Replacing LNT: The Integrated LNT-Hormesis Model.

Many scientists and regulators utilize the linear no-threshold (LNT) relationship to estimate the likelihood of carcinogenesis. The LNT model is incorrect and was adopted based upon false pretenses. The use of the model has been corrupted by many to claim that even the smallest ionizing radiation dose may initiate carcinogenesis. This claim has resulted in societal harm.

A MLC-based inversely optimized 3D spatially fractionated grid radiotherapy technique.

This study presents a MLC-based, 3D grid-therapy technique with characteristics of both 3D-conformal-radiotherapy and grid-therapy. It generates a brachytherapy-like dose distribution, with D50% of 20, 9.8, 5.4 and 2.9-Gy, for the spheres, target, 1 cm-outershell and 2 cm-outershell, respectively. It may provide a strategy to deliver ablative doses to large tumors safely.

Can radiation risks to patients be reduced without reducing radiation exposure? The status of chemical radioprotectants.

OBJECTIVE: Medical radiation exposure has increased sixfold since 1980 and is the largest controllable source of exposure. Many efforts have been devoted to reducing dose or eliminating unnecessary examinations but with limited success. The concern regarding nuclear terrorism has focused a large amount of attention on radioprotective drugs. The purpose of this article is twofold: to review the current concepts, potential, and limitations of chemical radioprotectants in reducing stochastic and deterministic effects and to assess the potential application to diagnostic and interventional medical radiation procedures. CONCLUSION: There are a wide variety of chemical compounds that have been studied for radioprotective effects. Although there is promising research, chemical radioprotectants have not been shown to be very effective and, with one limited exception, are not the standard of care in medicine.

Helper-independent piggyBac plasmids for gene delivery approaches: strategies for avoiding potential genotoxic effects.

Efficient integration of functional genes is an essential prerequisite for successful gene delivery such as cell transfection, animal transgenesis, and gene therapy. Gene delivery strategies based on viral vectors are currently the most efficient. However, limited cargo capacity, host immune response, and the risk of insertional mutagenesis are limiting factors and of concern. Recently, several groups have used transposon-based approaches to deliver genes to a variety of cells. The piggyBac (pB) transposase in particular has been shown to be well suited for cell transfection and gene therapy approaches because of its flexibility for molecular modification, large cargo capacity, and high transposition activity. However, safety considerations regarding transposase gene insertions into host genomes have rarely been addressed. Here we report our results on engineering helper-independent pB plasmids. The single-plasmid gene delivery system carries both the piggyBac transposase (pBt) expression cassette as well as the transposon cargo flanked by terminal repeat element sequences. Improvements to the helper-independent structure were achieved by developing new plasmids in which the pBt gene is rendered inactive after excision of the transposon from the plasmid. As a consequence, potentially negative effects that may develop by the persistence of an active pBt gene posttransposition are eliminated. The results presented herein demonstrate that our helper-independent plasmids represent an important step in the development of safe and efficient gene delivery methods that should prove valuable in gene therapy and transgenic approaches.

piggyBac transposon/transposase system to generate CD19-specific T cells for the treatment of B-lineage malignancies.

Nonviral integrating vectors can be used for expression of therapeutic genes. piggyBac (PB), a transposon/transposase system, has been used to efficiently generate induced pluripotent stems cells from somatic cells, without genetic alteration. In this paper, we apply PB transposition to express a chimeric antigen receptor (CAR) in primary human T cells. We demonstrate that T cells electroporated to introduce the PB transposon and transposase stably express CD19-specific CAR and when cultured on CD19(+) artificial antigen-presenting cells, numerically expand in a CAR-dependent manner, display a phenotype associated with both memory and effector T cell populations, and exhibit CD19-dependent killing of tumor targets. Integration of the PB transposon expressing CAR was not associated with genotoxicity, based on chromosome analysis. PB transposition for generating human T cells with redirected specificity to a desired target such as CD19 is a new genetic approach with therapeutic implications.

Lithium chloride protects retinal neurocytes from nutrient deprivation by promoting DNA non-homologous end-joining.

Lithium chloride is a therapeutic agent for treatment of bipolar affective disorders. Increasing numbers of studies have indicated that lithium has neuroprotective effects. However, the molecular mechanisms underlying the actions of lithium have not been fully elucidated. This study aimed to investigate whether lithium chloride produces neuroprotective function by improving DNA repair pathway in retinal neurocyte. In vitro, the primary cultured retinal neurocytes (85.7% are MAP-2 positive cells) were treated with lithium chloride, then cultured with serum-free media to simulate the nutrient deprived state resulting from ischemic insult. The neurite outgrowth of the cultured cells increased significantly in a dose-dependent manner when exposed to different levels of lithium chloride. Genomic DNA electrophoresis demonstrated greater DNA integrity of retinal neurocytes when treated with lithium chloride as compared to the control. Moreover, mRNA and protein levels of Ligase IV (involved in DNA non-homologous end-joining (NHEJ) pathway) in retinal neurocytes increased with lithium chloride. The end joining activity assay was performed to determine the role of lithium on NHEJ in the presence of extract from retinal neurocytes. The rejoining levels in retinal neurocytes treated with lithium were significantly increased as compared to the control. Furthermore, XRCC4, the Ligase IV partner, and the transcriptional factor, CREB and CTCF, were up-regulated in retinal cells after treating with 1.0mM lithium chloride. Therefore, our data suggest that lithium chloride protects the retinal neural cells from nutrient deprivation in vitro, which may be similar to the mechanism of cell death in glaucoma. The improvement in DNA repair pathway involving in Ligase IV might have an important role in lithium neuroprotection. This study provides new insights into the neural protective mechanisms of lithium chloride.

Use of intracytoplasmic sperm injection (ICSI) to generate transgenic animals.

Even though intracytoplasmic sperm injection (ICSI) has been widely used for the production of offspring in human infertility clinics and in reproductive research laboratories using mice, many researchers engaged in animal transgenesis still consider it somewhat cumbersome. The greatest advantage of ICSI-mediated transgenesis is that it allows introduction of very large DNA transgenes (e.g., yeast artificial chromosomes), with relatively high efficiency into the genomes of hosts, as compared to pronuclear injection. Recently, we have developed an active form of intracytoplasmic sperm injection-mediated transgenesis (ICSI-Tr) with fresh sperm utilizing transposons. The transgenic efficiencies rival all transgenic techniques except that of lentiviral methods.

Medical countermeasures against nuclear threats: radionuclide decorporation agents.

Exposure to radionuclides disseminated by a radiological dispersion device or deposited as fallout after a nuclear power plant accident or detonation of an improvised nuclear device could result in internal contamination of a significant number of individuals. Internalized radionuclides may cause both acute and chronic radiation injury and increase an individual's risk of developing cancer. This damage and risk can be mitigated by the use of decorporation agents that reduce internal contamination. Unfortunately, most effective agents decorporate only a limited range of radionuclides, and some are formulated in ways that would make administration in mass casualty situations challenging. There is a need for new radionuclide decorporation agents, reformulations of existing agents, and/or expansion of the labeled indications for existing treatments. Researchers developing novel or improved decorporation agents should also understand the regulatory pathway for these products. This workshop, the first in nearly half a century to focus exclusively on radionuclide decorporation, brought together researchers and scientific administrators from academia, government and industry as well as senior regulatory affairs officers and U.S. Food and Drug Administration personnel. Meeting participants reviewed recent progress in the development of decorporation agents and contemplated the future of the field.

Medical countermeasures for radiation combined injury: radiation with burn, blast, trauma and/or sepsis. report of an NIAID Workshop, March 26-27, 2007.

Non-clinical human radiation exposure events such as the Hiroshima and Nagasaki bombings or the Chernobyl accident are often coupled with other forms of injury, such as wounds, burns, blunt trauma, and infection. Radiation combined injury would also be expected after a radiological or nuclear attack. Few animal models of radiation combined injury exist, and mechanisms underlying the high mortality associated with complex radiation injuries are poorly understood. Medical countermeasures are currently available for management of the non-radiation components of radiation combined injury, but it is not known whether treatments for other insults will be effective when the injury is combined with radiation exposure. Further research is needed to elucidate mechanisms behind the synergistic lethality of radiation combined injury and to identify targets for medical countermeasures. To address these issues, the National Institute of Allergy and Infectious Diseases convened a workshop to make recommendations on the development of animal models of radiation combined injury, possible mechanisms of radiation combined injury, and future directions for countermeasure research, including target identification and end points to evaluate treatment efficacy.

Steps toward targeted insertional mutagenesis with class II transposable elements.

Insertional mutagenesis can be achieved by a variety of approaches, including both random and targeted methods. In contrast to chemical mutagenesis, insertional mutagens provide a molecular tag, thereby allowing rapid identification of the mutated genomic region. Integration into defined genomic locations has great utility for both gene insertion and mutagenesis. Our laboratories have explored targeted integration through the use of transposases coupled to defined DNA-binding domains. This technology holds great promise for targeted insertional mutagenesis by biasing integration events to regions recognized by the chosen DNA-binding domain. Herein, we provide a brief background on targeted transposon integration and detailed protocols for testing chimeric transposases in both mammalian cell culture and insect embryos.

The role of mTOR inhibition in augmenting radiation induced autophagy.

Radiation affects both tumor and normal tissues, limiting the total delivered radiation dose. Therefore, novel ways to exploit molecular targets and improve the therapeutic ratio are continually being investigated. Autophagy plays an important role in cancer cell death decisions, particularly in solid tumors. This is counterbalanced by its function in cellular energy preservation. Recent studies have attempted to exploit autophagy in order to improve therapeutic ratio. However, direct inhibition of autophagy has been demonstrated to promote cancer cell death or survival dependent on cell type and condition. The mammalian target of rapamycin (mTOR) also regulates autophagy, as well as cell survival and proliferation pathways. Therefore, inhibition at this level of signaling would represent an excellent therapeutic target as it would limit cell growth, decrease cell proliferation, and boost autophagocytosis. Current investigations of mTOR inhibitors in combination with radiation appear to potentiate radiation's ability to induce autophagy. Further studies are necessary to fully elucidate which tumors have the most robust induction of autophagy in response to mTOR inhibition and radiation.

CXCR4 down-regulation by small interfering RNA inhibits invasion and tubule formation of human retinal microvascular endothelial cells.

The progressive alterations to the retinal microvasculature in diabetic retinopathy are known to cause vision loss. Chemokines are characterized by their ability to induce cell invasion, adhesion and migration. In this study, we used double siRNA transfection to transiently and selectively decrease the level of the endogenous CXCR4 in human retinal microvascular endothelial cells (HRMECs). The functional consequences of silencing CXCR4 expression in HRMECs were investigated using an endothelial cell migration assay and tubule formation in Matrigel. When CXCR4 expression was decreased with siRNA, HRMECs were less invasive and also resulted in markedly diminished vascular networks on Matrigel as compared to the controls. Additionally, hypoxia and VEGF, the factors affecting microvascular, regulate the expression level of CXCR4 in HRMECs, respectively, which have synergistic, additive effect in the HRMECs. As such, CXCR4 antagonists may become a therapeutic target for the treatment of retinal angiopathies.

Active integration: new strategies for transgenesis.

This paper presents novel methods for producing transgenic animals, with a further emphasis on how these techniques may someday be applied in gene therapy. There are several passive methods for transgenesis, such as pronuclear microinjection (PNI) and Intracytoplasmic Sperm Injection-Mediated Transgenesis (ICSI-Tr), which rely on the repair mechanisms of the host for transgene (tg) insertion. ICSI-Tr has been shown to be an effective means of creating transgenic animals with a transfection efficiency of approximately 45% of animals born. Furthermore, because this involves the injection of the transgene into the cytoplasm of oocytes during fertilization, limited mosaicism has traditionally occurred using this technique. Current active transgenesis techniques involve the use of viruses, such as disarmed retroviruses which can insert genes into the host genome. However, these methods are limited by the size of the sequence that can be inserted, high embryo mortality, and randomness of insertion. A novel active method has been developed which combines ICSI-Tr with recombinases or transposases to increase transfection efficiency. This technique has been termed "Active Transgenesis" to imply that the tg is inserted into the host genome by enzymes supplied into the oocyte during tg introduction. DNA based methods alleviate many of the costs and time associated with purifying enzyme. Further studies have shown that RNA can be used for the transposase source. Using RNA may prevent problems with continued transposase activity that can occur if a DNA transposase is integrated into the host genome. At present piggyBac is the most effective transposon for stable integration in mammalian systems and as further studies are done to elucidate modifications which improve piggyBac's specificity and efficacy, efficiency in creating transgenic animals should improve further. Subsequently, these methods may someday be used for gene therapy in humans.

piggyBac is a flexible and highly active transposon as compared to sleeping beauty, Tol2, and Mos1 in mammalian cells.

A nonviral vector for highly efficient site-specific integration would be desirable for many applications in transgenesis, including gene therapy. In this study we directly compared the genomic integration efficiencies of piggyBac, hyperactive Sleeping Beauty (SB11), Tol2, and Mos1 in four mammalian cell lines. piggyBac demonstrated significantly higher transposition activity in all cell lines whereas Mos1 had no activity. Furthermore, piggyBac transposase coupled to the GAL4 DNA-binding domain retains transposition activity whereas similarly manipulated gene products of Tol2 and SB11 were inactive. The high transposition activity of piggyBac and the flexibility for molecular modification of its transposase suggest the possibility of using it routinely for mammalian transgenesis.

Functional zinc finger/sleeping beauty transposase chimeras exhibit attenuated overproduction inhibition.

The sleeping beauty (SB) transposon system has potential utility in gene transfer applications but lacks specificity for genomic integration and exhibits overproduction inhibition which limits in vivo activity. Targeting transposition may be possible by coupling a specific DNA binding domain to the SB transposase, but it is not known if this strategy will preserve or disrupt activity of the system. We engineered and tested chimeric SB transposases with two different human zinc finger DNA binding domain elements, Sp1 and zinc finger 202 (ZNF202). Addition of Sp1 to the C-terminus abolished transposase activity whereas N-terminal addition of either Sp1 or ZNF202 did not. Transposition activity exhibited by N-terminal chimeras was increased to levels similar to native SB through the use of a hyperactive transposase (SB12) and activating transposon mutations. Importantly, addition of DNA binding domains to the transposase N-terminus resulted in attenuation of overproduction inhibition, a major limitation of this system. These findings suggest that SB transposase chimeras may have specific advantages over the native enzyme.

Site-directed genome modification: derivatives of DNA-modifying enzymes as targeting tools.

The modification of mammalian genomes is an important goal in gene therapy and animal transgenesis. To generate stable genetic and biochemical changes, the therapeutic genes or transgenes need to be incorporated into the host genome. Ideally, the integration of the foreign gene should occur at sites that ensure their continual expression in the absence of any unwanted side effects on cellular metabolism. In this article, we discuss the opportunities provided by natural DNA-modifying enzymes, such as transposases, recombinases and integrases, to mediate the stable integration of foreign genes into host genomes. In addition, we discuss the approaches that have been taken to improve the efficiency and to modify the site-specificity of these enzymes.

Site-directed genome modification: nucleic acid and protein modules for targeted integration and gene correction.

A variety of technological advances in recent years have made permanent genetic manipulation of an organism a technical possibility. As the details of natural biological processes for genome modification are elucidated, the enzymes catalyzing these events (transposases, recombinases, integrases and DNA repair enzymes) are being harnessed or modified for the purpose of intentional gene modification. Targeted integration and gene repair can be mediated by the DNA-targeting specificity inherent to a particular enzyme, or rely on user-designed specificities. Integration sites can be defined by using DNA base-pairing or protein-DNA interaction as a means of targeting. This review will describe recent progress in the development of 'user-targetable' systems, particularly highlighting the application of custom DNA-binding proteins or nucleic acid homology to confer specificity.

The controversial abscopal effect.

The abscopal effect is potentially important for tumor control and is mediated through cytokines and/or the immune system, mainly cell-mediated immunity. It results from loss of growth stimulatory and/or immunosuppressive factors from the tumor. Until recently, the abscopal effect referred to the distant effects seen after local radiation therapy. However, the term should now be used interchangeably with distant bystander effect. Through analysis of distant bystander effects of other local therapies, we discuss the poorly understood and researched radiation-induced abscopal effect. Although the abscopal effect has been described in various malignancies, it is a rarely recognized clinical event. The abscopal effect is still extremely controversial with known data that both support and refute the concept.