The blood–brain barrier is formed by tightly connected cells lining the brain’s blood vessels, supported by pericytes, astrocytes and specialized molecular transport systems. Its primary role is protective: it limits the entry of toxins, pathogens and many medicines from the bloodstream into neural tissue. That same selectivity, however, makes it difficult to deliver gene therapies to the brain. Injecting a treatment directly into brain tissue or the fluid surrounding the spinal cord can bypass the barrier, but those approaches are invasive and may not distribute genetic material evenly throughout the central nervous system. An intravenous treatment that could circulate through the body and selectively reach neurons would therefore represent a major advance.

AAVs are among the leading vehicles for gene therapy because they can carry genetic instructions into cells while generally producing relatively mild immune reactions compared with some other viral platforms. AAV particles consist of a protein capsid surrounding a DNA payload. The capsid determines, in part, which tissues the particle can bind to, enter and persist within. Researchers can also alter the promoter, a regulatory DNA sequence that controls when and where the delivered gene is expressed. In this study, the team examined both components together, asking not only which capsids reached the brain most efficiently, but also whether promoter choice could reduce unwanted gene activity in organs outside the nervous system.

The researchers administered the candidate vectors intravenously to mice and used reporter genes to track delivery and expression. Reporters are molecular markers that produce readily measured signals, allowing scientists to map where a vector has traveled and where its genetic cargo has become active. The experiments used the broadly active CAG promoter as well as the neuron-specific hSyn promoter. CAG is commonly used when strong expression across many cell types is desired. By contrast, hSyn is associated primarily with neuronal gene activity, making it useful for testing whether a vector that reaches multiple organs can nevertheless restrict transgene production mainly to neurons.

Compared with AAV9, the two capsids PHP.eB and CNSRCV300 showed enhanced penetration of the blood–brain barrier and stronger transduction of brain tissue. Transduction refers to the process by which a viral vector introduces genetic material into a cell and enables that material to function. The study also found that these capsids displayed a predominant neuronal tropism, meaning that their activity in the brain favored neurons over other neural or tissue cell types. This distinction matters because many neurological disorders arise from defects in neurons, although other cells—including astrocytes, oligodendrocytes and microglia—can also be important therapeutic targets. A capsid that enters the brain efficiently but reaches the wrong cell population may still be poorly suited to a particular disease.

The promoter results revealed a trade-off between potency and selectivity. Relative to CAG-driven expression, the neuron-specific hSyn promoter slightly reduced cerebral transgene expression. In other words, the brain signal was somewhat weaker when the genetic payload was placed under neuronal control rather than the more broadly active promoter. Yet hSyn markedly reduced expression in peripheral tissues. This indicates that promoter engineering can provide an additional layer of biological targeting after a vector has entered a cell. The capsid influences where the particle goes, while the promoter helps determine whether the payload is switched on in that location.

That distinction became critical when the team looked beyond reporter expression and examined the physical distribution of the vectors. Immunofluorescence, quantitative polymerase chain reaction and Western blotting all provided evidence that AAV remained in peripheral tissues, including the liver. Immunofluorescence uses labeled antibodies to visualize proteins or cellular signals in tissue sections. Quantitative PCR measures the abundance of specific DNA sequences, allowing researchers to estimate how much vector-derived genetic material is present. Western blotting detects particular proteins and can help establish whether a delivered gene is producing its intended product. Together, these tests indicated that a low level of peripheral gene expression does not necessarily mean that the viral particles themselves have been eliminated from organs outside the brain.

Among the candidates, CNSRCV300 produced what the researchers described as the most favorable balance: robust central nervous system transduction with minimal peripheral accumulation. That combination could give the capsid a stronger safety profile than vectors that reach the brain but distribute more heavily to other organs. The liver is especially important in systemic AAV therapy because intravenously delivered particles commonly pass through and accumulate there. Hepatic exposure can create safety concerns through immune responses, unintended expression, cellular stress or difficulty controlling the biological effects of the therapy. The study does not establish that CNSRCV300 is safe for human use, but it identifies a measurable design goal for future vector development: maximizing brain delivery while minimizing the amount of vector deposited elsewhere.

The findings also challenge a tempting assumption about tissue-specific promoters. A neuron-specific promoter can reduce off-target expression, but it cannot prevent a capsid from physically reaching or remaining in peripheral organs. This means safety cannot be assessed solely by measuring where the therapeutic protein is produced. Researchers must also quantify vector genomes and examine the persistence of the capsid or its genetic payload in tissues throughout the body. The authors’ conclusion is that AAV targeting is co-regulated by capsid properties and promoter characteristics, rather than controlled by either element alone. In practical terms, an effective brain-directed therapy may require coordinated optimization of the viral shell, the regulatory DNA, the therapeutic payload and the dose.

The work provides a systematic framework for screening AAV vectors intended for disorders of the central nervous system, while underscoring the gap between promising mouse data and clinical application. Biology that enables a capsid to cross the mouse blood–brain barrier may not translate directly to humans, whose vascular architecture, receptor distribution and immune responses differ. Some engineered capsids can also behave differently across species, making human-relevant testing essential. The study was performed under approved animal protocols and was supported by the Lingang Laboratory Project. Its most important message is therefore not that a universal brain-delivery vector has been found, but that future gene therapies will need to treat distribution and safety as inseparable engineering problems. Better access to the brain is valuable only when it is accompanied by precise control over where the vector travels, where the gene is expressed and how long both remain in the body.

Intranasal delivery of AAVs represents a novel approach that circumvents barriers associated with traditional systemic administration. Traditional systemic routes often lead to substantial peripheral exposure, where therapeutic agents accumulate in non-target tissues. In contrast, intranasal delivery directly accesses the olfactory bulb, enabling AAVs to bypass the blood-brain barrier more effectively. This anatomical advantage may be crucial for treatments aimed at conditions like Alzheimer’s disease, Parkinson’s disease, and other neurodegenerative disorders.

The study conducted by Chukwu, Yuan, and Chen involved a meticulous comparison of both delivery routes in murine models to elucidate their respective efficiencies. By quantifying the brain-targeting efficacy and peripheral exposure of AAVs delivered through these two routes, the research team aimed to delineate the most effective delivery method for therapeutic genes. As the area of gene therapy continues to evolve, understanding these differences could significantly influence future therapeutic strategies.

One of the study’s focal points was the evaluation of how each delivery method impacts viral distribution in the brain. The researchers utilized various techniques, including quantitative PCR and fluorescence microscopy, to assess the localization and spread of AAVs post-delivery. The outcomes of these methodologies underscored that intranasal delivery resulted in a more favorable distribution pattern within specific brain regions associated with cognition and motor function.

On the other hand, intravenous delivery, while a widely accepted method in many therapeutic contexts, presented challenges in this comparative analysis. The research highlighted that, although intravenous administration might facilitate broader systemic circulation, it often leads to lower concentrations of AAVs in the targeted brain regions. This finding raises important questions about the trade-offs between delivery efficiency and the potential risks associated with increased peripheral exposure, which can lead to unintended immune responses or cytotoxic effects.

Chukwu et al. also explored the dynamics of tissue targeting and clearance post-delivery. Understanding how AAVs are processed by the body following their administration is crucial, as it directly affects the longevity and effectiveness of the therapeutic genes they carry. The researchers observed that intranasal delivery not only decreased peripheral exposure but also enhanced retention times in target brain areas, suggesting a maximized therapeutic window for sustained effects.

Another significant aspect of this study was the immune response elicited by each delivery method. Intravenous AAV delivery has historically been associated with a more pronounced immunogenic response, which can dampen the therapeutic efficacy of gene therapy protocols. In contrast, the intranasal route minimized immune activation, a finding that could be pivotal for developing safe and effective gene therapies with fewer side effects.

As the research unfolds, implications for clinical applications are increasingly apparent. The ability to effectively target the brain via intranasal routes suggests that this method could revolutionize treatment paradigms for neurological disorders, providing a less invasive and potentially more effective alternative to current therapies. The growing body of evidence supports the notion that optimized delivery systems are essential for advancing therapies and improving patient outcomes.

In addition to the neurological applications, the implications of this research extend beyond the brain. Understanding the comparative efficiencies of these delivery routes could pave the way for similar techniques in addressing other diseases where gene therapy holds promise, including cancer and inherited disorders. Intranasal delivery methods, if proven effective in human trials, could open new avenues for disease modification and management.

This comparative analysis ultimately emphasizes the need for a shift in perspective regarding AAV delivery methods. While traditional intravenous routes have been the standard, emerging evidence advocates for a reevaluation of intranasal routes. By focusing on brain-targeting efficiency without compromising safety, researchers may redefine standards for viral delivery systems in gene therapy.

As innovative strategies continue to emerge in the field of gene therapy, future studies must prioritize not only the efficacy of delivery methods but also their safety profiles and biological implications. The ongoing dialogue surrounding these advancements will likely lead to paradigm shifts in how therapies are administered and their trajectory in clinical practice.

Overall, the insights gleaned from this study by Chukwu and colleagues offer a glimpse into the future of gene therapy, demonstrating the intricacies of delivery methods and the importance of customized approaches tailored to specific therapeutic needs. Researchers and clinicians alike must adapt to these findings, which bear the potential to change therapeutic landscapes in profound ways for neurological and other diseases.

In conclusion, understanding the comparative advantages and limitations of intranasal versus intravenous AAV delivery is critical for harnessing the full potential of gene therapy. As research in this area expands, the findings promise not only to inform future studies but also to guide clinical decision-making processes in the pursuit of effective and safe gene therapies for patients suffering from debilitating conditions.

Subject of Research: Adeno-Associated Virus (AAV) Delivery Methods

Article Title: Intranasal versus intravenous AAV delivery: A comparative analysis of brain-targeting efficiency and peripheral exposure in mice

Article References:

Chukwu, C., Yuan, J. & Chen, H. Intranasal versus intravenous AAV delivery: A comparative analysis of brain-targeting efficiency and peripheral exposure in mice.
Gene Ther (2025). https://doi.org/10.1038/s41434-025-00585-y

Image Credits: AI Generated

DOI: 08 December 2025

Keywords: Gene therapy, AAV delivery, intranasal delivery, intravenous delivery, brain targeting, neurological disorders, immune response.