The Nanotechnology-Based Approaches against Kirsten Rat Sarcoma-Mutated Cancers.

Kirsten rat sarcoma (KRAS) is a small GTPase which acts as a molecular switch to regulate several cell biological processes including cell survival, proliferation, and differentiation. Alterations in KRAS have been found in 25% of all human cancers, with pancreatic cancer (90%), colorectal cancer (45%), and lung cancer (35%) being the types of cancer with the highest mutation rates. KRAS oncogenic mutations are not only responsible for malignant cell transformation and tumor development but also related to poor prognosis, low survival rate, and resistance to chemotherapy. Although different strategies have been developed to specifically target this oncoprotein over the last few decades, almost all of them have failed, relying on the current therapeutic solutions to target proteins involved in the KRAS pathway using chemical or gene therapy. Nanomedicine can certainly bring a solution for the lack of specificity and effectiveness of anti-KRAS therapy. Therefore, nanoparticles of different natures are being developed to improve the therapeutic index of drugs, genetic material, and/or biomolecules and to allow their delivery specifically into the cells of interest. The present work aims to summarize the most recent advances related to the use of nanotechnology for the development of new therapeutic strategies against KRAS-mutated cancers.

Influence of initial clinical suspicion on the diagnostic yield of laboratory enzymatic testing in lysosomal storage disorders. Experience from a multispecialty hospital.

Lysosomal storage disorders (LSD) are a group of inherited metabolic diseases mainly caused by a deficiency of lysosomal hydrolases, resulting in a gradual accumulation of non-degraded substrates in different tissues causing the characteristic clinical manifestations of such disorders. Confirmatory tests of suspected LSD individuals include enzymatic and genetic testing. A well-oriented clinical suspicion can improve the cost-effectiveness of confirmatory tests and reduce the time expended to achieve the diagnosis. Thus, this work aims to retrospectively study the influence of clinical orientation on the diagnostic yield of enzymatic tests in LSD by retrieving clinical, biochemical, and genetic data obtained from subjects with suspicion of LSD. Our results suggest that the clinical manifestations at the time of diagnosis and the initial clinical suspicion can have a great impact on the diagnostic yield of enzymatic tests, and that clinical orientation performed in specialized clinical departments can contribute to improve it. In addition, the analysis of enzymatic tests as the first step in the diagnostic algorithm can correctly guide subsequent confirmatory genetic tests, in turn increasing their diagnostic yield. In summary, our results suggest that initial clinical suspicion plays a crucial role on the diagnostic yield of confirmatory enzymatic tests in LSD.

Clinical features and health-related quality of life in adult patients with mucopolysaccharidosis IVA: the Spanish experience.

BACKGROUND: Mucopolysaccharidosis (MPS) IVA or Morquio A syndrome is a progressive and disabling disease characterized by a deficiency of the enzyme N-acetylgalactosamine-6-sulphate sulphatase. Its clinical presentation is very heterogeneous and poorly understood in adults. The aim of this study was to describe the clinical manifestations of MPS IVA in adult patients in Spain and to assess their health-related quality of life (HRQoL). RESULTS: Thirty-three patients from nine reference centres participated in the study. The median age was 32 (interquartile range [IQR]: 20.5-40.5) years. The phenotype was classical in 54.5% of patients, intermediate in 33.3% of patients, and non-classical in 12.1% of patients. The most common clinical manifestation was bone dysplasia, with a median height of 118 (IQR: 106-136) cm. Other frequent clinical manifestations were hearing loss (75.7%), ligamentous laxity (72.7%), odontoid dysplasia (69.7%), limb deformities that required orthopaedic aids (mainly hip dysplasia and genu valgus) (63.6%), and corneal clouding (60.6%). In addition, 36.0% of patients had obstructive sleep apnoea/hypopnoea syndrome and 33.3% needed non-invasive ventilation. Cervical surgery and varisation osteotomy were the most common surgical interventions (36.4% each). Almost 80% of patients had mobility problems and 36.4% used a wheelchair at all times. Furthermore, 87.9% needed help with self-care, 33.3% were fully dependent, and 78.8% had some degree of pain. HRQoL according to the health assessment questionnaire was 1.43 (IQR: 1.03-2.00) in patients with the non-classical phenotype, but 2.5 (IQR: 1.68-3.00) in those with the classical phenotype. Seven patients were initiated on enzyme replacement therapy (ERT), but two of them were lost to follow-up. Lung function improved in four patients and slightly worsened in one patient. The distance achieved in the six-minute walk test increased in the four patients who could perform it. HRQoL was better in patients treated with elosulfase alfa, with a median (IQR) of 1.75 (1.25-2.34) versus 2.25 (1.62-3.00) in patients not treated with ERT. CONCLUSIONS: The study provides real-world data on patients with MPS IVA. Limited mobility, difficulties with self-care, dependence, and pain were common, together with poor HRQoL. The severity and heterogeneity of clinical manifestations require the combined efforts of multidisciplinary teams.

Biomarkers in Fabry Disease. Implications for Clinical Diagnosis and Follow-up.

Fabry disease (FD) is a lysosomal storage disorder caused by deficient alpha-galactosidase A activity in the lysosome due to mutations in the GLA gene, resulting in gradual accumulation of globotriaosylceramide and other derivatives in different tissues. Substrate accumulation promotes different pathogenic mechanisms in which several mediators could be implicated, inducing multiorgan lesions, mainly in the kidney, heart and nervous system, resulting in clinical manifestations of the disease. Enzyme replacement therapy was shown to delay disease progression, mainly if initiated early. However, a diagnosis in the early stages represents a clinical challenge, especially in patients with a non-classic phenotype, which prompts the search for biomarkers that help detect and predict the evolution of the disease. We have reviewed the mediators involved in different pathogenic mechanisms that were studied as potential biomarkers and can be easily incorporated into clinical practice. Some accumulation biomarkers seem to be useful to detect non-classic forms of the disease and could even improve diagnosis of female patients. The combination of such biomarkers with some response biomarkers, may be useful for early detection of organ injury. The incorporation of some biomarkers into clinical practice may increase the capacity of detection compared to that currently obtained with the established diagnostic markers and provide more information on the progression and prognosis of the disease.

Extracellular vesicles from recombinant cell factories improve the activity and efficacy of enzymes defective in lysosomal storage disorders.

In the present study the use of extracellular vesicles (EVs) as vehicles for therapeutic enzymes in lysosomal storage disorders was explored. EVs were isolated from mammalian cells overexpressing alpha-galactosidase A (GLA) or N-sulfoglucosamine sulfohydrolase (SGSH) enzymes, defective in Fabry and Sanfilippo A diseases, respectively. Direct purification of EVs from cell supernatants was found to be a simple and efficient method to obtain highly active GLA and SGSH proteins, even after EV lyophilization. Likewise, EVs carrying GLA (EV-GLA) were rapidly uptaken and reached the lysosomes in cellular models of Fabry disease, restoring lysosomal functionality much more efficiently than the recombinant enzyme in clinical use. In vivo, EVs were well tolerated and distributed among all main organs, including the brain. DiR-labelled EVs were localized in brain parenchyma 1 h after intra-arterial (internal carotid artery) or intravenous (tail vein) administrations. Moreover, a single intravenous administration of EV-GLA was able to reduce globotriaosylceramide (Gb3) substrate levels in clinically relevant tissues, such kidneys and brain. Overall, our results demonstrate that EVs from cells overexpressing lysosomal enzymes act as natural protein delivery systems, improving the activity and the efficacy of the recombinant proteins and facilitating their access to organs neglected by conventional enzyme replacement therapies.

Nanotechnology-based approaches for treating lysosomal storage disorders, a focus on Fabry disease.

Lysosomal storage disorders (LSDs) are a group of rare diseases in which the defect of a lysosomal protein results in a pathogenic accumulation of nonmetabolized products within the cells. The main treatment for LSDs is enzyme replacement therapy (ERT), consisting in the exogenous administration a recombinant protein to replace the defective one. Although several diseases such as Gaucher, Fabry, and Pompe are treated following this approach, ERT is limited to LSDs without severe neuronal affectation because recombinant enzymes do not cross the blood-brain barrier. Moreover, ERT shows additional drawbacks, including enzyme low half-life, poor bioavailability, and immunogenic responses. In this scenario, nanotechnology-based drug delivery systems (DDS) have been proposed as solution to overcome these limitations and improve the efficacy of ERT. The present review summarizes distinct approaches followed by our group and collaborators on the use of DDS for restoring lysosomal enzymes in disease-affected cells. During the last decade, we have been exploring different synthetic nanoparticles, from electrolytic complexes, to liposomes and aggresomes, for the delivery of α-galactosidase A (GLA) enzyme. Studies were mainly conducted on Fabry disease models, but results can be also extrapolated to other LSDs, as well as to other diseases treated with alternative therapeutic proteins. The advantages and disadvantages of different DDS, the difficulties from working with very labile and highly glycosylated enzymes and the relevance of using appropriate targeting moieties is thoroughly discussed. Finally, the use of natural DDS, namely extracellular vesicles (EVs) is also introduced. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Neurological Disease Therapeutic Approaches and Drug Discovery > Nanomedicine for Cardiovascular Disease Therapeutic Approaches and Drug Discovery > Emerging Technologies.

Agreement between results of meta-analyses from case reports and clinical studies, regarding efficacy and safety of idursulfase therapy in patients with mucopolysaccharidosis type II (MPS-II). A new tool for evidence-based medicine in rare diseases.

BACKGROUND: A preliminary exploratory study shows solid agreement between the results of case reports and clinical study meta-analyses in mucopolysaccharidosis Type I (MPS-I) adult patients. The aim of the present study is to confirm previous results in another patient population, suffering from mucopolysaccharidosis Type II (MPS-II). METHODS: A systematic review and meta-analysis of case reports published by April 2018 was conducted for MPS-II patients treated with enzyme replacement therapy (ERT). The study is reported in accordance with PRISMA and MOOSE guidelines (PROSPERO database code CRD42018093408). The assessed population and outcomes were the same as previously analyzed in a meta-analysis of MPS-II clinical studies. The primary endpoint was the percent of clinical cases showing improvement in efficacy outcome, or no harm in safety outcome after ERT initiation. A restrictive procedure to aggregate case reports, by selecting standardized and well-defined outcomes, was proposed. Different sensitivity analyses were able to evaluate the robustness of results. RESULTS: Every outcome classified as "acceptable evidence group" in our case report meta-analysis had been graded as "moderate strength of evidence" in the aforementioned meta-analysis of clinical studies. Sensitivity, specificity, and positive-negative predictive values for results of both meta-analyses reached 100%, and were deemed equivalent. CONCLUSIONS: Aggregating case reports quantitatively, rather than analyzing them qualitatively, may improve conclusions in rare diseases and personalized medicine. Additionally, we propose some methods to evaluate publication bias and heterogeneity of the included studies in a meta-analysis of case reports.

Acute telomere deprotection prevents ongoing BFB cycles and rampant instability in p16INK4a-deficient epithelial cells.

Telomere dysfunction drives chromosome instability through endless breakage-fusion-bridge (BFB) cycles that promote the formation of highly rearranged genomes. However, reactivation of telomerase or ALT-pathway is required for genome stabilisation and full malignant transformation. To allow the unrestricted proliferation of cells at risk of transformation, we have established a conditional system of telomere deprotection in p16INK4a-deficient MCF-10A cells with modified checkpoints. After sustained expression of a dominant negative form of the shelterin protein TRF2 (TRF2ΔBΔM), cells with telomere fusion did progress to anaphase but no signs of ongoing BFB cycles were observed, thus anticipating proliferation defects. Indeed, 96 h TRF2ΔBΔM expression resulted in noticeable growth proliferation defects in the absence of cell cycle disturbances. Further transient periods of 96 h telomere uncapping did not result in cell cycle disturbances either. And reduction of the telomere damage to short acute deprotection periods did not in any case engender cells with a reorganised karyotype. Strikingly, the growth arrest imposed in cells showing dysfunctional telomeres was not accompanied by an activation of the DNA damage response at cellular level, or by the presence of visible markers of senescence or apoptosis. We propose that the deprotection of many telomeres simultaneously, even for a short time, results in a local activation of the cellular stress response which consequently triggers gradual cell withdrawal from cell cycle, restraining the onset of genomic instability.

Agreement between the results of meta-analyses from case reports and from clinical studies regarding the efficacy of laronidase therapy in patients with mucopolysaccharidosis type I who initiated enzyme replacement therapy in adult age: An example of case reports meta-analyses as an useful tool for evidence-based medicine in rare diseases.

BACKGROUND: Case reports might have a prominent role in the rare diseases field, due to the small number of patients affected by one such disease. A previous systematic review regarding the efficacy of laronidase therapy in patients with mucopolysaccharidosis type I (MPS-I) who initiated enzyme replacement therapy (ERT) in adult age has been published. The review included a meta-analysis of 19 clinical studies and the description of eleven case reports. It was of interest to perform a meta-analysis of those case reports to explore the role of such meta-analyses as a tool for evidence-based medicine in rare diseases. METHODS: The study included all case reports with standard treatment regimen. Primary analysis was the percentage of case reports showing an improvement in a specific outcome. Only when that percentage was statistically higher than 5%, the improvement was confirmed as such. The outcomes that accomplished this criterion were ranked and compared to the GRADE criteria obtained by those same outcomes in the previous meta-analysis of clinical studies. RESULTS: There were three outcomes that had a significant improvement: Urine glycosaminoglycans, liver volume and 6-minute walking test. Positive and negative predictive values, sensitivity and specificity for the results of the meta-analysis of case reports as compared to that of clinical studies were 100%, 88.9%, 75% and 100%, respectively. Accordingly, absolute (Rho=0.82, 95%CI: 0.47 to 0.95) and relative agreement (Kappa=0.79, 95%CI: 0.593 to 0.99) between the number of case reports with improvement in a specific outcome and the GRADE evidence score for that outcome were good. Sensitivity analysis showed that agreement between the meta-analysis of case reports and that of the clinical studies were good only when using a strong confirmatory strategy for outcome improvement in case reports. CONCLUSIONS: We found an agreement between the results of meta-analyses from case reports and from clinical studies in the efficacy of laronidase therapy in patients with MPS-I who initiated ERT in adult age. This agreement suggests that combining case reports quantitatively, rather than analyzing them separately or qualitatively, may improve conclusions in the field of rare diseases.

Efficacy of laronidase therapy in patients with mucopolysaccharidosis type I who initiated enzyme replacement therapy in adult age. A systematic review and meta-analysis.

BACKGROUND: The efficacy of starting enzyme replacement therapy (ERT) in adults with Muchopolysaccharidosis Type I (MPS-I) is controversial. Evaluating the benefits reported by patients initiating ERT with laronidase at adult age might help physicians decide whether the use of ERT in these patients is worthwhile from a clinical point of view. OBJECTIVE: To assess every effectiveness variable modified in MPS-I patients who initiated laronidase at adult age. METHODS: A systematic search of the literature, from inception to July 2016, was conducted using MEDLINE, EMBASE, CENTRAL and LILACS to identify randomized trials or observational studies including ≥1 MPS-I patients with ERT initiated in adult age (≥18years) and evaluating ERT efficacy. A meta-analysis of studies evaluating the same effectiveness outcome was performed and the evidence was rated according to GRADE criteria. Heterogeneity was assessed by the Chi-squared test and the I-squared statistic. Case reports were excluded from meta-analysis but their main outcomes were separately evaluated. The decrease in urine glycosaminoglycans (uGAGs) levels as patient percentage with reduction in uGAGs and with normalization was the primary outcome. RESULTS: Nineteen clinical studies and 12 case reports were selected. ERT decreased uGAG levels (high evidence) and liver volume (high), improved 6-min walking test (6MWT) (moderate) and increased blood anti-ERT antibody levels (high). There was no conclusive results (low or very low evidence) regarding improvement/stabilization of respiratory function, change in shoulder flexion, cardiac improvement/stabilization, improvement in symptoms of nocturnal hypoventilation and sleep apnea, improvement in quality of life, visual acuity, otolaryngologic function, bone mineral density or effectiveness of intrathecal therapy. LIMITATIONS: Excluding case reports, there was no study conducted specifically in the target population (ERT ≥18years). Data were from subgroup analyses of selected studies. There was a great heterogeneity between designs and clinical outcomes evaluated. CONCLUSIONS: ERT improves uGAGs and liver volume in MPS-I patients initiating therapy as adults, although the putative clinical benefit associated to these improvements is unclear. Moderate evidence was shown for improvement in 6MWT. Systematic review registration number (PROSPERO): 42,016,041,306.

Assessment of Bone Health in Patients With Type 1 Gaucher Disease Using Impact Microindentation.

Gaucher disease (GD), one of the most common lysosomal disorders (a global population incidence of 1:50,000), is characterized by beta-glucocerebrosidase deficiency. Some studies have demonstrated bone infiltration in up to 80% of patients, even if asymptomatic. Bone disorder remains the main cause of morbidity in these patients, along with osteoporosis, avascular necrosis, and bone infarcts. Enzyme replacement therapy (ERT) has been shown to improve these symptoms. This cross-sectional study included patients with type 1 Gaucher disease (GD1) selected from the Catalan Study Group on GD. Clinical data were collected and a general laboratory workup was performed. Bone mineral density (BMD) was measured at the lumbar spine and hip using dual-energy X-ray absorptiometry (DXA). Patients with bone infarcts or any other focal lesion in the area of indentation visible on imaging were excluded. Bone Material Strength index (BMSi) was measured by bone impact microindentation using an Osteoprobe instrument. Analysis of covariance (ANCOVA) models were fitted to adjust for age, sex, weight, and height. Sixteen patients with GD1 and 29 age- and sex-matched controls were included. GD1 was associated with significantly lower BMSi (adjusted beta -9.30; 95% CI, -15.18 to -3.42; p = 0.004) and reduced lumbar BMD (adjusted beta -0.14; 95% CI, -0.22 to -0.06; p = 0.002) and total hip BMD (adjusted beta -0.09; 95% CI, -0.15 to -0.03; p = 0.006), compared to GD1-free controls. Chitotriosidase levels were negatively correlated with BMSi (linear R2 = 51.6%, p = 0.004). Bone tissue mechanical characteristics were deteriorated in patients with GD1. BMSi was correlated with chitotriosidase, the marker of GD activity. Bone disorder requires special consideration in this group of patients, and microindentation could be an appropriate tool for assessing and managing their bone health. © 2017 American Society for Bone and Mineral Research.