Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Aug 12:13:148.
doi: 10.3389/fnmol.2020.00148. eCollection 2020.

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

Joseph Edward Rittiner  1   2   3 , Malik Moncalvo  1   2   3 , Ornit Chiba-Falek  4   5 , Boris Kantor  1   2   3
Affiliations

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

Joseph Edward Rittiner et al. Front Mol Neurosci. .

Abstract

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).

Keywords: AAV vectors; CRISPR-Cas 9 system; epigenetics (DNA methylation; gene editing; histone modifications); lentiviral (LV) vector; neurodegenarative diseases.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Lentivirus basics. (A) Simplified schematic of the wild-type human immunodeficiency virus type-1 (HIV-1) genome. (B) Lentivirus particle structure. (C) Plasmids used in the current (3rd generation) lentivirus packaging system. See the main text for a detailed description of the lentivirus packaging system; see Table 1 for lentivirus envelope proteins.
Figure 2
Figure 2
Adeno-Associated Virus (AAV)basics. (A) Simplified schematic of the wild-type AAV genome. (B) Plasmids used in the current AAV packaging system. See the main text for a detailed description of the AAV packaging system; see Table 2 for a comparison of common AAV serotypes.
Figure 3
Figure 3
Comparison of recombinant Integrase-Competent Lentivirus (ICLV), Integrase-Deficient Lentivirus (IDLV), and AAV life cycles. ICLV, IDLV, and AAV bind and enter target cells (i). AAV particles escape from endosomes into the cytoplasm (iia), then enter the nucleus and un-coat (iiia). After un-coating, a host polymerase performs second strand synthesis, leading to circularization and concatemerization of the AAV vector; a small percent integrates randomly into the host genome. Transcription occurs from all forms of the AAV transgene (iva). Uncoating and reverse transcription of ICLVs and IDLVs occur in the cytoplasm (iib). The dsDNA product is then imported into the nucleus (iiib). Some of this DNA integrates into the host genome, while the majority recombines into one- or two-LTR circles and remains episomal (ivb). Transcription occurs from all forms of the transgene, but rates of integration and circle formation differ between IDLV and ICLV (see Table 3).
Figure 4
Figure 4
Applications of clustered regularly interspaced short palindromic repeats (CRISPR) technology (A) Active Cas9 introduces a double-stranded DNA break, which is repaired via non-homologous end joining (NHEJ), creating indels. Alternatively, if a dsDNA donor template is provided, the dsDNA break can be repaired by homologous recombination, resulting in a targeted insertion. (B) Cytosine Base Editors catalyze the conversion of all cytosines within a 5–6 nucleotide window to uracils. Uracil is then read as thymine during replication, completing the C:G to T:A conversion. (C) Similarly, Adenosine Base Editors (ABEs) catalyze the conversion of all adenosines within a 5–6 nucleotide window to inosines. Inosine is then read as guanine during replication, completing the A:T to G:C conversion.
Figure 5
Figure 5
Proposed mechanism of prime editing. First, the 5’ end of the pegRNA binds to the protospacer of the target DNA and the protospacer-adjacent motif (PAM) strand is nicked (i). The nicked PAM strand then hybridizes with the primer binding site (PBS) at the far 3’ end of the pegRNA (ii). The interior of the pegRNA then serves as a template for reverse transcription, which extends from the free 3’-OH of the PAM strand (iii). The prime editing complex then disengages, leaving the target site with two redundant PAM strands, or “flaps” (iv). The unedited 5’ flap is preferentially degraded by cellular endonucleases, allowing the edited 3’ flap to hybridize with the non-PAM strand. Finally, DNA repair mechanisms transfer the desired edits to the non-PAM strand (v).
Figure 6
Figure 6
Strategies for epigenetic repression of risk-factor genes using Cas9 fusion proteins. Fusions containing the catalytic domain of a DNA methyltransferase cause targeted methylation of CpG sites and the recruitment of inhibitory methyl-CpG-binding proteins (i). Alternatively, a transcriptional repression domain (TRD) can be fused to Cas9, leading to the direct recruitment of transcriptional repression complexes (ii). Finally, multiple forms of inhibitory histone-modifying enzymes can be fused to Cas9, altering histone acetylation/methylation patterns and causing the formation of closed chromatin (iii).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Adli M. (2018). The CRISPR tool kit for genome editing and beyond. Nat. Commun. 9:1911. 10.1038/s41467-018-04252-2 - DOI - PMC - PubMed
    1. Alzheimer’s Dementia . (2020). 2020 Alzheimer’s disease facts and figures. Alzheimers Dement. 16, 391–460. 10.1201/b15134-4 - DOI - PubMed
    1. Amir R. E., Van den Veyver I. B., Wan M., Tran C. Q., Francke U., Zoghbi H. Y. (1999). Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat. Genet. 23, 185–188. 10.1038/13810 - DOI - PubMed
    1. Anders C., Niewoehner O., Duerst A., Jinek M. (2014). Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513, 569–573. 10.1038/nature13579 - DOI - PMC - PubMed
    1. Anzalone A. V., Randolph P. B., Davis J. R., Sousa A. A., Koblan L. W., Levy J. M., et al. . (2019). Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157. 10.1038/s41586-019-1711-4 - DOI - PMC - PubMed

LinkOut - more resources