RESUMO
BACKGROUND: Neonatal hypoxic-ischemic brain injury (HIBI) is a significant contributor to neonatal mortality and long-term neurodevelopmental disability, characterized by massive neuronal loss and reactive astrogliosis. Current therapeutic approaches for neonatal HIBI have been limited to general supportive therapy because of the lack of methods to compensate for irreversible neuronal loss. This study aimed to establish a feasible regenerative therapy for neonatal HIBI utilizing in vivo direct neuronal reprogramming technology. METHODS: Neonatal HIBI was induced in ICR mice at postnatal day 7 by permanent right common carotid artery occlusion and exposure to hypoxia with 8% oxygen and 92% nitrogen for 90 min. Three days after the injury, NeuroD1 was delivered to reactive astrocytes of the injury site using the astrocyte-tropic adeno-associated viral (AAV) vector AAVShH19. AAVShH19 was engineered with the Cre-FLEX system for long-term tracking of infected cells. RESULTS: AAVShH19-mediated ectopic NeuroD1 expression effectively converted astrocytes into GABAergic neurons, and the converted cells exhibited electrophysiological properties and synaptic transmitters. Additionally, we found that NeuroD1-mediated in vivo direct neuronal reprogramming protected injured host neurons and altered the host environment, i.e., decreased the numbers of activated microglia, reactive astrocytes, and toxic A1-type astrocytes, and decreased the expression of pro-inflammatory factors. Furthermore, NeuroD1-treated mice exhibited significantly improved motor functions. CONCLUSIONS: This study demonstrates that NeuroD1-mediated in vivo direct neuronal reprogramming technology through AAV gene delivery can be a novel regenerative therapy for neonatal HIBI.
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Developing a cutting-edge system capable of ensuring long-lasting functionality of therapeutic agents and implementing diverse delivery modes is challenging. A quasi-spherical triple-layered capsule containing suspended liquid droplets and allowing multi-modal delivery of therapeutic agents in the aqueous phase was developed, primarily by adopting the core principles for creating liquid marbles. A naturally derived wettable polysaccharide-pectin-was utilized as a liquid-air interfacial barrier to keep the liquid droplets in the core zone. To tailor the pectin-coated droplet as a therapeutic agent carrier, anionic alginate and cationic chitosan layers were sequentially formed via additional interactions: physically stacking substances with structural chirality (pectin-alginate) and inducing electrostatic association to create the reversible complex coacervates (alginate-chitosan). The resulting system, which is called a Chitosan-Alginate-Pectin-coated Suspended-Liquid-Encapsulating (CAPSuLE) marble, had sufficient mechanical strength to resist external harsh environments and exhibited unique features: ecofriendly sustainability, responsiveness to external stimuli, coacervate-driven coalescence for linking adjacent marbles, and a self-repairing ability. The proposed CAPSuLE system can facilitate the adoption of the liquid-marble concept to biomedical fields, extending its applicability in the fields of biology and applied engineering.
Assuntos
Quitosana , Pectinas , Alginatos , Carbonato de Cálcio , Eletricidade EstáticaRESUMO
Developing gene carriers with improved affinities for target cells and the simultaneous diversification of their delivery modes will be pivotal for upgrading gene therapy technologies. In this study, a simple and versatile adeno-associated virus (AAV) conjugation platform using the cross-linker 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) is proposed. Depending on the quantity of the DTSSP molecules, the AAV-DTSSP complexes could either be linked with the relevant biomolecules for altering cellular tropisms or further form a self-assembled AAV-DTSSP pellet capable of mimicking a polymeric gene delivery system. At lower quantities of DTSSP, the AAV-DTSSP complexes were conjugated with aminated l-fucose molecules, whose levels are typically upregulated in pancreatic cancer cells, resulting in enhanced gene delivery efficiencies in pancreatic cancer cells. At higher concentrations of DTSSP, visible solid forms of the AAV-DTSSP pellets were formed, and the AAV pellets demonstrated the capability to induce a localized, sustained gene expression pattern comparable to that of conventional biomaterial-based approaches. Thus, a multipurpose AAV cross-linking platform, which can enable AAV vector systems that are capable of altering cellular tropisms and simultaneously inducing solid-phase delivery, will provide crucial insights into vector design for further upgrading of gene delivery technologies.
RESUMO
In addition to the ability to boost gene delivery efficiency in many therapeutically relevant cells, the capability of circumventing neutralizing antibody (NAb) inactivation is a key prerequisite that gene carriers must fulfill for their extensive applications as therapeutic agents in many gene therapy trials, especially for cancer treatments. This study revealed that a genetically engineered adeno-associated virus (AAV) variant, AAVr3.45, inherently possesses dual beneficial properties as a gene carrier: (i) efficiently delivering therapeutic genes to many clinically valuable cells (e.g., stem or cancer cells) and (ii) effectively bypassing immunoglobulin (IgG) neutralization. Detailed interpretation of the structural features of AAVr3.45, which was previously engineered from AAV2, demonstrated that the LATQVGQKTA peptide at the heparan sulfate proteoglycan binding domain, especially the presence of cationic lysine on the peptide, served as a key motif for dramatically enhancing its gene delivery capabilities, ultimately broadening its tropisms for many cancer cell lines. Furthermore, the substitution of valine on the AAV2 capsid at the amino acid 719 site to methionine functioned as a coordinator for promoting viral resistance against IgG inactivation. The NAb-resistant characteristics of AAVr3.45 were possibly associated with the LATQVGQKTA sequence itself, indicating that its synergistic cooperation with the point mutation (V719M) is required for maximizing its ability to evade NAb inactivation. The potential of AAVr3.45 as a cancer gene therapy agent was confirmed by provoking apoptosis in breast adenocarcinoma by efficiently delivering a pro-apoptotic gene, BIM (Bcl-2-like protein 11), under high titers of human IgG. Thus, the superior aspects of the NAb-resistant AAVr3.45 as a potential therapeutic agent for systemic injection approaches, especially for cancer gene therapy, were highlighted in this study.