Exploring the potential of cell-derived vesicles for transient delivery of gene editing payloads.

Gene editing technologies have the potential to correct genetic disorders by modifying, inserting, or deleting specific DNA sequences or genes, paving the way for a new class of genetic therapies. While gene editing tools continue to be improved to increase their precision and efficiency, the limited efficacy of in vivo delivery remains a major hurdle for clinical use. An ideal delivery vehicle should be able to target a sufficient number of diseased cells in a transient time window to maximize on-target editing and mitigate off-target events and immunogenicity. Here, we review major advances in novel delivery platforms based on cell-derived vesicles - extracellular vesicles and virus-like particles - for transient delivery of gene editing payloads. We discuss major findings regarding packaging, in vivo biodistribution, therapeutic efficacy, and safety concerns of cell-derived vesicles delivery of gene-editing cargos and their potential for clinical translation.

Isolation of Adeno-associated Viral Vectors Through a Single-step and Semi-automated Heparin Affinity Chromatography Protocol.

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. However, the commonly used methods to purify AAVs make use of cesium chloride or iodixanol density gradient ultracentrifugation. Despite their advantages, these methods are time-consuming, have limited scalability, and often result in vectors with low purity. To overcome these constraints, researchers are turning their attention to chromatography techniques. Here, we present an optimized heparin-based affinity chromatography protocol that serves as a universal capture step for the purification of AAVs. This method relies on the intrinsic affinity of AAV serotype 2 (AAV2) for heparan sulfate proteoglycans. Specifically, the protocol entails the co-transfection of plasmids encoding the desired AAV capsid proteins with those of AAV2, yielding mosaic AAV vectors that combine the properties of both parental serotypes. Briefly, after the lysis of producer cells, a mixture containing AAV particles is directly purified following an optimized single-step heparin affinity chromatography protocol using a standard fast protein liquid chromatography (FPLC) system. Purified AAV particles are subsequently concentrated and subjected to comprehensive characterization in terms of purity and biological activity. This protocol offers a simplified and scalable approach that can be performed without the need for ultracentrifugation and gradients, yielding clean and high viral titers.

A new protocol for whole-brain biodistribution analysis of AAVs by tissue clearing, light-sheet microscopy and semi-automated spatial quantification.

Recombinant adeno-associated virus (rAAV) has become one of the most promising gene delivery systems for both in vitro and in vivo applications. However, a key challenge is the lack of suitable imaging technologies to evaluate delivery, biodistribution and tropism of rAAVs and efficiently monitor disease amelioration promoted by AAV-based therapies at a whole-organ level with single-cell resolution. Therefore, we aimed to establish a new pipeline for the biodistribution analysis of natural and new variants of AAVs at a whole-brain level by tissue clearing and light-sheet fluorescence microscopy (LSFM). To test this platform, neonatal C57BL/6 mice were intravenously injected with rAAV9 encoding EGFP and, after sacrifice, brains were processed by standard immunohistochemistry and a recently released aqueous-based clearing procedure. This clearing technique required no dedicated equipment and rendered highly cleared brains, while simultaneously preserving endogenous fluorescence. Moreover, three-dimensional imaging by LSFM allowed the quantitative analysis of EGFP at a whole-brain level, as well as the reconstruction of Purkinje cells for the retrieval of valuable morphological information inaccessible by standard immunohistochemistry. In conclusion, the pipeline herein described takes the AAVs to a new level when coupled to LSFM, proving its worth as a bioimaging tool in tropism and gene therapy studies.

ULK overexpression mitigates motor deficits and neuropathology in mouse models of Machado-Joseph disease.

Machado-Joseph disease (MJD) is a fatal neurodegenerative disorder clinically characterized by prominent ataxia. It is caused by an expansion of a CAG trinucleotide in ATXN3, translating into an expanded polyglutamine (polyQ) tract in the ATXN3 protein, that becomes prone to misfolding and aggregation. The pathogenesis of the disease has been associated with the dysfunction of several cellular mechanisms, including autophagy and transcription regulation. In this study, we investigated the transcriptional modifications of the autophagy pathway in models of MJD and assessed whether modulating the levels of the affected autophagy-associated transcripts (AATs) would alleviate MJD-associated pathology. Our results show that autophagy is impaired at the transcriptional level in MJD, affecting multiple AATs, including Unc-51 like autophagy activating kinase 1 and 2 (ULK1 and ULK2), two homologs involved in autophagy induction. Reinstating ULK1/2 levels by adeno-associated virus (AAV)-mediated gene transfer significantly improved motor performance while preventing neuropathology in two in vivo models of MJD. Moreover, in vitro studies showed that the observed positive effects may be mainly attributed to ULK1 activity. This study provides strong evidence of the beneficial effect of overexpression of ULK homologs, suggesting these as promising instruments for the treatment of MJD and other neurodegenerative disorders.

miRNA-Mediated Knockdown of ATXN3 Alleviates Molecular Disease Hallmarks in a Mouse Model for Spinocerebellar Ataxia Type 3.

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder caused by the expansion of a CAG repeat in the ATXN3 gene. This mutation leads to a toxic gain of function of the ataxin-3 protein, resulting in neuronal dysfunction and atrophy of specific brain regions over time. As ataxin-3 is a dispensable protein in rodents, ataxin-3 knockdown by gene therapy may be a powerful approach for the treatment of SCA3. In this study, we tested the feasibility of an adeno-associated viral (AAV) vector carrying a previously described artificial microRNA against ATXN3 in a striatal mouse model of SCA3. Striatal injection of the AAV resulted in good distribution throughout the striatum, with strong dose-dependent ataxin-3 knockdown. The hallmark intracellular ataxin-3 inclusions were almost completely alleviated by the microRNA-induced ATXN3 knockdown. In addition, the striatal lesion of dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32) in the SCA3 mice was rescued by ATXN3 knockdown, indicating functional rescue of neuronal signaling and health upon AAV treatment. Together, these data suggest that microRNA-induced ataxin-3 knockdown is a promising therapeutic strategy in the treatment of SCA3.

Protocol for the Characterization of the Cytosine-Adenine-Guanine Tract and Flanking Polymorphisms in Machado-Joseph Disease: Impact on Diagnosis and Development of Gene-Based Therapies.

Polyglutamine spinocerebellar ataxias (SCAs) constitute a group of autosomal dominantly inherited neurodegenerative disorders with considerable phenotypic overlap. Definitive diagnoses rely on the detection of a mutation in each associated locus, comprising the abnormal expansion of the trinucleotide cytosine-adenine-guanine (CAG) in coding exons. Assessment of single nucleotide polymorphisms associated with the CAG expansion in the context of SCAs is also relevant for improving molecular diagnosis and for generating novel therapeutic strategies. The current study is focused on Machado-Joseph disease/SCA type 3, with the aim of developing a protocol for the accurate determination of the CAG length in exon 10 of the human ATXN3 gene and to characterize flanking polymorphisms. A single pair of primers was designed and validated, and two complementary PCR-based methods were established. In method I, PCR amplicons were cloned and sequenced, allowing the assessment of three single nucleotide polymorphisms in the vicinity of the CAG repeat (C987GG/G987GG, TAA1118/TAC1118, and C1178/A1178), which can constitute potential targets for personalized gene-based therapies. Method II combines PCR, capillary electrophoresis, and a size correction formula, enabling a time and cost-effective determination of the number of CAGs. The established protocol paves the way to overcome technical difficulties related to the molecular characterization of the CAG motif and intragenic polymorphisms in the context of Machado-Joseph disease/SCA type 3 and may prove useful when applied to other polyglutamine SCAs.

Antisense oligonucleotide therapeutics in neurodegenerative diseases: the case of polyglutamine disorders.

Polyglutamine (polyQ) disorders are a group of nine neurodegenerative diseases that share a common genetic cause, which is an expansion of CAG repeats in the coding region of the causative genes that are otherwise unrelated. The trinucleotide expansion encodes for an expanded polyQ tract in the respective proteins, resulting in toxic gain-of-function and eventually in neurodegeneration. Currently, no disease-modifying therapies are available for this group of disorders. Nevertheless, given their monogenic nature, polyQ disorders are ideal candidates for therapies that target specifically the gene transcripts. Antisense oligonucleotides (ASOs) have been under intense investigation over recent years as gene silencing tools. ASOs are small synthetic single-stranded chains of nucleic acids that target specific RNA transcripts through several mechanisms. ASOs can reduce the levels of mutant proteins by breaking down the targeted transcript, inhibit mRNA translation or alter the maturation of the pre-mRNA via splicing correction. Over the years, chemical optimization of ASO molecules has allowed significant improvement of their pharmacological properties, which has in turn made this class of therapeutics a very promising strategy to treat a variety of neurodegenerative diseases. Indeed, preclinical and clinical strategies have been developed in recent years for some polyQ disorders using ASO therapeutics. The success of ASOs in several animal models, as well as encouraging results in the clinic for Huntington's disease, points towards a promising future regarding the application of ASO-based therapies for polyQ disorders in humans, offering new opportunities to address unmet medical needs for this class of disorders. This review aims to present a brief overview of key chemical modifications, mechanisms of action and routes of administration that have been described for ASO-based therapies. Moreover, it presents a review of the most recent and relevant preclinical and clinical trials that have tested ASO therapeutics in polyQ disorders.