miRNAs AS POTENTIAL REGULATORS OF MYELIN BASIC PROTEIN RECOVERY DURING DEVELOPMENT IN A MURINE MODEL OF PHENYLKETONURIA

Untreated phenylketonuria (PKU) patients and PKU animal models show hypomyelination in the central nervous system and white matter damages. These cerebral alterations are accompanied by myelin basic protein (MBP) impairment, which could be the reason of the clinical traits mentioned above, as MBP is crucial for the correct assembling of the myelin sheath. In this study, we analyzed MBP protein and mRNA expression on brains of WT and phenylketonuric (ENU2) mice during post-natal development (14-60-180-270-360-540 post-natal days, PND). The results showed a progressive MBP protein expression recovery during post-natal development, together with an unaltered MBP mRNA expression. Furthermore, for the same time intervals, a significant decrease of the phenylalanine concentration in the bloodstream of PKU mice was detected, as well as in the PKU mice brains from 14 to 60 PND. To try to explain this scenario, we hypothesized a hindrance during MBP translation in the early development, leading us to perform a microRNA microarray analysis on 60 PND mice. Microarray output and the following in silico analyses underlined the potential role of microRNAs in the PKU cerebral outcomes. In addition, in order to link predictive analysis with concrete data, we performed a proteomic assay on ENU2 brains of 60 and 360 PND. Taken together, we assessed miR-218-1-3p, miR-1231-3p and miR-217-5p as the most promising microRNAs, since that an alteration on their predicted and downregulated targets (MAG, CNTNAP2 and ANLN, respectively) could indirectly lead to a low MBP protein expression. Moreover, their expression shows an opposite trend to that observed for MBP protein during development, except for miR-217-5p. Furthermore, target proteins revealed a complete normalization in aged ENU2 mice. In conclusion, these results provide a new perspective on the PKU pathophysiology understanding and treatment, emphasizing the possible role of differentially expressed microRNAs in PKU brains, especially during early development

Preclinical developments of enzyme-loaded red blood cells

Therapeutic enzymes are currently used in the treatment of several diseases. In most cases, the benefits are limited due to poor in vivo stability, immunogenicity, and drug-induced inactivating antibodies. A partial solution to the problem is obtained by masking the therapeutic protein by chemical modifications. Unfortunately, this is not a satisfactory solution because frequent adverse events, including anaphylaxis, can arise

Preclinical and clinical developments in enzyme-loaded red blood cells: an update

Introduction: We have previously described the preclinical developments in enzyme-loaded red blood cells to be used in the treatment of several rare diseases, as well as in chronic conditions. Area covered: Since our previous publication we have seen further progress in the previously discussed approaches and, interestingly enough, in additional new studies that further strengthen the idea that red blood cell-based therapeutics may have unique advantages over conventional enzyme replacement therapies in terms of efficacy and safety. Here we highlight these investigations and compare, when possible, the reported results versus the current therapeutic approaches. Expert opinion: The continuous increase in the number of new potential applications and the progress from the encapsulation of a single enzyme to the engineering of an entire metabolic pathway open the field to unexpected developments and confirm the role of red blood cells as cellular bioreactors that can be conveniently manipulated to acquire useful therapeutic metabolic abilities. Positioning of these new approaches versus newly approved drugs is essential for the successful transition of this technology from the preclinical to the clinical stage and hopefully to final approval

Myelin basic protein recovery during PKU mice lifespan and the potential role of microRNAs on its regulation

Untreated phenylketonuria (PKU) patients and PKU animal models show hypomyelination in the central nervous system and white matter damages, which are accompanied by myelin basic protein (MBP) impairment. Despite many assumptions, the primary explanation of the mentioned cerebral outcomes remains elusive. In this study, MBP protein and mRNA expression on brains of wild type (WT) and phenylketonuric (ENU2) mice were analyzed throughout mice lifespan (14-60-180-270-360-540 post-natal days, PND). The results confirmed the low MBP expression at first PND times, while revealed an unprecedented progressive MBP protein expression recovery in aged ENU2 mice. Unexpectedly, unaltered MBP mRNA expression between WT and ENU2 was always observed. Additionally, for the same time intervals, a significant decrease of the phenylalanine concentration in the peripheral blood and brain of ENU2 mice was detected, to date, for the first time. In this scenario, a translational hindrance of MBP during initial and late cerebral development in ENU2 mice was hypothesized, leading to the execution of a microRNA microarray analysis on 60 PND brains, which was followed by a proteomic assay on 60 and 360 PND brains in order to validate in silico miRNA-target predictions. Taken together, miR-218-1-3p, miR-1231-3p and miR-217-5p were considered as the most impactful microRNAs, since a downregulation of their potential targets (MAG, CNTNAP2 and ANLN, respectively) can indirectly lead to a low MBP protein expression. These miRNAs, in addition, follow an opposite expression trend compared to MBP during adulthood, and their target proteins revealed a complete normalization in aged ENU2 mice. In conclusion, these results provide a new perspective on the PKU pathophysiology understanding and on a possible treatment, emphasizing the potential modulating role of differentially expressed microRNAs in MBP expression on PKU brains during PKU mouse lifespan