Next Generation Exon 51 Skipping Antisense Oligonucleotides for Duchenne Muscular Dystrophy.

In the last two decades, antisense oligonucleotides (AONs) that induce corrective exon skipping have matured as promising therapies aimed at tackling the dystrophin deficiency that underlies the severe and progressive muscle fiber degeneration in Duchenne muscular dystrophy (DMD) patients. Pioneering first generation exon 51 skipping AONs like drisapersen and eteplirsen have more recently been followed up by AONs for exons 53 and 45, with, to date, a total of four exon skipping AON drugs having reached (conditional) regulatory US Food and Drug Administration (FDA) approval for DMD. Nonetheless, considering the limited efficacy of these drugs, there is room for improvement. The aim of this study was to develop more efficient [2'-O-methyl-modified phosphorothioate (2'OMePS) RNA] AONs for DMD exon 51 skipping by implementing precision chemistry as well as identifying a more potent target binding site. More than a hundred AONs were screened in muscle cell cultures, followed by a selective comparison in the hDMD and hDMDdel52/mdx mouse models. Incorporation of 5-methylcytosine and position-specific locked nucleic acids in AONs targeting the drisapersen/eteplirsen binding site resulted in 15-fold higher exon 51 skipping levels compared to drisapersen in hDMDdel52/mdx mice. However, with similarly modified AONs targeting an alternative site in exon 51, 65-fold higher skipping levels were obtained, restoring dystrophin up to 30% of healthy control. Targeting both sites in exon 51 with a single AON further increased exon skipping (100-fold over drisapersen) and dystrophin (up to 40%) levels. These dystrophin levels allowed for normalization of creatine kinase (CK) and lactate dehydrogenase (LDH) levels, and improved motor function in hDMDdel52/mdx mice. As no major safety observation was obtained, the improved therapeutic index of these next generation AONs is encouraging for further (pre)clinical development.

Chromate-Free Corrosion Protection Strategies for Magnesium Alloys-A Review: Part III-Corrosion Inhibitors and Combining Them with Other Protection Strategies.

Owing to the unique active corrosion protection characteristic of hexavalent chromium-based systems, they have been projected to be highly effective solutions against the corrosion of many engineering metals. However, hexavalent chromium, rendered a highly toxic and carcinogenic substance, is being phased out of industrial applications. Thus, over the past few years, extensive and concerted efforts have been made to develop environmentally friendly alternative technologies with comparable or better corrosion protection performance to that of hexavalent chromium-based technologies. The introduction of corrosion inhibitors to a coating system on magnesium surface is a cost-effective approach not only for improving the overall corrosion protection performance, but also for imparting active inhibition during the service life of the magnesium part. Therefore, in an attempt to resemble the unique active corrosion protection characteristic of the hexavalent chromium-based systems, the incorporation of inhibitors to barrier coatings on magnesium alloys has been extensively investigated. In Part III of the Review, several types of corrosion inhibitors for magnesium and its alloys are reviewed. A discussion of the state-of-the-art inhibitor systems, such as iron-binding inhibitors and inhibitor mixtures, is presented, and perspective directions of research are outlined, including in silico or computational screening of corrosion inhibitors. Finally, the combination of corrosion inhibitors with other corrosion protection strategies is reviewed. Several reported highly protective coatings with active inhibition capabilities stemming from the on-demand activation of incorporated inhibitors can be considered a promising replacement for hexavalent chromium-based technologies, as long as their deployment is adequately addressed.

Chromate-Free Corrosion Protection Strategies for Magnesium Alloys-A Review: Part II-PEO and Anodizing.

Although hexavalent chromium-based protection systems are effective and their long-term performance is well understood, they can no longer be used due to their proven Cr(VI) toxicity and carcinogenic effect. The search for alternative protection technologies for Mg alloys has been going on for at least a couple of decades. However, surface treatment systems with equivalent efficacies to that of Cr(VI)-based ones have only begun to emerge much more recently. It is still proving challenging to find sufficiently protective replacements for Cr(VI) that do not give rise to safety concerns related to corrosion, especially in terms of fulfilling the requirements of the transportation industry. Additionally, in overcoming these obstacles, the advantages of newly introduced technologies have to include not only health safety but also need to be balanced against their added cost, as well as being environmentally friendly and simple to implement and maintain. Anodizing, especially when carried out above the breakdown potential (technology known as Plasma Electrolytic Oxidation (PEO)) is an electrochemical oxidation process which has been recognized as one of the most effective methods to significantly improve the corrosion resistance of Mg and its alloys by forming a protective ceramic-like layer on their surface that isolates the base material from aggressive environmental agents. Part II of this review summarizes developments in and future outlooks for Mg anodizing, including traditional chromium-based processes and newly developed chromium-free alternatives, such as PEO technology and the use of organic electrolytes. This work provides an overview of processing parameters such as electrolyte composition and additives, voltage/current regimes, and post-treatment sealing strategies that influence the corrosion performance of the coatings. This large variability of the fabrication conditions makes it possible to obtain Cr-free products that meet the industrial requirements for performance, as expected from traditional Cr-based technologies.

Chromate-Free Corrosion Protection Strategies for Magnesium Alloys-A Review: PART I-Pre-Treatment and Conversion Coating.

Corrosion protection systems based on hexavalent chromium are traditionally perceived to be a panacea for many engineering metals including magnesium alloys. However, bans and strict application regulations attributed to environmental concerns and the carcinogenic nature of hexavalent chromium have driven a considerable amount of effort into developing safer and more environmentally friendly alternative techniques that provide the desired corrosion protection performance for magnesium and its alloys. Part I of this review series considers the various pre-treatment methods as the earliest step involved in the preparation of Mg surfaces for the purpose of further anti-corrosion treatments. The decisive effect of pre-treatment on the corrosion properties of both bare and coated magnesium is discussed. The second section of this review covers the fundamentals and performance of conventional and state-of-the-art conversion coating formulations including phosphate-based, rare-earth-based, vanadate, fluoride-based, and LDH. In addition, the advantages and challenges of each conversion coating formulation are discussed to accommodate the perspectives on their application and future development. Several auspicious corrosion protection performances have been reported as the outcome of extensive ongoing research dedicated to the development of conversion coatings, which can potentially replace hazardous chromium(VI)-based technologies in industries.

Water-Mediated Epoxy/Surface Adhesion: Understanding the Interphase Region.

Epoxy resins coatings are commonly found in corrosion protection coatings but the presence of water can affect their adhesion to the substrate, often weakening the adhesion of the coating to the solid, reducing its efficiency. Nevertheless, small amounts of water can enhance the epoxy/substrate interactions. In this work, the interphase region of an epoxy precursor and metal oxide substrates is investigated using molecular simulations and it is found that water accumulates between the epoxy layer and the solid substrate. At high water concentrations (9 wt %) the interaction between the epoxy precursor and the solid surface is weakened regardless of the nature of the solid, but at low water concentrations the nature of the solid surface becomes important. For hematite, the presence of water decreases the strength of adhesion but for goethite the presence of a small amount of water (3 wt %) enhances the adhesion to the surface resulting in a densification at the interface.

A Cost-Consequence Analysis of Different Screening Procedures in Alzheimer's Disease: Results from the MOPEAD Project.

BACKGROUND: For care planning and support, under-detection and late diagnosis of Alzheimer's disease (AD) is a great challenge. Models of Patient-Engagement for Alzheimer's Disease (MOPEAD) is an EU-funded project aiming at testing different strategies to improve this situation. OBJECTIVE: To make a cost-consequence analysis of MOPEAD. METHODS: Four screening strategies were tested in five countries (Germany, the Netherlands, Slovenia, Spain, and Sweden): 1) a web-approach; 2) Open-House initiative; 3) in primary care; and 4) by diabetes specialists. Persons-at-risk of AD in all strategies were offered referral to a hospital-based specialist. The primary health-economic outcome was the cost per true-positive case (TP) of AD from the screened population. RESULTS: Of 2,847 screened persons, 1,121 screened positive (39%), 402 were evaluated at memory clinics (14%), and 236 got an AD diagnosis (8%). The cost per TP of those screened was €3,115 with the web-approach, €2,722 with the Open-House, €1,530 in primary care, and €1,190 by diabetes specialists. Sensitivity analyses that more likely reflect the real-world situation confirmed the results. The number-needed-to-screen was 30 with the web-approach, 8 with the Open-House and primary care, and 6 with the diabetes specialists.There were country differences in terms of screening rates, referrals to memory clinics, staff-types involved, and costs per TP. CONCLUSION: In primary care and by the diabetes specialist, the costs per TP/screened population were lowest, but the capacity of such settings to identify cases with AD-risk must be discussed. Hence new diagnostic strategies such as web-solutions and Open-House initiatives may be valuable after modifications.

Cyclic Peptides to Improve Delivery and Exon Skipping of Antisense Oligonucleotides in a Mouse Model for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a severe, progressive muscle wasting disorder caused by reading frame disrupting mutations in the DMD gene. Exon skipping is a therapeutic approach for DMD. It employs antisense oligonucleotides (AONs) to restore the disrupted open reading frame, allowing the production of shorter, but partly functional dystrophin protein as seen in less severely affected Becker muscular dystrophy patients. To be effective, AONs need to be delivered and effectively taken up by the target cells, which can be accomplished by the conjugation of tissue-homing peptides. We performed phage display screens using a cyclic peptide library combined with next generation sequencing analyses to identify candidate muscle-homing peptides. Conjugation of the lead peptide to 2'-O-methyl phosphorothioate AONs enabled a significant, 2-fold increase in delivery and exon skipping in all analyzed skeletal and cardiac muscle of mdx mice and appeared well tolerated. While selected as a muscle-homing peptide, uptake was increased in liver and kidney as well. The homing capacity of the peptide may have been overruled by the natural biodistribution of the AON. Nonetheless, our results suggest that the identified peptide has the potential to facilitate delivery of AONs and perhaps other compounds to skeletal and cardiac muscle.

Intracellular Distribution and Nuclear Activity of Antisense Oligonucleotides After Unassisted Uptake in Myoblasts and Differentiated Myotubes In Vitro.

Clinical efficacy of antisense oligonucleotides (AONs) for the treatment of neuromuscular disorders depends on efficient cellular uptake and proper intracellular routing to the target. Selection of AONs with highest in vitro efficiencies is usually based on chemical or physical methods for forced cellular delivery. Since these methods largely bypass existing natural mechanisms for membrane passage and intracellular trafficking, spontaneous uptake and distribution of AONs in cells are still poorly understood. Here, we report on the unassisted uptake of naked AONs, so-called gymnosis, in muscle cells in culture. We found that gymnosis works similarly well for proliferating myoblasts as for terminally differentiated myotubes. Cell biological analyses combined with microscopy imaging showed that a phosphorothioate backbone promotes efficient gymnosis, that uptake is clathrin mediated and mainly results in endosomal-lysosomal accumulation. Nuclear localization occurred at a low level, but the gymnotically delivered AONs effectively modulated the expression of their nuclear RNA targets. Chloroquine treatment after gymnotic delivery helped increase nuclear AON levels. In sum, we demonstrate that gymnosis is feasible in proliferating and non-proliferating muscle cells and we confirm the relevance of AON chemistry for uptake and intracellular trafficking with this method, which provides a useful means for bio-activity screening of AONs in vitro.

Feasibility of SPECT-CT Imaging to Study the Pharmacokinetics of Antisense Oligonucleotides in a Mouse Model of Duchenne Muscular Dystrophy.

Antisense oligonucleotides (AONs) are promising candidates for treatment of Duchenne muscular dystrophy (DMD), a severe and progressive disease resulting in premature death. However, more knowledge on the pharmacokinetics of new AON drug candidates is desired for effective application in the clinic. We assessed the feasibility of using noninvasive single-photon emission computed tomography-computed tomography (SPECT-CT) imaging to determine AON pharmacokinetics in vivo. To this end, a 2'-O-methyl phosphorothioate AON was radiolabeled with 123I or 111In, and administered to mdx mice, a rodent model of DMD. SPECT-CT imaging was performed to determine AON tissue levels, and the results were compared to data obtained with invasive analysis methods (scintillation counting and a ligation-hybridization assay). We found that SPECT-CT data obtained with 123I-AON and 111In-AON were qualitatively comparable to data derived from invasive analytical methods, confirming the feasibility of using SPECT-CT analysis to study AON pharmacokinetics. Notably, also AON uptake in skeletal muscle, the target tissue in DMD, could be readily quantified using SPECT-CT imaging, which was considered a particular challenge in mice, due to their small size. In conclusion, our results demonstrate that SPECT-CT imaging allows for noninvasive characterization of biodistribution and pharmacokinetics of AONs, thereby enabling quantitative comparisons between different radiolabeled AON drug candidates and qualitative conclusions about the corresponding unmodified parent AONs. This technology may contribute to improved (pre)clinical drug development, leading to drug candidates with optimized characteristics in vivo.

Reproducibility of hippocampal atrophy rates measured with manual, FreeSurfer, AdaBoost, FSL/FIRST and the MAPS-HBSI methods in Alzheimer's disease.

The purpose of this study is to assess the reproducibility of hippocampal atrophy rate measurements of commonly used fully-automated algorithms in Alzheimer disease (AD). The reproducibility of hippocampal atrophy rate for FSL/FIRST, AdaBoost, FreeSurfer, MAPS independently and MAPS combined with the boundary shift integral (MAPS-HBSI) were calculated. Back-to-back (BTB) 3D T1-weighted MPRAGE MRI from the Alzheimer's Disease Neuroimaging Initiative (ADNI1) study at baseline and year one were used. Analysis on 3 groups of subjects was performed - 562 subjects at 1.5T, a 75 subject group that also had manual segmentation and 111 subjects at 3T. A simple and novel statistical test based on the binomial distribution was used that handled outlying data points robustly. Median hippocampal atrophy rates were -1.1%/year for healthy controls, -3.0%/year for mildly cognitively impaired and -5.1%/year for AD subjects. The best reproducibility was observed for MAPS-HBSI (1.3%), while the other methods tested had reproducibilities at least 50% higher at 1.5T and 3T which was statistically significant. For a clinical trial, MAPS-HBSI should require less than half the subjects of the other methods tested. All methods had good accuracy versus manual segmentation. The MAPS-HBSI method has substantially better reproducibility than the other methods considered.

Evaluation of 2'-Deoxy-2'-fluoro Antisense Oligonucleotides for Exon Skipping in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder typically caused by frame-shifting mutations in the DMD gene. Restoration of the reading frame would allow the production of a shorter but partly functional dystrophin protein as seen in Becker muscular dystrophy patients. This can be achieved with antisense oligonucleotides (AONs) that induce skipping of specific exons during pre-mRNA splicing. Different chemical modifications have been developed to improve AON properties. The 2'-deoxy-2'-fluoro (2F) RNA modification is attractive for exon skipping due to its ability to recruit ILF2/3 proteins to the 2F/pre-mRNA duplex, which resulted in enhanced exon skipping in spinal muscular atrophy models. In this study, we examined the effect of two different 2'-substituted AONs (2'-F phosphorothioate (2FPS) and 2'-O-Me phosphorothioate (2OMePS)) on exon skipping in DMD cell and animal models. In human cell cultures, 2FPS AONs showed higher exon skipping levels than their isosequential 2OMePS counterparts. Interestingly, in the mdx mouse model, 2FPS was less efficient than 2OMePS and suggested safety issues as evidenced by increased spleen size and weight loss. Our results do not support a clinical application for 2FPS AON.

The corrosion protection of AA2024-T3 aluminium alloy by leaching of lithium-containing salts from organic coatings.

Lithium carbonate and lithium oxalate were incorporated as leachable corrosion inhibitors in model organic coatings for the protection of AA2024-T3. The coated samples were artificially damaged with a scribe. It was found that the lithium-salts are able to leach from the organic coating and form a protective layer in the scribe on AA2024-T3 under neutral salt spray conditions. The present paper shows the first observation and analysis of these corrosion protective layers, generated from lithium-salt loaded organic coatings. The scribed areas were examined by scanning and transmission electron microscopy before and after neutral salt spray exposure (ASTM-B117). The protective layers typically consist of three different layered regions, including a relatively dense layer near the alloy substrate, a porous middle layer and a flake-shaped outer layer, with lithium uniformly distributed throughout all three layers. Scanning electron microscopy and white light interferometry surface roughness measurements demonstrate that the formation of the layer occurs rapidly and, therefore provides an effective inhibition mechanism. Based on the observation of this work, a mechanism is proposed for the formation of these protective layers.

Peptide conjugation of 2'-O-methyl phosphorothioate antisense oligonucleotides enhances cardiac uptake and exon skipping in mdx mice.

Antisense oligonucleotide (AON)-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy that is currently being tested in various clinical trials. This approach is based on restoring the open reading frame of dystrophin transcripts resulting in shorter but partially functional dystrophin proteins as found in patients with Becker muscular dystrophy. After systemic administration, a large proportion of AONs ends up in the liver and kidneys. Therefore, enhancing AON uptake by skeletal and cardiac muscle would improve the AONs' therapeutic effect. For phosphorodiamidate morpholino oligomer, AONs use nonspecific positively charged cell penetrating peptides to enhance efficacy. However, this is challenging for negatively charged 2'-O-methyl phosphorothioate oligomer. Therefore, we screened a 7-mer phage display peptide library to identify muscle and heart homing peptides in vivo in the mdx mouse model and found a promising candidate peptide capable of binding muscle cells in vitro and in vivo. Upon systemic administration in dystrophic mdx mice, conjugation of a 2'-O-methyl phosphorothioate AON to this peptide indeed improved uptake in skeletal and cardiac muscle, and resulted in higher exon skipping levels with a significant difference in heart and diaphragm. Based on these results, peptide conjugation represents an interesting strategy to enhance the therapeutic effect of exon skipping with 2'-O-methyl phosphorothioate AONs for Duchenne muscular dystrophy.

Critical assessment of intramodality 3D ultrasound imaging for prostate IGRT compared to fiducial markers.

PURPOSE: A quantitative 3D intramodality ultrasound (US) imaging system was verified for daily in-room prostate localization, and compared to prostate localization based on implanted fiducial markers (FMs). METHODS: Thirteen prostate patients underwent multiple US scans during treatment. A total of 376 US-scans and 817 matches were used to determine the intra- and interoperator variability. Additionally, eight other patients underwent daily prostate localization using both US and electronic portal imaging (EPI) with FMs resulting in 244 combined US-EPI scans. Scanning was performed with minimal probe pressure and a correction for the speed of sound aberration was performed. Uncertainties of both US and FM methods were assessed. User variability of the US method was assessed. RESULTS: The overall US user variability is 2.6 mm. The mean differences between US and FM are: 2.5 ± 4.0 mm (LR), 0.6 ± 4.9 mm (SI), and -2.3 ± 3.6 mm (AP). The intramodality character of this US system mitigates potential errors due to transducer pressure and speed of sound aberrations. CONCLUSIONS: The overall accuracy of US (3.0 mm) is comparable to our FM workflow (2.2 mm). Since neither US nor FM can be considered a gold standard no conclusions can be drawn on the superiority of either method. Because US imaging captures the prostate itself instead of surrogates no invasive procedure is required. It requires more effort to standardize US imaging than FM detection. Since US imaging does not involve a radiation burden, US prostate imaging offers an alternative for FM EPI positioning.

Active breathing control in combination with ultrasound imaging: a feasibility study of image guidance in stereotactic body radiation therapy of liver lesions.

PURPOSE: Accurate tumor positioning in stereotactic body radiation therapy (SBRT) of liver lesions is often hampered by motion and setup errors. We combined 3-dimensional ultrasound imaging (3DUS) and active breathing control (ABC) as an image guidance tool. METHODS AND MATERIALS: We tested 3DUS image guidance in the SBRT treatment of liver lesions for 11 patients with 88 treatment fractions. In 5 patients, 3DUS imaging was combined with ABC. The uncertainties of US scanning and US image segmentation in liver lesions were determined with and without ABC. RESULTS: In free breathing, the intraobserver variations were 1.4 mm in left-right (L-R), 1.6 mm in superior-inferior (S-I), and 1.3 mm anterior-posterior (A-P). and the interobserver variations were 1.6 mm (L-R), 2.8 mm (S-I), and 1.2 mm (A-P). The combined uncertainty of US scanning and matching (inter- and intraobserver) was 4 mm (1 SD). The combined uncertainty when ABC was used reduced by 1.7 mm in the S-I direction. For the L-R and A-P directions, no significant difference was observed. CONCLUSION: 3DUS imaging for IGRT of liver lesions is feasible, although using anatomic surrogates in the close vicinity of the lesion may be needed. ABC-based breath-hold in midventilation during 3DUS imaging can reduce the uncertainty of US-based 3D table shift correction.

Dose reduction in LDR brachytherapy by implanted prostate gold fiducial markers.

PURPOSE: The dosimetric impact of gold fiducial markers (FM) implanted prior to external beam radiotherapy of prostate cancer on low dose rate (LDR) brachytherapy seed implants performed in the context of combined therapy was investigated. METHODS: A virtual water phantom was designed containing a single FM. Single and multi source scenarios were investigated by performing Monte Carlo dose calculations, along with the influence of varying orientation and distance of the FM with respect to the sources. Three prostate cancer patients treated with LDR brachytherapy for a recurrence following external beam radiotherapy with implanted FM were studied as surrogate cases to combined therapy. FM and brachytherapy seeds were identified on post implant CT scans and Monte Carlo dose calculations were performed with and without FM. The dosimetric impact of the FM was evaluated by quantifying the amplitude of dose shadows and the volume of cold spots. D(90) was reported based on the post implant CT prostate contour. RESULTS: Large shadows are observed in the single source-FM scenarios. As expected from geometric considerations, the shadows are dependent on source-FM distance and orientation. Large dose reductions are observed at the distal side of FM, while at the proximal side a dose enhancement is observed. In multisource scenarios, the importance of shadows appears mitigated, although FM at the periphery of the seed distribution caused underdosage (

Evaluation of the antibacterial spectrum of drosocin analogues.

Drosocin is a 19-mer, cationic antimicrobial peptide from Drosophila melanogaster. The aim of the study was to examine the antibacterial spectrum of unglycosylated drosocin analogues. Furthermore, the amino acid sequence of DnaK, drosocin's intracellular target, from susceptible species was aligned and studied for sequence homology. From this a panel of 31 bacterial strains, including Salmonella strains with truncated lipopolysaccharide structures, was tested for susceptibility towards three drosocin analogues. Available bacterial DnaK amino acid sequences were retrieved from the ExPASy proteomics server of the Swiss Institute of Bioinformatics studied for sequence homology. Seventeen of the 31 strains tested were susceptible for the drosocin analogues. Minimal inhibitory concentration values against mainly Gram-negative bacteria ranged from 3.1 to 100 microm. With the exception of Micrococcus luteus and Xanthomonas campestris all drosocin analogue-susceptible strains were Enterobacteriaceae showing a high DnaK amino acid sequence homology.

Biological evaluation of Tyr6 and Ser7 modified drosocin analogues.

An array of analogues of the cationic antimicrobial peptide drosocin was synthesized containing substitutions of Tyr6 and Ser7 in order to increase the proteolytic stability. Stabilizing the N-terminus with unnatural amino acids increased the serum stability of analogues by almost a factor 30 over an 8 h period.

The potential of virus-induced gene silencing for speeding up functional characterization of plant genes.

Virus-induced gene silencing (VIGS) has been shown to be of great potential in plant reverse genetics. Advantages of VIGS over other approaches, such as T-DNA or transposon tagging, include the circumvention of plant transformation, methodological simplicity and robustness, and speedy results. These features make VIGS an attractive alternative instrument in functional genomics, even in a high throughput fashion. The system is already well established in Nicotiana benthamiana; however, efforts are being made to improve VIGS in other species, including monocots. Current research is focussed on unravelling the mechanisms of post-transcriptional gene silencing and VIGS, as well as on finding novel viral vectors in order to broaden the host species spectrum. We examined how VIGS has been used to assess gene functions in plants, including molecular mechanisms involved in the process, available methodological elements, such as vectors and inoculation procedures, and we looked for examples in which the system has been applied successfully to characterize gene function in plants.