Therapeutic approaches for lysosomal storage diseases: a patent update.

Lysosomal Storage Diseases (LSDs) are inherited metabolic disorders caused by specific lysosomal protein deficiencies, which lead to abnormal storage of macromolecular substrates. Most LSDs are characterized by central nervous system (CNS) pathology, intracellular deposition and protein aggregation, events also found in age-related neurodegenerative diseases. Over the past two decades, a few approaches for the cure of these disorders have been approved for clinical use, i.e. enzyme replacement therapy (ERT) and substrate reduction therapy (SRT). However, these treatments are hampered by major limitations, such as the poor biodistribution in the CNS for ERT and severe side effects for SRT. Several additional therapeutic strategies have been proposed. In particular clinical trials are ongoing based on enzyme enhancement by pharmacological chaperones, i.e. small molecule compounds able to increases the residual activity of the lysosomal enzyme, and gene therapy approaches. In addition, recent patents in the field provide evidence that many efforts are currently dedicated to i) improve the properties of enzymes used for ERT, ii) find new pharmacological chaperones without inhibitory effects on enzyme activity and iii) combine gene therapy approaches with genome editing methods.

MicroRNAs and Molecular Mechanisms of Neurodegeneration.

During the last few years microRNAs (miRNAs) have emerged as key mediators of post-transcriptional and epigenetic regulation of gene expression. MiRNAs targets, identified through gene expression profiling and studies in animal models, depict a scenario where miRNAs are fine-tuning metabolic pathways and genetic networks in both plants and animals. MiRNAs have shown to be differentially expressed in brain areas and alterations of miRNAs homeostasis have been recently correlated to pathological conditions of the nervous system, such as cancer and neurodegeneration. Here, we review and discuss the most recent insights into the involvement of miRNAs in the neurodegenerative mechanisms and their correlation with significant neurodegenerative disorders.

Lipid-based nanocarriers for CNS-targeted drug delivery.

Nanotechnology exerts an increasing impact on the development of more effective tools for the diagnosis and treatment of human diseases. This applies in particular to central nervous system (CNS) disorders. Development of therapeutics for CNS is, in fact, one of the most challenging areas in drug development, mainly due to the presence of the blood-brain barrier (BBB) which separates the blood from the cerebral parenchyma thus limiting the brain uptake of the vast majority of neurotherapeutic agents. Among the several strategies which have been developed over the last years in order to overcome this problem, nanotechnology-based approaches have gained increasing attention as the most promising strategies for CNS targeted drug delivery. Nanocarriers offer several advantages such as the possibility to maintain drug levels in a therapeutically desirable range, as well as the increase of half-lives, solubility, stability and permeability of drugs. Furthermore, the system can be designed in such a way as to release the drug in a controlled way or in a triggered way. This review focuses on lipid-based nanocarriers and more specifically on liposomes, lipid-core micelles, and lipid nanocapsules, and provides an update on their composition and use, including recent patents in the field.

Recent developments in therapeutic approaches for lysosomal storage diseases.

Genetic mutations that cause specific lysosomal protein deficiencies account for more than 45 Lysosomal Storage Diseases (LSDs), mostly pre-adult disorders which are associated with neurological symptoms and mental retardation. Interestingly, such diseases are often characterized by intracellular deposition and protein aggregation, events also found in age-related neurodegenerative diseases. During the past twenty years, different approaches have been introduced for the treatment of these disorders, several of which are now in routine clinical use or clinical trials. Among them, enzyme replacement therapy (ERT) represented a major progress. However, the usefulness of ERT is limited due to the fact that enzyme distribution is insufficient and treatment costs are very high. A further novel therapeutic option for LSDs is based on the use of small molecules, that can either inhibit a key enzyme which is responsible for substrate synthesis (substrate reduction) or act as a chaperone to increase the residual activity of the lysosomal enzyme (pharmacological chaperones). In addition, recently various gene therapy approaches have been developed, mostly based on adeno-associated and lentiviral vectors, and strategies based on stem cells administration are beginning their route. This review provides an update of the status of research on LSDs therapeutic approaches, including recent patents in the field.

Fibroblasts from PS1 mutated pre-symptomatic subjects and Alzheimer's disease patients share a unique protein levels profile.

In Alzheimer's disease (AD), a major goal is to improve early detection, as the diagnosis cannot be made until patients exhibit a noticeable decline in cognition and the brain is irreversibly damaged. With this aim in mind, we performed proteome analysis of familial AD fibroblasts from both demented and pre-symptomatic subjects, using a 2D-PAGE based approach and then identifying proteins by mass spectrometry. We compared primary fibroblast cultures from skin biopsy of presenilin 1 (PS1) mutated patients, pre-symptomatic subjects carrying mutations in the PS1 gene but healthy at the time of skin biopsy, and age-matched individuals as control. 15 differentially expressed proteins were identified in PS1 mutated fibroblasts, related to cell adhesion and cytoskeleton, energy and glucose metabolism, stress response and ubiquitin-proteasome system, and signal transduction. Interestingly, many of these proteins have been previously associated with AD and neurodegeneration. Overall results indicated that a unique protein profile can be identified by peripheral cell analysis of PS1 mutated individuals, and showed that fibroblasts are a useful cell model for pathological investigations as well as identification of potential biomarkers for AD diagnosis at early stages.

New perspectives for the diagnosis of Alzheimer's disease.

Alzheimer's disease is the most common cause of dementia in the elderly. Currently its clinical assessment is based on the exclusion of other forms of dementia and a definitive diagnosis requires a confirmation by examination of post-mortem brains. Therefore, there is a strong need to find easy measurable AD biomarkers that could facilitate the early diagnosis and monitoring the efficacy of the few therapies currently available. This would favor the development of further therapeutic approaches. Recently, dozens of biomarkers altered in peripheral tissues and body fluids have been patented by a variety of approaches, including transcriptomics, proteomics and peptidomics. However, assays for the routine laboratory diagnosis of AD are not available yet. The validation of these biomarkers is hindered by the fact that patient classification relies on clinical diagnosis that is not always accurate and this problem obstacles the enrollment of well characterized large patient cohorts needed for confirmation. This review provides an update of the status of research on AD peripheral biomarkers in the current post-genomic era, including recent patents in the field.

Lack of association between Alzheimer's disease and the promoter region polymorphisms of the nicastrin gene.

The biological analysis of nicastrin (NCSTN) shows its crucial role in gamma-cleavage of the amyloid precursor protein. Inhibition of NCSTN demonstrated altered gamma-cleavage activity, suggesting its potential implication in Alzheimer's disease (AD). We sequenced the NCSTN gene promoter region and found two promoter single nucleotide polymorphisms (SNPs) at putative transcription binding sites, -796T/G and -1216C/A. The association study using the promoter SNPs showed no significant genetic effect upon the development of AD. Haplotype analysis with the promoter SNPs and coding SNPs demonstrated no significant difference between familial AD cases and controls. Moreover, the genotype of each promoter SNP did not have an association with age-at-onset in AD. Our investigation suggests that the two promoter SNPs are unrelated to the development of AD, however, further investigation at the promoter region of NCSTN may be necessary to address its potential implication of gene expression in AD.

Association analysis between Alzheimer's disease and the Nicastrin gene polymorphisms.

The biological study of the Nicastrin protein shows its crucial role in the pathogenesis of Alzheimer's disease (AD). We tested the hypothesis that the Nicastrin (NCSTN) gene might be genetically associated with AD. The association analysis of two single nucleotide polymorphisms (SNPs) in the coding region (cSNPs) of NCSTN were performed in an Italian population. No evidence of association was obtained between the two SNPs investigated in sporadic and familial AD cases under the stratification of currently known genetic risk factors including the apolipoprotein E (APOE), the presenilins and the beta-amyloid precursor protein. The result suggests no apparent synergic interaction between the NCSTN and APOE epsilon 4 in the risk to develop the late onset sporadic form of AD. But considering its biological effects, the result can not exclude the NCSTN as candidate for genetic factor in AD. Further genetic study of the NCSTN would be necessary to evaluate the potential genetic involvement in AD.

The expression of the dodecameric ferritin in Listeria spp. is induced by iron limitation and stationary growth phase.

The Gram-positive bacterium Listeria innocua possesses an authentic ferritin with an unusual dodecameric assemblage that resembles the quaternary structure of the DNA-binding proteins designated Dps (DNA-binding proteins from starved cells). The L. innocua gene encoding the above protein, termed ferritin from Listeria innocua (fri), has been localized on a 3-kb HindIII chromosomal fragment cloned in the Escherichia coli strain DH5alphaF'. DNA sequence analysis reveals an open reading frame of 468 nucleotides matching perfectly the amino acid sequence of the protein. Primer extension analysis indicates the presence of two transcriptional startpoints located 36 (proximal) and 85 nt (distal) upstream the fri start codon, respectively. Each transcriptional startpoint is preceded by suitably located -10 and -35 elements, which match the sigma(A) (proximal) and sigma(B) (distal) consensus sequences.In L. innocua and Liseria monocytogenes, fri expression increases both upon entry into stationary phase and, more markedly, under low-iron growth conditions. The effect of iron is apparent in the exponential and stationary phases of growth. An up-regulation by iron limitation has never been observed in other proven ferritins and bacterioferritins, but has been reported for several members of the Dps family. The unusual regulation by iron of the Listeria ferritin gene provides further support to the evolutionary link with the Dps family and suggests that the iron storage function may not be the unique role of ferritin in the physiology of this bacterium.