Gene-Editing Technologies Paired With Viral Vectors for Translational Research Into Neurodegenerative Diseases.

Diseases of the central nervous system (CNS) have historically been among the most difficult to treat using conventional pharmacological approaches. This is due to a confluence of factors, including the limited regenerative capacity and overall complexity of the brain, problems associated with repeated drug administration, and difficulties delivering drugs across the blood-brain barrier (BBB). Viral-mediated gene transfer represents an attractive alternative for the delivery of therapeutic cargo to the nervous system. Crucially, it usually requires only a single injection, whether that be a gene replacement strategy for an inherited disorder or the delivery of a genome- or epigenome-modifying construct for treatment of CNS diseases and disorders. It is thus understandable that considerable effort has been put towards the development of improved vector systems for gene transfer into the CNS. Different viral vectors are of course tailored to their specific applications, but they generally should share several key properties. The ideal viral vector incorporates a high-packaging capacity, efficient gene transfer paired with robust and sustained expression, lack of oncogenicity, toxicity and pathogenicity, and scalable manufacturing for clinical applications. In this review, we will devote attention to viral vectors derived from human immunodeficiency virus type 1 (lentiviral vectors; LVs) and adeno-associated virus (AAVs). The high interest in these viral delivery systems vectors is due to: (i) robust delivery and long-lasting expression; (ii) efficient transduction into postmitotic cells, including the brain; (iii) low immunogenicity and toxicity; and (iv) compatibility with advanced manufacturing techniques. Here, we will outline basic aspects of LV and AAV biology, particularly focusing on approaches and techniques aiming to enhance viral safety. We will also allocate a significant portion of this review to the development and use of LVs and AAVs for delivery into the CNS, with a focus on the genome and epigenome-editing tools based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas 9) and the development of novel strategies for the treatment of neurodegenerative diseases (NDDs).

The mechanistic role of alpha-synuclein in the nucleus: impaired nuclear function caused by familial Parkinson's disease SNCA mutations.

Alpha-synuclein SNCA has been implicated in the etiology of Parkinson's disease (PD); however, the normal function of alpha-synuclein protein and the pathway that mediates its pathogenic effect is yet to be discovered. We investigated the mechanistic role of SNCA in the nucleus utilizing isogenic human-induced pluripotent stem cells-derived neurons from PD patients with autosomal dominant mutations, A53T and SNCA-triplication, and their corresponding corrected lines by genome- and epigenome-editing. Comparisons of shape and integrity of the nuclear envelope and its resistance to stresses found that both mutations result in similar nuclear envelope perturbations that were reversed in the isogenic mutation-corrected cells. Further mechanistic studies showed that SNCA mutation has adverse effects on the nucleus by trapping Ras-related nuclear protein (RAN) and preventing it from transporting key nuclear proteins such as, DNMT3A, for maintaining normal nuclear function. For the first time, we proposed that α-syn interacts with RAN and normally functions in the nucleocytoplasmic transport while exerts its pathogenic effect by sequestering RAN. We suggest that defects in the nucleocytoplasmic transport components may be a general pathomechanistic driver of neurodegenerative diseases.

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models.

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson's disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.