Trends and challenges of AAV-delivered gene editing therapeutics for CNS disorders: Implications for neurodegenerative disease

Abstract

Recent advances in gene-editing technologies offer new opportunities for drug development to treat unmet medical needs in central nervous system (CNS) disorders including neurogenerative diseases of the aging brain. The adeno-associated virus (AAV) is a promising and most widely utilized vector for gene therapy application including the CNS. AAV is characterized by high transduction efficiency in both dividing and non-dividing cells, low immunogenicity and toxicity, and exceptional tissue specificity. The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-based technologies has revolutionized all aspects of modern sciences and created an innovative therapeutic toolkit with the potential to address a wide range of neurological diseases, including Alzheimer's (AD) and Parkinson's (PD) diseases. However, AAV limitations for delivering CRISPR modalities continue to impede viable therapeutic interventions targeting the brain. This review highlights challenges and strategies to deliver AAV-CRISPR-based therapeutic cargos for gene therapy applications in the CNS, with a particular focus on AD and PD preclinical studies.

Keywords: APOE; Alzheimer’s disease; MT: Delivery Strategies; Parkinson’s disease; SNCA; adeno-associated vector; all-in-one delivery system; clustered regularly interspaced short-palindromic repeats/CRISPR-associated protein; epigenome-based editing; gene editing; transcriptional repressor.

Graphical abstract

Figure 1

The AAV genome (A) Schematic organization of the AAV genome. The viral genome consists of 4.7 kb of ssDNA and carries two ORFs, rep and cap; which are flanked by a pair of 146-bp inverted terminal repeats (ITRs). A total of eight polypeptides are produced from these ORFs; four enzymatic proteins encoded by rep (Rep78/68 and Rep52/40), and three structural capsid proteins (VP1, VP2, and VP3) and an assembly activating protein (AAP), encoded by cap. The two larger Rep proteins involved in wild-type virus integration and replication, and the shorter proteins mediate the packaging of the viral DNA into the particles. The capsid VP1, VP2, and VP3 proteins are generated at a ratio of 1:1:10, respectively, 60 total copies of these structural proteins create a nonenveloped icosahedral AAV particle. Last, the aforementioned AAP protein is responsible for transporting VP1-3 proteins to the nucleolus, which then enables viral packaging and assembly. (B) Production of AAV vectors. The wild type of the vector’s genome is split into three cassettes: the expression cassette carries a transgene-of-interest, which is flanked by 5′- and 3′-ITRs to ensure the packaging. The packaging cassette supplies rep-cap genes, and the helper cassette provides E2a, E4org6, and VA RNA gene products. The CMV promoter is used to express helper and packaging plasmids’ genes as shown.

Figure 2

AAV administration for CNS disorders The strategies of the AAV optimization for neurodegenerative diseases’ treatment are highlighted. (1) The selection of an optimal route-of-administration (ROA) is highlighted. In this regard, the following main ROAs are outlines for the CNS delivery: 1. intracisternal (via injections into intra-cisterna magna [ICM]); 2. intrathecal (IT); and 3. intracerebroventricularly (ICV). (2) The approaches related to the development of the viral capsids/serotypes that are crossing the BBB are discussed in the main text. (3) The optimization in the viral ITRs and other cis-acting elements within the expression cassettes are discussed in the main text. (4) The self-complementary approach aimed to decrease the unpackaging time and increase gene expression of the viral genome is highlighted in the text. (5) miRNA-detargeting is covered in the main text as well.

Figure 3

APOE-targeted therapeutics (A) Schematic organization of the APOE gene locus. Apolipoprotein E (ApoE) is encoded by the APOE gene positioned on chromosome 19q13.32 (GRCh 38/hg38: chr19:44,905,795–44,909,392). Two common coding SNPs in exon 4 change amino acids at positions 112 and 158 and give rise to three protein isoforms, APOEε2, APOEε3, and APOEε4 (Cys-Cys, Cys-Arg, Arg-Arg, respectively). A rare SNP, Christchurch, on the background of APOEε3—APOEε3ch—encodes a protein isoform that differs at amino acid position R136S (Arg-Ser). (B) A schematic diagram of the APOE-targeted epigenome therapy system. In individuals, carriers of the APOEε4 allele, the therapeutics vector will repress the expression of the ε4 allele and mitigates its pathogenic effects. Left, a representation of the all-in-one AAV-epigenome editing platform. Middle, the expressed platform targets the APOE gene, red circle designates the ε4-SNP (rs429358). Right, the targeting leads to specific reduction in APOEe4 expression. Lower panel, the concept of the technology. The repressor mediates its effect via closing the chromatin structure at the gene regulatory region.^,

Figure 4

SNCA intron 1 targeted epigenome-editing platform (A) A schematic diagram of the SNCA-targeted epigenome therapy platform. Upper panel, SNCA gene structure; lower left, an example of the all-in-one lentivirus (LV)-epigenome editing vector targeting dopaminergic neurons via expression driven by TH regulatory sequences; middle, the expressed platform targets intron 1 of SNCA gene; right, the effects of the all-in-one LV-repressor vector on the reduction of SNCA-mRNA and protein levels. Lower right, diagram of the components of the all-in-one vector. (B) A schematic description of the targeted region in SNCA intron 1. The gRNA sequences are marked in red; the PAM sequences are designated in bold, and the CpG nucleotides are numbered in blue. The SNCA transcription start site (TSS) is highlighted with an arrow. Two epigenome-editing repressor units used to repress SNCA gene expression are highlighted: KRAB-MeCP2(TRD) and DNMT3A. The locations of these repressors paired with the respective gRNA1 and 2, respectively, are marked at the intron 1 region.