A poly(ethylene) glycolylated peptide for ocular delivery compacts DNA into nanoparticles for gene delivery to post‐mitotic tissues in vivo

Background

We have previously shown that a novel synthetic peptide for ocular delivery (POD) can efficiently compact DNA and deliver it to cells in vitro. This observation prompted us to develop use of POD as a nonviral vector in vivo.

Methods

POD peptide was modified using poly(ethylene) glycol (PEG‐POD) and used to compact DNA into nanoparticles that were then analysed using electron microscopy, dynamic light scattering, and fluorescent labeling. Transfection efficiency and localization were determined 48 h post‐injection into the subretinal space of the mouse eye using luciferase and LacZ, respectively. Efficiency of ocular transfection was compared to two other PEGylated peptides: PEG‐TAT and PEG‐CK30.

Results

PEG‐POD can compact DNA and form discrete nanoparticles of approximately 136 nm that can penetrate and transduce the retinal pigment epithelium (RPE) in vivo. PEG‐POD significantly increased expression of plasmid DNA by 215‐fold, PEG‐TAT by 56.52‐fold, and PEG‐CK30 by 24.73‐fold relative to DNA injected alone. In all cases β‐galactosidase was observed primarily in the RPE layer after subretinal injection. Electrophysiological analyses of PEG‐POD transduced retina indicates an absence of PEG‐POD‐mediated toxicity. PEG‐POD can protect plasmid DNA from DNaseI digestion, resulting in significant transfection of the lung after intravenous injection in mice.

Conclusions

PEG‐POD was found to significantly increase gene delivery relative to both DNA alone and other pegylated peptides. These findings highlight the use of pegylated peptides, and specifically PEG‐POD, as novel gene delivery vectors. Copyright © 2009 John Wiley & Sons, Ltd.     

Non-viral in vivo immune gene therapy of cancer: combined strategies for treatment of systemic disease

Many patients with various types of cancers have already by the time of presentation, micrometastases in their tissues and are left after treatment in a minimal residual disease state [Am J Gastroenterol 95(12), 2000]. To prevent tumour recurrence these patients require a systemic based therapy, but current modalities are limited by toxicity or lack of efficacy. We have previously reported that immune reactivity to the primary tumour is an important regulator of micrometastases and determinant of prognosis. This suggests that recruitment of specific anti-tumour mechanisms within the primary tumour could be used advantageously for tumour control as either primary or neo-adjuvant treatments. Recently, we have focused on methods of stimulating immune eradication of solid tumours and minimal residual disease using gene therapy approaches. Gene therapy is now a realistic prospect and a number of delivery approaches have been explored, including the use of viral and non-viral vectors. Non-viral vectors have received significant attention since, in spite of their relative delivery inefficiency, they may be safer and have greater potential for delivery of larger genetic units. By in vivo electroporation of the primary tumour with plasmid expressing GM-CSF and B7-1, we aim to stimulate immune eradication of the treated tumour and associated metastases. In this symposium report, we describe an effective gene based approach for cancer immunotherapy by inducing cytokine and immune co-stimulatory molecule expression by the growing cells of the primary tumour using a plasmid electroporation gene delivery strategy. We discuss the potential for enhancement of this therapy by its application as a neoadjuvant to surgical excision and by its use in combination with suppressor T cell depletion.This article is a symposium paper from the Annual Meeting of the “International Society for Cell and Gene Therapy of Cancer”, held in Shenzhen, China, on 9–11 December 2005.     

Comparative LCA of treatment options for US scrap tires: material recycling and tire-derived fuel combustion

Purpose

This life cycle assessment (LCA) study compares two prevalent end-of-life (EOL) treatment methods for scrap tires: material recycling and energy recovery. The primary intended use of the study results is to inform stakeholders of the relative environmental burdens and trade-offs associated with these two EOL vehicle tire treatment methods. The study supports prioritization of the waste treatment hierarchy for this material stream in the US.

Methods

This LCA compares (1) material recycling through ambient-temperature mechanical processing and (2) energy recovery through co-incineration of both whole and preprocessed scrap tires at a cement kiln. The avoided burden recycling methodology reflects the substitution of virgin synthetic rubber used in asphalt modification with the ground tire rubber from material recycling and the substitution of conventional kiln fuels with the tire-derived fuel (TDF). Both attributional (ALCA) and consequential (CLCA) methodologies are used: the ALCA assesses the environmental profiles of the treatment methods and the CLCA examines the potential effects of shifting more scrap tires to material recycling. The attributional portion of the LCA study was conducted in accordance with ISO standards 14044 series.

Results

The results in both methodological approaches indicate that the material recycling scenario provides greater impact reductions than the energy recovery scenario in terms of the examined environmental impact potentials: energy demand, iron ore consumption, global warming potential, acidification, eutrophication, smog formation, and respiratory effects. The additional impact reductions from material recycling are significant, and the establishment of new infrastructure required for a shift to material recycling incurs relatively insignificant burdens. Sensitivity analyses indicate that this conclusion does not change for (1) a range of TDF heating values, (2) a decrease in the mixed scrap tire rubber-to-steel composition ratio, (3) two alternative electricity grid fuel mixes with higher and lower carbon dioxide emission rankings than that of the baseline scenario, or (4) a comparison of material recycling to energy recovery when TDF is used in pulp and paper mills instead of cement kilns.

Conclusions

These results provide a basis for more informed decision-making when prioritizing scrap tire waste treatment hierarchy.     

Binding of bovine T194A PrPC by PrPSc-specific antibodies

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases that are based on the misfolding of a cellular prion protein (PrP^C) into an infectious, pathological conformation (PrP^Sc). There is proof-of-principle evidence that a prion vaccine is possible but this is tempered with concerns of the potential dangers associated with induction of immune responses to a widely-expressed self-protein. By targeting epitopes that are specifically exposed upon protein misfolding, our group developed a vaccine that induces PrP^Sc-specific antibody responses. Here we consider the ability of this polyclonal antibody (SN6b) to bind to a mutant of PrP^C associated with spontaneous prion disease. Polyclonal antibodies were selected to mimic the vaccination outcome and also explore all possible protein conformations of the recombinant bovine prion protein with mutation T194A [bPrP(T194A)]. This mutant is a homolog of the human T183A mutation of PrP^C that is associated with early onset of familial dementia. With nanopore analysis, under non-denaturing conditions, we observed binding of the SN6b antibody to bPrP(T194A). This interaction was confirmed through ELISAs as well as immunoprecipitation of the recombinant and cellularly expressed forms of bPrP(T194A). This interaction did not promote formation of a protease resistant conformation of PrP in vitro. Collectively, these findings support the disease-specific approach for immunotherapy of prion diseases but also suggest that the concept of conformation-specific immunotherapy may be complicated in individuals who are genetically predisposed to PrP^C misfolding.     

Vitamin D Binding Protein Genotype Is Associated with Serum 25-Hydroxyvitamin D and PTH Concentrations,as Well as Bone Health in Children and Adolescents in Finland

Vitamin D binding protein (DBP)/group-specific component (Gc), correlates positively with serum vitamin D metabolites, and phenotype influences serum 25-hydroxyvitamin D (S-25(OH)D) concentration. The protein isoform has been associated with decreased bone mineral density (BMD) and increased fracture risk. We examined the role of GC genotypes in S-25(OH)D status and BMD in 231 Finnish children and adolescents aged 7−19 yr. BMD was measured with DXA from lumbar spine (LS), total hip, and whole body, and for 175 subjects, radial volumetric BMD was measured with pQCT. Background characteristic and total dietary intakes of vitamin D and calcium were collected. The concentrations of 25(OH)D, parathyroid hormone (PTH), calcium and other markers of calcium homeostasis were determined from blood and urine. Genotyping was based on single-nucleotide polymorphism (rs4588) in the GC gene. The genotype distribution was: GC 1/1 68%, GC 1/2 26% and GC 2/2 6%. A significant difference emerged in 25(OH)D and PTH concentrations between the genotypes, (p = 0.001 and 0.028 respectively, ANCOVA). There was also a linear trend in: Gc 2/2 had the lowest 25(OH)D and PTH concentrations (p = 0.025 and 0.012, respectively). Total hip bone mineral content was associated with GC genotype (BMC) (p = 0.05, ANCOVA) in boys. In regression analysis, after adjusting for relevant covariates, GC genotype was associated with LS BMC and strength and strain index (SSI) Z-score in both genders, and LS BMD in boys. In conclusion, the present study demonstrates the association between GC genotypes and S-25(OH)D and PTH concentrations. The results show the influence of DBP genetic variation on bone mass accrual in adolescence.     

A non membrane-targeted human soluble CD59 attenuates choroidal neovascularization in a model of age related macular degeneration

Age related macular degeneration (AMD) is the most common cause of blindness amongst the elderly. Approximately 10% of AMD patients suffer from an advanced form of AMD characterized by choroidal neovascularization (CNV). Recent evidence implicates a significant role for complement in the pathogenesis of AMD. Activation of complement terminates in the incorporation of the membrane attack complex (MAC) in biological membranes and subsequent cell lysis. Elevated levels of MAC have been documented on choroidal blood vessels and retinal pigment epithelium (RPE) of AMD patients. CD59 is a naturally occurring membrane bound inhibitor of MAC formation. Previously we have shown that membrane bound human CD59 delivered to the RPE cells of mice via an adenovirus vector can protect those cells from human complement mediated lysis ex vivo. However, application of those observations to choroidal blood vessels are limited because protection from MAC- mediated lysis was restricted only to the cells originally transduced by the vector. Here we demonstrate that subretinal delivery of an adenovirus vector expressing a transgene for a soluble non-membrane binding form of human CD59 can attenuate the formation of laser-induced choroidal neovascularization and murine MAC formation in mice even when the region of vector delivery is distal to the site of laser induced CNV. Furthermore, this same recombinant transgene delivered to the intravitreal space of mice by an adeno-associated virus vector (AAV) can also attenuate laser-induced CNV. To our knowledge, this is the first demonstration of a non-membrane targeting CD59 having biological potency in any animal model of disease in vivo. We propose that the above approaches warrant further exploration as potential approaches for alleviating complement mediated damage to ocular tissues in AMD.     

Inhibition of human leukocyte 5-lipoxygenase by 15-HPETE and related eicosanoids

The inhibition of human leukocyte 5-lipoxygenase by 15-hydroperoxyeicosatetraenoic acid and its chemical or enzymatic rearrangement products was investigated. 15-Hydroperoxyeicosatetraenoic acid was the most potent inhibitor tested. The inhibition was found to be time dependent and is not due to chemical or enzymatic decomposition products nor metabolism of 15-hydroperoxyeicosatetraenoic acid to 5,15-dihydroperoxyeicosatetraenoic acid.     

General practitioner participation in intranatal care in the northern region in 1983.

In 1983 a quarter of general practitioners in the Northern region of England cared for obstetric deliveries and half of these for a minimum of 10 deliveries a year. Most expected their intranatal work to remain at the same level or increase in the next 10 years. Most participating general practitioners did their own forceps deliveries and initiated inductions. Most out of hours deliveries were attended by the mother''s own general practitioner or a partner. A quarter of all respondents had cared for planned and unplanned home births. Few were happy about attending them, but most would provide planned home care if urged to do so.