CNS-wide repopulation by hematopoietic-derived microglia-like cells corrects progranulin deficiency in mice.

Hematopoietic stem cell transplantation can deliver therapeutic proteins to the central nervous system (CNS) through transplant-derived microglia-like cells. However, current conditioning approaches result in low and slow engraftment of transplanted cells in the CNS. Here we optimized a brain conditioning regimen that leads to rapid, robust, and persistent microglia replacement without adverse effects on neurobehavior or hematopoiesis. This regimen combines busulfan myeloablation and six days of Colony-stimulating factor 1 receptor inhibitor PLX3397. Single-cell analyses revealed unappreciated heterogeneity of microglia-like cells with most cells expressing genes characteristic of homeostatic microglia, brain-border-associated macrophages, and unique markers. Cytokine analysis in the CNS showed transient inductions of myeloproliferative and chemoattractant cytokines that help repopulate the microglia niche. Bone marrow transplant of progranulin-deficient mice conditioned with busulfan and PLX3397 restored progranulin in the brain and eyes and normalized brain lipofuscin storage, proteostasis, and lipid metabolism. This study advances our understanding of CNS repopulation by hematopoietic-derived cells and demonstrates its therapeutic potential for treating progranulin-dependent neurodegeneration.

Novel human recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in a glyco-engineered Escherichia coli strain.

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disease caused by mutations in the gene encoding the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS), resulting in the accumulation of keratan sulfate (KS) and chondroitin-6-sulfate (C6S). Previously, it was reported the production of an active human recombinant GALNS (rGALNS) in E. coli BL21(DE3). However, this recombinant enzyme was not taken up by HEK293 cells or MPS IVA skin fibroblasts. Here, we leveraged a glyco-engineered E. coli strain to produce a recombinant human GALNS bearing the eukaryotic trimannosyl core N-glycan, Man3GlcNAc2 (rGALNSoptGly). The N-glycosylated GALNS was produced at 100 mL and 1.65 L scales, purified and characterized with respect to pH stability, enzyme kinetic parameters, cell uptake, and KS clearance. The results showed that the addition of trimannosyl core N-glycans enhanced both protein stability and substrate affinity. rGALNSoptGly was capture through a mannose receptor-mediated process. This enzyme was delivered to the lysosome, where it reduced KS storage in human MPS IVA fibroblasts. This study demonstrates the potential of a glyco-engineered E. coli for producing a fully functional GALNS enzyme. It may offer an economic approach for the biosynthesis of a therapeutic glycoprotein that could prove useful for MPS IVA treatment. This strategy could be extended to other lysosomal enzymes that rely on the presence of mannose N-glycans for cell uptake.

Laronidase-loaded liposomes reach the brain and other hard-to-treat organs after noninvasive nasal administration.

Mucopolysaccharidosis type I (MPS I) is caused by a lack of the lysosomal enzyme α-L-iduronidase (IDUA), responsible for the degradation of the glycosaminoglycans (GAGs) dermatan and heparan sulfate, leading to multisystemic signs and symptoms. Enzyme replacement therapy (ERT) is a treatment that consists of weekly intravenous administrations of laronidase, a recombinant version of IDUA. However, ERT has limited access to certain tissues, such as bone, cartilage, and brain, and laronidase fails to trespass the BBB. In this sense, this study reports the development and characterization of laronidase-loaded liposomes for the treatment of MPS I mice. Liposomal complexes were obtained by the thin film formation method followed by microfluidization. The main characterization results showed mean vesicle size of 103.0 ± 3.3 nm, monodisperse populations of vesicles, zeta potential around + 30.0 ± 2.1 mV, and mucoadhesion strength of 5.69 ± 0.14 mN. Treatment of MPS I mice fibroblasts showed significant increase in enzyme activity. Nasal administration of complexes to MPS I mice resulted in significant increase in laronidase activity in the brain cortex, heart, lungs, kidneys, eyes, and serum. The overall results demonstrate the feasibility of nasal administration of laronidase-loaded liposomes to deliver enzyme in difficult-to-reach tissues, circumventing ERT issues and bringing hope as a potential treatment for MPS I.

Cardiovascular testing recovery in Latin America one year into the COVID-19 pandemic: An analysis of data from an international longitudinal survey.

Background: The COVID-19 pandemic disproportionately impacted Latin America (LATAM), significantly disrupting cardiovascular testing. This study evaluated cardiac procedure recovery in LATAM one year after the outbreak. Methods: The International Atomic Energy Agency (IAEA) surveyed 669 centers in 107 countries worldwide, including 135 facilities in 19 LATAM countries, to assess cardiovascular procedure volumes in March 2019, April 2020, and April 2021, and changes in center practices and staffing conditions one year into the COVID-19 pandemic. Findings: LATAM centers reported a 21 % decrease in procedure volumes in April 2021 from pre-pandemic-baseline, vs. a 0 % change in the rest of the world (RoW), and greater volume reductions for almost all procedure types. Centers in Central America and Mexico reported the largest procedure reductions (47 % reduction) compared to the Caribbean (15 %), and South America (14 %, p = 0.01), and this LATAM region was a significant predictor of lower procedure recovery in multivariable regression. More LATAM centers reported reduced salaries and increased layoffs of clinical staff compared to RoW, and LATAM respondents estimated that half of physician and non-physician staff experienced excess psychological stress related to the pandemic, compared to 25 % and 30 % in RoW (p < 0.001). Conclusions: Cardiovascular testing recovery in LATAM trailed behind RoW for most procedure types, with centers in Central America and Mexico reporting the greatest volume reductions. This study found lasting impacts of COVID-19 on cardiovascular care in LATAM and the need for mental health support for LATAM healthcare workers in current and future pandemics.

Effect of genistein and coenzyme Q10 in oxidative damage and mitochondrial membrane potential in an attenuated type II mucopolysaccharidosis cellular model.

Mucopolysaccharidosis type II (MPS II) is an inborn error of the metabolism resulting from several possible mutations in the gene coding for iduronate-2-sulfatase (IDS), which leads to a great clinical heterogeneity presented by these patients. Many studies demonstrate the involvement of oxidative stress in the pathogenesis of inborn errors of metabolism, and mitochondrial dysfunction and oxidative stress can be related since most of reactive oxygen species come from mitochondria. Cellular models have been used to study different diseases and are useful in biochemical research to investigate them in a new promising way. The aim of this study is to develop a heterozygous cellular model for MPS II and analyze parameters of oxidative stress and mitochondrial dysfunction and investigate the in vitro effect of genistein and coenzyme Q10 on these parameters for a better understanding of the pathophysiology of this disease. The HP18 cells (heterozygous c.261_266del6/c.259_261del3) showed almost null results in the activity of the IDS enzyme and presented accumulation of glycosaminoglycans (GAGs), allowing the characterization of this knockout cellular model by MPS II gene editing. An increase in the production of reactive species was demonstrated (p < .05 compared with WT vehicle group) and genistein at concentrations of 25 and 50 µm decreased in vitro its production (p < .05 compared with HP18 vehicle group), but there was no effect of coenzyme Q10 in this parameter. There was a tendency for lysosomal pH change in HP18 cells in comparison to WT group and none of the antioxidants tested demonstrated any effect on this parameter. There was no increase in the activity of the antioxidant enzymes superoxide dismutase and catalase and oxidative damage to DNA in HP18 cells in comparison to WT group and neither genistein nor coenzyme q10 had any effect on these parameters. Regarding mitochondrial membrane potential, genistein induced mitochondrial depolarization in both concentrations tested (p < .05 compared with HP18 vehicle group and compared with WT vehicle group) and incubation with coenzyme Q10 demonstrated no effect on this parameter. In conclusion, it is hypothesized that our cellular model could be compared with a milder MPS II phenotype, given that the accumulation of GAGs in lysosomes is not as expressive as another cellular model for MPS II presented in the literature. Therefore, it is reasonable to expect that there is no mitochondrial depolarization and no DNA damage, since there is less lysosomal impairment, as well as less redox imbalance.

Comparison of different DNA preservation solutions for oral cytological samples.

OBJECTIVE: The objective of this study was to compare the DNA preservation capacity of buccal mucosa exfoliated cells when stored in different solutions under varying time and temperature conditions. DESIGN: DNA preservation solutions, including Dimethyl sulphoxide disodium-EDTA-saturated NaCl (DESS), Tris-EDTA-NaCl-Tween20 buffer (TENT), Nucleic Acid Preservation Buffer (NAP), and phosphate-buffered saline (PBS), were prepared. Buccal mucosa cells from a single patient were collected, dispensed into these solutions, and stored at room temperature (RT) and 4 °C for 24 h, 72 h, 30 days, 90 days, and 180 days. DNA was extracted using the salting-out method and the QIAamp DNA Mini Kit. DNA concentration and purity were determined using the QuBit device and NanoDrop, while DNA integrity was assessed using the Agilent 4200 TapeStation system. The ability to amplify the IFNA primer was also evaluated by PCR. RESULTS: The salting-out method yielded better concentration and purity results, with PBS, TENT, and DESS buffers demonstrating superior concentration values when stored at 4 °C, resulting in mean values exceeding 10 ng/µL for up to 30 days. DESS consistently exhibited the best integrity values over time for both temperature conditions. Amplification capacity was enhanced when samples were stored at 4 °C. When stored at RT, PBS achieved 100% amplification within 24 h. NAP yielded the poorest results. CONCLUSION: In the context of long-term preservation, the DESS buffer emerges as the most effective solution, maintaining requisite DNA quality and quantity standards for up to 30 days at RT and up to 3 months at 4 °C.

CNS Repopulation by Hematopoietic-Derived Microglia-Like Cells Corrects Progranulin deficiency.

Hematopoietic stem cell transplantation can deliver therapeutic proteins to the CNS through donor-derived hematopoietic cells that become microglia-like cells. However, using standard conditioning approaches, hematopoietic stem cell transplantation is currently limited by low and slow engraftment of microglia-like cells. We report an efficient conditioning regimen based on Busulfan and a six-day course of microglia depletion using the colony-stimulating factor receptor 1 inhibitor PLX3397. Combining Busulfan-myeloablation and transient microglia depletion results in robust, rapid, and persistent microglia replacement by bone marrow-derived microglia-like cells throughout the CNS. Adding PLX3397 does not affect neurobehavior or has adverse effects on hematopoietic reconstitution. Through single-cell RNA sequencing and high-dimensional CyTOF mass cytometry, we show that microglia-like cells are a heterogeneous population and describe six distinct subpopulations. Though most bone-marrow-derived microglia-like cells can be classified as homeostatic microglia, their gene signature is a hybrid of homeostatic/embryonic microglia and border associated-macrophages. Busulfan-myeloablation and transient microglia depletion induce specific cytokines in the brain, ultimately combining myeloid proliferative and chemo-attractive signals that act locally to repopulate microglia from outside the niche. Importantly, this conditioning approach demonstrates therapeutic efficacy in a mouse model of GRN deficiency. Transplanting wild-type bone marrow into Grn-/- mice conditioned with Busulfan plus PLX3397 results in high engraftment of microglia-like cells in the brain and retina, restoring GRN levels and normalizing lipid metabolism.

Characterization of heart disease in mucopolysaccharidosis type II mice.

Mucopolysaccharidosis type II (MPSII) is a progressive lysosomal storage disease caused by mutations in the IDS gene, that leads to iduronate 2-sulfatase (IDS) enzyme deficiency. The enzyme catalyzes the first step of degradation of two glycosaminoglycans (GAGs), heparan sulfate (HS) and dermatan sulfate (DS). The consequences of MPSII are progressively harmful and can lead to death by cardiac failure. The aim of this study was to characterize the cardiovascular disease in MPSII mice. Thus, we evaluated the cardiovascular function of MPSII male mice at 6, 8, and 10 months of age, through functional, histological, and biochemical analyzes. Echocardiographic analyses showed a progressive loss in cardiac function, observed through parameters such as reduction in ejection fraction (46% in control versus 28% in MPS II at 10 months, P < .01) and fractional area change (31% versus 23%, P < .05). Similar results were found in parameters of vascular competence, obtained by echo Doppler. Both aortic dilatation and an increase in pulmonary resistance were observed at all time points in MPSII mice. The histological analyses showed an increase in the thickness of the heart valves (2-fold thicker than control values at 10 months). Biochemical analyzes confirmed GAG storage in these tissues, with a massive elevation of DS in the myocardium. Furthermore, an important increase in the activity of proteases such as cathepsin S and B (up to 5-fold control values) was found and could be related to the progressive loss of cardiac function observed in MPSII mice. In this work, we demonstrated that loss of cardiac function in MPSII mice started at 6 months of age, although its global cardiac capacity was still preserved at this time. Disease progressed at later time points leading to heart failure. The MPSII mice at later times reproduce many of the cardiovascular events found in patients with Hunter's disease.

Does exposure to moonlight affect day/night changes in melatonin and metabolic parameters in Amazonian fish?

Lunar cycle modulates the rhythmic activity patterns of many animals, including fish. The effect of the moonlight cycle on daily melatonin and metabolic parameters was evaluated in matrinxã (Brycon amazonicus) subjected to external natural lighting. Eighty juvenile were distributed in 4 tanks of 1m3 (20 fish/tank) and divided into two groups. One group was exposed to the full moon and the other group to the new moon for 30 days, which corresponds to the duration of the lunar period. At the end of the lunar phase, 6 fish from each group were anesthetized to collect blood, tissue and eye samples at midday and midnight. The comparison between the light and dark periods revealed a significant increase in plasma and ocular melatonin in the last period. However, there was no significant difference for plasma melatonin between moons. Ocular melatonin presented higher concentrations during the new moon. Glucose, total proteins, cortisol, liver glutathione and gill lipid peroxidation were higher in the full moon compared to in the new moon. Plasma triglyceride was higher during the night for the full moon, and the opposite was found for the new moon. Total cholesterol values were higher at night regardless the moon phase. Glutathione in the gills and lipid peroxidation in the liver showed no significant differences. These results highlight the importance of considering both the day and lunar cycles for melatonin and metabolic parameters in species of commercial interest and susceptible to stressful situations in rearing conditions.

Daily rhythms in the behavioural stress response of the zebrafish Danio rerio.

In nature, animals are exposed to stressors that occur with different likelihood throughout the day, such as risk of predation and human disturbance. Hence, the stress response is expected to vary plastically to adaptively match these challenges. Several studies have supported this hypothesis in a wide range of vertebrate species, including some teleost fish, mostly through evidence of circadian variation in physiology. However, in teleost fish, circadian variation in behavioural stress responses is less understood. Here, we investigated the daily rhythm of stress response at the behavioural level in the zebrafish Danio rerio. We exposed individuals and shoals to an open field test every 4 h over a 24 h cycle, recording three behavioural indicators of stress and anxiety levels in novel environments (thigmotaxis, activity and freezing). Thigmotaxis and activity significantly varied throughout the day with a similar pattern, in line with a stronger stress response in the night phase. The same was suggested by analysis of freezing in shoals, but not in individual fish, in which variation appeared mostly driven by a single peak in the light phase. In a control experiment, we observed a set of subjects after familiarisation with the open-field apparatus. This experiment indicated that activity and freezing might present a daily rhythmicity that is unrelated to environmental novelty, and thus to stress responses. However, the thigmotaxis was constant through the day in the control condition, suggesting that the daily variation of this indicator is mostly attributable to the stress response. Overall, this research indicates that behavioural stress response of zebrafish does follow a daily rhythm, although this may be masked using behavioural indicators other than thigmotaxis. This rhythmicity can be relevant to improve welfare in aquaculture and reliability of behavioural research in fish models.

Recirculating packed-bed biofilm photobioreactor combined with membrane ultrafiltration as advanced wastewater treatment.

Packed-bed biofilm photobioreactor combined with ultrafiltration membrane was investigated for intensifying the process for secondary wastewater effluent treatment. Cylindrical glass carriers were used as supporting material for the microalgal-bacterial biofilm, which developed from indigenous microbial consortium. Glass carriers allowed adequate growth of the biofilm with limited suspended biomass. Stable operation was achieved after a start-up period of 1000 h, where supernatant biopolymer clusters were minimized and complete nitrification was observed. After that time, biomass productivity was 54 ± 18 mg·L-1·day-1. Green microalgae Tetradesmus obliquus and several strains of heterotrophic nitrification-aerobic denitrification bacteria and fungi were identified. Combined process exhibited COD, nitrogen and phosphorus removal rates of 56 ± 5%, 12 ± 2% and 20 ± 6%, respectively. Membrane fouling was mainly caused by biofilm formation, which was not effectively mitigated by air-scouring aided backwashing.

Circadian rhythm of preferred temperature in fish: Behavioural thermoregulation linked to daily photocycles in zebrafish and Nile tilapia.

Ectothermic vertebrates, e.g. fish, maintain their body temperature within a specific physiological range mainly through behavioural thermoregulation. Here, we characterise the presence of daily rhythms of thermal preference in two phylogenetically distant and well-studied fish species: the zebrafish (Danio rerio), an experimental model, and the Nile tilapia (Oreochromis niloticus), an aquaculture species. We created a non-continuous temperature gradient using multichambered tanks according to the natural environmental range for each species. Each species was allowed to freely choose their preferred temperature during the 24h cycle over a long-term period. Both species displayed strikingly consistent temporal daily rhythms of thermal preference with higher temperatures being selected during the second half of the light phase and lower temperatures at the end of the dark phase, with mean acrophases at Zeitgeber Time (ZT) 5.37 h (zebrafish) and ZT 12.5 h (tilapia). Interestingly, when moved to the experimental tank, only tilapia displayed consistent preference for higher temperatures and took longer time to establish the thermal rhythms. Our findings highlight the importance of integrating both light-driven daily rhythm and thermal choice to refine our understanding of fish biology and improve the management and welfare of the diversity of fish species used in research and food production.

Psychotherapists' experiences of telepsychotherapy during the COVID-19 pandemic in Argentina: impact on therapy setting, therapeutic relationship and burden.

In order to prevent the spread of the coronavirus in Argentina, quarantine and distancing measures were adopted. This has led to a large increase in the use of online psychotherapy. Several international studies have already examined the transition from face-to-face therapy to telepsychotherapy during the COVID-19 pandemic from a therapist perspective, but within these studies, the Latin American context is not represented. To fill this research gap, this qualitative study explored the Argentinean psychotherapists' experiences with online psychotherapy during the COVID-19 pandemic by means of in-depth interviews and content analysis. Based on the international literature, an interview guideline was created which covered three main topics: setting, therapeutic relationship, and burden among therapists. For the analysis of the therapists' answers, a detailed codebook was developed. Effects were identified in all three areas investigated. The nine interviewed psychotherapists described unfavorable therapy conditions which affected the therapeutic setting, the psychotherapeutic methods and contents. Positive effects in the therapeutic relationship included patients being more open, but also negative effects such as discontinuation of therapies were reported. Therapists experienced telepsychotherapy as more exhausting than in-person psychotherapy, but they considered it a valuable tool during the pandemic, and the transition an enriching experience. These results, which are partially consistent with international findings, have practical implications for the practice of telepsychotherapy in Argentina. Technical conditions should be improved and patients' privacy during the session should be ensured. Treatment techniques and diagnostics need to be adapted for their use in telepsychotherapy.

Behavioural response to toxic elements, detoxification and organ accumulation are time-of-day-dependent in zebrafish.

Toxic elements, such as mercury (Hg) and arsenic (As), are major pollutants in aquatic environments, posing ecological threats to living organisms due to their toxicity and bioaccumulation. This paper investigated whether zebrafish response to Hg and As displayed day/night differences. Fish were exposed to either 35 µg/L of mercury chloride for 6 h or 65 mg/L of sodium arsenate for 4 h, at two different times of the day: mid-light (day; ML) and mid-darkness (night; MD). Fish were video-recorded to investigate their behavioural response and at the end of each trial, gills and liver samples were collected for gene expression measurement. Gills, liver and brain samples were also obtained to determine Hg and As concentration. A control group (non-exposed) was video-recorded and sampled too. The effect of Hg and As on zebrafish swimming activity and the expression of antioxidant and metallothionein genes was time-of-day-dependent, with a stronger response being observed during the day than at night. However, the neurobehavioural effect of Hg was more affected by the time of exposure than the effect of As. In addition, Hg concentration in the gills was significantly higher in zebrafish exposed at ML than at MD. Altogether, these findings suggest that zebrafish response to Hg and As is time-of-day-dependent and remark the importance of considering toxicity rhythms when using this fish species as a model in toxicological research.

Evaluation of oxidative stress and mitochondrial function in a type II mucopolysaccharidosis cellular model: in vitro effects of genistein and coenzyme Q10.

Mucopolysaccharidosis type II (MPS II or Hunter Syndrome) is a lysosomal disease caused by deficient degradation of glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate due to the deficiency of the enzyme iduronate-2-sulfatase. The main treatment for MPS II is the administration of the recombinant form of the enzyme, in a process known as enzyme replacement therapy (ERT). Oxidative damage can contribute to the pathophysiology of MPS II and treatment with ERT can reduce the effects of oxidative stress. For a better understanding of pathophysiology of MPS II, we evaluated biomarkers of mitochondrial dysfunction, DNA (Deoxyribonucleic acid) damage, antioxidant defenses, reactive species production and lysosomal size in IDS-deficient HEK 293 cells and investigate the in vitro effect of genistein and coenzyme Q10 (CoQ) on these biomarkers. An increase in the production of reactive species was demonstrated, as well as an increase in the activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT). Also, an increase in lysosomal volume and oxidative damage to DNA were verified. There was no evidence of a change in mitochondrial function in this cell model. In the HEK 293 (human embryonic kidney 293) knockout (KO) HP10 cell model we found that genistein at concentrations of 25 and 50 µm decreased in vitro the production of reactive species and the activity of the SOD enzyme, showing an antioxidant protective effect. Still, in these cells we verified that the coenzyme Q10 in the concentrations of 5 and 10 µm decreased in vitro the activity of the SOD enzyme and in the concentration of 10 µm decreased in vitro the DNA damage, also demonstrating antioxidant protection. In conclusion, MPS II knockout cells demonstrated oxidative stress and DNA damage and genistein, as well as coenzyme Q10, have been shown to have an important protective effect in vitro against these oxidative damages.

Administration time-dependent effects of poly (I:C) on antioxidant and immune responses along the diurnal time scale in zebrafish.

The circadian clock of vertebrates regulates many biological processes, including the immune system. This paper investigated whether responsiveness to poly (I:C), a synthetic analog of double-stranded RNA used as an immunostimulant, exhibits day/night differences in zebrafish. Fish were intraperitoneally (IP) injected with either phosphate-buffered saline (PBS) or poly (I:C) at two different time points: "Zeitgeber Time" (ZT) 4 (day) and ZT16 (night). Then, 6 h later, fish were euthanized, and tissue samples (skin, liver and kidney) were collected. A control group (intact fish) was also sampled at the same time points. The effect of poly (I:C) on the expression of antioxidant and immune genes was time-of-day-dependent, and the response was stronger following poly (I:C) administration in the day than at night. Time-dependent differences were observed for some genes in the PBS and control groups. However, these differences were tissue-specific. In liver, almost all the genes were affected by time of day. In kidney, poly (I:C) affected the expression of all the gene markers regardless of administration time. These findings highlight the importance of considering the time to administer poly (I:C) when evaluating the fish immune response.

Improved engraftment and therapeutic efficacy by human genome-edited hematopoietic stem cells with Busulfan-based myeloablation.

Autologous hematopoietic stem cell transplantation using genome-edited cells can become a definitive therapy for hematological and non-hematological disorders with neurological involvement. Proof-of-concept studies using human genome-edited hematopoietic stem cells have been hindered by the low efficiency of engraftment of the edited cells in the bone marrow and their modest efficacy in the CNS. To address these challenges, we tested a myeloablative conditioning regimen based on Busulfan in an immunocompromised model of mucopolysaccharidosis type 1. Compared with sub-lethal irradiation, Busulfan conditioning enhanced the engraftment of edited CD34+ cells in the bone marrow, as well the long-term homing and survival of bone-marrow-derived cells in viscera, and in the CNS, resulting in higher transgene expression and biochemical correction in these organs. Edited cell selection using a clinically compatible marker resulted in a population with low engraftment potential. We conclude that conditioning can impact the engraftment of edited hematopoietic stem cells. Furthermore, Busulfan-conditioned recipients have a higher expression of therapeutic proteins in target organs, particularly in the CNS, constituting a better conditioning approach for non-hematological diseases with neurological involvement.

Brain and visceral gene editing of mucopolysaccharidosis I mice by nasal delivery of the CRISPR/Cas9 system.

BACKGROUND: Mucopolysaccharidosis type I (MPS I) is an inherited disease caused by deficiency of the enzyme alpha-l-iduronidase (IDUA). MPS I affects several tissues, including the brain, leading to cognitive impairment in the severe form of the disease. Currently available treatments do not reach the brain. Therefore, in this study, we performed nasal administration (NA) of liposomal complexes carrying two plasmids encoding for the CRISPR/Cas9 system and for the IDUA gene targeting the ROSA26 locus, aiming at brain delivery in MPS I mice. METHODS: Liposomes were prepared by microfluidization, and the plasmids were complexed to the formulations by adsorption. Physicochemical characterization of the formulations and complexes, in vitro permeation, and mucoadhesion in porcine nasal mucosa (PNM) were assessed. We performed NA repeatedly for 30 days in young MPS I mice, which were euthanized at 6 months of age after performing behavioral tasks, and biochemical and molecular aspects were evaluated. RESULTS: Monodisperse mucoadhesive complexes around 110 nm, which are able to efficiently permeate the PNM. In animals, the treatment led to a modest increase in IDUA activity in the lung, heart, and brain areas, with reduction of glycosaminoglycan (GAG) levels in serum, urine, tissues, and brain cortex. Furthermore, treated mice showed improvement in behavioral tests, suggesting prevention of the cognitive damage. CONCLUSION: Nonviral gene editing performed through nasal route represents a potential therapeutic alternative for the somatic and neurologic symptoms of MPS I and possibly for other neurological disorders.

Genome editing in lysosomal disorders.

Lysosomal disorders are a group of heterogenous diseases caused by mutations in genes that encode for lysosomal proteins. With exception of some cases, these disorders still lack both knowledge of disease pathogenesis and specific therapies. In this sense, genome editing arises as a technique that allows both the creation of specific cell lines, animal models and gene therapy protocols for these disorders. Here we explain the main applications of genome editing for lysosomal diseases, with examples based on the literature. The ability to rewrite the genome will be of extreme importance to study and potentially treat these rare disorders.

In Situ Gene Therapy.

Gene therapy is a technique that aims at the delivery of nucleic acids to cells, to obtain a therapeutic effect. In situ gene therapy consists of the administration of the gene product to a specific site. It possesses several advantages, such as the reduction in potential side effects, the need for a lower vector dose, and, as a consequence, reduced costs, compared to intravenous administration. Different vectors, administration routes and doses involving in situ gene transfer have been tested both in animal models and humans, with in situ gene therapy drugs already approved in the market. In this review, we present applications of in situ gene therapy for different diseases, ranging from monogenic to multifactorial diseases, focusing mainly on therapies designed for the intra-articular and intraocular compartments, as well as gene therapies for the central nervous system (CNS) and for tumors. Gene therapy finally seems to blossom as a viable therapeutic approach. The growth in the number of clinical protocols shown here is evident, and the positive outcomes observed in several clinical trials indicate that more products based on in situ gene therapy should reach the market in the next years.