vector-infected cells is important for the prolongation of the transduction efficiency of Ad vectors.

Adenoviruses, Human↗
PubMed2006

Engineering conditionally replication-competent adenoviral vectors carrying the cytosine deaminase gene increases the infectivity and therapeutic effect for breast cancer gene therapy.

We constructed a conditionally replication-competent adenoviral vector Ad.Lp-CD-IRES-E1A(control) in which the expression of both the prodrug-activating cytosine deaminase gene and the viral replication E1A gene were driven by the L-plastin tumor-specific promoter. In order to overcome the low infectivity of the adenoviral vectors for breast cancer cells, and to increase the safety and efficacy for cancer gene therapy, this vector was further modified on a transductional level by simultaneously ablating the native tropism of the vector to the primary CAR receptor and inserting a RGD-4C peptide into the HI loop of the fiber, which allows the vector to use the alphavbeta3 and alphavbeta5 receptors as alternative receptors. The resulting vector was named Ad.Lp-CD-IRES-E1A(MRGD). The transduction efficiency of the vector for breast cancer cell lines which have low expression level of CAR was increased both in vitro and in vivo. The Ad.Lp-CD-IRES-E1A(MRGD) vector produces a higher vector particle yield and a greater cytotoxic effect in tumor cells which have a low expression level of CAR, than did the Ad.Lp-CD-IRES-E1A(control) vector. Intratumoral injection of the Ad.Lp-CD-IRES-E1A(MRGD) vector following the intraperitoneal injection of 5FC into xenotransplanted human breast cancer cell lines which have low expression level of CAR led to greater degree of tumor regression in vivo than did the intratumoral injection of control adenoviral vectors not so modified.

Adenoviridae↗
PubMed1999

Efficacy and safety analyses of a recombinant human immunodeficiency virus type 1 derived vector system.

Lentiviruses infect both dividing and nondividing cells. In this study we characterized a lentiviral vector system consisting of a packaging vector (pHP) and a transducing vector (pTV) derived from a recombinant human immunodeficiency virus type 1 (HIV-1). In pHP, the long terminal repeats (LTRs), the 5' untranslated leader and portions of the env and nef genes were deleted. The leader sequence of pHP was substituted with a modified Rous sarcoma virus (RSV) 59 bp leader containing a mutated RSV gag AUG and a functional 5' splice site. The pHP construct was found to direct Gag-Pol synthesis as efficiently as wild-type HIV-1. The pTV construct contains sequences required for RNA packaging, reverse transcription and integration, but lacks viral genes. Co-transfection of pHP, pTV and a vesicular stomatitis virus G (VSV-G) envelope plasmid produced vectors at titers of 10(5)-10(6) transducing units per milliliter in 48 h. Replication-competent virus (RCV) was not detected when deletions were made in the env gene in pHP. The ability of this vector system to transduce dividing and nondividing cell in vitro and in vivo was also demonstrated. Compared with a Moloney murine leukemia virus (MLV) vector, the HP/TV vectors transduced human muscle-, kidney-, liver-derived cell lines and CD34+ primary hematopoietic progenitor cells more efficiently. Although the levels of the pTV transgene expression were high soon after transduction, the expression tended to decrease with time due either to the loss of proviral DNA or to the inactivation of promoter activity, which was found to be cell type-dependent. Analyses of extrachromosomal DNA showed that the unintegrated proviral DNA of lentiviral vectors survived much longer than that of the retroviral vectors. We demonstrate that the HP/TV vector is capable of high efficiency transduction and that long-term expression of lentiviral vectors is dependent on target cell type, the internal promoter and the transgene itself in the transducing vector.

Animals↗
PubMed2000

High efficiency retroviral vectors that contain no viral coding sequences.

Almost all currently available retroviral vectors based on murine leukemia virus (MLV) contain one or more viral coding sequences. Because these sequences are also present in the packaging genome, it has been suggested that homologous recombination may occur between the same nucleotide sequence in the packaging genome and the vector, resulting in the production of replication competent retrovirus (RCR). Up until now, it has been difficult to completely remove viral coding sequences since some were thought to be involved in the optimum function of the retroviral vector. For example, the gag coding sequence present in almost all available retroviral vectors has been believed to be necessary for efficient viral packaging, while the pol coding sequence present in the highly efficient vector MFG has been thought to be involved in achieving the high levels of gene expression. However, we have now developed a series of retroviral vectors that are absent of any retroviral coding sequences but produce even higher levels of gene expression without compromising viral titer. In these vectors, the intron and exon sequences from heterologous cellular or viral genes are present. When compared with the well-known MLV-based vectors, some of these newly developed vectors have been shown to produce significantly higher levels of gene expression for a longer period. In an experimental system that can maximize the production of RCR, our newly constructed vectors produced an absence of RCR. These vectors should prove to be safer than other currently available retroviral vectors containing one or more viral coding sequences.

3T3 Cells↗
PubMed2000

Adeno-associated virus vector mediated gene transfer to pancreatic beta cells.

Insulin-dependent diabetes mellitus (IDDM) or type 1 diabetes is an autoimmune disease that results in destruction of the insulin-producing pancreatic islet beta cells. Several factors induce the invasion of immune cells into islets and trigger inflammation. Gene therapy approaches targeting the islet cells could be an effective treatment to prevent the onset or reverse type 1 diabetes. Allogeneic islet transplantation provides short-term treatment. However, genetically modified islets, which resist the host immune response, could provide long-term solutions. Adeno-associated virus (AAV) is emerging as a prominent vector system for delivering therapeutic genes for human gene therapy. AAV vector can transduce nondividing cells and provide long-term gene expression by integrating into host chromosome. Therefore, it is an appropriate vector system for islet cell gene therapy. To test the efficacy of AAV vector to transduce pancreatic endocrine cells, we constructed AAV vectors using plasmid pSub201. Wild-type AAV DNA analogue from plasmid psub201 was subcloned into a cloning plasmid pSP72 and AAV vectors were constructed by inserting the transgenes with heterologous promoter in place of AAV open reading frames (rep and cap). In this report we demonstrate the transduction of pancreatic islet cells with AAV vectors encoding bacterial -galactosidase enzyme or enhanced green fluorescent protein (EGFP) as reporter gene. Dispersed porcine and rat islet cells can be transduced by AAV vector, with an efficiency of 47% and 38%, respectively. In particular porcine islet insulin producing beta cells were transduced with an efficiency of 39%. Intact rat islet cells were transduced with an efficiency of 26% as estimated by FACS analysis following transduction with an AAV vector encoding EGFP. Transduction of intact rat islets with an AAV vector did not alter glucose-induced insulin secretion. AAV vector transduction was higher in transformed islet cell lines INS-1 and RIN m5F with an efficiency of 65% and 57%, respectively. These new results suggest that AAV vectors will provide an improved method of gene delivery to pancreatic islets and isolated pancreatic beta cells.

Animals↗
PubMed2002

Induction of cyclo-oxygenase-2 in non-small cell lung cancer cells by infection with DeltaE1, DeltaE3 recombinant adenovirus vectors.

Infection of epithelial-derived cells by adenovirus vectors has myriad effects on cellular behavior and function. Some are relevant to the desired effect of the encoded transgene and therapeutic goals of gene therapy approach. The current experiments describe the induction of COX-2 protein and PGE-2 production by non-small cell lung cancer (NSCLC) cells following infection with a first generation (DeltaE1, DeltaE3) Ad vector. COX-2 overexpression by malignant cells has been shown to enhance cellular invasion, induce angiogenesis, regulate anti-apoptotic cellular defenses and augment immunologic resistance through production of PGE-2. Data show DeltaE1, DeltaE3, Ad5 vector infection induces dose-dependent increases in PGE-2 production by NSCLC cell lines. Data with UV/psoralen inactivated vectors and control vectors show this effect is dependent on Ad vector gene expression, but independent of the transgene expressed. Selective blockade of ERK with PD98029 abrogated induction of PGE-2 by Ad vectors. Consistent with these data, detectable increases in COX-2 protein were seen at 48 h after infection by Western blot that were paralleled by increases in the phosphorylation of ERK-1/2. UV/psoralen-inactivated vector did not induce COX-2 protein or ERK phosphorylation at 48 h. Further, an inhibitor of NF-kappa B (NFkappaB) translocation to the nucleus, SN50, had no effect on PGE-2 levels. In contrast, Ad vector infection did induce NFkappaB activity measured by NFkappaB-luciferase reporter plasmid, transfected into a NSCLC cell line. Collectively the data indicate DeltaE1, DeltaE3, Ad5 vector infection leads to ERK phosphorylation with parallel increases in COX-2 protein and PGE-2 production. These effects appear unrelated to NFkappaB and are dependent on gene expression by the vector. This information may need to be considered when defining targets for cancer gene therapy and/or the choice of viral vector.

Adenoviridae↗
PubMed2003

Fetal muscle gene transfer is not enhanced by an RGD capsid modification to high-capacity adenoviral vectors.

High levels of alpha(v) integrin expression by fetal muscle suggested that vector re-targeting to integrins could enhance adenoviral vector-mediated transduction, thereby increasing safety and efficacy of muscle gene transfer in utero. High-capacity adenoviral (HC-Ad) vectors modified by an Arg-Gly-Asp (RGD) peptide motif in the HI loop of the adenoviral fiber (RGD-HC-Ad) have demonstrated efficient gene transfer through binding to alpha(v) integrins. To test integrin targeting of HC-Ad vectors for fetal muscle gene transfer, we compared unmodified and RGD-modified HC-Ad vectors. In vivo, unmodified HC-Ad vector transduced fetal mouse muscle with four-fold higher efficiency compared to RGD-HC-Ad vector. Confirming that the difference was due to muscle cell autonomous factors and not mechanical barriers, transduction of primary myogenic cells isolated from murine fetal muscle in vitro demonstrated a three-fold better transduction by HC-Ad vector than by RGD-HC-Ad vector. We hypothesized that the high expression level of coxsackievirus and adenovirus receptor (CAR), demonstrated in fetal muscle cells both in vitro and in vivo, was the crucial variable influencing the relative transduction efficiencies of HC-Ad and RGD-HC-Ad vectors. To explore this further, we studied transduction by HC-Ad and RGD-HC-Ad vectors in paired cell lines that expressed alpha(v) integrins and differed only by the presence or absence of CAR expression. The results increase our understanding of factors that will be important for retargeting HC-Ad vectors to enhance gene transfer to fetal muscle.

Adenoviridae↗
PubMed2004

Tight regulation from a single tet-off rAAV vector as demonstrated by flow cytometry and quantitative, real-time PCR.

Vectors suitable for delivery of therapeutic genes to the CNS for chronic neurodegenerative diseases will require regulatable transgene expression. In this study, three self-regulating rAAV vectors encoding humanized green fluorescent protein (hGFP) were made using the tetracycline (tet)-off system. Elements were cloned in different orientations relative to each other and to the AAV internal terminal repeat (ITRs). The advantage of this vector system is that all infected cells will carry both the 'therapeutic' gene and the tet-regulator. To compare the efficiency of the vectors, 293T cells infected by each vector were grown in the presence or absence of the tet-analog doxycycline (dox). Cells were analyzed by flow cytometry for hGFP protein expression, and quantitative RT-PCR (QRT-PCR) for levels of hGFP mRNA and the tet-activator (tTA) mRNA. In the presence of dox, cells infected with one of the vectors, rAAVS3, showed less than 2% total fluorescent intensity and mRNA copy number than cells grown without dox. The other two vectors were significantly more leaky. Levels of tTA mRNA were not affected by dox. The S3 vector also displayed tight regulation in HeLa and HT1080 cells. To assess regulation in the brain, the S3 vector was injected into rat striatum and rats maintained on regular or dox-supplemented water. At 1 month after vector injection, numerous positive cells were observed in rats maintained on regular water whereas only rare positive cells with very low levels of fluorescence were observed in rats maintained on water containing dox. The QRT-PCR analysis showed that dox inhibited expression of hGFP mRNA in brain by greater than 99%. These results demonstrate that exceedingly tight regulation of transgene expression is possible using the tet-off system in the context of a self-regulating rAAV vector and that the specific orientation of two promoters relative to each other and to the ITRs is important. Regulatable vectors based on this design are ideal for therapeutic gene delivery to the CNS.

Animals↗
PubMed2004

CTLA4Ig delivered by high-capacity adenoviral vector induces stable expression of dystrophin in mdx mouse muscle.

Adenoviral (Ad) vector-mediated gene delivery of normal, full-length dystrophin to skeletal muscle provides a promising strategy for the treatment of Duchenne muscular dystrophy (DMD), an X-linked recessive, dystrophin-deficient muscle disease. Studies in animal models suggest that successful DMD gene therapy by Ad vector-mediated gene transfer would be precluded by cellular and humoral immune responses induced by vector capsid and transgene proteins. To address the immunity induced by Ad vector-mediated dystrophin gene delivery to dystrophic muscle, we developed high-capacity adenoviral (HC-Ad) vectors expressing mouse dystrophin driven by the muscle creatine kinase promoter (AdmDys) and mCTLA4Ig (AdmCTLA4Ig) individually, or together from one vector (AdmCTLA4Ig/mDys). We found stable expression of dystrophin protein in the tibialis anterior muscles of mdx mice, coinjected with AdmCTLA4Ig and AdmDys, or injected alone with AdmCTLA4Ig/mDys, whereas the expression of dystrophin protein in the control group coinjected with AdmDys and an empty vector decreased by at least 50% between 2 and 8 weeks after administration. Additionally, we observed reductions in Ad vector-induced Th1 and Th2 cytokines, Ad vector-specific cytotoxic T lymphocyte activation and neutralizing anti-Ad antibodies in both experimental groups that received a mCTLA4Ig-expressing vector as compared to the control group. This study demonstrates that the coexpression of mCTLA4Ig and dystrophin in skeletal muscle provided by HC-Ad vector-mediated gene transfer can provide stable expression of dystrophin in immunocompetent, adult mdx mouse muscle and applies a potentially powerful strategy to overcome adaptive immunity induced by Ad vector-mediated dystrophin gene delivery toward the ultimate goal of treatment for DMD.

Abatacept↗
PubMed2007

Lentiviral vector transduction of a dominant-negative Rev gene into human CD34+ hematopoietic progenitor cells potently inhibits human immunodeficiency virus-1 replication.

Gene therapy for human immunodeficiency virus (HIV)-1 may be performed by introducing into hematopoietic stem cells genes that inhibit replication of HIV-1 using lentiviral vectors. However, production of lentiviral vectors derived from HIV-1 may be inhibited by the gene being carried to inhibit HIV-1 and these vectors could be mobilized by wild-type HIV-1 infecting transduced cells. This study investigates these problems for the delivery of a dominant-negative rev gene humanized revM10 (huM10) by a lentiviral vector. Although most packaging plasmids suffered inhibition of expression of HIV-1 virion proteins by vectors expressing huM10, the packaging plasmids that expressed the highest levels of HIV-1 virion proteins produced vectors at titers that would be sufficient for clinical applications. The vectors carrying huM10 were used to transduce primary human CD34(+) hematopoietic progenitor cells and yielded high-level transduction without toxicity and conferred potent inhibition of HIV-1. The use of lentiviral vectors with deletion of the enhancers and promoter from the LTR (self-inactivating (SIN) vectors) decreased the frequency of vector mobilization by wild-type HIV-1; SIN vectors carrying huM10 were not mobilized detectably. These studies indicate that lentiviral vectors can be made effective for use in gene therapy for HIV-1.

Antigens, CD34↗