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      CRISPR/Cas9 Delivery Potentials in Alzheimer’s Disease Management: A Mini Review

      review-article
      1 , 2 , * , 3 , 1
      Pharmaceutics
      MDPI
      CRISPR, Alzheimer’s disease, drug delivery, gene editing

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          Abstract

          Alzheimer’s disease (AD) is the most common dementia disorder. While genetic mutations account for only 1% of AD cases, sporadic AD resulting from a combination of genetic and risk factors constitutes >90% of the cases. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein (Cas9) is an impactful gene editing tool which identifies a targeted gene sequence, creating a double-stranded break followed by gene inactivation or correction. Although CRISPR/Cas9 can be utilized to irreversibly inactivate or correct faulty genes in AD, a safe and effective delivery system stands as a challenge against the translation of CRISPR therapeutics from bench to bedside. While viral vectors are efficient in CRISPR/Cas9 delivery, they might introduce fatal side effects and immune responses. As non-viral vectors offer a better safety profile, cost-effectiveness and versatility, they can be promising for the in vivo delivery of CRISPR/Cas9 therapeutics. In this minireview, we present an overview of viral and non-viral vector based CRISPR/Cas9 therapeutic strategies that are being evaluated on pre-clinical AD models. Other promising non-viral vectors that can be used for genome editing in AD, such as nanoparticles, nanoclews and microvesicles, are also discussed. Finally, we list the formulation and technical aspects that must be considered in order to develop a successful non-viral CRISPR/Cas9 delivery vehicle.

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          Most cited references47

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          A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.

          Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
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            Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage

            Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. 1,2 Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus from the cellular response to dsDNA breaks. 1,2 Here we report the development of base editing, a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting “base editors” convert cytidines within a window of approximately five nucleotides (nt), and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor (UGI), and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favor desired base-editing outcomes, resulting in permanent correction of ∼15-75% of total cellular DNA with minimal (typically ≤ 1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.
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              Search-and-replace genome editing without double-strand breaks or donor DNA

              Summary Most genetic variants that contribute to disease 1 are challenging to correct efficiently and without excess byproducts 2–5 . Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed >175 edits in human cells including targeted insertions, deletions, and all 12 types of point mutations without requiring double-strand breaks or donor DNA templates. We applied prime editing in human cells to correct efficiently and with few byproducts the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA), to install a protective transversion in PRNP, and to precisely insert various tags and epitopes into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer byproducts than homology-directed repair, complementary strengths and weaknesses compared to base editing, and much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle can correct up to 89% of known genetic variants associated with human diseases.
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                Author and article information

                Journal
                Pharmaceutics
                Pharmaceutics
                pharmaceutics
                Pharmaceutics
                MDPI
                1999-4923
                25 August 2020
                September 2020
                : 12
                : 9
                : 801
                Affiliations
                [1 ]Department of Pharmaceutics, Institute of Pharmacy, University of Bonn, 53121 Bonn, Germany; alf.lamprecht@ 123456uni-bonn.de
                [2 ]Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, Pharos University in Alexandria, Alexandria 21615, Egypt
                [3 ]Department of Neuropathology, University of Bonn Medical Center, 53105 Bonn, Germany; susanne.schoch@ 123456uni-bonn.de
                Author notes
                [* ]Correspondence: amira.sayed@ 123456pua.edu.eg or amirahanafy@ 123456uni-bonn.de ; Tel.: +20-3-3877394
                Author information
                https://orcid.org/0000-0002-1938-3393
                https://orcid.org/0000-0002-2046-2670
                Article
                pharmaceutics-12-00801
                10.3390/pharmaceutics12090801
                7559557
                32854251
                932a85af-0c7b-4ded-a668-5c619a28fb99
                © 2020 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 14 July 2020
                : 20 August 2020
                Categories
                Review

                crispr,alzheimer’s disease,drug delivery,gene editing
                crispr, alzheimer’s disease, drug delivery, gene editing

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