Brain-derived neurotrophic factor (BDNF) plays a key role in brain development, synaptic plasticity, and cognitive processes such as learning and memory. Preclinical studies have shown that inhibiting the anti-sense strand of BDNF (Bdnf-AS), a long non-coding RNA (lncRNA) that regulates Bdnf, can enhance Bdnf expression and promote functional plasticity, highlighting its therapeutic potential in neurodegenerative and neurodevelopment diseases. This study explores both systemic and localised delivery of short hairpin RNA (shRNA) targeting Bdnf-AS in a rat model using two delivery approaches: lipid nanoparticle (LNP)-mediated delivery and direct lentiviral injection.
A total of 48 rats (24 male and 24 female) underwent intravenous catheter implantation for injections and blood collection. The rats were assigned to receive one of the following treatments: LNPs carrying a plasmid vector expressing shRNA targeting Bdnf-AS (sh-AsBdnf, n = 15), shRNA targeting GFP (sh-GFP, n = 11), or saline as an untreated control (n = 22). The LNPs were designed to deliver plasmid vectors to liver cells where they are hypothesised to release Extracellular Vesicles (EVs) loaded with the shRNAs. These EVs are expected to cross the blood-brain barrier and, in the sh-AsBdnf group, downregulate Bdnf-AS expression in the brain to influence learning and memory-related behaviours.
In a separate experiment targeting localised brain regions, 16 additional male and female rats underwent stereotaxic surgery for bilateral lentiviral injections in the prefrontal cortex (PFC) and hippocampus (HPC), allowing region-specific manipulation of Bdnf-AS. Experimental groups included an empty vector control (n = 4), a vector expressing shRNA targeting GFP (n = 4), and a vector expressing shRNA targeting Bdnf-AS (n = 8).
Behavioural assessments, including novel object recognition, novel place recognition, fear conditioning, fear extinction learning and reinforcement learning were performed over several weeks following LNP injection and the stereotaxic surgery in order to determine if Bdnf-AS rats showed any changes in learning and memory. Blood samples were obtained at two different time points for detection of circulating mature shRNA using qPCR. Additionally, following euthanasia, tissues (blood, liver, spleen, and brain) were collected and stored at -80°C for molecular analyses.
This study presents a dual-delivery platform to investigate the role of Bdnf-AS in brain function, with the broader aim of informing RNA-based therapeutics for neurodegenerative and neurodevelopmental disorders. While analysis is ongoing, this work underscores the therapeutic potential of targeting lncRNAs like Bdnf-AS in modulating Bdnf expression.