Liliana Mendonça

Transferrin Receptor-Targeted Liposomes Encapsulating anti-BCR-ABL siRNA or asODN for Chronic Myeloid Leukemia Treatment

The present work aimed at the development and application of transferrin receptor (TrfR)-targeted sterically stabilized liposomes encapsulating anti-BCR-ABL siRNA or asODN. Transferrin was coupled to the surface of liposomes encapsulating siRNA or asODN through the postinsertion method. Cell association and internalization were assessed by flow cytometry and confocal microscopy, respectively. BCR-ABL mRNA and Bcr-Abl protein levels were evaluated by qRT-PCR and Western blot, respectively. Cell viability was assessed using the resazurin reduction method. The amount of coupled transferrin and the size and stability over time of the liposomes were very satisfactory and reproducible. The siRNA encapsulation yield was dependent on the concentration of the encapsulation buffer used (20 or 300 mM), as opposed to asODN encapsulation yield which was high for both concentrations tested. Cell association and internalization studies were performed in leukemia cell lines treated with liposomes coupled to Trf (Trf-liposomes) or albumin (BSA-liposomes) or with nontargeted liposomes (NT-liposomes) encapsulating fluorescently labeled siRNA (Cy3-siRNA). These experiments clearly indicated that BSA- and NT-liposomes have no ability to promote the delivery of the encapsulated nucleic acids and that the Trf-liposomes deliver the nucleic acids by a Trf receptor-dependent mechanism. The Trf-liposomes encapsulating siRNA or asODN promote sequence-specific down-regulation of the BCR-ABL mRNA, although a certain extent of nonspecific sequence effects at the protein and cell viability level were observed. Overall, our results indicate that Trf-liposomes encapsulating gene silencing tools allow combining molecular and cellular targeting, which is a valuable approach for cancer treatment.

Tumor-targeted Chlorotoxin-coupled Nanoparticles for Nucleic Acid Delivery to Glioblastoma Cells: A Promising System for Glioblastoma Treatment

The present work aimed at the development and application of a lipid-based nanocarrier for targeted delivery of nucleic acids to glioblastoma (GBM). For this purpose, chlorotoxin (CTX), a peptide reported to bind selectively to glioma cells while showing no affinity for non-neoplastic cells, was covalently coupled to liposomes encapsulating antisense oligonucleotides (asOs) or small interfering RNAs (siRNAs). The resulting targeted nanoparticles, designated CTX-coupled stable nucleic acid lipid particles (SNALPs), exhibited excellent features for in vivo application, namely small size (<180 nm) and neutral surface charge. Cellular association and internalization studies revealed that attachment of CTX onto the liposomal surface enhanced particle internalization into glioma cells, whereas no significant internalization was observed in noncancer cells. Moreover, nanoparticle-mediated miR-21 silencing in U87 human GBM and GL261 mouse glioma cells resulted in increased levels of the tumor suppressors PTEN and PDCD4, caspase 3/7 activation and decreased tumor cell proliferation. Preliminary in vivo studies revealed that CTX enhances particle internalization into established intracranial tumors. Overall, our results indicate that the developed targeted nanoparticles represent a valuable tool for targeted nucleic acid delivery to cancer cells. Combined with a drug-based therapy, nanoparticle-mediated miR-21 silencing constitutes a promising multimodal therapeutic approach towards GBM.

Transplantation of cerebellar neural stem cells improves motor coordination and neuropathology in Machado-Joseph disease mice

Machado-Joseph disease is a neurodegenerative disease without effective treatment. Patients with Machado-Joseph disease exhibit significant motor impairments such as gait ataxia, associated with multiple neuropathological changes including mutant ATXN3 inclusions, marked neuronal loss and atrophy of the cerebellum. Thus, an effective treatment of symptomatic patients with Machado-Joseph disease may require cell replacement, which we investigated in this study. For this purpose, we injected cerebellar neural stem cells into the cerebellum of adult Machado-Joseph disease transgenic mice and assessed the effect on the neuropathology, neuroinflammation mediators and neurotrophic factor levels and motor coordination. We found that upon transplantation into the cerebellum of adult Machado-Joseph disease mice, cerebellar neural stem cells differentiate into neurons, astrocytes and oligodendrocytes. Importantly, cerebellar neural stem cell transplantation mediated a significant and robust alleviation of the motor behaviour impairments, which correlated with preservation from Machado-Joseph disease-associated neuropathology, namely reduction of Purkinje cell loss, reduction of cellular layer shrinkage and mutant ATXN3 aggregates. Additionally, a significant reduction of neuroinflammation and an increase of neurotrophic factors levels was observed, indicating that transplantation of cerebellar neural stem cells also triggers important neuroprotective effects. Thus, cerebellar neural stem cells have the potential to be used as a cell replacement and neuroprotective approach for Machado-Joseph disease therapy.

Co-Encapsulation of Anti-BCR-ABL siRNA and Imatinib Mesylate in Transferrin Receptor-Targeted Sterically Stabilized Liposomes for Chronic Myeloid Leukemia Treatment

Chronic myeloid leukemia (CML) is triggered by the BCR‐ABL oncogene. Imatinib is the first‐line treatment of CML; however imatinib resistance and intolerance have been detected in many patients. Therefore, new therapeutic approaches are required. The present work aimed at the development and application of transferrin receptor (TrfR) targeted liposomes co‐encapsulating anti‐BCR‐ABL siRNA and imatinib at different molar ratios. The encapsulation yields and drug loading of each molecule was evaluated. Anti‐leukemia activity of the developed formulations co‐encapsulating siRNA and imatinib and of the combination of Trf‐liposomes carrying siRNA and free imatinib under two different treatment schedules of pre‐sensitization was assessed. The results obtained demonstrate that the presence of imatinib significantly decreases the encapsulation yields of siRNA, whereas imatinib encapsulation yields are increased by the presence of siRNA. Cytotoxicity assays demonstrate that the formulations co‐encapsulating siRNA and imatinib promote a 3.84‐fold reduction on the imatinib IC50 (from 3.49 to 0.91 µM), whereas a 8.71‐fold reduction was observed for the pre‐sensitization protocols (from 42.7 to 4.9 nM). It was also observed that the formulations with higher siRNA to imatinib molar ratios promote higher cell toxicity. Thus, the present work describes a novel triple targeting strategy with one single system: cellular targeting (through the targeting ligand, transferrin) and molecular targeting at the BCR‐ABL mRNA and Bcr‐Abl protein level. Biotechnol. Bioeng. 2010;107: 884–893.

Re-establishing ataxin-2 downregulates translation of mutant ataxin-3 and alleviates Machado–Joseph disease

Machado-Joseph disease is a progressive neurodegenerative disorder associated with the polyQ-expanded ataxin-3 (encoded by ATXN3), for which no therapy is available. With the aim of clarifying the mechanism of neurodegeneration, we hypothesized that the abnormally long polyQ tract would interact aberrantly with ataxin-2 (encoded by ATXN2), another polyQ protein whose function has recently been linked to translational regulation. Using patients samples and cellular and animals models we found that in Machado-Joseph disease: (i) ataxin-2 levels are reduced; and (ii) its subcellular localization is changed towards the nucleus. Restoring ataxin-2 levels by lentiviral-mediated overexpression: (i) reduced mutant ataxin-3 levels; and (ii) rescued behaviour defects and neuropathology in a transgenic mouse model of Machado-Joseph disease. Conversely (i) mutating the ataxin-2 motif that enables binding to its natural interactor and translation activator poly(A)-binding protein; or (ii) overexpressing poly(A)-binding protein, had opposite effects, increasing mutant ataxin-3 translation and aggregation. This work suggests that in Machado-Joseph disease, mutant ataxin-3 drives an abnormal reduction of ataxin-2 levels, which overactivates poly(A)-binding protein, increases translation of mutant ataxin-3 and other proteins and aggravates Machado-Joseph disease. Re-establishment of ataxin-2 levels reduces mutant ataxin-3 and alleviates Machado-Joseph disease pathogenesis opening a new avenue for therapeutic intervention in this and potentially other polyQ disorders.

Motor Dysfunctions and Neuropathology in Mouse Models of Spinocerebellar Ataxia Type 2: A Comprehensive Review

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant ataxia caused by an expansion of CAG repeats in the exon 1 of the gene ATXN2, conferring a gain of toxic function that triggers the appearance of the disease phenotype. SCA2 is characterized by several symptoms including progressive gait ataxia and dysarthria, slow saccadic eye movements, sleep disturbances, cognitive impairments, and psychological dysfunctions such as insomnia and depression, among others. The available treatments rely on palliative care, which mitigate some of the major symptoms but ultimately fail to block the disease progression. This persistent lack of effective therapies led to the development of several models in yeast, C. elegans, D. melanogaster, and mice to serve as platforms for testing new therapeutic strategies and to accelerate the research on the complex disease mechanisms. In this work, we review 4 transgenic and 1 knock-in mouse that exhibit a SCA2-related phenotype and discuss their usefulness in addressing different scientific problems. The knock-in mice are extremely faithful to the human disease, with late onset of symptoms and physiological levels of mutant ataxin-2, while the other transgenic possess robust and well-characterized motor impairments and neuropathological features. Furthermore, a new BAC model of SCA2 shows promise to study the recently explored role of non-coding RNAs as a major pathogenic mechanism in this devastating disorder. Focusing on specific aspects of the behavior and neuropathology, as well as technical aspects, we provide a highly practical description and comparison of all the models with the purpose of creating a useful resource for SCA2 researchers worldwide.

Safety profile of the intravenous administration of brain-targeted stable nucleic acid lipid particles.

In a clinical setting, where multiple administrations of the therapeutic agent are usually required to improve the therapeutic outcome, it is crucial to assess the immunogenicity of the administered nanoparticles. In this data work, we investigated the safety profile of the repeated intravenous administration of brain-targeted stable nucleic acid lipid particles (RVG-9r-targeted SNALPs). To evaluate local activation of the immune system, we performed analysis of mouse tissue homogenates and sections from cerebellum. To investigate peripheral activation of the immune system, we used serum of mice that were intravenously injected with RVG-9r-targeted SNALPs. These data are related and were discussed in the accompanying research article entitled “Intravenous administration of brain-targeted stable nucleic acid lipid particles alleviates Machado–Joseph disease neurological phenotype” (Conceição et al., in press) [1].

Stem Cell-Based Therapies for Polyglutamine Diseases

Polyglutamine (polyQ) diseases are a family of neurodegenerative disorders with very heterogeneous clinical presentations, although with common features such as progressive neuronal death. Thus, at the time of diagnosis patients might present an extensive and irreversible neuronal death demanding cell replacement or support provided by cell-based therapies. For this purpose stem cells, which include diverse populations ranging from embryonic stem cells (ESCs), to fetal stem cells, mesenchymal stromal cells (MSCs) or induced pluripotent stem cells (iPSCs) have remarkable potential to promote extensive brain regeneration and recovery in neurodegenerative disorders. This regenerative potential has been demonstrated in exciting pre and clinical assays. However, despite these promising results, several drawbacks are hampering their successful clinical implementation. Problems related to ethical issues, quality control of the cells used and the lack of reliable models for the efficacy assessment of human stem cells. In this chapter the main advantages and disadvantages of the available sources of stem cells as well as their efficacy and potential to improve disease outcomes are discussed.

Cordycepin activates autophagy through AMPK phosphorylation to reduce abnormalities in Machado–Joseph disease models

Abstract Machado‐Joseph disease (MJD) is a neurodegenerative disorder caused by an abnormal expansion of citosine‐adenine‐guanine trinucleotide repeats in the disease‐causing gene. This mutation leads to an abnormal polyglutamine tract in the protein ataxin‐3 (Atx3), resulting in formation of mutant Atx3 aggregates. Despite several attempts to develop a therapeutic option for MJD, currently there are no available therapies capable of delaying or stopping disease progression. Recently, our group reported that reducing the expression levels of mutant Atx3 lead to a mitigation of several MJD‐related behavior and neuropathological abnormalities. Aiming a more rapid translation to the human clinics, in this study we investigate a pharmacological inhibitor of translation—cordycepin—in several preclinical models. We found that cordycepin treatment significantly reduced (i) the levels of mutant Atx3, (ii) the neuropathological abnormalities in a lentiviral mouse model, (iii) the motor and neuropathological deficits in a transgenic mouse model and (iv) the number of ubiquitin aggregates in a human neural model. We hypothesize that the effect of cordycepin is mediated by the increase of phosphorylated adenosine monophosphate‐activated protein kinase (AMPK) levels, which is accompanied by a reduction in the global translation levels and by a significant activation of the autophagy pathway. Overall, this study suggests that cordycepin might constitute an effective and safe therapeutic approach for MJD, and probably for the other polyglutamine diseases.

487. Non-Viral Silencing of Machado-Joseph Disease Through the Systemic Route

Tese de doutoramento em Ciencias Farmaceuticas, na area de especializacao de Biotecnologia Farmaceutica, apresentada a Faculdade de Farmacia da Universidade de Coimbra