Successful Gene Transfer Research Articles

APOE ε3 Gene Transfer Attenuates Brain Damage after Experimental Stroke

Apolipoprotein E (apoE, protein; APOE, gene) is the major lipid-transport protein in the brain and plays an important role in modulating the outcome and regenerative processes after acute brain injury. The aim of the present study was to determine if gene transfer of the epsilon3 form of APOE improves outcome in a murine model of transient focal cerebral ischaemia. Mice received an intrastriatal injection of vehicle, a second-generation adenoviral vector containing the green fluorescent protein gene (Ad-GFP) or a vector containing the APOE epsilon3 gene (Ad-APOE) 3 days before 60 mins focal ischaemia. Green fluorescent protein expression was observed in cells throughout the striatum and subcortical white matter indicating successful gene transfer and expression. ApoE levels in the brain were significantly increased after Ad-APOE compared with Ad-GFP or vehicle treatment. Ad-APOE treatment reduced the volume of ischaemic damage by 50% compared with Ad-GFP or vehicle treatment (13+/-3 versus 29+/-4 versus 27+/-5 mm(3)). The extent of postischaemic apoE immunoreactivity was enhanced in Ad-APOE compared with Ad-GFP or vehicle treated mice. These results show the ability of APOE gene transfer to markedly improve outcome after cerebral ischaemia and suggest that modulating apoE levels may be a potential strategy in human stroke therapy.

Stem Cell Collection and Gene Transfer in Fanconi Anemia

Fanconi anemia (FA) is a rare genetic syndrome characterized by progressive bone marrow failure (BMF), congenital anomalies, and a predisposition to malignancy. Successful gene transfer into hematopoietic stem cells (HSCs) could reverse BMF in this disease. We developed clinical trials to determine whether a sufficient number of CD34(+) stem cells could be collected for gene modification and to evaluate the safety and efficacy of HSC-corrective gene transfer in FA genotype A (FANCA) patients. Here, we report that FA patients have significant depletion of their BM CD34(+) cell compartment even before severe pancytopenia is present. However, oncoretroviral-mediated ex vivo gene transfer was efficient in clinical scale in FA-A cells, leading to reversal of the cellular phenotype in a significant percentage of CD34(+) cells. Re-infusion of gene-corrected products in two patients was safe and well tolerated and accompanied by transient improvements in hemoglobin and platelet counts. Gene correction was transient, likely owing to the low dose of gene-corrected cells infused. Our early experience shows that stem cell collection is well tolerated in FA patients and suggests that collection be considered as early as possible in patients who are potential candidates for future gene transfer trials.

Direct AAV-mediated gene delivery to the temporomandibular joint

Successful intra-temporomandibular joint gene transfer is a prerequisite for gene therapy to induce mandibular condylar growth. This study was carried out to investigate the expression of recombinant adeno-associated virus (rAAV) based vector-mediated enhanced green fluorescence protein (eGFP) in the mandibular condyle. The transduction efficiencies for primary chondrocytes using different hybrids of rAAV vectors were identified by fluorescence microscopy and FACS. Sprague-Dawley rats were injected with either rAAV(2/2)-eGFP construct or saline into both mandibular condyles. The spatial and temporal transgene expression was detected by in situ hybridization, RT-PCR and real-time PCR. Our result showed that rAAVs were capable of infecting rat chondrocytes. rAAV(2/2) was the best serotype to infect chondrocytes in vitro. By in situ hybridization, eGFP expression was clearly detected in the deeper layer of mandibular condyle as early as 7 days after injection. By real-time PCR, the transgene expression reached a peak at 21 days. This study was the first report to explore that rAAV-mediated genes could be transferred to mandibular condyle in vivo. Our results strongly suggest that rAAV(2/2) mediated gene delivery is a promising approach to deliver candidate genes for future regulating mandibular condylar growth.

Effects of ex vivo transduction of mesencephalic reaggregates with bcl-2 on grafted dopamine neuron survival

Survival rates of dopamine (DA) neurons grafted to the denervated striatum are extremely poor (5–20%). Gene transfer of survival promoting factors, such as the anti-apoptotic protein bcl-2, to mesencephalic DA neurons prior to transplantation (ex vivo transduction) offers a novel approach to increase graft survival. However, specific criteria to assess the efficacy of various vectors must be adhered to in order to reasonably predict successful gene transfer with appropriate timing and levels of protein expression. Cell culture results utilizing three different herpes simplex virus (HSV) vectors to deliver the reporter β-galactosidase gene (lacZ) indicate that transduction of mesencephalic cells with a helper virus-free HSV amplicon (HF HSV-TH9lac) that harbors the 9-kb tyrosine hydroxylase (TH) promoter to drive lacZ gene expression elicits the transduction of the highest percentage (≈50%) of TH-immunoreactive (THir) neurons without significant cytotoxic effects. This transduction efficiency and limited cytotoxicity was superior to that observed following transduction with helper virus-containing HSV (HC HSVlac) and helper virus-free HSV amplicons (HF HSVlac) expressing lacZ under the transcriptional control of the HSV immediate-early 4/5 gene promoter. Subsequently, we assessed the ability of HSV-TH9lac and the bcl-2 expressing HSV-TH9bcl-2 amplicon to transduce mesencephalic reaggregates. Although an increase in bcl-2 and β-galactosidase protein was induced by transduction, amplicon-mediated overexpression of bcl-2 did not lead to an increase in grafted THir neuron number. Even with highly efficient viral vector-mediated transduction, our results demonstrate that ex vivo gene transfer of bcl-2 to mesencephalic reaggregates is ineffective in increasing grafted DA neuron survival.

AAV-mediated gene transfer for retinal diseases

Vectors based on the adeno-associated virus (rAAV) are able to transduce the retina of animal models, including non-human primates, for a long-term period, safely and at sustained levels. The ability of the various rAAV serotypes to transduce retinal target cells has been exploited to successfully transfer genes to photoreceptors, retinal pigment epithelium and the inner retina, which are affected in many inherited and non-inherited blinding diseases. rAAV-mediated, constitutive and regulated gene expression at therapeutic levels has been achieved in the retina of animal models, thus providing proof-of-principle of gene therapy efficacy and safety in models of dominant and recessive retinal disorders. In addition, gene transfer of molecules with either neurotrophic or antiangiogenic properties provides useful alternatives to the classic gene replacement for treatment of both mendelian and complex traits affecting the retina. Years of successful rAAV-mediated gene transfer to the retina have resulted in restoration of vision in dogs affected with congenital blindness. This has paved the way to the first attempts at treating inherited retinal diseases in humans with rAAV. Although the results of rAAV clinical trials for non-retinal diseases give a warning that the outcome of viral-mediated gene transfer in humans may be different from that predicted based on results in other species, the immune privilege of the retina combined with the versatility of rAAV serotypes may ultimately provide the first successful treatment of human inherited diseases using rAAV.

Polyclonality of Retroviral Insertion Sites Following Successful Retroviral-Mediated Gene Transfer of CD18 in Canine Leukocyte Adhesion Deficiency.

Canine leukocyte adhesion deficiency (CLAD) represents a large-animal model for the human disease leukocyte adhesion deficiency, type 1 (LAD-1). Both CLAD and LAD are characterized by life-threatening bacterial infections and are due to mutations in the leukocyte integrin CD18. We recently reported successful gene therapy in six dogs with CLAD following infusion of autologous CD34+ bone marrow hematopoietic stem cells transduced with the retroviral vector PG13/MSCV-cCD18. The level of CD18+ gene corrected leukocytes ranged from 1.3% to 8.4% at one year post-transplant in the six CLAD dogs. We continue to monitor four of the six dogs for long-term follow-up. To date no gene transfer-related adverse events have been observed in any of the dogs. However, verification of polyclonality of insertion sites in the gene-corrected cells and characterization of the genomic sites of retroviral integration remain critical safety issues. To this end, we have demonstrated the polyclonality of contributing retrovirus-transduced cells in all six dogs using linear-amplification mediated PCR (LAM-PCR) with DNA obtained from peripheral blood leukocytes up to 2 years following infusion of gene-corrected cells. Multiple clones were shown to contribute to hematopoiesis; no predominant clones emerged over time. Retroviral insertion sites in the six dogs were identified using ligation-mediated PCR (LM-PCR) followed by sequencing of the genomic sequence adjacent to the integrated viral 3′ LTR. Mapping of insertion sites was facilitated by the recent completion of the annotated dog genome sequence. To date, we have identified 341 unique retroviral insertion sites. Analysis of these insertion sites indicated that 46% of the sites are located within gene-coding regions of the genome (introns or exons). Another 35% of the sites were located within 50 kb of the transcription start site of a gene-coding sequence. These observations match previous reports of preferential integration of MLV retroviral vectors near coding sequences. The insertion sites were well-distributed across all chromosomal locations, with no evidence for insertional hotspots or preferential growth of clones containing common insertion sites: only six pairs of insertion sites were located within 5 kb of each other. Further analysis was performed on a sub-group of 294 genes that had insertion sites located either in them or within 50 kb of the transcription start site. 19 of these genes (6.5%) were present in either or both of two cancer gene databases (Sanger Institute Cancer Gene Census Table, Mouse Retrovirus Tagged Cancer Gene Database). Functional annotation of the 294 genes revealed some groups of interest: 17% of the genes were involved in phosphorylation, 4% were proto-oncogenes, 12% encoded zinc-finger proteins, 23% were involved in signal transduction, 2% encoded transforming proteins, 5% were involved in cell proliferation, 8% were involved in transcriptional regulation and 4% encoded protein kinases. We are continuing to sequence and characterize a large number of retroviral insertion sites from the gene-corrected CLAD dogs. Analysis of these insertion sites should provide considerable insight into the risk of genotoxicity through insertional mutagenesis in a disease-specific large-animal model of a human hematopoietic disease.

Spinal gene transfer using ultrasound and microbubbles

Spinal gene therapy is a promising option for treating various spinal-related disorders. Several previous studies using viral vectors reported successful transfer of therapeutic genes into the spinal nerve system. However, because of the considerable immunogenicity related to the use of viruses, non-viral gene transfer still needs to be developed. One possible approach is the combined use of ultrasound and echo-contrast microbubbles. The present study shows that this method can be applied for targeted intrathecal gene delivery. We intrathecally injected a mixture of plasmid-DNA encoded with luciferase and commercially available albumin microbubbles by needle puncture at the lower lumbar intervertebral space in mice. Subsequent percutaneous ultrasonication on the lumbar vertebrae significantly enhanced the luciferase expression, analyzed by imaging luciferin bioluminescence, in the dorsal meningeal cells at the insonated region. No apparent neurological damages were induced by the present spinal interventions. In addition to the general benefits of the combined use of ultrasound and microbubbles, our approach can offer some advantages specific to spinal gene transfection including minimal invasiveness of simple percutaneous dural puncture, targetability due to the limited access of ultrasound waves through anatomical apertures of the vertebrae, and possible paracrine delivery of therapeutic molecules to the spinal nerve system.

Nucleocytoplasmic Transport of Plasmid DNA: A Perilous Journey from the Cytoplasm to the Nucleus

Nonviral vectors represent a promising approach for the safe delivery of therapeutic DNA in genetic and acquired human diseases. Before synthetic vector systems can be used for clinical applications, their limited efficacy must be addressed. At the cellular level, successful gene transfer is dependent on several additional factors including DNA uptake, release from the DNA-vector complex, and nucleocytoplasmic transport. This paper reviews the major metabolic and physical impediments that plasmid DNA vectorized by synthetic vectors encounters between the cytosol and the nucleus. Plasmid DNA that escapes the endolysosomal compartment encounters the diffusional and metabolic barriers of the cytoplasm, reducing the number of intact plasmids that reach the nuclear envelope. Nuclear translocation of DNA requires either the disassembly of the nuclear envelope during cell division or active nuclear transport via the nuclear pore complex. In the nucleus, plasmid DNA is relatively stable, but its transcription and its fate during cell division are still debated. A better understanding of the cellular and molecular basis of nonviral gene transfer during nucleocytoplasmic trafficking may provide strategies to overcome those obstacles that limit the efficiency of nonviral gene delivery. We review some of the current methods of gene transfer mediated by synthetic vectors, highlighting systems that exploit our actual knowledge of the nucleocytoplasmic transport of plasmid DNA.

A perfusion protocol for highly efficient transduction of intact pancreatic islets of Langerhans

Successful gene transfer to pancreatic islets might be a powerful tool for dissecting the biological pathways involved in the functional impairment and destruction of beta cells in type 1 diabetes. In the long run, such an approach may also prove useful for promoting islet graft survival after transplantation in diabetic patients. However, efficient genetic modification of primary insulin-producing cells is limited by the specific compact structure of the pancreatic islet. We present here a whole-pancreas perfusion-based transduction procedure for genetic modification of intact pancreatic islets. We used flow cytometry analysis and confocal microscopy to evaluate the efficiency of in vitro and perfusion-based transduction protocols that use adenoviral and lentiviral vectors expressing green fluorescent protein. Islet cell viability was assessed by fluorescence microscopy and beta cell function was determined via glucose-stimulated insulin secretion. In intact rat and human pancreatic islets, adenoviral and lentiviral vectors mediated gene transfer to about 30% of cells, but they did not reach the inner cellular mass within the islet core. Using the whole-pancreas perfusion protocol, we demonstrate that at least in rodent models the centrally located insulin-producing cells can be transduced with high efficiency, while preserving the structural integrity of the islet. Moreover, islet cell viability and function are not impaired by this procedure. These results support the view that perfusion-based transduction protocols may significantly improve the yield of successfully engineered primary insulin-producing cells for diabetes research.

Naked plasmid DNA transfer to the porcine liver using rapid injection with large volume

The naked plasmid DNA transfer method of rapid injection with large volume has been useful for gene therapy in experimental study. However, only small animals like rodents have usually been reported on. In this study, the authors attempted to transfect naked plasmid DNA to the porcine liver by modified hydrodynamic method. We decided to transfer plasmid DNA to a part of the liver using the angio-catheter to reduce the liver damage. To discern the condition of injection, naked plasmid DNA-encoding green fluorescent protein (GFP) was transferred for use as a marker gene. The GFP gene expression was markedly observed in gene-transferred pig livers. In large animals, not only the naked gene quantity, the solution volume containing the plasmid DNA and the injection speed, but also the additional treatments of the portal vein and the hepatic artery preparation were crucial. We found that the following injection condition were needed: plasmid DNA, 3 mg; the solution volume, 150 ml and the injection speed, 5 ml/s. The portal vein and the hepatic artery were clamped during gene delivery and the blood flow of the portal vein was flushed out using normal saline. Cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4-Ig) gene was used to test for secretory protein. CTLA4-Ig gene was injected with a large volume of solution via the hepatic vein to the left outer lobe of the liver selectively. CTLA4-Ig was detected in the pig blood at a maximum serum level of 161.7 ng/ml 1 day after gene transfer, and the CTLA4-Ig was detected for several weeks. Our new technique of inserting a catheter into only a selected portion of the liver reduced liver toxicity and increased gene transfer efficiency. This is the first report of successful gene transfer, using a hydrodynamic method, to the segmental liver in pigs, and achieved more than enough secretory protein for the clinically therapeutic level in pigs.

High volume hydrodynamic injection of plasmid DNA via the hepatic artery results in a high level of gene expression in rat hepatocellular carcinoma induced by diethylnitrosamine

Hydrodynamic injection of naked plasmid DNA (pDNA) via the tail vein is a safe and effective method of gene transfer to the liver. However, successful gene transfer has yet to be shown for hepatocellular carcinoma (HCC); therefore, we investigated the feasibility and efficacy of hydrodynamic injection via the tail vein and hepatic artery in a diethylnitrosamine (DEN)-induced HCC model in rats. HCC was induced in Sprague-Dawley rats by 100 ppm DEN in drinking water. pCMV-SPORT-beta-galactosidase (beta-gal, 400 microg) was injected (i) via the tail vein in a volume of 0.1 ml/g in 30 s or (ii) via the hepatic artery in a volume of 5 or 10 ml at 1 ml/s, either with or without temporary occlusion of the inferior vena cava (IVC) and portal vein (PV). The liver was harvested 24 h after administration, and beta-gal expression was evaluated with X-gal staining and measurement of enzymatic activity in tissue homogenates. Hydrodynamic injection via the tail vein achieved transgene expression only in non-cancerous tissue (tumor: 0.16 +/- 0.04%, non-tumor: 5.07 +/- 1.66%). Hydrodynamic injection via the hepatic artery was tolerated, but failed to produce efficient transgene expression in tumor and non-tumor cells. On the other hand, concomitant use of temporary IVC/PV occlusion with hydrodynamic injection via the hepatic artery dramatically increased transgene expression in cancer cells, but tumor-selective gene transfer was not achieved with this procedure (tumor: 7.38 +/- 3.66%, non-tumor: 7.77 +/- 1.06%). High-volume hydrodynamic injection of a pDNA solution via the hepatic artery with IVC/PV occlusion achieved a high level of gene expression in a HCC rat model. This gene transfer technique may have potential in clinical gene therapy for HCC.

Sustained Transgene Expression in Intervertebral Disc Cells In Vivo Mediated by Microbubble-Enhanced Ultrasound Gene Therapy

In vivo studies using a rat model were performed to determine the feasibility of microbubble-enhanced ultrasound gene transfer technique to the intervertebral disc. 1) To establish this microbubble-enhanced ultrasound gene therapy technique for intervertebral disc cells in vivo without using viral vectors and 2) to estimate the duration of transgene expression in vivo. Intervertebral disc degeneration and associated spinal disorders remain a formidable problem. Although gene therapy approaches have been reported as having promising therapeutic potential to regenerate the disc, concerns over safety issues using recombinant viral vectors limits its application. Successful gene transfer using ultrasound has been reported in muscle and cardiovascular systems in vivo. Two different reporter plasmid DNA encoding green fluorescent protein (GFP) and firefly luciferase were used. Plasmid DNA was mixed with ultrasonography contrast agent (microbubbles) and injected into coccygeal intervertebral discs of Sprague-Dawley rats. The therapeutic ultrasound was irradiated on the surface of injected discs. Rats were killed 1, 3, 6, 12, and 24 weeks after gene transduction. Harvested nucleus pulposus tissues were used for evaluation of transgene expression. The intact discs were used as a control. Seven days after gene transfection, considerable numbers of GFP-positive cells were observed in nucleus pulposus from the GFP-transfected group. Luciferase assay revealed that the ultrasound group demonstrated approximately an 11-fold increase in luciferase activity over the plasmid DNA-only group. Furthermore, transgene expression mediated by this method was observed, at least up to 24 weeks. Our study indicated that ultrasound transfection method with microbubbles significantly enhanced transfection efficiency of plasmid DNA into the nucleus pulposus cells in vivo. Furthermore, the sustained transgene expression in vivo was possible up to 24 weeks. The long-term gene expression mediated by simple and safe procedure has important clinical applications, including the treatment of chronic types of disease such as degenerative disc diseases.

Gene therapy with the interleukin-1 receptor antagonist for the treatment of arthritis

Local gene therapy is a promising approach for the treatment of arthritis. The first clinical trial of this strategy involved nine postmenopausal women requiring unilateral sialistic implant arthroplasty of metacarpophalangeal (MCP) joints 2–5 for the treatment of rheumatoid arthritis (RA). Autologous synoviocytes were recovered and transduced with a retrovirus, MFG-IRAP, which carries human interleukin-1 receptor antagonist (IL-1Ra) cDNA. In a dose-escalation, double-blind fashion, two MCP joints on each subject’s hand were injected with genetically modified cells, and the other two MCPs were injected with unmodified cells. After 1 week, the injected joints underwent replacement arthroplasty. Examination of retrieved synovia confirmed successful gene transfer and intra-articular transgene expression. A subsequent, similar study involving two subjects with RA provided clear evidence of a marked clinical response to the gene therapy. No adverse events occurred during either study. These highly promising fi...

Collateral artery enlargement is impaired in eNOS−/− mice due to a greater increase in oxidative stress in eNOS−/− than in wild‐type mice after induction of hindlimb ischemia and can not be reversed by eNOS gene transfer

Nitric oxide (NO) derived from endothelial nitric oxide synthase (eNOS) is critical to vascular homeostasis. Oxidative stress increases the production of reactive oxygen species (ROS), causing local tissue damage and depleting tetrahydrobiopterin (BH4). Thus, eNOS activity and the resultant NO bioavailability depends on the redox state of the surrounding tissue. Using wild-type C57Bl/6 (WT) and eNOS−/− mice, we surgically induced hindlimb ischemia and attempted to increase NO bioavailability by adenoviral mediated eNOS gene transfer. AdeNOS injection significantly increased collateral artery enlargement in wild-type, but not eNOS−/− mice, in spite of successful eNOS gene transfer. We hypothesized that this failure to reverse the eNOS−/− phenotype was due to an increase in oxidative stress in these mice that inhibited normal eNOS activity. Thigh muscle protein extracts from either eNOS−/− or WT mice were added to positive controls in a NOS activity assay. Ischemic eNOS−/−, but not WT tissue significantly inhibited the NOS activity of these reactions. In addition, the Amplex Red assay for hydrogen peroxide in ischemic thigh muscle showed a greater increase in eNOS−/− mice than WT controls. These results show a greater increase in oxidative stress in eNOS−/− mice after induction of muscle ischemia that cannot be reversed by eNOS gene transfer, suggesting that endothelial dysfunction plays a critical role in collateral artery enlargement. RO1HL75353-01.

Improvement of pulmonary hypoplasia associated with congenital diaphragmatic hernia by in utero CFTR gene therapy

Congenital diaphragmatic hernia (CDH) may be an ideal candidate disease for in utero gene therapy as disrupted fetal lung growth plays a significant role in disease outcome. We previously demonstrated that transient in utero overexpression of CFTR during fetal development resulted in lung epithelial proliferation and differentiation. We hypothesized that gene therapy with CFTR would improve the pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH). CDH was induced by the herbicide 2,4-dichlorophenyl-4-nitrophyl ether (nitrofen) following maternal ingestion at either 10 or 13 days gestation. In utero gene transfer of the CFTR gene was subsequently performed at 16 days gestation. Examination of the fetuses at 22 days gestation revealed little improvement in the CFTR-treated lungs following induction of hernias with nitrofen at 10 days gestation. However, the CFTR gene treatment significantly improved internal surface area, saccular density, overall saccular number, and amount of saccular air space in the lungs that were treated with nitrofen at 13 days gestation. RT-PCR demonstrated that gene transfer occurred following treatment at 13 days gestation but not in the lungs treated with nitrofen at 10 days gestation, despite gene transfer at the same gestational age (16 days) in both groups. As disruption of lung development correlates with the gestational stage at which nitrofen exposure occurs, these results confirmed previous findings that in utero gene transfer efficiency depends on the stage of lung development. Lung development may be significantly delayed in human CDH to allow for successful gene transfer later in gestation, providing a substantial therapeutic window.

Recombinant polymer‐protein fusion: a promising approach towards efficient and targeted gene delivery

Synthetic vectors such as polymers have the potential to reduce the safety problems associated with viral vectors; however, their low transfection efficiency limits their clinical utility. To study the critical steps involved in an efficient transgene expression, there is a need for creative approaches that allow a systematic correlation between gene carrier structure and properties necessary for successful gene transfer. Using recombinant techniques a prototype vector comprised of tandem repeating units fused to a targeting moiety was biosynthesized to mediate gene transfer in mammalian cell lines. The carrier was designed to have the structure of (KHKHKHKHKK)6-FGF2 where lysine (K) residues would allow complexation with plasmid DNA, basic fibroblast growth factor (FGF2) to target cells over-expressing FGF2 receptors (FGFR), and histidine (H) residues to facilitate escape from the endosomal compartments. The gene carrier was biosynthesized in E. coli, purified using a Ni-NTA column, characterized, complexed with pDNA, and the complexes were used to transfect NIH 3T3, T-47D and COS-1 mammalian cell types known to express FGFR. Results demonstrate the successful cloning and expression of the gene carrier with over 95% purity. The molecular weight of the gene carrier was determined by MALDI-TOF to be 27 402. Amino acid content analysis and Western blot confirmed the expression of the gene carrier in E. coli. The vector was able to condense pDNA, induce cell proliferation in NIH 3T3 fibroblasts, and mediate transgene expression in NIH 3T3, T-47D and COS-1 mammalian cell types. Genetic engineering techniques show promise for systematic investigation of structure-activity relationships of non-viral gene delivery vectors.

Transfer Gen Penanda Pada Kentang (Solanum tuberosum L.) CV Panda dan Atlantik dengan Bantuan Agrobacterium

This research was carried out to evaluate the efficiency of marker gene transfer of potato ( Solanum tuberosum L.) explant tissues, which were co-cultivated with Agrobacterium tumefaciens strain LBA 4404 carrying binary vector pBI121. Nodes and leaf segments from shoot cultures of Panda and Atlantic cultivars were used as explants. The marker gene transfer procedure was initiated with explant pre-conditioning, followed with co-cultivating and selecting of putative transformant cells. Explants were pre-conditioned over-night in callus induction medium containing basal medium of MS (Murashige & Skoog, 1962) supplemented with 5 mg/l naphthalene acetic acid (NAA) and 0.1 mg/l benzyl adenine. After 10 min. inoculation in A. tumefaciens suspension, explants were co-cultivated for 1, 3, 5, 7 and 9 days. After co-cultivation, explant tissues were placed on callus induction medium added with 150 mg/l kanamycin. Successful marker gene transfer was calculated as the percentage of total explants producing callus on selection medium. Results showed that more co-cultivated nodal explants became resistant to kanamycin than co-cultivated leaf explants. The highest efficiency of transformation (45%) of nodal explants was observed in Panda cultivar whereas that in Atlantik was only 30%. The most efficient transformation of leaf explants was observed in Panda cultivar (20%) whereas that in Atlantik was15%. The PCR analysis detected the presence of NPTII gene in transgenic tissues.

978. Reduced Immune Response to Adenovirus Vector Formulated with the Anti-Inflammatory Liposome DS/DOPE

Introduction: Successful gene transfer to lung has been hindered by immune responses elicited against gene transfer vectors and transgenes as well as inefficient gene transfer to target cells. We have previously demonstrated that, when delivered intranasally, adenoviral (AdV) vectors formulated with the anti-inflammatory cationic lipid dexamethasone-spermine (DS) and the neutral lipid dioleoylphosphatidylethanolamine (DOPE) target transgene expression to airway epithelia and reduce cellular infiltration to the lung.Methods: C57Bl/6 mice were administered with two doses of AdV vector expressing either β-galactosidase (LacZ) or human α- antitrypsin (AAT), with or without DS/DOPE, at days 0 and 14 respectively. At various time points cytokine responses, T-cell responses and B-cell responses were quantified.Results: We found that the six hour acute response to AdV vector alone has higher serum levels of many inflammatory cytokines and chemokines, such as KC/GROα(6-fold increase), TNF-α(5.7-fold increase), and IL-6 (2.7-fold increase). Furthermore, the production of neutralizing antibody against AdV at day 28 was significantly reduced when at least one dose of vector was formulated with DS/ DOPE (AdV,AdV=400; AdV+DS/DOPE,AdV=60; AdV+DS/ DOPE,AdV+DS/DOPE= 213). ELISPOT assays on splenocytes, isolated from vector treated mice, on days 14, 28, and 34 suggest altered kinetics of T-cell response based on formulation of AdV with DS/DOPE, though not necessarily a reduced response to the LacZ transgene. However, the localized pulmonary response to the vectors following double administration was significantly reduced for AdV formulated with DS/DOPE compared to AdV alone as indicated by quantification of CD4 (204.7 + 107.3 vs. 478.8 + 258.9) and CD8 (305.3 + 175.3 vs. 571.8 + 238.4) immunohistochemistry.Conclusion: Taken together our results indicate that formulating AdV vector with DS/DOPE targets transgene expression to airway epithelia while reducing the immune response to AdV. Introduction: Successful gene transfer to lung has been hindered by immune responses elicited against gene transfer vectors and transgenes as well as inefficient gene transfer to target cells. We have previously demonstrated that, when delivered intranasally, adenoviral (AdV) vectors formulated with the anti-inflammatory cationic lipid dexamethasone-spermine (DS) and the neutral lipid dioleoylphosphatidylethanolamine (DOPE) target transgene expression to airway epithelia and reduce cellular infiltration to the lung. Methods: C57Bl/6 mice were administered with two doses of AdV vector expressing either β-galactosidase (LacZ) or human α- antitrypsin (AAT), with or without DS/DOPE, at days 0 and 14 respectively. At various time points cytokine responses, T-cell responses and B-cell responses were quantified. Results: We found that the six hour acute response to AdV vector alone has higher serum levels of many inflammatory cytokines and chemokines, such as KC/GROα(6-fold increase), TNF-α(5.7-fold increase), and IL-6 (2.7-fold increase). Furthermore, the production of neutralizing antibody against AdV at day 28 was significantly reduced when at least one dose of vector was formulated with DS/ DOPE (AdV,AdV=400; AdV+DS/DOPE,AdV=60; AdV+DS/ DOPE,AdV+DS/DOPE= 213). ELISPOT assays on splenocytes, isolated from vector treated mice, on days 14, 28, and 34 suggest altered kinetics of T-cell response based on formulation of AdV with DS/DOPE, though not necessarily a reduced response to the LacZ transgene. However, the localized pulmonary response to the vectors following double administration was significantly reduced for AdV formulated with DS/DOPE compared to AdV alone as indicated by quantification of CD4 (204.7 + 107.3 vs. 478.8 + 258.9) and CD8 (305.3 + 175.3 vs. 571.8 + 238.4) immunohistochemistry. Conclusion: Taken together our results indicate that formulating AdV vector with DS/DOPE targets transgene expression to airway epithelia while reducing the immune response to AdV.

Comparative Analysis of Bacterial-Origin Genes for Plant Mitochondrial Ribosomal Proteins

Mitochondrial ribosomes contain bacterial-type proteins reflecting their endosymbiotic heritage, and a subset of these genes is retained within the mitochondrion in land plants. Variation in gene location is observed, however, because migration to the nucleus is still an ongoing evolutionary process in plants. To gain insights into adaptation events related to successful gene transfer, we have compiled data for bacterial-origin mitochondrial-type ribosomal protein genes from the completely sequenced Arabidopsis and rice genomes. Approximately 75% of such nuclear-located genes encode amino-terminal extensions relative to their Escherichia coli counterparts, and of that set, only about 30% have introns at (or near) the junction in support of an exon shuffling-type recruitment of upstream expression/targeting signals. We find that genes that were transferred to the nucleus early in eukaryotic evolution have, on average, about twofold higher density of introns within the core ribosomal protein sequences than do those that moved to the nucleus more recently. About 20% of such introns are at positions identical to those in human orthologs, consistent with their ancestral presence. Plant mitochondrial-type ribosomal protein genes have dispersed chromosomal locations in the nucleus, and about 20% of them are present in multiple unlinked copies. This study provides new insights into the evolutionary history of endosymbiotic bacterial-type genes that have been transferred from the mitochondrion to the nucleus.

Long-term correction of hyperbilirubinemia in the Gunn Rat by repeated intravenous delivery of naked plasmid DNA into muscle

We evaluated nonviral gene delivery into skeletal muscle via femoral artery and great saphenous vein for correction of hyperbilirubinemia in the Gunn rat, the animal model of Crigler–Najjar syndrome type I. A single injection of pDNA expressing hUGT1A1 under the CMV promoter resulted in excretion of bilirubin glucuronides in bile and a significant decrease in serum bilirubin for at least 2 or 4 weeks, respectively. Loss of metabolic effect was associated with a decrease in recombinant protein in muscle, while pDNA and transcript were detectable 4 weeks after gene delivery. Monthly intravenous gene delivery maintained metabolic correction for at least 5 months. Fibrosis around vessels in the arterial group limited the number of successful repeat gene transfer sessions to 3. Animals expressing hUGT1A1 developed anti-hUGT1A1 antibodies and lymphocytic infiltrate in muscle. Immunosuppression abrogated antibody response, ameliorated lymphocytic inflammation, and enhanced metabolic correction but did not prevent a decrease in the amount of recombinant protein. In conclusion, repeated intravenous delivery of pDNA into muscle enables long-term correction of hyperbilirubinemia in the Gunn rat. The procedure is safe and simple, with great clinical potential. Further studies are needed to explain the mechanisms of loss and improve the stability of recombinant hUGT1A1 in muscle.