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      Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins

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          Summary

          Methods to deliver gene editing agents in vivo as ribonucleoproteins could offer safety advantages over nucleic acid delivery approaches. We report the development and application of engineered DNA-free virus-like particles (eVLPs) that efficiently package and deliver base editor or Cas9 ribonucleoproteins. By engineering VLPs to overcome cargo packaging, release, and localization bottlenecks, we developed fourth-generation eVLPs that mediate efficient base editing in several primary mouse and human cell types. Using different glycoproteins in eVLPs alters their cellular tropism. Single injections of eVLPs into mice support therapeutic levels of base editing in multiple tissues, reducing serum Pcsk9 levels 78% following 63% liver editing, and partially restoring visual function in a mouse model of genetic blindness. In vitro and in vivo off-target editing from eVLPs was virtually undetected, an improvement over AAV or plasmid delivery. These results establish eVLPs as promising vehicles for therapeutic macromolecule delivery that combine key advantages of both viral and nonviral delivery.

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          Highlights

          • Engineered virus-like particles (eVLPs) overcome three bottlenecks to protein delivery

          • DNA-free eVLPs efficiently deliver gene editing proteins with minimal off-target editing

          • Base editor eVLPs reduced serum Pcsk9 levels 78% following 63% liver editing in mice

          • Base editor eVLPs improved visual function in a mouse model of genetic blindness

          Abstract

          Engineered, DNA-free virus-like particles efficiently deliver gene editing proteins, have minimal off-target effects, can be applied in vivo to deliver base editors to multiple organs, and are used to improve visual function in a mouse model of genetic blindness.

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          Most cited references91

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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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            Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage

            Summary The spontaneous deamination of cytosine is a major source of C•G to T•A transitions, which account for half of known human pathogenic point mutations. The ability to efficiently convert target A•T base pairs to G•C therefore could advance the study and treatment of genetic diseases. While the deamination of adenine yields inosine, which is treated as guanine by polymerases, no enzymes are known to deaminate adenine in DNA. Here we report adenine base editors (ABEs) that mediate conversion of A•T to G•C in genomic DNA. We evolved a tRNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR-Cas9. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs (e.g., ABE7.10), that convert target A•T to G•C base pairs efficiently (~50% in human cells) with very high product purity (typically ≥ 99.9%) and very low rates of indels (typically ≤ 0.1%). ABEs introduce point mutations more efficiently and cleanly than a current Cas9 nuclease-based method, induce less off-target genome modification than Cas9, and can install disease-correcting or disease-suppressing mutations in human cells. Together with our previous base editors, ABEs advance genome editing by enabling the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.
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              Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors

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                Author and article information

                Contributors
                Journal
                Cell
                Cell
                Cell
                Cell Press
                0092-8674
                1097-4172
                20 January 2022
                20 January 2022
                : 185
                : 2
                : 250-265.e16
                Affiliations
                [1 ]Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA
                [2 ]Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA
                [3 ]Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA
                [4 ]Gavin Herbert Eye Institute, Center for Translational Vision Research, Department of Ophthalmology, University of California, Irvine, CA, USA
                [5 ]Department of Pharmacology, Case Western Reserve University, Cleveland, OH, USA
                [6 ]Department of Physiology and Biophysics, University of California, Irvine, CA, USA
                [7 ]Cardiovascular Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA
                [8 ]Division of Cardiovascular Medicine, Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA
                [9 ]Department of Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA
                [10 ]Department of Pediatrics, Division of Blood and Marrow Transplant and Cellular Therapy, University of Minnesota, Minneapolis, MN, USA
                [11 ]Department of Chemistry, University of California, Irvine, CA, USA
                [12 ]Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, USA
                Author notes
                []Corresponding author drliu@ 123456fas.harvard.edu
                [13]

                These authors contributed equally

                [14]

                Lead contact

                Article
                S0092-8674(21)01484-7
                10.1016/j.cell.2021.12.021
                8809250
                35021064
                6da4bfcc-8a5e-4f07-8de2-101666fd5287
                © 2021 The Author(s)

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

                History
                : 10 October 2021
                : 23 November 2021
                : 15 December 2021
                Categories
                Article

                Cell biology
                genome editing,base editing,in vivo delivery,ribonucleoproteins,therapeutic gene editing,virus-like particles

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