Using interpretable boosting algorithms for modeling environmental and agricultural data.

We describe how interpretable boosting algorithms based on ridge-regularized generalized linear models can be used to analyze high-dimensional environmental data. We illustrate this by using environmental, social, human and biophysical data to predict the financial vulnerability of farmers in Chile and Tunisia against climate hazards. We show how group structures can be considered and how interactions can be found in high-dimensional datasets using a novel 2-step boosting approach.

Detailed Protocol for the Novel and Scalable Viral Vector Upstream Process for AAV Gene Therapy Manufacturing.

Recombinant adeno-associated viral (rAAV) vector-based gene therapy has been adapted for use in more than 100 clinical trials. This is mainly because of its excellent safety profile, ability to target a wide range of tissues, stable transgene expression, and significant clinical benefit. However, the major challenge is to produce a high-titer, high-potency vector to achieve a better therapeutic effect.

Inhibition of pathological brain angiogenesis through systemic delivery of AAV vector expressing soluble FLT1.

The soluble vascular endothelial growth factor (VEGF) receptor 1 (sFLT1) has been tested in both animals and humans for anti-angiogenic therapies, for example, age-related macular degeneration. We hypothesized that adeno-associated viral vector (AAV)-mediated sFLT1 expression could be used to inhibit abnormal brain angiogenesis. We tested the anti-angiogenic effect of sFLT1 and the feasibility of using AAV serotype 9 to deliver sFLT1 through intravenous injection (IV) to the brain angiogenic region.

Gene Therapies for Neovascular Age-Related Macular Degeneration.

Pathological neovascularization is a key component of the neovascular form (also known as the wet form) of age-related macular degeneration (AMD) and proliferative diabetic retinopathy. Several preclinical studies have shown that antiangiogenesis strategies are effective for treating neovascular AMD in animal models. Vascular endothelial growth factor (VEGF) is one of the main inducers of ocular neovascularization, and several clinical trials have shown the benefits of neutralizing VEGF in patients with neovascular AMD or diabetic macular edema.

Aurintricarboxylic acid increases yield of HSV-1 vectors.

Production of large quantities of viral vectors is crucial for the success of gene therapy in the clinic. There is a need for higher titers of herpes simplex virus-1 (HSV-1) vectors both for therapeutic use as well as in the manufacturing of clinical grade adeno-associated virus (AAV) vectors. HSV-1 yield increased when primary human fibroblasts were treated with anti-inflammatory drugs like dexamethasone or valproic acid.

Cyclin-dependent kinase 9 activity regulates neutrophil spontaneous apoptosis.

Neutrophils are the most abundant leukocyte and play a central role in the immune defense against rapidly dividing bacteria. However, they are also the shortest lived cell in the blood with a lifespan in the circulation of 5.4 days.

Anti-angiogenic effects of purine inhibitors of cyclin dependent kinases.

Small molecular inhibitors of Cyclin dependent kinases (Cdks) are currently being developed as anticancer therapeutics due to their antiproliferative properties. The purine Cdk specific inhibitor (R)-roscovitine (seliciclib, CYC202) represents one of the most promising of these compounds. It is currently evaluated in clinical trials concerning cancer therapy.

AAV5-mediated sFLT01 gene therapy arrests retinal lesions in Ccl2(-/-)/Cx3cr1(-/-) mice.

To test the effects of adeno-associated virus encoding sFLT01 (AAV5.sFLT01) on the retinal lesions in Ccl2(-/-)/Cx3cr1(-/-) mice, a model for age-related macular degeneration (AMD), AAV5.sFLT01 was injected into the subretinal space of the right eyes and the left eyes served as controls.

sFLT01: a novel fusion protein with antiangiogenic activity.

sFLT01 is a novel fusion protein that consists of the VEGF/PlGF (placental growth factor) binding domain of human VEGFR1/Flt-1 (hVEGFR1) fused to the Fc portion of human IgG(1) through a polyglycine linker. It binds to both human VEGF (hVEGF) and human PlGF (hPlGF) and to mouse VEGF (mVEGF) and mouse PlGF (mPlGF). In vitro, sFLT01 inhibited the proliferation of human umbilical vein endothelial cells and pericytes stimulated by either hVEGF or hPlGF.

Inhibition of choroidal neovascularization in a nonhuman primate model by intravitreal administration of an AAV2 vector expressing a novel anti-VEGF molecule.

Inhibition of vascular endothelial growth factor (VEGF) for the management of the pathological ocular neovascularization associated with diseases such as neovascular age-related macular degeneration is a proven paradigm; however, monthly intravitreal injections are required for optimal treatment. We have previously shown that a novel, secreted anti-VEGF molecule sFLT01 delivered by intravitreal injection of an AAV2 vector (AAV2-sFLT01) gives persistent expression and is efficacious in a murine model of retinal neovascularization. In the present study, we investigate transduction and efficacy of an intravitreally administered AAV2-sFLT01 in a nonhuman primate (NHP) model of choroidal neovascularization (CNV).

A novel roscovitine derivative potently induces G1-phase arrest in platelet-derived growth factor-BB-activated vascular smooth muscle cells.

Abnormal vascular smooth muscle cell (VSMC) proliferation contributes to the pathogenesis of restenosis. Thus, drugs interfering with cell cycle progression in VSMC are promising candidates for an antirestenotic therapy. In this study, we pharmacologically characterize N-5-(2-aminocyclohexyl)-N-7-benzyl-3-isopropyl-1(2)H-pyrazolo[4,3-d]pyrimidine-5,7-di-amine (LGR1406), a novel derivative of the cyclin-dependent kinase (CDK) inhibitor roscovitine (ROSC), in PDGF-BB-activated VSMC.

Novel anti-VEGF chimeric molecules delivered by AAV vectors for inhibition of retinal neovascularization.

Vascular endothelial growth factor (VEGF) is important in pathological neovascularization, which is a key component of diseases such as the wet form of age-related macular degeneration, proliferative diabetic retinopathy and cancer. One of the most potent naturally occurring VEGF binders is VEGF receptor Flt-1. We have generated two novel chimeric VEGF-binding molecules, sFLT01 and sFLT02, which consist of the second immunoglobulin (IgG)-like domain of Flt-1 fused either to a human IgG1 Fc or solely to the CH3 domain of IgG1 Fc through a polyglycine linker 9Gly.

Positron emission tomography-based imaging of transgene expression mediated by replication-conditional, oncolytic herpes simplex virus type 1 mutant vectors in vivo.

To evaluate the efficiency of gene delivery in gene therapy strategies for malignant brain tumors, it is important to determine the distribution and magnitude of transgene expression in target tumor cells over time. Here, we assess the time- and vector dose-dependent kinetics of recombinant herpes simplex virus (HSV)-1 vector-mediated gene expression and vector replication in culture and in vivo by a recently developed radiotracer method for noninvasive imaging of gene expression (J. G.

Isolation of a Brassica napus L. cDNA encoding a putative high-mobility-group HMG I/Y protein.

A cDNA encoding a high-mobility-group protein has been isolated from a microspore-specific library of Brassica napus. The 930 bp cDNA contains a 612 bp open reading frame encoding a protein of 203 amino acids residues exhibiting significant homology to HMG-I/Y protein from Arabidopsis thaliana (62%). The predicted protein contains four copies of the 'AT-hook' motif which is involved in binding A/T-rich DNA.

HSV-1 infected cell proteins influence tetracycline-regulated transgene expression.

Background: This study investigates elements of herpes simplex virus type 1 (HSV-1) which influence transgene expression in tetracycline-regulated expression systems.

Methods: Different HSV-1 mutants were used to infect Vero cells that had been transfected with plasmids containing the luciferase gene under the control of tet-off or tet-on tetracycline-regulation systems.

Results: The baseline level of luciferase expression was elevated after infection with HSV-1 mutants lacking one or more immediate early genes encoding transactivating factors: ICP27, ICP4 and ICP0.

High-molecular-mass complexes formed in vivo contain smHSPs and HSP70 and display chaperone-like activity.

Stress can have profound effects on the cell. The elicitation of the stress response in the cell is often accompanied by the synthesis of high-molecular-mass complexes, sometimes termed heat shock granules (HSGs). The presence of the complexes has been shown to be important for the survival of cells subjected to stress.

Combined HSV-1 recombinant and amplicon piggyback vectors: replication-competent and defective forms, and therapeutic efficacy for experimental gliomas.

Background: The versatility of HSV-1 vectors includes large transgene capacity, selective replication of mutants in dividing cells, and availability of recombinant virus (RV) and plasmid-derived (amplicon) vectors, which can be propagated in a co-dependent, 'piggyback', manner.

Methods: A replication-defective piggyback vector system was generated in which the amplicon carries either of two genes essential for virus replication, IE2 (ICP27) or IE3 (ICP4), as well as lacZ; the RV is deleted in both these genes, and vector stocks are propagated in cells transfected with one of the complementary genes. In the replication-competent system, the amplicon carries the IE2 and lacZ; the RV had a large deletion in the IE2; and stocks are propagated in untransfected cells.

Generation of an adenovirus vector lacking E1, e2a, E3, and all of E4 except open reading frame 3.

Toxicity and immunity associated with adenovirus backbone gene expression is an important hurdle to overcome for successful gene therapy. Recent efforts to improve adenovirus vectors for in vivo use have focused on the sequential deletion of essential early genes. Adenovirus vectors have been constructed with the E1 gene deleted and with this deletion in combination with an E2a, E2b, or E4 deletion.

Intraarterial delivery of adenovirus vectors and liposome-DNA complexes to experimental brain neoplasms.

This study investigated the intraarterial delivery of genetically engineered replication-deficient adenovirus vectors (AVs) and cationic liposome-plasmid DNA complexes (lipoDNA) to experimental brain tumors. Adenovirus or lipoDNA was injected into the internal carotid artery (ICA) of F344 rats harboring intracerebral 9L gliosarcomas, using bradykinin (BK) to selectively permeabilize the blood-tumor barrier (BTB). Brain and internal organs of the animals were collected 48 hr after vector injection and stained for expression of the marker gene product, beta-galactosidase (beta-Gal).

Pre-existing herpes simplex virus 1 (HSV-1) immunity decreases, but does not abolish, gene transfer to experimental brain tumors by a HSV-1 vector.

The influence of pre-existing anti-herpes simplex type 1 (HSV-1) immunity on HSV-1 vector-mediated gene transfer to glioma cells was analyzed in this gene marking study using intracranial D74 gliomas in syngeneic Fischer rats. The HSV-1 mutant virus used, hrR3, is defective in ribonucleotide reductase and bears the marker genes E. coli lacZ and HSV-1 thymidine kinase (HSVtk).

Targeting gene therapy vectors to CNS malignancies.

Gene therapy offers significant advantages to the field of oncology with the addition of specifically and uniquely engineered mechanisms of halting malignant proliferation through cytotoxicity or reproductive arrest. To confer a true benefit to the therapeutic ratio (the relative toxicity to tumor compared to normal tissue) a vector or the transgene it carries must selectively affect or access tumor cells. Beyond the selective toxicities of many transgene products, which frequently parallel that of contemporary chemotherapeutic agents, lies the potential utility of targeting the vector.

Green fluorescent protein as a reporter for retrovirus and helper virus-free HSV-1 amplicon vector-mediated gene transfer into neural cells in culture and in vivo.

Green fluorescent protein (GFP) is an effective marker for retrovirus and herpes virus vector-mediated gene transfer into various central nervous system-derived cells, both proliferative and non-proliferative, in culture and in vivo. Retrovirus vectors were used to stably transduce several rat and human glioma lines, and a multipotent mouse neural progenitor line in culture. Implantation of selected pools of transduced glioma cells into rodent brain allowed clear visualization of the tumor and the invading tumor edge.

Therapeutic efficiency and safety of a second-generation replication-conditional HSV1 vector for brain tumor gene therapy.

A second-generation replication-conditional herpes simplex virus type 1 (HSV) vector defective for both ribonucleotide reductase (RR) and the neurovirulence factor gamma34.5 was generated and tested for therapeutic safety and efficiency in two different experimental brain tumor models. In culture, cytotoxic activity of this double mutant HSV vector, MGH-1, for 9L gliosarcoma cells was similar to that of the HSV mutant, R3616, which is defective only for gamma34.

HSV/AAV hybrid amplicon vectors extend transgene expression in human glioma cells.

Novel hybrid vectors, which incorporate critical elements of both herpes simplex virus type 1 (HSV-1) amplicon vectors and adeno-associated virus (AAV) vectors, are able to sustain transgene expression in dividing glioma cells for over 2 weeks. These vectors combine the high infectibility and large transgene capacity of HSV-1 vectors with the potential for episomal amplification and chromosomal integration of AAV vectors. The hybrid vectors contain the HSV-1 origin of DNA replication, oriS, and the DNA cleavage/packaging signal, pac, which allow amplicon replication and packaging in HSV-1 virions.

Human fetal astrocytes as an ex vivo gene therapy vehicle for delivering biologically active nerve growth factor.

The therapeutic use of neurotrophic factors for neurodegenerative diseases is promising, however, optimal methods for continuous delivery of these substances to the human central nervous system (CNS) remains problematic. One approach would be to graft genetically engineered human cells that continuously secrete high levels of a biologically produced and processed neurotrophic factor. This ex vivo gene therapy approach has worked well in animal models of neurodegenerative diseases using a variety of nonneuronal cell types to deliver the transgene.