Hurdles of environmental risk assessment procedures for advanced therapy medicinal products: comparison between the European Union and the United States.

An environmental risk assessment (ERA) consists of an analysis of the risks to human health and the environment that a medicinal product may cause due to its release during clinical development or after entering the market. Regulators in European Union (EU) and the United States (US) require that advanced therapy medicinal products (ATMPs) that are also genetically modified organisms (GMOs) undergo an ERA in order to be approved for marketing authorization. This work aims to review the regulatory issues that need to be taken into consideration for carrying out an ERA, comparing the EU and the US. The European regulatory framework for environmental procedures and the dissimilarities in its implementation across the Member States and its implications at a logistical level are analyzed in detail. In addition, this review provides a brief insight into the non-clinical and clinical assessments that should be carried out during the development of the product in order to conduct a successful ERA, and thus facilitate its marketing authorization and post-marketing monitoring. Finally, the need for a European harmonization regarding environmental procedures for ATMPs is discussed.

Molecular signature of the immune and tissue response to non-coding plasmid DNA in skeletal muscle after electrotransfer.

Electrotransfer of plasmid DNA in skeletal muscle is a common non-viral delivery method for both therapeutic genes and DNA vaccines. Yet, despite the similar approaches, an immune response is detrimental in gene therapy, but desirable for vaccines. However, the full nature of the immune and tissue responses to nucleic acids and electrotransfer in skeletal muscle has not been addressed. Here we used microarray analysis, fluorescence-activated cell sorting and quantitative polymerase chain reaction to obtain the molecular and cellular signature of the tissue and immune response to electrotransfer of saline and non-coding plasmid DNA. Saline electrotransfer resulted in limited infiltration and induction of a moderate damage-repair gene expression pattern not involving innate immune activation. However, plasmid electrotransfer augmented expression of the same genes in addition to inducing a strong innate immune response associated with pro-inflammatory infiltration. In particular, the inflammasome, Toll-like receptor 9 and other pattern recognition receptors able to respond to cytoplasmic DNA were upregulated. Several key differences in the nature of the inflammatory infiltrate and the kinetics of gene expression were also identified when comparing electrotransfer of conventional and CpG-free plasmids. Our data provide insights into the mechanisms of DNA detection and response in muscle that has relevance for non-viral gene therapy and DNA vaccination.

In vivo genetic engineering of murine pancreatic beta cells mediated by single-stranded adeno-associated viral vectors of serotypes 6, 8 and 9.

AIMS/HYPOTHESIS: The genetic engineering of pancreatic beta cells could be a powerful tool for examining the role of key genes in the cause and treatment of diabetes. Here we performed a comparative study of the ability of single-stranded (ss) adeno-associated viral vectors (AAV) of serotypes 6, 8 and 9 to transduce the pancreas in vivo. METHODS: AAV6, AAV8 and AAV9 vectors encoding marker genes were delivered to the pancreas via intraductal or systemic administration. Transduced cells were analysed by immunostaining. AAV9 vectors encoding hepatocyte growth factor (HGF) were delivered intraductally to a transgenic mouse model of type 1 diabetes and glycaemia was monitored. RESULTS: AAV6, AAV8 and AAV9 mediated efficient and long-term transduction of beta cells, with AAV6 and AAV8 showing the highest efficiency. However, alpha cells were poorly transduced. Acinar cells were transduced by the three serotypes tested and ductal cells only by AAV6. In addition, intraductal delivery resulted in higher AAV-mediated transduction of the pancreas than did systemic administration. As proof of concept, intraductal delivery of AAV9 vectors encoding for the beta cell anti-apoptotic and mitogenic HGF preserved beta cell mass, diminished lymphocytic infiltration of the islets and protected mice from autoimmune diabetes. CONCLUSIONS/INTERPRETATION: Intraductal administration of AAV6, AAV8 and AAV9 is an efficient way to genetically manipulate the pancreas in vivo. This technology may prove useful in the study of islet physiopathology and in assessment of new gene therapy approaches designed to regenerate beta cell mass during diabetes.

Switches in 6-phosphofructo-2-kinase isoenzyme expression during rat sperm maturation.

Here we analyzed Pfkfb3 and Pfkfb4 gene expression in rat testis development, isolated testicular cells and spermatozoa. Real time RT-PCR analysis during testis development showed the maximum expression of Pfkfb3 in pre-puber samples and of Pfkfb4 in adult samples. Western blot analysis showed that uPFK-2 protein, a product of Pfkfb3 gene, was present in all the cell types forming the seminiferous epithelium (Sertoli, interstitial and spermatogenic cells). In contrast, tPFK-2, a product of Pfkfb4 gene, was restricted to spermatogenic cells. Confocal analyses by indirect immunofluorescence also corroborated this expression pattern. Immunoblotting studies of isolated spermatozoa demonstrated the presence of uPFK-2 only in immature sperm and once spermatozoa became fully functional this isozyme was replaced by the testicular isozyme tPFK-2. Moreover, immunostaining confirmed that tPFK-2 was localized mainly in the acrosomal region of the sperm head and in the mid-piece of the flagellum, where other spermatogenic cell-specific glycolytic enzymes have been found.

Rapid human skin permeation and topical anaesthetic activity of a new amethocaine microemulsion.

We developed a fast-acting, topical, 4% (w/w) amethocaine microemulsion and tested its in vitro permeation in isolated human skin. Comparison with a commercial amethocaine gel (Ametop((R)) ) was performed using Franz diffusion cells. Permeability coefficient (k(p)), flux (J) and percentage permeation after 10 h of microemulsion application were, in all cases, 1.5 times higher than those of the gel. The values obtained for the P(1) parameter [1], 1.06.10(-2) cm (microemulsion) and 0.724.10(-2) cm (gel) indicate that the microemulsion excipients favour amethocaine deposition in the skin, increasing the permeability coefficient, amount of drug retained in the skin, and the flux achieved. Analgesic activity was also examined in rats made hyperalgesic or allodynic after carrageenan-induced inflammation. The rats were distributed into four groups (n = 5-9 per group), each group receiving topically either amethocaine microemulsion, amethocaine gel (Ametop), amethocaine subcutaneous infiltration or nothing (controls). In edematous paws, anti-hyperalgesic activity appeared at 4.2 and 13.8 min after application of amethocaine microemulsion and gel, respectively. These effects are lower than after 0.5% w/w amethocaine infiltration. Amethocaine microemulsion was the only topical formulation with an anti-allodynic effect, although this effect was less than with amethocaine infiltration. These results suggest that microemulsion could be a valuable formula for improving amethocaine permeation and thus bringing rapid pain relief.

Bacterial and viral etiology of serious infections in very young Filipino infants.

OBJECTIVE: Pneumonia, meningitis and other serious infections are leading causes of death in developing countries. As part of a multicenter study we aimed to determine the etiology of pneumonia, meningitis and other serious infections in a cohort of Filipino infants ages 90 days or younger. METHOD: During a 2-year period, 2053 infants age 90 days or younger presenting to 1 of 3 Manila community hospitals were screened; 873 had signs or symptoms suggestive of an infectious illness, and 608 were judged to have clinical features suggestive of severe infection and had laboratory workup including blood for culture and white blood cell count, nasopharyngeal aspirate for virology, cerebrospinal fluid culture when indicated and chest radiograph. Chest radiographs were read independently by 3 radiologists without knowledge of clinical findings. RESULTS: Of the 873 enrolled infants, 81 died (91%). After exclusion of presumed contaminants, positive bacterial culture from blood and/or cerebrospinal fluid was obtained in 35 infants (5.8%; 95% confidence interval 4%, 8%), 9 of whom died. The organisms responsible for meningitis were Acinetobacter spp. (4), Streptococcus pneumoniae (2), Escherichia coli (2), Enterobacter spp. (1), Pseudomonas aeruginosa (1), Haemophilus influenzae (1) and Staphylococcus aureus (1); those responsible for the other clinical diagnoses were Salmonella spp. (6), Enterobacter spp. (3), Streptococcus pyogenes (3), other Gram-negative organisms (8), S. pneumoniae (1) and Staphylococcus aureus (2). In 685 infants examined for viral causes of their illness, 223 viruses were isolated from 219 infants (32%; 95% confidence interval 28%, 36%). Enteroviruses were the most common potential pathogens identified (22% of infants studied), followed by respiratory syncytial virus (17%), rhinovirus (10%) and adenovirus (4%). Concomitant virus identification occurred in 10 of those with positive bacterial culture (29%; 95% confidence interval, 15%, 46%), with enterovirus being found in 7 of these cases. CONCLUSION: Many young Filipino infants with life-threatening illness were evaluated in this study. Thirty-five had infections attributable to bacteria, with Salmonella spp. being the most common, followed by Gram-negative organisms. Pneumococcus was an unusual cause.

Postpubertal disease status in diabetes and factor structure anomaly on the WISC-R.

Diabetic children have been found to display an anomalous factor structure on the Wechsler Intelligence Scale for Children-Revised (WISC-R) (Holmes, Cornwell, Dunlap, Chen, & Lee, 1992). The present study sought to extend this finding with a larger cross-regional sample of children to determine which, if any, demographic or disease factor(s) might be related to the anomalous structure. Results revealed that groups of older (> = 12 years) children and those with late disease onset (> = 5 years) exhibited an anomalous four-factor structure in which the traditional Perceptual Organization factor (II) split into two factors: Picture Completion and Picture Arrangement formed a visual discrimination factor; and Block Design and Object Assembly created a spatial conceptual factor. It is postulated that diabetic performance on this visual discrimination factor may reflect mild visual neuropathies, often associated with adolescence and postpubertal disease status.