lacZ Vector Research Articles

Modified Adenovirus Reduces De Novo Peritoneal Adhesions in Rats and Limits Off-Target Transfection. Role of EZH2 in Adhesion Formation

ABSTRACTAim of the study: Adenovector encoding tissue plasminogen activator (tPA) was shown to reduce experimental peritoneal adhesion. We investigated the targeting potential of our modified adenovector, its ability to reduce adhesions and the epigenetic role of histone methyltransferase EZH2 in adhesion formation. Materials and methods: Control lacZ, nonmodified tPA or modified tPA vectors were instilled in the peritoneal cavity after injury in de novo adhesions or after lysis of adhesions in recurrent adhesions. Adhesion severity was scored and adhesions and liver tissues were examined for adenovirus E4 gene and tPA mRNA expression. Levels of tPA, plasminogen activator inhibitor-1 (PAI-1), transforming growth factor-β1 (TGF-β1), and EZH2 expression were measured. Results: E4 transcripts were detected in adhesions of nonmodified and modified and in livers of nonmodified but not in livers of modified de novo adhesions. Both nonmodified (p = 0.021) and modified vectors (p = 0.036) reduced the severity of de novo adhesions compared to lacZ vector. Levels of tPA in nonmodified (p = 0.021) and modified adhesions (p = 0.001) were elevated while PAI-1 (p = 0.013 and p = 0.001, respectively) and TGF-β1 levels (p = 0.002 and p = 0.016, respectively) were reduced compared with lacZ group. All vectors were not expressed in recurrent adhesions and severity score were not different among groups. EZH2 levels were elevated in de novo nontreated (p = 0.001) and was further increased in recurrent (p = 0.001) nontreated adhesions compared with noninjured peritoneum. Conclusion: Modified adenovirus successfully targeted de novo adhesions but not liver tissues and reduced the severity of de novo adhesions. EZH2 is involved in the development and progression of peritoneal adhesions.

A Guided Inquiry‐Based Animal Cell Culture Laboratory Course for Undergraduates

The revised version of this course, Tissue Culture (CHE470), includes a series of animal cell culture‐based experiments and lectures on Biochemistry & Molecular Biology (BMB) aspects of cell culture, including cellular transformation and metabolism, growth kinetics, transfection, inducible gene expression and biochemical assays. After receiving lectures on BMB aspects of cell culture and training in fundamental animal cell culture techniques, student undertake a unified series of experiments consists of (1) animal cell transfection by electroporation, (2) clonal selection, isolation and expansion, (3) induction of gene expression with tetracycline, (4) genomic DNA (gDNA) isolation from clonal cell populations and PCR screening of clonal gDNAs, (5) RNA isolation and Real Time‐PCR, (6) protein isolation for western blotting, and (7) beta‐galactosidase enzyme activity assays. In the revised version of this course, first executed in Fall 2015, students were provided with two plasmid DNA samples for transfection into CHO T‐RExTM cells. Each student received a known sample of an unaltered tetracyclin‐inducible beta‐galactosidase expression vector (pcDNA4/TO/mycHIS‐LacZ) and one of four pcDNA4/TO/mycHIS‐lacZ vectors altered by site‐directed mutagenesis. Students were not informed about the nature of the mutation in the mutant vector provided to them. Mutations in the four altered vectors include single nucleotide mutations (SNP) that inactivate the LacZ ATG start codon (ATG‐>TTG), or one of two missense mutations in the beta‐galactosidase active site (E461K and Y503C), or a nonsense mutation that produces a truncated beta‐galactosidase protein (Y503X). After separately transfecting their two vectors into CHO T‐RExTM cells, student used antibiotic selection and isolated clonal cell populations that they molecular characterized. Students utilized their experimental data acquired from TaqMan RT‐PCR, PCR screening of clones and direct DNA sequencing, western blotting and beta‐galactosidase activity assays to determine the exact nature of the mutation in their pcDNA4/TO/mycHIS‐LacZ mutant vector provided. This guided experimental approach provides opportunities for inquiry‐based learning through students’ acquiring and analyses of experimental observations and data. Course grades are based upon a written BMB peer‐review journal style manuscript report of their experimental data and conclusions from their semester‐long series of experiments, three in‐class exams, an oral presentation of their data and findings, and some animal cell culture laboratory exercises. Student outcomes were measured by anonymous feedback through an online survey system using a numeric rating scale (5 = strongly agree; 1 = strongly disagree). Combined Fall 2013 and 2014 survey data from CHE470 student cohorts (n = 7) reported they “know more about this subject” (mean = 5.00, dev. = 0.000), that their “skills in this area have improved” (mean = 5.00, dev. = 0.000), their “appreciation of this subject increased” (mean = 5.00, dev. = 0.000), and that “learning objectives of the course were met” (mean = 4.86, dev. = 0.378). Survey data are consistent with students finding this course informative and beneficial.Support or Funding InformationThis project was supported through generous financial support from the University of Tampa Department of Chemistry, Biochemistry & Physics.

An efficient microinjection method for unfertilized eggs of Asian amphioxus Branchiostoma belcheri

Amphioxus is a promising model animal for evolutionary and developmental studies. However, as an emerging model organism, amphioxus lacks most molecular techniques applied in other well-developed model animals. Microinjection is a powerful technique for gene manipulation, and thus it undoubtedly is one of useful approaches in the studies of gene function and embryonic development. Although the method has been exploited in Florida and European amphioxus, it still remains to be optimized and introduced into other amphioxus species. In order to introduce the technique into our lab, we followed and optimized the previous description and successfully performed microinjection on unfertilized eggs of Asian amphioxus Branchiostoma belcheri. We made six solutions for practice: 200 mM KCl, 100 ng/μl actin-LacZ or 100 ng/μl actin-RFP vectors, LacZ or RFP vectors without promoter and RFP capped mRNA. More than 99.2 % of eggs injected with KCl were able to be fertilized, 94.3 % of them could hatch normally and 55.9 % survived until 2-day larvae, all of which were nearly equivalent to those obtained from normally fertilized eggs. Embryos injected with two plasmid constructs also showed very high fertilizing and hatching ratios, but normally developing ratios were slightly lower than that of KCl injection. Of those injected embryos, 91.8 % expressed exogenous gene LacZ and 80.5 % exhibited foreign RFP expression, which were driven by a promoter from amphioxus β-actin gene. The data indicated a successful modified microinjection method for the unfertilized eggs of Asian amphioxus, and those modifications improved the feasibility and efficiency of microinjection on amphioxus.

Tyrosine hydroxylase gene transfections to different sites of striatum in the rat model of Parkinson’s disease

The use of gene therapy has been intensively studied as a potential method to treat Parkinson’s disease (PD) and other degenerative brain diseases. However, the effects of experimental measures and approaches on the outcome of gene delivery or on the physiological state of target tissues have not been analyzed as much and systematically. Therefore, we have infused adenovirus vectors expressing either a therapeutic tyrosine hydroxylase (TH) gene or a lacZ reporter gene into striatum in a rat model of PD. The experimental procedures were tested using the Ad lacZ vector in order to optimize concentrations, volumes, infusion speeds and transfection times. The expression of Ad lacZ vector was lower and declined earlier in the lesioned than unlesioned striatum suggesting that the lesion affects on the transfection efficiency and outcome of gene transfection. The effect of three different approaches of Ad TH vector transfection was compared: 1) the delivery of Ad TH gene vector alone into one single site of striatum, 2) the delivery of Ad TH gene vector alone into multiple sites of striatum, and 3) the delivery of Ad TH gene vector into one site of striatum followed by a continuous infusion of tetrahydrobiopterin (BH4) cofactor with a mini pump. There was a small and transient unsignificant decrease in the turning behavior when the Ad TH vector was delivered into one site of the striatum. Simultaneous infusion into several sites or together with BH4 cofactor did not improve more the effect of gene delivery. Thus, although the effects were unsignificant, the Ad TH transfection seemed to decrease the turning behavior in the rat model of PD and the optimal effect was seen at some specific doses and time points. Furthermore, the outcome of gene therapy could depend in addition to the amount and efficacy of gene vectors also on the physiological state and experimental strategies.

Intravenous Delivery of Adeno-Associated Viral Vector Serotype 9 Mediates Effective Gene Expression in Ischemic Stroke Lesion and Brain Angiogenic Foci

Adeno-associated viral vector (AAV) is a powerful tool for delivering genes to treat brain diseases. Intravenous delivery of a self-complementary but not single-stranded AAV9 (ssAAV9) mediates robust gene expression in the adult brain. We tested if ssAAV9 effectively mediates gene expression in the ischemic stroke lesion and angiogenic foci. Focal ischemic stroke was induced by permanent occlusion of the left middle cerebral artery (MCAO) and focal angiogenesis was induced by injecting an AAV expressing vascular endothelial growth factor (AAV-VEGF) into the basal ganglia. ssAAV vectors that have cytomegalovirus (CMV) promoter driving (AAV-CMVLacZ) or hypoxia response elements controlling (AAV-H9LacZ) LacZ expression were packaged in AAV9 or AAV1 capsid and injected into mice through the jugular vein 1 hour after MCAO or 4 weeks after the induction of angiogenesis. LacZ gene expression was analyzed in the brain and other organs 5 days after LacZ vector injection. LacZ expression was detected in the peri-infarct region of AAV9-CMVLacZ and AAV9-H9LacZ-injected MCAO mice and the brain angiogenic foci of AAV9-CMVLacZ-injected mice. Minimum LacZ expression was detected in the brain of AAV1-CMVLacZ-injected mice. Robust LacZ expression was found in the liver and heart of AAV-CMVLacZ-injected mice, but not in AAV9-H9LacZ-injected mice. ssAAV9 could be a useful tool to deliver therapeutic genes to the ischemic stroke lesion or brain angiogenic foci.

Direct rAAV SOX9 administration for durable articular cartilage repair with delayed terminal differentiation and hypertrophy in vivo

Direct gene transfer strategies are of promising value to treat articular cartilage defects. Here, we tested the ability of a recombinant adeno-associated virus (rAAV) SOX9 vector to enhance the repair of cartilage lesions in vivo. The candidate construct was provided to osteochondral defects in rabbit knee joints vis-à-vis control (lacZ) vector treatment and to cells relevant of the repair tissue (mesenchymal stem cells, chondrocytes). Efficient, long-term transgene expression was noted within the lesions (up to 16 weeks) and in cells in vitro (21 days). Administration of the SOX9 vector was capable of stimulating the biological activities in vitro and over time in vivo. SOX9 treatment in vivo was well tolerated, leading to improved cartilage repair processes with enhanced production of major matrix components. Remarkably, application of rAAV SOX9 delayed premature terminal differentiation and hypertrophy in the newly formed cartilage, possible due to contrasting effects of SOX9 on RUNX2 and β-catenin osteogenic expression in this area. Most strikingly, SOX9 treatment improved the reconstitution of the subchondral bone in the defects, possibly due to an increase in RUNX2 expression in this location. These findings show the potential of direct rAAV gene delivery as an efficient tool to treat cartilage lesions.

Role of circulating fibroblast growth factor-2 in lipopolysaccharide-induced acute kidney injury in mice

Fibroblast growth factor-2 (FGF-2) is an angiogenic growth factor involved in renal growth and regeneration. Previous studies in rodents revealed that single intrarenal injections of FGF-2 improved the outcome of acute kidney injury (AKI). Septic children usually show elevated plasma levels of FGF-2, and are at risk of developing AKI. However, the role of circulating FGF-2 in the pathogenesis of AKI is not well understood. We have developed a mouse model to determine how FGF-2 released into the circulation modulates the outcome of AKI induced by lipopolysaccharide (LPS). Young FVB/N mice were infected with adenoviruses carrying a secreted form of human FGF-2 or control LacZ vectors. Subsequently, when the circulating levels of FGF-2 were similar to those seen in septic children, mice were injected with a non-lethal dose of LPS or control buffer. All mice injected with LPS developed hypotension and AKI, from which they recovered after 5 days. FGF-2 did not improve the outcome of AKI, and induced more significant renal proliferative and apoptotic changes during the recovery phase. These findings suggest that circulating FGF-2 may not necessarily prevent the development or improve the outcome of AKI. Moreover, the renal accumulation of FGF-2 might cause further renal damage.

AAV based gene delivery to myocardium in rodents and pigs

Objective: To optimize transgene expression levels after AAV‐mediated gene transfer different delivery methods were compared in a rat (A) and porcine (B) heterotopic heart transplantation model.Methods: (A) Heterotopic abdominal heart transplantations were performed in male Lewis rats. After harvesting the donor hearts, the viral vectors were delivered to the graft by the following methods: (1) 0.35 ml saline solution containing AAV2/9‐LacZ (2 × 1011 vector genome, vg) was injected directly into the myocardium (apex) immediately after reperfusion. (2) cardioplegic solution (0.3 ml) containing AAV‐2(HBSD), 2/9‐LacZ vectors was rapidly injected into the aortic root with the pulmonary trunk clamped. Before transplantation the transfected heart was incubated for 20 min in iced cardioplegia. (3) A reperfusion system was applied: For 20 min a cold solution of cardioplegia (5 ml) and AAV‐2(HBSD) or AAV2/9‐LacZ vectors were recirculated through the donor heart. Transplanted grafts were explanted after 3 weeks. To detect and measure marker gene expression X‐gal staining or a luciferase assay was performed.In a second series we compared the effects of the transduction of PD‐L1 to LacZ using the optimum method (intraaortic root injection) in the same heart transplantation model.(B) Heterotopic abdominal HTX was performed in pigs (Landrace, 10–18 kg) following vector application to the donor heart in an in situ‐Langendorff perfusion system (AAV2/GFP and AAV2/Luciferase). Recipients were given tacrolimus (0.3 mg/kg BW), after 21 days the transplanted hearts were explanted for transgene expression analysis. In a second series we compared AAV2/9‐mediated transduction of PD‐L1 and LacZ in the in situ‐Langendorff model and consequent allogeneic heterotopic abdominal heart transplantation.Results: (A) Highest transfection efficiency was observed in the grafts treated with intracoronary infusion of AAV2/9 at the higher dosage, and the expression pattern was global and homogenous in the grafts. hPD‐L1 transduction resulted in no significant difference of survival time and signs of rejection after allogeneic rat heart transplantation.(B) AAV2‐mediated gene delivery was unable to yield sufficient transgene expression after in situ‐Langendorff perfusion of porcine hearts. AAV2/9 based gene transfer of LacZ and hPD‐L1 led to excellent myocardial gene expression lasting up to 2 months. Due to species incompatibility no protective effects were observed in our allogeneic porcine transplantation model.Conclusions: (A) We demonstrated that infusion of AAV vectors into the aortic root with the pulmonary trunk clamped is a simple and efficient method for gene delivery to the donor heart in an allogeneic rat heart transplantation setting. Gene transfer of hPD‐L1 was ineffective to protect against allorejection, on the contrary there was a trend to aggravated rejection. Therefore the exact mechanism of hPD‐L1 in allograft rejection needs further investigation.(B) The in situ‐Langendorff model was developed to allow isolated target organ perfusion with high vector concentrations under physiological conditions. Using this method AAV2/9 mediated gene delivery into porcine hearts proved effective to induce excellent marker gene expression. Expression of hPD‐L1 could also be achieved but was ineffective in pig allotransplantation due to species incompatibility. Double transgenic pig hearts (e.g. Gal‐KO+CD46) can now efficiently be transduced with hPD‐L1 or hCTLA4‐Ig to further optimize long‐term survival after pig‐to‐primate cardiac xenotransplantation.

Decreased Insulin Secretion and Accumulation of Triglyceride in .BETA. Cells Overexpressing a Dominant-negative Form of AMP-activated Protein Kinase

Adenosine 5' -monophosphate-activated protein kinase (AMPK) has been implicated in the regulation of energy metabolism, although its role in the pancreatic beta cells remains unclear. In the present, we have overexpressed a dominant negative form of AMPKalpha1 subunit (Asp57Ala) tagged with c-myc epitope (AMPKalpha1-DN) in INS-1D cells with an adenoviral vector. After 48 h of adenoviral infection, overexpression of AMPKalpha1-DN in INS-1D cells was confirmed by Western blot analysis with anti-c-myc antibody. Phosphorylation of the Thr172 in AMPKalpha1/alpha2 subunit was progressively decreased in parallel with increasing number of adenoviral titers. Glucose-stimulated insulin secretion in response to 30 mmol/L glucose was decreased in INS-1D cells overexpressing AMPKalpha1-DN as compared to control cells infected with adeno- LacZ vector. Neither cellular insulin content nor insulin mRNA level was changed between the two groups. Phosphorylation of acetyl-CoA carboxylase (ACC), a down-stream substrate of AMPK, was decreased, indicating that ACC activity was increased, due to the decreased AMPK activity. In fact, intracellular triglyceride content was increased as compared to control cells. The beta-oxidation of palmitate was decreased at 30 mmol/L glucose. Insulin secretion in response to potassium chloride or glibenclamide was also decreased as compared to control cells. In conclusion, suppression of AMPK activity in beta-cells inhibited insulin secretion in response to glucose, potassium chloride or glibenclamide without altering insulin content. Accumulation of triglyceride subsequent to the activation of ACC by suppression of AMPK activity, was suggested to be, at least in part, responsible for the impaired insulin secretion through so-called lipotoxicity mechanism.

Long-Term Protection Against Human Papillomavirus E7-Positive Tumor by a Single Vaccination of Adeno-Associated Virus Vectors Encoding a Fusion Protein of Inactivated E7 of Human Papillomavirus 16/18 and Heat Shock Protein 70

We investigated a gene vaccine strategy against human papillomavirus (HPV)-induced cancer and premalignant diseases, using adeno-associated virus (AAV) vector encoding the viral E7 oncoproteins as the tumor antigens from HPV serotypes 16 (HPV16) and 18 (HPV18). Genetically inactivated E7 proteins were fused with a heat shock protein 70 (hsp70) to minimize the risk of cell transformation and enhance immune responses. The fusion protein gene was packaged in AAV serotype 1 or 2 (AAV1 or 2) for efficient in vivo gene expression. Our results showed that after a single intramuscular injection, the AAV1 vector elicited stronger HPV-specific cytotoxic T lymphocyte (CTL) responses and interferon-gamma secretion when compared with the AAV2 vector. Prophylactic immunization with AAV1 protected 100% of the mice from tumor growth for more than 1 year, whereas all the control mice immunized with either a LacZ vector or saline grew large tumors and died within 6 weeks after inoculation of E7-positive tumor cell line TC-1. In addition, this single-dose AAV1 vaccination completely protected the mice against second and third challenges with higher numbers of TC-1 cells. Despite lower CTL responses against the E7 antigens, AAV2 vector prophylactic immunization was also sufficient to protect 100% of the mice against the initial and second tumor challenges and 70% of the mice against the third challenge. In addition, therapeutic immunization with AAV1 after palpable tumor formation inhibited tumor growth and caused tumor regression in some mice. Thus, our studies support the potential of AAV vectors as a genetic vaccine for the prevention and treatment of HPV-induced malignancies.

Recombinant adeno-associated viruses (rAAV2) facilitate the intraperitoneal gene delivery to cancer cells

Peritoneal dissemination of cancer cells is characteristic of advanced stages of ovarian, breast and lung cancers, and is associated with poor patient survival. The presence of cancer cells in effusions complicates treatment protocols, while cell eradication is seriously limited. One of the novel options available is cancer gene therapy with recombinant adeno-associated viruses. This combination represents the most promising gene delivery vehicles to neoplasmatic cells within serosal cavities due to their unique properties that include the ability to infect proliferating cells of broad host range, as well as the potential of long-term expression. Recombinant infectious adeno-associated virus serotype 2 particles (rAAV2) were produced in a helper-free system using an AAV-293 packaging cell line, and quantitatively analyzed by real-time PCR. Balb/c mice intraperitoneally pre-injected with L1 cancer cells were treated with different doses of rAAV2. Subsequently, the mice were sacrificed and intraperitoneal effusions were analyzed for rAAV presence and rAAV/β-galactosidase (LacZ) vector efficiency in order to infect cancer cells within the peritoneal cavity. We reported an efficient infection of L1 cancer cells disseminated into the peritoneal cavity by rAAV2. The expression of reporter genes (GFP and LacZ) attributable to the rAAV cell uptake was closely dependent on an applied multiplicity of infection ratio (MOI). The highest infection efficiency was observed at a MOI of 50 and 200. Our study confirmed the ability of adeno-associated viruses to facilitate gene transferability to cancer cells disseminated in the serosal cavity, as well as the potential usefulness of these viruses as a new approach in cancer gene therapy.

Sample purification improves the analysis of nonviral in vivo gene transfection

Nonviral gene delivery has gained a lot of interest as a promising approach for gene therapy. Despite intensive studies and much progress the outcome of nonviral vectors has remained significantly weaker than that of viral vectors. A weak transfection efficiency of nonviral gene transfection is still limiting their in vivo use. We have tested the possibility to improve the measurement of transfection efficiency by increasing the sensitivity of analysis with sample purification. The BPVlacZ and CMVlacZ plasmids were transfected by i.v. infusion of the PEI/DNA complexes in the rats. An adenovirus lacZ vector was used as a reference. The transfection efficiency was analysed from the lungs and brain. Tissue samples were minced and homogenized for preparation of crude homogenates and for further purification of crude homogenates with a DEAE anion exchange chromatography. The beta-galactosidase activity was measured using a luminometric assay. The obtained activity of beta-galactosidase was higher in the purified than nonpurified samples and the analysis of transfection efficiency as beta-galactosidase activity was improved more than 1000-fold by the purification of samples from perfused target tissues. An increased sensitivity of analysis by sample preparation may be a useful and inexpensive strategy to detect and estimate a low transfection efficiency or transgene expression often associated with a nonviral in vivo gene delivery.

Inhibition of HIV-1 by DpT-Based Iron Chelators

HIV-1 transcription is activated by HIV-1 Tat protein, which recruits transcriptional co-activators to the HIV-1 promoter. Elongation of HIV-1 transcription is mediated by the interaction of Tat with host cell cycle-dependent kinase 9 (CDK9)/cyclin T1, which phosphorylates the C-terminal domain of RNA polymerase II. Tat itself is phosphorylated by host cell cycle-dependent kinase 2 (CDK2) [1] and inhibition of CDK2 by tridentate iron chelators such as 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone, (311) or ICL670 (deferasirox) inhibits HIV-1 transcription [2]. In addition to the inhibition of CDK2, 311 and ICL670 also prevent association of CDK9 with cyclin T1 [2], which could lead to inhibition of CDK9 activity and also to inhibition of HIV-1 transcription. Recently, a group of novel di-2-pyridylketone thiosemicarbazone (DpT) based tridentate iron chelators were shown to exhibit marked antiproliferative activity in vivo [3]. Here we screened DpT-based and also 2-benzoylpyridine thiosemicarbazone (BpT)-based tridentate iron chelators and identified three chelators, Dp44mT, Bp4eT and Bp4aT, that inhibited HIV-1 transcription but were not cytotoxic as determined by propidium iodide uptake, LDH release and calcein-AM uptake. The inhibition of HIV-1 transcription was observed in CEM HIV-1 LTR-GFP cells infected with Adeno-Tat and in 293T cells transiently transfected with HIV-1-LTR LacZ and Tat-expressing vectors with IC50s in the mid-nanomolar range. These new iron chelators also inhibited HIV-1 replication in CEM and THP-1 cells at 10 mM concentration. Analysis of the molecular mechanism of HIV-1 inhibition revealed that the DpT- and BpT-based iron chelators inhibited the activities of both CDK9/cyclin T1 and CDK2. The CDK9/cyclin T1 complex was disrupted in the cells treated with iron chelators, suggesting a possible mechanism for the inhibition of CDK9. In conclusion, our findings provide further evidence that iron chelators may inhibit HIV-1 transcription by deregulating CDK2 and CDK9. The projected therapeutic index of the selected DpT-based iron chelators was over 103 suggesting their potential usefulness as future anti-retroviral therapeutics.

A Hybrid Vector System Expands Adeno-associated Viral Vector Packaging Capacity in a Transgene-independent Manner

The trans-splicing (ts) and overlapping (ov) vectors expand the packaging capacity of adeno-associated virus (AAV). But their application depends on the inherent properties of the target gene. The ts vectors require an optimal gene-splitting site and the ov vectors require a highly recombinogenic domain. In order to overcome these limitations, we developed a hybrid dual (hd) vector system. In the hd vectors, we inserted a highly recombinogenic alkaline phosphatase (AP) sequence in the ts vectors to allow for transgene-independent reconstitution through homologous recombination of the AP sequences. We first tested the hybrid system with the LacZ gene. Both in the cell line (in vitro) and in the mouse muscle (in vivo), the hd vectors significantly outperformed the ts and ov vectors. In muscle, the transduction efficiency of the hybrid vectors reached 80% of that from the single intact vector. Southern blot confirmed AP sequence-mediated transgene reconstitution. In order to validate the hybrid system, we split the 6 kilobase (kb) mini-dystrophin gene at the exon 55/56 junction, a predicted poor site for the ts approach. In dystrophic mdx mouse muscle, the hd vectors yielded 5.6-fold higher transduction than the ts vectors did. Taken together, these data suggest that the hybrid system efficiently expresses large therapeutic genes that are poor candidates for the ts and ov approaches.

Analysis of Tumors Arising in Male B6C3F1 Mice with and without AAV Vector Delivery to Liver

The present study reports on the frequency of liver tumors observed in a gene therapy study with AAV vectors in male mice of the B6C3F1 hybrid background, which are known to have a high frequency of spontaneous liver tumors. Male mice with mutations in their Otc gene and their wild-type siblings received AAV vectors expressing either the murine Otc or the LacZ gene. Untreated control animals were included in the study. All experimental groups, including wild-type and OTC-deficient animals not treated with vector, developed liver nodules, which in some cases were due to hepatocellular carcinoma. Vector DNA was lower in tumors than in adjacent normal liver. A statistical analysis of the data did not show an association between treatment with Otc vectors and formation of tumors in OTC-deficient mice. However, mice treated with LacZ vectors showed increased risks of tumor formation and hepatocellular carcinoma relative to untreated animals or animals that had received vectors with Otc as the transgene. It appears that AAV vectors alone do not contribute to the formation of tumors in these strains of mice although the expression of LacZ alone or in combination with vector may be problematic.

Novel cystogenic role of basic fibroblast growth factor in developing rodent kidneys

Basic fibroblast growth factor (bFGF) is a heparin-binding growth factor that is accumulated in human dysplastic and cystic renal diseases. Previous studies have shown that bFGF can modulate the growth of developing renal tubules; however, its role in the pathogenesis of renal cyst formation is not clearly understood. Here, we tested the hypothesis that overexpression of bFGF in developing rodent kidneys induces cyst formation in vivo. We used two different adenoviral-mediated gene-transferring approaches to overexpress bFGF in developing rodent kidneys. Initially, metanephric kidney (MK) explants harvested from embryonic day 15 Sprague-Dawley rats were infected with adenoviral vectors (rAd) encoding human bFGF or LacZ genes and transplanted under the renal capsule of adult female rats. Subsequently, to determine whether bFGF could induce renal cysts in developing kidneys with an intact renal collecting system, we injected rAd-bFGF or LacZ vectors in the retroorbital plexus of newborn mice. Basic FGF induced a more efficient integration of the MK explants into the host kidneys and increased the vascularization and proliferation of developing tubules, leading to tubular dilatation and rapid formation of renal cysts. In addition, we successfully expressed human bFGF in the kidney of newborn mice in vivo and induced tubular dilatation and renal cysts. In contrast, mice injected with rAd-lacZ did not develop tubular dilatation or renal cysts. To the best of our knowledge, these experiments show for the first time that overexpression of bFGF in developing rodent kidneys can induce the formation of renal cysts in vivo.

ALADIN I482S causes selective failure of nuclear protein import and hypersensitivity to oxidative stress in triple A syndrome

Triple A syndrome is an autosomal recessive neuroendocrinological disease caused by mutations in a gene that encodes 546 amino acid residues. The encoded protein is the nucleoporin ALADIN, a component of nuclear pore complex (NPC). We identified a mutant ALADIN(I482S) that fails to target NPC and investigated the consequences of mistargeting using cultured fibroblasts (I482Sf) from a patient with triple A syndrome. ALADIN(I482S) affected a karyopherin-alpha/beta-mediated import pathway and decreased nuclear accumulations of aprataxin (APTX), a repair protein for DNA single-strand breaks (SSBs), and of DNA ligase I in I482Sf. This decrease was restored by wild-type ALADIN. ALADIN(I482S) had no effect on imports of M9/kap-beta2, BIB/kap-beta3, histone H1/importin 7, the ubiquitin conjugating enzyme UbcM2/importin 11, or the spliceosome protein U1A, indicating that ALADIN(I482S) selectively impaired transport of discrete import complexes through NPC. Cell survival assay showed hypersensitivity of I482Sf to l-buthionine-(S,R)-sulfoximine (BSO), a glutathione-depleting agent. BSO decreased nuclear APTX and ligase I levels in I482Sf and normal control fibroblasts, but increased SSBs only in I482Sf. These observations implied that I482Sf are hypersensitive to BSO and no longer sufficiently repair SSBs. Consistent with this notion, I482Sf transfected with both APTX and ligase I had increased resistance to BSO, whereas I482Sf transfected with LacZ vector remained hypersensitive to BSO. We propose that oxidative stress aggravates nuclear import failure, which is already compromised in patient cells. Consequent DNA damage, beyond the limited capacity of DNA repair proteins, i.e., APTX and ligase I, may participate in triggering cell death.

412. A Novel Friedreich's Ataxia Model and In Vivo Gene Rescue Using HSV-1 Amplicon Vectors in Transgenic Mice

There is currently no effective cure or treatment for Friedreich's ataxia (FA), the most common of the hereditary ataxias. The disease is caused by mutations in FRDA, the frataxin gene, which result in drastically reduced expression levels. In whole animal models, one of the key difficulties in mimicking the human disease is obtaining the precise levels of frataxin expression with the appropriate tissue distribution to provoke pathology but not result in lethality. In order to develop strategies to circumvent these problems, conditional (floxed) frataxin transgenic mice have been generated by the laboratory of Helene Puccio and Michel Koenig (Strassbourg). We have generated CRE-expressing HSV-1 amplicon vectors and present results showing loss of frataxin protein levels in neurons from these loxP[frda] mice infected by these vectors. Using stereotactic injection, we have also achieved in vivo delivery of these CRE-expressing vectors into the olivocerebellar circuit of loxP[frda] mice to generate a localised gene knockout model. These mice develop a behavioural deficit in the rotarod assay detectable after 4 weeks, and with a stable difference with respect to lacZ vector-injected control mice up to the latest time point measured (16 weeks). We have also generated viral vectors expressing human frataxin cDNA and present results showing correction of this ataxic phenotype in our novel FA model. When frataxin-expressing vectors were injected into mice rendered ataxic 4 weeks previously, behavioural recovery was detectable as soon as 4 weeks after the second injection and with a stable difference up to 12 weeks with respect to mice re-injected with lacZ vector. Our novel FA mouse model and our successful in vivo gene rescue strategy will serve to rapidly and specifically address the neurological aspects of this neurodegenerative disease, both in understanding the pathogenic mechanisms as well as developing therapies.

740. A Novel Hybrid System Efficiently Expands AAV Packaging Capacity

The small packaging capacity is a long-standing hurdle limiting broad application of adeno-associated virus (AAV) in gene therapy. The trans-splicing and overlapping methods were developed recently to overcome this barrier. However, the trans-splicing method is highly dependent on the existence of an optimal gene splitting site and the overlapping method only works in genes that carry a highly recombinogenic domain in the middle of the gene. To overcome these limitations, we developed a novel trans-splicing/overlapping hybrid vector system that can efficiently reconstitute any large gene. In the hybrid system, we engineered a 0.8kb highly recombinogenic alkaline phosphatase (AP) gene fragment at the end of the splice donor and beginning of the splice acceptor sequences in the trans-splicing vectors. We hypothesize that the addition of the AP gene fragment will facilitate homologous recombination in the trans-splicing vectors and increase the chance of transgene reconstitution. To test this hypothesis, we compared transduction efficiency of the intact gene, trans-splicing, overlapping and hybrid LacZ vectors, all delivered in AAV-6 capsids. In MO59K cells at a moi of 10,000 expression cassettes/cell, |[beta]|-galactosidase activity in the intact gene infected cells was 1086 |[plusmn]| 88 unit/mg protein at 45 hrs post-infection. Activity from overlapping and trans-splicing vectors reached only 20% (238 |[plusmn]| 21 unit/mg protein) and 60% (654 |[plusmn]| 40 unit/mg protein) of that from the intact gene vector, respectively. Surprisingly, activity in hybrid vector infected cells (1137 |[plusmn]| 82 unit/ mg protein) was identical to that of intact LacZ infected cells. To determine the molecular pathways of the transgene reconstitution in hybrid vectors, we performed Southern blot. Consistent with our initial hypothesis, vector genomes formed both ITR-mediated (trans- splicing pathway) and AP-mediated (overlapping pathway) concatamers in hybrid vector co-infected cells. We next examined in vivo efficiency of the hybrid vectors in the tibialis anterior muscles of 6-week-old C57Bl10 mice (10e10 particles of each vector). LacZ expression was determined six weeks post-infection. Amazingly, exactly the same expression profile as we saw in MO59K cells was reproduced in muscle. The levels of |[beta]|-galactosidase activity were 1948 |[plusmn]| 137, 1288 |[plusmn]| 40, 556 |[plusmn]| 76, and 1674 |[plusmn]| 86 unit/mg protein for the intact gene, trans-splicing, overlapping and hybrid vector infected muscles, respectively. In summary, we have developed a generic dual vector approach that can be used to efficiently express any large therapeutic gene.

Comparative analysis of antitumor activity of CD40L, RANKL, and 4‐1BBL in vivo following intratumoral administration of viral vectors or transduced dendritic cells

The tumor necrosis factor (TNF) family comprises a group of ligands that regulate cell proliferation, differentiation, activation, maturation and apoptosis through interaction with the corresponding TNF receptor family members. In this study, we have evaluated whether adenovirus-mediated intratumoral gene transfer of CD40L, RANKL, or 4-1BBL elicits an immune response to established murine MC38 and TS/A tumors. Intratumoral administration of the recombinant adenoviral vectors expressing CD40L, RANKL or 4-1BBL 7 days post-tumor cell inoculation resulted in significant inhibition of MC38 tumor growth for all three ligands when compared with control groups treated with either saline or control adenovirus. However, intratumoral injection of Ad-4-1BBL or Ad-CD40L resulted in a significantly stronger inhibition of TS/A tumor progression than did Ad-RANKL treatment. We also demonstrated that intratumoral administration of dendritic cells (DC) transduced with adenoviral vectors encoding the TNF-related ligands resulted in a significant inhibition of MC38 tumor growth as compared with control groups treated with Ad-LacZ-transduced DC or saline-treated DC. In addition, DC overexpressing CD40L secreted considerably more IL-12 and expressed higher levels of the co-stimulatory molecules, CD80, CD86 and CD40, than did DC overexpressing LacZ, 4-1BBL or RANKL. We have also demonstrated that DC/CD40L, DC/4-1BBL, and DC/RANKL survived significantly longer than control DC or DC infected with the LacZ vector. Taken together, these results demonstrate that adenoviral gene transfer of CD40L, RANKL or 4-1BBL elicit a significant antitumor effect in two different tumor models, with CD40L gene transfer inducing the strongest antitumor effect.