Gene transfer techniques

Despite efforts to use adeno-associated viral (AAV) vector-mediated gene therapy for treatment of pancreatic ductal adenocarcinoma (PDAC), transduction efficiency remains a limiting factor and thus improvement of AAV delivery would significantly facilitate the treatment of this malignancy. Site-directed mutagenesis of specific tyrosine (Y) residues to phenylalanine (F) on the surface of various AAV serotype capsids has been reported as a method for enhancing gene transfer efficiencies. In the present studies, we determine whether Y-to-F mutations could also enhance AAV8 gene transfer in the pancreas to facilitate gene therapy for PDAC. Three different Y-to-F mutant vectors (a single-mutant, Y733F; a double-mutant, Y447F+Y733F; and a triple-mutant, Y275F+Y447F+Y733F) and wild-type AAV8 (WT-AAV8) were administered by intraperitoneal or tail-vein routes to Kras(G12D+/-), Kras(G12D+/-)/Pten(+/-), and wild-type mice. The transduction efficiency of these vectors expressing the mCherry rep...

Hepatoma‐derived growth factor (HDGF) participates in tumourigenesis but its role in breast cancer is unclear. We set out to elucidate the expression profile and function of HDGF during breast carcinogenesis. Immunoblot and immunohistochemical studies revealed elevated HDGF expression in human breast cancer cell lines and tissues. Nuclear HDGF labelling index was positively correlated with tumour grade, stage and proliferation index, but negatively correlated with survival rate in breast cancer patients. HDGF over‐expression was associated with lymph node metastasis and represented an independent prognostic factor for tumour recurrence. Gene transfer studies were performed to elucidate the influence of cellular HDGF level on the malignant behaviour and epithelial‐mesenchymal transition (EMT) of breast cancer cells. Adenovirus‐mediated HDGF over‐expression stimulated the invasiveness and colony formation of MCF‐7 cells. Moreover, HDGF over‐expression promoted breast cancer cell EMT b...

BackgroundHepatic fibrosis represents a process of healing and scarring in response to chronic liver injury. Effective therapies are lacking. We have previously demonstrated that α‐melanocyte‐stimulating hormone (α‐MSH) gene therapy protects against thioacetamide‐induced acute liver failure in mice. Recent reports showed that collagen metabolism is a novel target of α‐MSH. Therefore, the aim of this study is to investigate whether α‐MSH gene therapy possesses anti‐hepatic fibrogenic effect in mice.MethodsLiver fibrosis was induced in mice by administering carbon tetrachloride (CCl4) continuously for 10 weeks. α‐MSH expression plasmid was delivered via electroporation after liver fibrosis had been established. Histopathology, reverse‐transcription polymerase chain reaction (RT‐PCR), immunoblotting, and gelatin zymography were used to investigate its possible mechanisms of action.Resultsα‐MSH gene therapy reversed established liver fibrosis in CCl4‐treated mice. RT‐PCR revealed that α...

Adeno-associated viral (AAV) vectors (serotype 2) efficiently transduce skeletal muscle, and have been used as gene delivery vehicles for hemophilia B and for muscular dystrophies in experimental animals and humans. Recent reports suggest that AAV vectors based on serotypes 1, 5, and 7 transduce murine skeletal muscle much more efficiently than AAV-2, with reported increases in expression ranging from 2-fold to 1000-fold. We sought to determine whether this increased efficacy could be observed in species other than mice. In immunodeficient mice we saw 10-to 20-fold higher levels of human factor IX (hF.IX) expression at a range of doses, and in hemophilic dogs we observed approximately 50-fold higher levels of expression. The increase in transgene expression was due partly to higher gene copy number and a larger number of cells transduced at each injection site. In all immunocompetent animals injected withAAV-1, inhibitory antibodies to F.IX developed, but in immunocompetent mice treated with high doses of vector, inhibitory antibodies eventually disappeared. These studies emphasize that the increased efficacy of AAV-1 vectors carries a risk of inhibitor formation, and that further studies will be required to define doses and treatment regimens that result in tolerance rather than immunity to F.IX.

Animal models of hemophilia and related diseases are important for development of novel treatments and to understand the pathophysiology of bleeding disorders in humans. Testing in animals with the equivalent human disorder provides informed estimates of doses and measures of efficacy, which aids in design of human trials. Many models of hemophilia A, hemophilia B, and von Willebrand disease have been developed from animals with spontaneous mutations (hemophilia A dogs, rats, sheep; hemophilia B dogs; and von Willebrand disease pigs and dogs), or by targeted gene disruption in mice to create hemophilia A, B, or VWD models. Animal models have been used to generate new insights into the pathophysiology of each bleeding disorder and also to perform pre-clinical assessments of standard protein replacement therapies as well as novel gene transfer technology. Both the differences between species and differences in underlying causative mutations must be considered in choosing the best animal for a specific scientific study Importance of Animal Models for Study of Human Bleeding Disorders Animal models are important for FDA-required pre-clinical assessment of safety prior to testing of novel therapeutics in humans. If testing can be done in animals with the equivalent of the human disease under study, better informed estimates of dose finding can be obtained (pharmacokinetics), and measures of efficacy can be tested (pharmacodynamics), which aid in pre-clinical assessment of the drug and design of human trials. Apart from drug development, animal models of human disease are also important for understanding the pathophysiology of those diseases.

The Pax6 gene plays an important role in eye morphogenesis throughout the animal kingdom. The Pax6 gene and its homologue could form ectopic eyes by targeted expression in Drosophila and Xenopus. Thus, this gene is a master gene for the eye morphogenesis at least in these animals. In the early development of the vertebrate eye, Pax6 is required for the instruction of multipotential progenitor cells of the neural retina (NR). Primitive retinal pigment epithelial (RPE) cells are able to switch their phenotype and differentiate into NR under exogenous intervention, including treatment with fibroblast growth factors (FGFs), and surgical removal of endogenous NR. However, the molecular basis of phenotypic switching is still controversial. Here, we show that Pax6 alone is sufficient to induce transdifferentiation of ectopic NR from RPE cells without addition of FGFs or surgical manipulation. Pax6-mediated transdifferentiation can be induced even at later stages of development. Both in vivo and in vitro studies show that the Pax6 lies downstream of FGF signaling, highlighting the central roles of Pax6 in NR transdifferentiation. Our results provide an evidence of retinogenic potential of nearly mature RPE and a cue for new therapeutic approaches to regenerate functional NR in patients with a visual loss.

MATERIALS AND METHODS Retroviral vectors. Four different retroviral vectors for expression of LNGFR as a reporter gene were generated using the fullcoding 1.5-kb Ssr I fragment of the human LNGFR cDNA." The NSV-N and NTK-N vectors were obtained by cloning the LNGFR cDNA into the unique Hind111 and Bgl I 1 sites of the NSV and NTK

The purpose of this study was to develop a clinically applicable high-efficiency percutaneous means of therapeutic gene delivery to the failing heart. Background Substantial advances in the understanding of the cellular and molecular basis of heart failure (HF) have recently fostered interest in the potential utility of gene and cell therapy as novel therapeutic approaches. However, successful clinical translation is currently limited by the lack of safe, efficient, and selective delivery systems. Methods We developed a novel percutaneous closed-loop recirculatory system that provides homogeneous myocardial delivery for gene transfer in the failing large animal heart. After 4 weeks' rapid pacing in adult sheep to induce HF, the animals were randomly allocated to receive either adenovirus expressing a pseudophosphorylated mutant (AdS16E) of phospholamban (PLN) or Ad-␤-galactosidase (AdLacZ). Results Two weeks after gene delivery, in the presence of continued pacing, left ventricular (LV) ejection fraction had significantly improved in the AdS16E-treated animals (27 Ϯ 3% to 50 Ϯ 4%; p Ͻ 0.001), whereas a further decline occurred in the AdLacZ group (34 Ϯ 4% to 27 Ϯ 3%; p Ͻ 0.05). In conjunction, AdS16E delivery resulted in significant reductions in LV filling pressures and end-diastolic diameter (both p Ͻ 0.05). In conjunction, AdS16Etreated animals showed significant improvement in the expression of PLN and Ca 2ϩ-adenosine triphosphatase activity. In separate animals, recirculating AdLacZ delivery was shown to achieve superior myocardial gene expression in contrast to intracoronary delivery and was associated with lower systemic expression. Conclusions We report the development of a novel closed-loop system for cardiac gene therapy. Using this approach delivery of AdS16E reversed HF progression in a large animal HF model.

BACKGROUND Current techniques for delivery of gene therapy, deliver the vector to the target organ and also to the systemic circulation. Targeted gene therapy aims at delivering the vector to specific and restricted cell populations, thus sparing all other cells of the 51 PROCEEDINGS INAUGURAL PERFUSION DOWNUNDER

Polyethylenimine (PEI) based polymers are efficient agents for cell transfection. However, their use has been hampered due to high cell death associated with transfection thereby resulting in low efficiency of gene delivery within the cells. To circumvent the problem of cellular toxicity, metal binding peptides were linked to PEI. Eight peptide-PEI derivatives were synthesized to improve cell survival and transfection efficiency. TAT linked PEI was used as a control polymer. Peptides linked with PEI amines formed nano gels as shown by electron microscopy and atomic force microscopic measurements. Polymers were characterized by spectroscopic methods and their ability to form complexes with plasmids was tested using electrophoretic studies. These modifications improved polymer biocompatibility as well as cell survival markedly when compared to PEI alone. A subset of the modified peptide-polymers also showed significantly higher transfection efficiency in primary human cells with respect to the widely used transfection agent, lipofectamine. Study of the underlying mechanism of the observed phenomena revealed lower levels of 'reactive oxygen species' (ROS) in presence of the peptide-polymers when compared to PEI alone. This was further corroborated with global gene expression analysis which showed upregulation of multiple genes and pathways involved in regulating intracellular oxidative stress.

Liver gene transfer with adeno-associated viral (AAV) 2/8 vectors is being considered for therapy of systemic diseases like mucopolysaccharidosis type VI (MPS VI), a lysosomal storage disease due to deficiency of arylsulfatase B (ARSB). We have previously reported that liver gene transfer with AAV2/8 results in sustained yet variable expression of ARSB. We hypothesized that the variability we observed could be due to pre-existing immunity to wild-type AAV8. To test this, we compared the levels of AAV2/8-mediated transduction in MPS VI cats with and without pre-existing immunity to AAV8. In addition, since levels of lysosomal enzymes as low as 5% of normal are expected to be therapeutic, we evaluated the impact of pre-existing immunity on MPS VI phenotypic rescue. AAV2/8 administration to MPS VI cats without pre-existing neutralizing antibodies to AAV8 resulted in consistent and dose-dependent expression of ARSB, urinary glycosaminoglycan (GAG) reduction, and femur length amelioration. Conversely, animals with pre-existing immunity to AAV8 showed low levels of ARSB expression and limited phenotypic improvement. Our data support the use of AAV2/8-mediated gene transfer for MPS VI and other systemic diseases, and highlight that pre-existing immunity to AAV8 should be considered in determining subject eligibility for therapy.

Liver gene transfer with adeno-associated viral (AAV) 2/8 vectors is being considered for therapy of systemic diseases like mucopolysaccharidosis type VI (MPS VI), a lysosomal storage disease due to deficiency of arylsulfatase B (ARSB). We have previously reported that liver gene transfer with AAV2/8 results in sustained yet variable expression of ARSB. We hypothesized that the variability we observed could be due to pre-existing immunity to wild-type AAV8. To test this, we compared the levels of AAV2/8-mediated transduction in MPS VI cats with and without pre-existing immunity to AAV8. In addition, since levels of lysosomal enzymes as low as 5% of normal are expected to be therapeutic, we evaluated the impact of pre-existing immunity on MPS VI phenotypic rescue. AAV2/8 administration to MPS VI cats without pre-existing neutralizing antibodies to AAV8 resulted in consistent and dose-dependent expression of ARSB, urinary glycosaminoglycan (GAG) reduction, and femur length amelioration. Conversely, animals with pre-existing immunity to AAV8 showed low levels of ARSB expression and limited phenotypic improvement. Our data support the use of AAV2/8-mediated gene transfer for MPS VI and other systemic diseases, and highlight that pre-existing immunity to AAV8 should be considered in determining subject eligibility for therapy.

The Fifth Bioengineering and Imaging Research Opportunities Workshop (BIROW V) was held on January 18-19, 2008. As with previous BIROW meetings, the purpose of BIROW V was to identify and characterize research and engineering opportunities in biomedical engineering and imaging. The topic of this BIROW meeting was Imaging and Characterizing Structure and Function in Native and Engineered Tissues. Under this topic, four areas were explored in depth: 1) Heterogeneous single-cell measurements and their integration into tissue and organism models; 2) Functional, molecular, and structural imaging of engineered tissue in vitro and in vivo; 3) New technologies for characterizing cells and tissues in situ; 4) Imaging for targeted cell, gene, and drug delivery. ' 2008 International Society for Advancement of Cytometry Key terms tissue engineering, functional, molecular and structural imaging, imaging of engineered tissues, targeted cell, gene, and drug delivery, single-cell measurements, emerging imaging technologies THE Fifth Bioengineering and Imaging Research Opportunities Workshop (BIROW V) was held on January 18-19 in North Bethesda, Maryland. The name of the workshop was changed slightly from preceding BIROW workshops (which were known as Biomedical Imaging Research Opportunities Workshops) to emphasize biomedical engineering as well as imaging. BIROW V was sponsored by the Academy of Radiology Research (ARR), American Association of Physicists in Medicine (AAPM), American Institute for Medical and Biological Engineering (AIMBE), International Society for Analytical Cytology (ISAC), and the Society for Imaging Informatics in Medicine (SIIM). Partial financial support for the meeting was furnished by the National Institute of Biomedical Imaging and Bioengineering. The purpose of BIROW V (as of BIROWs I-IV which were held in 2003, 2004, 2005, and 2006) (1-4) was to identify and characterize opportunities for scientific research and engineering development in biomedical engineering and imaging. The topic of BIROW V was Imaging and Characterizing Structure and Function in Native and Engineered Tissues. The meeting focused on four areas of scientific research that offer opportunities for major developments in biomedical engineering and imaging. The four areas are as follows:

Study Design-In vitro experiment using human intervertebral disc (IVD) cells and adenovirustherapeutic gene constructs. Objectives-To examine the biologic effect of "cocktail" therapeutic gene transfer to human IVD cells in three dimensional cultures. Summary of Background Data-. Gene therapy is regarded as a potential option for the treatment of degenerative disc disease. Although various anabolic genes have previously been introduced for this purpose, cocktail gene transfer of anabolic genes to IVD cells has never been attempted. Materials and Methods-Human IVDs were harvested during surgical disc procedures and cultured. We prepared recombinant adenovirus constructs bearing the TGF-β1 gene (Ad/TGF-β1), the IGF-1 gene (Ad/IGF-1), and the BMP-2 gene (Ad/BMP-2). Transgene expression was detected by luciferase assays, enzyme linked immuno-sorbent assays, and Western blot analysis. Newly synthesized proteoglycan was measured by 35 S-sulfate incorporation on Sephadex G-25M in PD 10 columns. Human IVD cells were transduced by single, double, and triple combination of Ad/TGF-β1, Ad/IGF-1, Ad/BMP-2 with an MOI of 75, then cultured three-dimensionally in alginate beads. Results-Transgene expression was detected at 18 hours after viral transduction. IVD cultures with Ad/TGF-β1, Ad/IGF-1, Ad/BMP-2 (MOI of 75) showed 2.9, 1.8, and 1.9 fold increases, respectively, in proteoglycan synthesis compared to control. Human IVD cultures with double gene combination (MOI of 75) showed 3.2 to 3.9 fold increases of proteoglycan synthesis. Lastly, Human IVD cultures with triple gene combination (TGF-β1+IGF-1+BMP-2 genes with an MOI of 75) transfer demonstrated 4.7 fold increase in proteoglycan synthesis compared control. Conclusion-Combination or "cocktail" gene therapy offers a promising mechanism for maximizing matrix synthesis with low dose of adenoviral mixtures, circumventing systemic, local toxic effect, and immune response.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by a single gene mutation, a reciprocal translocation that originates the Bcr-Abl gene with constitutive tyrosine kinase activity. As a monogenic disease, it is an optimum target for RNA silencing therapy. We developed a siRNA-based therapeutic approach in which the siRNA is delivered by pepM or pepR, two cell-penetrating peptides (CPPs) derived from the dengue virus capsid protein. These peptides have a dual role: siRNA delivery into cells and direct action as bioportides, i.e. intracellularly bioactive CPPs, targetting cancer-related signaling processes. Both pepM and pepR penetrate the positive Bcr-Abl(+) Cell Line (BV173). Five in silico designed anti-Bcr-Abl siRNA were selected for in vitro analysis after thorough screening. The Bcr-Abl downregulation kinetics (48h to 168h) was followed by quantitative PCR. The bioportide action of the peptide vectors was evaluated by genome-wide microarray analysis and furt...

Live attenuated bacterial vectors, such as Salmonella typhimurium, have shown promise as delivery vehicles for DNA. We have examined two new strains of S. typhimurium and their impact on dendritic cell maturation (CD12-sifA/aroC mutant and WT05-ssaV/aroC, both in TML background). Strain WT05 matured dendritic cells in a more efficient way; caused higher release of cytokines TNF-alpha, IL-12, IL-1beta; and was efficient for gene transfer. These findings suggest that the genetic background of the attenuation can influence the pattern of inflammatory immune response to Salmonella infection.

Transient expression and stable integration and expression of trans offspring of certain leguminous plants after electroporation of DNA The method described in this article thus allows the study of transgene expression in tissues differentiating from meristematic cells present in the treated buds. In addition, transgenic plants can be recovered in the Index Entries: Electroporation, nodal meristem; transgenic plants.

Industrial biotechnology has been defined as the use and application of biotechnology for the sustainable processing and production of chemicals, materials and fuels. It makes use of biocatalysts such as microbial communities, whole-cell microorganisms or purified enzymes. In the review these processes are described. Drug design is an iterative process which begins when a chemist identifies a compound that displays an interesting biological profile and ends when both the activity profile and the chemical synthesis of the new chemical entity are optimized. Traditional approaches to drug discovery rely on a stepwise synthesis and screening program for large numbers of compounds to optimize activity profiles. Over the past ten to twenty years, scientists have used computer models of new chemical entities to help define activity profiles, geometries and relativities. This article introduces inter alia the concepts of molecular modelling and contains references for further reading.

The cricket (Gryllus bimaculatus) is a hemimetabolous insect that is emerging as a model organism for the study of neural and molecular mechanisms of behavioral traits. However, research strategies have been limited by a lack of genetic manipulation techniques that target the nervous system of the cricket. The development of a new method for efficient gene delivery into cricket brains, using in vivo electroporation, is described here. Plasmid DNA, which contained an enhanced green fluorescent protein (eGFP) gene, under the control of a G. bimaculatus actin (Gb′-act) promoter, was injected into adult cricket brains. Injection was followed by electroporation at a sufficient voltage. Expression of eGFP was observed within the brain tissue. Localized gene expression, targeted to specific regions of the brain, was also achieved using a combination of local DNA injection and fine arrangement of the electroporation electrodes. Further studies using this technique will lead to a better understanding of the neural and molecular mechanisms that underlie cricket behaviors.

Phosphodiesterase-6 (PDE6) is the key effector enzyme of the vertebrate phototransduction pathway in rods and cones. Rod PDE6 catalytic core is composed of two distinct subunits, PDE6␣ and PDE6␤, whereas two identical PDE6␣Ј subunits form the cone PDE6 catalytic core. It is not known whether this difference in PDE6 catalytic subunit identity contributes to the functional differences between rods and cones. To address this question, we expressed cone PDE6␣Ј in the photoreceptor cells of the retinal degeneration 10 (rd10) mouse that carries a mutation in rod PDE␤ subunit. We show that adeno-associated virus-mediated subretinal delivery of PDE6␣Ј rescues rod electroretinogram responses and preserves retinal structure, indicating that cone PDE6␣Ј can couple effectively to the rod phototransduction pathway. We also show that restoration of light sensitivity in rd10 rods is attributable to assembly of PDE6␣Ј with rod PDE6␥. Single-cell recordings revealed that, surprisingly, rods expressing cone PDE6␣Ј are twofold more sensitive to light than wild-type rods, most likely because of the slower shutoff of their light responses. Unlike in wild-type rods, the response kinetics in PDE6␣Ј-treated rd10 rods accelerated with increasing flash intensity, indicating a possible direct feedback modulation of cone PDE6␣Ј activity. Together, these results demonstrate that cone PDE6␣Ј can functionally substitute for rod PDE␣␤ in vivo, conferring treated rods with distinct physiological properties.

Type 1 diabetes results from the T cell‐mediated destruction of pancreatic beta cells. Islet transplantation has recently become a potential therapeutic approach for patients with type 1 diabetes. However, islet‐graft failure appears to be a challenging issue to overcome. Thus, complementary gene therapy strategies are needed to improve the islet‐graft survival following transplantation. Immune modulation through gene therapy represents a novel way of attacking cytotoxic T cells targeting pancreatic islets. Various death ligands of the TNF family such as FasL, TNF, and TNF‐Related Apoptosis‐Inducing Ligand (TRAIL) have been studied for this purpose. The over‐expression of TNF or FasL in pancreatic islets exacerbates the onset of type 1 diabetes generating lymphocyte infiltrates responsible for the inflammation. Conversely, the lack of TRAIL expression results in higher degree of islet inflammation in the pancreas. In addition, blocking of TRAIL function using soluble TRAIL receptors...

Type 1 diabetes (T1D), characterized by permanent destruction of insulin-producing beta cells, is lethal unless conventional exogenous insulin therapy or whole-organ transplantation is employed. Although pancreatic islet transplantation is a safer and less invasive method compared with whole-organ transplant surgery, its treatment efficacy has been limited by islet graft malfunction and graft failure. Thus, ex vivo genetic engineering of beta cells is necessary to prolong islet graft survival. For this reason, a novel gene therapy approach involving adenovirus-mediated TRAIL gene delivery into pancreatic islets was tested to determine whether this approach would defy autoreactive T cell assault in streptozotocin (STZ)-induced diabetic rats. To test this, genetically modified rat pancreatic islets were transplanted under the kidney capsule of STZ-induced diabetic rats, and diabetic status (blood sugar and body weight) was monitored after islet transplantation. STZ-induced diabetic rats carrying Ad5hTRAIL-infected islets experienced prolonged normoglycemia compared with animals grafted with mock-infected or AdCMVLacZ-infected islets. In addition, severe insulitis was detected in animals transplanted with mock-infected or AdCMVLacZ-infected islets, whereas the severity of insulitis was reduced in animals engrafted with Ad5hTRAIL-infected islets. Thus, TRAIL overexpression in pancreatic islets extends allograft survival and function, leading to a therapeutic benefit in STZ-induced diabetic rats.

Objective: The Shal (or Kv4) gene family has been proposed to be responsible for primary subunits of the transient outward potassium current (I). More precisely, Kv4.2 and Kv4.3 have been suggested to be the most likely molecular correlates for I in rat cells. The to to purpose of the present study was to compare the properties of the rat Kv4.3 gene product when expressed in a human cell line (HEK293 cells) with that of I recorded from rat ventricular cells. Methods: The cDNA encoding the rat Kv4.3 potassium channel was cloned into to the pHook2 mammalian expression vector and expressed into HEK293. Patch clamp experiments using the whole cell configuration were used to characterise the electrophysiological parameters of the current induced by Kv4.3 in comparison with the rat ventricular myocyte I current. Results: The transfection of HEK293 cells with rat Kv4.3 resulted in the expression of a time-and voltage-dependent outward to potassium current. The current activated for potentials positive to 240 mV and the steady-state inactivation curve had a midpoint of 247.460.3 mV and a slope of 5.960.2 mV. Rat ventricular I current was activated at potentials positive to 220 mV and inactivated with to a half-inactivation potential and a Boltzmann factor of 229.160.7 mV and 4.560.5 mV, respectively. The time course of recovery from inactivation of rat Kv4.3 expressed in HEK293 cells and of I recorded from native rat ventricular cells were exponentials with time to constants of 213.264.1 msec and 23.61.5 msec, respectively. Pharmacologically, I of rat myocytes showed a greater sensitivity to to 4-aminopyridine than Kv4.3 since half-maximal effects were obtained with 1.5460.13 mM and 0.1460.02 mM on Kv4.3 and I , to respectively. In both Kv4.3 and I , 4-aminopyridine appears to bind to the closed state of the channel. Finally, although a higher level of to expression was observed in the atria compared to the ventricle, the distribution of the Kv4.3 gene across the ventricles appeared to be homogenous. Conclusion: The results of the present study show that Kv4.3 channel may play a major role in the molecular structure of the rat cardiac I current. Furthermore, because the distribution of Kv4.3 across the ventricle is homogenous, the blockade of this channel to by specific drugs may not alter the normal heterogeneity of I current.

Ad.CMV-CD is a replication incompetent adenoviral vector carrying a cytomegalovirus (CMV)-driven transcription unit of the cytosine deaminase (CD) gene. The CD transcription unit in this vector catalyzes the deamination of the nontoxic pro-drug, 5-fluorocytosine (5-FC), thus converting it to the cytotoxic drug 5-fluorouracil (5-FU). This adenoviral vector prodrug activation system has been proposed for use in selectively sensitizing breast cancer cells, which may contaminate collections of autologous stem cells products from breast cancer patients, to the toxic effects of 5-FC, without damaging the reconstitutive capability of the normal hematopoietic cells. This system could conceivably kill even the nondividing breast cancer cells, because the levels of 5-FU generated by this system are 10 to 30 times that associated with systemic administration of 5-FU. The incorporation of 5-FU into mRNA at these high levels is sufficient to disrupt mRNA processing and protein synthesis so that ...

In mammalian species, mitochondrial DNA (mtDNA) with pathogenic mutations that induce mitochondrial respiration defects has been proposed to be involved in tumor phenotypes via induction of enhanced glycolysis under normoxic conditions (the Warburg effects). However, because both nuclear DNA and mtDNA control mitochondrial respiratory function, it is difficult to exclude the possible contribution of nuclear DNA mutations to mitochondrial respiration defects and the resultant expression of tumor phenotypes. Therefore, it is important to generate transmitochondrial cybrids sharing the same nuclear DNA background but carrying mtDNA with and without the mutations by using intercellular mtDNA transfer technology. Our previous studies isolated transmitochondrial cybrids and showed that specific mtDNA mutations enhanced tumor progression as a consequence of overproduction of reactive oxygen species (ROS). This study assessed whether mtDNA mutations inducing ROS overproduction always enhance tumor progression. We introduced mtDNA from senescence-accelerated mice P1 (SAMP1) into C57BL/6J (B6) micederived Lewis lung carcinoma P29 cells, and isolated new transmitochondrial cybrids (P29mtSAMP1 cybrids) that overproduced ROS. The inoculation of the cybrids into B6 mice unexpectedly showed that mtDNA from SAMP1 mice conversely induced tumor suppression. Moreover, the tumor suppression of P29mtSAMP1 cybrids in B6 mice occurred as a consequence of innate immune responses of the host B6 mice. Enzyme pretreatment experiments of P29mtSAMP1 cybrids revealed that some peptides encoded by mtDNA and expressed on the cell surface of P29mtSAMP1 cybrids induce increased IL-6 production from innate immune cells (dendritic cells) of B6 mice, and mediate augmented inflammatory responses around the tumor-inoculated environment. These observations indicate presence of a novel role of mtDNA in tumor phenotype, and provide new insights into the fields of mitochondrial tumor biology and tumor immunology.

Background We have investigated a simple strategy for enhancing transgene expression specificity by leveraging genetic silencer elements. The approach serves to restrict transgene expression to a tissue of interest - the nervous system in the example provided here - thereby promoting specific/exclusive targeting of discrete cellular subtypes. Recent innovations are bringing us closer to understanding how the brain is organized, how neural circuits function, and how neurons can be regenerated. Fluorescent proteins enable mapping of the 'connectome', optogenetic tools allow excitable cells to be short-circuited or hyperactivated, and targeted ablation of neuronal subtypes facilitates investigations of circuit function and neuronal regeneration. Optimally, such toolsets need to be expressed solely within the cell types of interest as off-site expression makes establishing causal relationships difficult. To address this, we have exploited a gene 'silencing' system that p...

Different methods are being deployed for non-viral DNA/RNA delivery. However non-viral formulations for DNA/RNA-delivery are often accompanied by severe toxicity and thus low efficiency. Particular costly cell culture media are required as well. Here we introduce sapofection as a valuable enhancing method for non-viral DNA/RNA delivery. Sapofection is based on the application of DNA/RNA nanoplexes and sapofectosid, a plant derived natural transfection reagent. Sapofectosid was produced from plant raw material by chromatographic methods and characterized by tandem mass spectrometry and intensive one and two dimensional NMR-spectroscopy. Sapofectosid did enhance the transfection efficiency of different DNA-and RNA-nanoplexes formulated with liposomes, polyethylenimine (PEI) or targeted and non-targeted oligo-lysine peptides. All nanoplexes were characterized physicochemically and the influence of sapofectosid on the nanoplex integrity was determined by DNA complexation assays. The nanoplexes and sapofectosid were administered to a variety of cancer cell lines and the transfection efficiency was investigated by flow cytometry and confocal microscopy. Dependent on the cell line the transfection efficiencies varied from 6 to 76%. The saponin-and receptor-mediated endocytosis of nanoplexes was investigated by flow cytometry. As demonstrated by impedance based live cell imaging sapofection was non-toxic. The findings show the great potential of sapofection to be used as an effective and nontoxic transfection enhancing method. Recently, we have reported that a complex extract of natural

We report a series of studies that assess the feasibility and sensitivity of imaging of herpes virus type one thymidine kinase (HSV1-tk) gene transfer and expression with [124I]-5-iodo-2'-fluoro-1-beta-D-arabinofuranosyluracil ([124I]-FIAU) and positron emission tomography (PET) and the ability of [124I]-FIAU-PET imaging to discriminate different levels of HSV1-tk gene expression. Studies were performed in rats bearing multiple s.c. tumors derived from W256 rat carcinoma and RG2 rat glioma cells. In the first set, we tested the sensitivity of [124I]-FIAU-PET imaging to detect low levels of HSV1-tk gene expression after retroviral-mediated gene transfer. HSV1-tk gene transduction of one of preestablished wild-type W256 tumor in each animal was accomplished by direct intratumoral injection of retroviral vector-producer cells (W256-->W256TK* tumors). Tumors produced from W256 and W256TK+ cells served as the negative and positive control in each animal. Highly specific images of ...

Background Cell penetrating peptides have been successfully used to mediate the intracellular delivery of a wide variety of molecules of pharmacological interest. The main aim of the present work was to evaluate the potential of the S4 13-PV cell penetrating peptide to mediate the intracellular delivery of plasmid DNA, aiming at its use in gene therapy applications. The S4 13-PV cell penetrating peptide is a chimeric peptide that results from the combination of a cell penetrating sequence derived from the Dermaseptin S4 peptide with the nuclear localization signal present in the Simian Virus 40 (SV40) large T antigen. Methods S4 13-PV cell penetrating peptide and cationic liposomes composed of 1,2-dioleoyl-3-trimethylammonium-propane:1,2-dioleoyl-snglycero-3-phosphoethanolamine were complexed with pDNA at different charge ratios. Complexation of pDNA was assessed by gel electrophoresis. Luciferase assay, fluorescence microscopy and fluorescence-activated cell sorting analysis were used to evaluate reporter gene delivery to TSA and HeLa cells. Cytotoxicity of the pDNA complexes was assessed by Alamar blue assay. Results Complexes obtained through electrostatic association of the S4 13-PV cell penetrating peptide with plasmid DNA are able to very efficiently mediate transfection, particularly at high peptide/DNA charge ratios. Additionally, our results clearly demonstrate that, both in HeLa and TSA cells, ternary complexes, resulting from association of cationic liposomes to peptide/DNA complexes, are significantly more efficient in mediating transfection than the corresponding peptide/DNA or cationic liposome/DNA complexes. Conclusions Overall, our data highlight the potential of cell penetrating peptides for the development of improved nonviral gene delivery systems.

We created the FAST (Flexible Accelerated STOP TetO-knockin) system, an efficient method for manipulating gene expression in vivo to rapidly screen animal models of disease. A single gene targeting event yields 2 distinct knockin mice-STOP-tetO and tetO knockin-which permit generation of multiple strains with variable expression patterns: 1) knockout, 2) Cre-mediated rescue; 3) tTA-mediated misexpression; 4) tTA-mediated overexpression; and 5) tTS-mediated conditional knockout/knockdown. Using the FAST system, multiple gain-and loss-of-function strains can therefore be generated on a timescale not previously achievable. These strains can then be screened for clinically-relevant abnormalities. We demonstrate the flexibility and broad applicability of the FAST system by targeting several genes encoding proteins implicated in neuropsychiatric disorders: Mlc1, Neuroligin 3, the serotonin 1A receptor, and the serotonin 1B receptor. Keywords genetics; gene targeting; animal model; mouse; conditional modulation of gene expression; developmental change Gain-of-function and loss-of-function studies are commonly used to examine gene function in vivo, particularly in attempts to model human disease in animals. Developing animal models of disease is key to the process of elucidating neuropsychiatric disease pathophysiology, in turn leading to drug discovery and translation to patient populations.

IL-5 is induced locally in the lung and systemically in the circulation during allergic airways eosinophilic inflammation both in humans and experimental animals. However, the precise role of local and systemic IL-5 in the development of allergic airways eosinophilia remains to be elucidated. In our current study, we demonstrate that compared with their IL-5 ϩրϩ counterparts, IL-5 Ϫ / Ϫ mice lacked an IL-5 response both in the lung and peripheral blood, yet they released similar amounts of IL-4, eotaxin, and MIP-1 ␣ in the lung after ovalbumin (OVA) sensitization and challenge. At cellular levels, these mice failed to develop peripheral blood and airways eosinophilia while the responses of lymphocytes, neutrophils, and macrophages remained similar to those in IL-5 ϩրϩ mice. To dissect the relative role of local and systemic IL-5 in this model, we constructed a gene transfer vector expressing murine IL-5. Intramuscular IL-5 gene transfer to OVA-sensitized IL-5 Ϫ / Ϫ mice led to raised levels of IL-5 compartmentalized to the circulation and completely reconstituted airways eosinophilia upon OVA challenge, which was associated with reconstitution of eosinophilia in the bone marrow and peripheral blood. Significant airways eosinophilia was observed for at least 7 d in these mice. In contrast, intranasal IL-5 gene transfer, when rendered to give rise to a significant but compartmentalized level of transgene protein IL-5 in the lung, was unable to reconstitute airways eosinophilia in OVA-sensitized IL-5 Ϫ / Ϫ mice upon OVA-challenge, which was associated with a lack of eosinophilic responses in bone marrow and peripheral blood. Our findings thus provide unequivocal evidence that circulating but not local lung IL-5 is critically required for the development of allergic airways eosinophilia. These findings also provide the rationale for developing strategies to target circulating IL-5 and/or its receptors in bone marrow to effectively control asthmatic airways eosinophilia. (J.

Background-Therapeutic angiogenesis by the administration of recombinant vascular endothelial growth factor (rVEGF) is a novel strategy for the treatment of ischemic disorders. rVEGF has been delivered as a protein, by plasmid DNA, and by genetically engineered cells with different pharmacokinetic and physiological properties. In the present study, we examined a new method for delivery of rVEGF using implantable bioartificial muscle (BAM) tissues made from genetically modified primary skeletal myoblasts. Our goal was to determine whether the rVEGF delivered by this technique promoted controlled angiogenesis in nonischemic and/or ischemic adult mouse tissue. Methods and Results-Primary adult mouse myoblasts were retrovirally transduced to secrete human or mouse rVEGF and tissue-engineered into implantable 1ϫ10 to 15-mm BAMs containing parallel arrays of postmitotic myofibers. In vitro, they secreted 290 to 511 ng of bioactive mouse or human VEGF/BAM per day. rVEGF BAMs implanted subcutaneously into syngeneic mice caused a 30-fold increase in the number of CD31-positive capillary cells within the BAM by 1 week compared with control BAMs. Implantation of rVEGF-secreting BAMs into ischemic hindlimbs resulted in a 2-to 3-fold increase in capillary density of neighboring host muscle by 1 week and out to 4 weeks with no evidence of hemangioma formation. Conclusions-Local delivery of rVEGF from BAMs rapidly increases capillary density both within the BAM itself and in adjacent ischemic muscle tissue. Genetically engineered muscle tissue provides a method for therapeutic protein delivery in a dose-regulated fashion. (Circulation. 2001;104:594-599.

CD31 is a member of the Ig superfamily expressed on various cell types of the vasculature, including a certain subpopulation of T lymphocytes. Previous reports suggest that interaction of CD31 with its heterophilic ligand on T cells (T cell CD31 ligand) plays a regulatory role in T lymphocyte activation. Here we demonstrate that a soluble rCD31-receptorglobulin (CD31Rg) specifically down-regulated the proliferation of human peripheral blood CD31− T lymphocytes stimulated via CD3 and CD28 mAbs. Notably, engagement of the T cell CD31 ligand by CD31Rg during primary stimulation also induced a prolonged unresponsive state in T cells. Retroviral transduction of CD31 into CD31− Th clones resulted in a significant inhibition of their proliferative capacity. When cocultured with purified CD31− T lymphocytes, irradiated CD31-transduced Th clones counterregulated the CD3/CD28-mediated activation of these cells. Furthermore, primary stimulation in the presence of CD31-transduced Th clones indu...

Encapsulation of plasmid DNA in nanoparticle is expected to enhance the stability of DNA, reproducibility and frequency of the genetic transformation in plants. Here we report the formulation of HMG Co-A reductase gene loaded calcium phosphate nanoparticles (Cap nanoparticles) and their in-vitro, in-vivo characterization. The developed Cap nanoparticles were characterized by DSC, FT-IR, and XRD. Developed Cap nanoparticles were spherical in shape having the particle size and zeta potential in the range of 10.86±0.09nm to 33.42±0.18nm and-25.5±0.07mV to-31.7±0.07mV (for Cap-I to Cap-IV). DNA releasing in acidic media showed, initially slow release followed by fast release with a maximum release of Cap-I (95.77±1.39%)> Cap-II (87.32±2.07%)> Cap-III (76.54±2.01%)> Cap-IV (72.93±1.75%) over 60min. Cap nanoparticles were quite stable at storage condition of 40±0.5°C/75±5%RH, 25±0.5°C/60±RH, 4±0.5°C/ambient humidity and the integrity of pDNA encapsulated was confirmed by gel electrophoresis. Compared to wild type C. intybus, transformation efficiency and enhanced biosynthesis of esculin with the DNA nanoparticles in C. intybus were about 10% and 71%, respectively. Antioxidant activity capacity of the biotransformed plants was significantly higher than the normal plant due to high accumulation of esculin.

Background-Cell specific gene transfer and sustained transgene expression are goals of cutaneous gene therapy. Pseudotyping strategy with AAV vectors has the potential to confer unique cellular tropism and transduction efficiency. We hypothesize that pseudotyped AAV vectors have differential tropism and transduction efficiency under normal and wound conditions in dermal fibroblasts. Materials and Methods-AAV2 genome with GFP reporter was packaged in capsids of other serotypes AAV5, AAV7, and AAV8, producing pseudotyped vectors AAV2/5, AAV2/7, and AAV2/8 respectively. Murine and human dermal fibroblasts were transduced by the different pseudotypes for 24 hours at multiplicities of infection (MOI)10 2 , 10 3 , 10 4 , and 10 5 . Transduction efficiency was assessed at days 3 and 7. Experiments were repeated in a simulated wound environment by adding 10ng/ml PDGF-B to culture media. Results-Transduction efficiency of the pseudotyped AAV vectors is dose dependent. MOI 10 5 results in significantly higher gene transfer. Under normal culture conditions, the pseudotyping strategy confers differential transduction of dermal fibroblasts, with significantly enhanced transduction of murine cells by AAV2/5 and AAV2/8 compared to AAV2/2. AAV2/8 is more efficacious in transducing human cells. Under wound conditions, transduction efficiency of AAV2/2, 2/5 and 2/8 is significantly lower in murine fibroblasts. At day 3 under wound conditions, all vectors demonstrate similar transduction efficiency, but by day 7 the three pseudotyped vectors transduce significantly more murine cells compared to AAV2/2. However, in human cells, there is no significant difference in transduction efficiency of each pseudotype between normal and wound conditions at both 3 and 7 days. Conclusions-The AAV pseudotyping strategy represents a gene transfer technology that can result in differential transduction of dermal fibroblasts. The differences in transduction efficiency

Several mutations causing both photoreceptor degeneration and malfunction have been identified in humans and animals. Although intraocular injection of trophic factors has been shown to reduce photoreceptor death in a few conditions of rapid photoreceptor loss, it is unclear whether long-term beneficial changes in functional properties of affected photoreceptors can be obtained by treatment with these factors. The rds/rds mouse is a spontaneous mutant bearing a null mutation in the rds/peripherin gene, which is linked to many forms of dominant retinal degenerations in humans. Here, we report that intraocular adenovirus-mediated gene transfer of ciliary neurotrophic factor (CNTF) in this mutant reduces photoreceptor loss, causes a significant increase in the length of photoreceptor segments, and results in a redistribution and an increase in the retinal content of the photopigment rhodopsin. These effects are accompanied by a significant increase in the amplitude of the a- and b-waves of the scotopic electroretinogram. These results suggest that continuous administration of CNTF could potentially be useful for the treatment of some forms of retinal degeneration.

During the last two decades the fields of molecular and cellular cardiology, and more recently molecular cardiac surgery, have developed rapidly. The concept of delivering cDNA encoding a therapeutic gene to cardiomyocytes using a vector system with substantial cardiac tropism, allowing for long-term expression of a therapeutic protein, has moved from hypothesis to bench to clinical application. However, the clinical results to date are still disappointing. The ideal gene transfer method should be explored in clinically relevant animal models of heart disease to evaluate the relative roles of specific molecular pathways in disease pathogenesis, helping to validate the potential targets for therapeutic intervention. Successful clinical cardiovascular gene therapy also requires the use of nonimmunogenic cardiotropic vectors capable of expressing the requisite amount of therapeutic protein in vivo and in situ. Depending on the desired application either regional or global myocardial ge...

Objective: Therapeutic safety and efficacy are the basic prerequisites for clinical gene therapy. We investigated the effect of high-dose molecular cardiac surgery with recirculating delivery (MCARD)-mediated adenoassociated virus 9 (AAV9)/sarcoplasmic reticulum Ca 2þ adenosine triphosphatase (SERCA2a) gene delivery on clinical parameters, oxidative stress, humoral and cellular immune responses, and cardiac remodeling. Methods: Ischemic cardiomyopathy was generated in a sheep model. The sheep were assigned to 1 of 2 groups: control (n ¼ 10) and study (MCARD, n ¼ 6). The control group underwent no intervention and the study group received 10 14 genome copies of AAV9/SERCA2a 4 weeks after infarction.

Despite the great interest in identifying the cell-of-origin for different cancers, little knowledge exists regarding the extent to which the specific origin of a tumor contributes to its properties. To directly examine this question, we expressed identical oncogenes in two types of glial progenitor cells, glial-restricted precursor (GRP) cells and oligodendrocyte/type-2 astrocyte progenitor cells (O-2A/OPCs), and in astrocytes of the mouse CNS (either directly purified or generated from GRP cells).In vitro, expression of identical oncogenes in these cells generated populations differing in expression of antigens thought to identify tumor initiating cells, generation of 3D aggregates when grown as adherent cultures, and sensitivity to the chemotherapeutic agent BCNU.In vivo, cells differed in their ability to form tumors, in malignancy and even in the type of host-derived cells infiltrating the tumor mass. Moreover, identical genetic modification of these different cells yielded ben...

Due to the absence of blood vessels and nerves, the regenerative potential of articular cartilage is significantly constrained. This implies that the impact of a ruptured cartilage extends to the entire joint. Osteoarthritis, a health condition, may arise due to injury and the progressive breakdown of joint tissues. The progression of osteoarthritis can be accelerated by the extensive degradation of articular cartilage, which is ranked as the third most prevalent musculoskeletal disorder necessitating rehabilitation, following low back pain and fractures. The existing therapeutic interventions for cartilage repair exhibit limited efficacy and seldom achieve complete restoration of both functional capacity and tissue homeostasis. Emerging technological advancements in the field of tissue engineering hold significant promise for the development of viable substitutes for cartilage tissue, capable of exhibiting functional properties. The overarching strategy involves ensuring that the cell source is enriched with bioactive molecules that facilitate cellular differentiation and/or maturation. This review provides a comprehensive summary of recent advancements in the field of cartilage tissue engineering. Additionally, it offers an overview of recent clinical trials that have been conducted to examine the latest research developments and clinical applications pertaining to weakened articular cartilage and osteoarthritis.