RESUMO
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.
Assuntos
Doença de Parkinson , Animais , Doença de Parkinson/genética , Doença de Parkinson/terapia , Leucócitos Mononucleares , Encéfalo/metabolismo , Terapia Genética/métodos , Transgenes , Vetores Genéticos , Dependovirus/genéticaRESUMO
The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis (VL), a potentially fatal disease if left untreated. Given the limitations of current therapies, there is an urgent need for new, safe, and effective drugs. To discover novel antileishmanial compounds from previously unexplored chemical spaces, we conducted a high-throughput screening (HTS) of 2562 natural compounds, assessing their activity against L. donovani promastigotes and intracellular amastigotes. Utilizing the criteria of ≥70% parasite growth inhibition and ≥70% host cell (THP-1) viability, we selected 100 inhibitors for half-maximal inhibitory concentration (IC50) value determination. Twenty-six compounds showed activities in both forms of Leishmania with a selectivity index of over 3. Clustering analysis resulted in four chemical clusters with scaffolds of lycorine (cluster 1), 5-hydroxy-9,10-dihydro-4H,8H-pyrano[2,3-f]chromene-4,8-dione (cluster 2), and semi-synthetic derivatives of ansamycin macrolide (cluster 4). The enantiomer of lycorine, BMD-NP-00820, showed the highest anti-amastigote activity with an IC50 value of 1.74 ± 0.27 µM and a selectivity index (SI) > 29. In cluster 3, the most potent compound had an IC50 value of 2.20 ± 0.29 µM with an SI > 23, whereas in cluster 4, with compounds structurally similar to the tuberculosis drug rifapentine, BMD-NP-02085 had an IC50 value of 1.76 ± 0.28 µM, but the SI value was 7.5. Taken together, the natural products identified from this study are a potential source for the discovery of antileishmanial chemotypes for further development.
RESUMO
BACKGROUND: Parthanatos represents a critical molecular aspect of Parkinson's disease, wherein AIMP2 aberrantly activates PARP-1 through direct physical interaction. Although AIMP2 ought to be a therapeutic target for the disease, regrettably, it is deemed undruggable due to its non-enzymatic nature and predominant localization within the tRNA synthetase multi-complex. Instead, AIMP2 possesses an antagonistic splice variant, designated DX2, which counteracts AIMP2-induced apoptosis in the p53 or inflammatory pathway. Consequently, we examined whether DX2 competes with AIMP2 for PARP-1 activation and is therapeutically effective in Parkinson's disease. METHODS: The binding affinity of AIMP2 and DX2 to PARP-1 was contrasted through immunoprecipitation. The efficacy of DX2 in neuronal cell death was assessed under 6-OHDA and H2O2 in vitro conditions. Additionally, endosomal and exosomal activity of synaptic vesicles was gauged in AIMP2 or DX2 overexpressed hippocampal primary neurons utilizing optical live imaging with VAMP-vGlut1 probes. To ascertain the role of DX2 in vivo, rotenone-induced behavioral alterations were compared between wild-type and DX2 transgenic animals. A DX2-encoding self-complementary adeno-associated virus (scAAV) was intracranially injected into 6-OHDA induced in vivo animal models, and their mobility was examined. Subsequently, the isolated brain tissues were analyzed. RESULTS: DX2 translocates into the nucleus upon ROS stress more rapidly than AIMP2. The binding affinity of DX2 to PARP-1 appeared to be more robust compared to that of AIMP2, resulting in the inhibition of PARP-1 induced neuronal cell death. DX2 transgenic animals exhibited neuroprotective behavior in rotenone-induced neuronal damage conditions. Following a single intracranial injection of AAV-DX2, both behavior and mobility were consistently ameliorated in neurodegenerative animal models induced by 6-OHDA. CONCLUSION: AIMP2 and DX2 are proposed to engage in bidirectional regulation of parthanatos. They physically interact with PARP-1. Notably, DX2's cell survival properties manifest exclusively in the context of abnormal AIMP2 accumulation, devoid of any tumorigenic effects. This suggests that DX2 could represent a distinctive therapeutic target for addressing Parkinson's disease in patients.