Brain-targeted delivery of neuroprotective survival gene minimizing hematopoietic cell contamination: implications for Parkinson's disease treatment

Abstract

Background: Neurodegenerative diseases, including Parkinson's disease, Amyotropic Lateral Sclerosis (ALS) and Alzheimer's disease, present significant challenges for therapeutic development due to drug delivery restrictions and toxicity concerns. Prevailing strategies often employ adeno-associated viral (AAV) vectors to deliver neuroprotective survival genes directly into the central nervous system (CNS). However, these methods have been limited by triggering immunogenic responses and risk of tumorigenicity, resulting from overexpression of survival genes in peripheral blood mononuclear cells (PBMC), thereby increasing the risk of tumorigenicity in specific immune cells. Thus, by coding selectively suppressive microRNA (miRNA) target sequences in AAV genome, we designed CNS-targeted neuroprotective gene expression vector system without leakage to blood cells.

Methods: To minimize the potential for transgene contamination in the blood, we designed a CNS-specific AAV system. Our system utilized a self-complementary AAV (scAAV), encoding a quadruple repeated target sequence of the hematopoietic cell-specific miR142-3p at the 3' untranslated region (UTR). As a representative therapeutic survival gene for Parkinson's disease treatment, we integrated DX2, an antagonistic splice variant of the apoptotic gene AIMP2, known to be implicated in Parkinson's disease, into the vector.

Results: This configuration ensured that transgene expression was stringently localized to the CNS, even if the vector found its way into the blood cells. A single injection of scAAV-DX2 demonstrated marked improvement in behavior and motor activity in animal models of Parkinson's disease induced by either Rotenone or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Importantly, comprehensive preclinical data adhering to Good Laboratory Practice (GLP) standards revealed no adverse effects in the treated animals.

Conclusions: Our CNS-specific vector system, which encodes a survival transgene DX2, signifies a promising avenue for safe gene therapy, avoiding unintended expression of survival gene in blood cells, applicable to various neurodegenerative diseases.

Keywords: AIMP2 splicing variants; Adeno-associated virus; DX2; Gene therapy; Parkinson’s disease; miRNA.

Fig. 1

Protective effect of DX2 against ROS-induced cell death in SH-SY5Y cells. A A graph showing a viable cell counting in SH-SY5Y cells overexpressing Flag-EV, Flag-AIMP2, and Flag-DX2, after screening with cell death-inducing chemicals. All chemicals were treated for 24 h after vector overexpression, at the following concentrations: H₂O₂ (100 μM), 6-OHDA (100 μM), TNF-α (10 μM), cycloheximide (CHX, 10 μM), actinomycin D (Act D, 10 nM), cisplatin (Cis, 10 μM), paclitaxel (Pac, 10 μM), and 5-fluorouracil (5-FU, 10 μM). B A graph comparing cell counts between AAV-GFP and AAV-DX2 infections. All cell countings were repeated three times. C–E Quantitative PCR graphs analyzing the expression patterns of mRNAs involved in cell survival by AAV-DX2 F–I Immunoblot assays confirming the Caspase-cascade pathway in cell survival induced by AAV-DX2. J Quantification of dot blot assay data for AAV-GFP and AAV-DX2

Fig. 2

Efficacy test of scAAV2-GFP and scAAV2-DX2 in vitro and in vivo A, B Transduction efficacy test of AAV2-GFP in Hela, Hek293 and SK-N-SH. A 1000MOI concentration of ssAAV2-GFP and scAAV2-GFP B 10000MOI cencentration of ssAAV2-GFP and scAAV2-GFP C GFP expression was observed with fluorescence microscopy in SH-SY5Y. D The percentage of GFP-positive cells was measured following dose-dependent infection for 48 h with ssAAV2-GFP and scAAV-GFP in the SH-SY5Y cell line. E scAAV2-GFP expression in the right striatum, scale bar: 500 μm and 100 μm. F scAAV2-GFP expression in the spinal cord of intrathecally injected mice. The lumbar region specified as L1 to L5 was injected and used for confocal imaging. G DX2 expression was checked on the day after injection at the isolated substantia nigra using real-time qPCR. After scAAV2-DX2 treatment in H₂O₂-induced apoptosis situations. H The difference in AIMP2 and DX2 expression levels in mouse organ

Fig. 3

Development of scAAV-DX2 with the microRNA142-3p target sequence A Screening of hematopoietic cell expressed microRNA. Among the miRNA, miR142-3p was more highly expressed than the others B The relative value of Luciferase assay result with hematopoietic cell expressed miRNA and its target plasmid. P Plasmid. C Scheme of DX2 vector with variously repeated miR142-3p target sequence and mutation form. The symbol of * is a single mutation sequence. D, E DX2 expression is inhibited by the miR142-3p target sequence in hematopoietic cells. F Scheme of scAAV-DX2 with miR142-3p target sequence. ns: no-significant; *: p < 0.05; **:p < 0.01; ***:p < 0.001. t-test. TS: miR142-3p target sequence; mut: mutation

Fig. 4

scAAV2-DX2-miR142-3p target sequence selectively expressed in the target organ A Comparison of transgene expression between scAAV-DX2 and scAAV2-DX2-miR142-3p target sequence plasmid in hematopoietic cells and neuronal cells. B Relative expression ratio of DX2 with or without miR142 inhibitor in THP-1 cells. ‘ + ’ means 10 pmol of miR142 inhibitor. C Body distribution test for scAAV-DX2 in intracranially injected mice. D–G Assessing scAAV-DX2 spreading and expression during intrathecal injection administration between lumbar and blood. D DNA level of DX2 in low-dose injected mice E DX2 level of DX2 in high-dose injected mice. F RNA level of DX2 in low-dose injected mice G RNA level of DX2 in high-dose injected mice. animal number: n = 6, Low dose: 2.4 × 10¹⁰vg/animal, High dose: 9.6 × 10¹⁰vg/animal. ns: no-significant; *: p < 0.05; **:p < 0.01; ***: p < 0.001 TS miR142-3p target sequence, SC spinal cord, CB celebellum, CL cerebral lobes, BS Brain stem, WB whole blood

Fig. 5

DX2 restores motor symptoms in MPTP-induced PD model A Scheme of scAAV2-DX2 transduction in MPTP-induced mouse model. scAAV2-DX2 was infused in the substantial nigra region with the stereotaxic machine and then subcutaneous injection with MPTP was performed once per day. Before measuring the behaviour test, the training of each test was continued. B scAAV2-DX2-treated mice showed slightly longer latency to fall in the rotarod test when compared with vehicle (scAAV2-GFP, GFP), indicating that scAAV2-DX2 attenuated damage towards dopaminergic neurons. C DX2-treated mice showed improved locomotor activity based on the SHIRPA test. D DX2-treated mice showed a relatively lower level of limb deficit. E Immunofluorescence image of TH-positive cells in the mouse substantia nigra. The white scale bar represents 500 µm F, G DX2 F and Bax G mRNA expression of the indicated mice brain. H–I Quantification of immunoblot assay for cleaved caspase-8 and cleaved caspase-9. animals: naive, n = 6; GFP, n = 9; DX2, n = 12. scAAV2: scAAV2-GFP, 4 × 10⁹ vg; scAAV2-DX2, 4 × 10⁹ vg. ns, non-significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; t-test

Fig. 6

scAAV2-DX2 has a therapeutic effect in rotenone-induced parkinsonism mice A scheme of the therapeutic model of rotenone-induced Parkinson’s disease using scAAV2-DX2 B The pole test. C The rotarod test. scAAV2-DX2 recovered motor coordination and balance in the rotenone-treated PD mouse model. D Immunohistochemistry and immunofluorescence image of the mouse substantia nigra. The upper panel shows TH-positive cells in the striatum and the lower panel indicates the distribution of an injected-GFP expressing virus. The white scale bar represents 50 µm. Animals; n = 5 (in each group), ns, no significant; *, P < 0.05; ***, P < 0.001; t-test. E Schemic image of regulation of therapeutic viral genome