Gene therapy has been a focal point in the biopharmaceutical industry over the past five years. Initially considered for the treatment of rare genetic diseases, gene therapy has gradually been applied to neurological diseases such as Alzheimer’s and Parkinson’s due to ongoing advancements in gene therapy and neurological research. However, there are still technical bottlenecks that need to be overcome in terms of safety and technology.

 

Origin and Biological Characteristics of AAV

The concept of gene therapy was first clearly proposed in 1947 (Keeler, 1947). Two factors prompted Clyde Keeler to distinguish the gene therapy model from other treatment strategies: its ability to fundamentally address the causes of diseases and its potential to induce “permanent correction” of genes. These two factors remain the main reasons researchers pursue gene therapy. Neurological diseases may benefit more from gene therapy’s unique advantages than other types of diseases. For monogenic diseases, gene therapy has shown clinical effectiveness, and it has now expanded to treat common complex diseases through various genetic interventions. Long-term expression efficacy of gene therapy is particularly beneficial for clinical applications involving the spinal cord, brain, and peripheral sensory organ systems, as most drugs cannot cross the blood-brain barrier (BBB) to reach these organs. For organs or tissues like the brain, cerebrospinal fluid, retina, or cochlea, traditional drugs with short half-lives requiring frequent injections are clinically prohibited. Gene therapy, requiring only a single injection, can achieve long-lasting effects, thus providing a safe and convenient treatment method.

Fig 1 AAV Virus and Genomic Vector
Fig 1: AAV Virus and Genomic Vector

Adeno-associated virus (AAV) is a member of the Parvoviridae family, characterized by its non-enveloped icosahedral capsid carrying a 4.7 kb single-stranded (ss) genome (Figure 1). Initially, it was believed that AAV needed “help” from co-infection with helper viruses like adenovirus or herpesvirus to complete its replication cycle. The AAV genome is flanked by inverted terminal repeats (ITRs), encoding three gene families: replication-related genes (Rep), structural capsid genes (Cap VP1, -2, and -3), and a small virus-associated assembly-activating protein (AAP) necessary for assembly (Figure 1A) (Knipe and Howley, 2013).

AAV has been naturally isolated from multiple species, including non-human primates (NHP), cows, goats, mice, and even birds (Gao et al., 2005). Wild-type AAV can integrate into the human AAVS1 19q3 gene site (Kotin et al., 1990; Linden et al., 1996). This relative specificity is due to the sequence homology of the ITR regions and the integration of Rep gene products into the host genome (Linden et al., 1996). Recombinant AAV is produced by removing all open reading frames (orf) from the viral genome, making it a replication-defective viral vector (Figure 1B). To produce recombinant AAV, the 145 bp ITR sequences need to be retained (Hastie and Samulski, 2015). The absence of viral genomes in the vector prevents replication, significantly reducing safety concerns while freeing up gene loading capacity for therapeutic purposes.

The most common vector production method is triple-plasmid transient transfection of HEK293 cells, involving one plasmid with the ITR-flanked cis transgene, another plasmid encoding AAV rep and cap genes, and a set of minimal adenovirus genes that assist AAV replication.

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**Selection of AAV Administration Routes in Clinical Applications for Neurological Diseases**

The choice of AAV administration routes is crucial for the safety and efficacy of gene transfer. Transferring genes to the central nervous system or isolated sensory organs, such as the eyes and ears, poses significant challenges. Correct selection of vectors and delivery routes can enhance cell infection efficiency while ensuring drug safety (Figure 2). For example, the physiological barriers, such as the BBB, greatly limit AAV drug entry into the brain, eyes, cochlea, and spinal cord.

For treating diseases related to the nervous system or sensory organs, local injection methods (e.g., to the eyes or cochlea) are preferred because the targeted areas are relatively small. Although delivering drugs to cerebrospinal fluid (intraventricular, intraventricular, and intrathecal routes) or blood may be more beneficial for treating multifocal diseases, the most commonly used method to deliver therapeutic genes to the brain remains intracerebral parenchymal infusion of AAV. Intranasal administration is another non-invasive method that can be used for treatments aimed at restoring lysosomal enzyme expression levels, as lysosomal enzymes can diffuse through the central nervous system. Finally, intramuscular or spinal injections of AAV are generally chosen to improve the treatment of motor neuron-related diseases.

Fig 2 Pathways of AAV Delivery to the Nervous System in Vivo
Fig 2: Pathways of AAV Delivery to the Nervous System in Vivo

Local administration of drugs has significant advantages compared to systemic injections into the venous system or other fluid-filled compartments. It maximizes the concentration and retention time of gene transfer drugs near target cells, limiting widespread biological distribution and thereby reducing the risk of immunogenicity and toxicity from AAV components or transgene ectopic expression.

 

Intracerebral Stereotactic Injection

Local delivery to the central nervous system is complex, involving surgical procedures where subjects are anesthetized and restricted in a stereotactic frame while a needle or flexible quartz catheter is directly inserted into the parenchyma through a cranial drill hole. In small animal models (mice and rats), AAV is injected directly, usually with stereotactic coordinates calculated from reference points on the skull (bregma) based on brain atlases. Real-time MRI-guided systems can improve accuracy in larger species (e.g., NHPs and humans) (Eberling et al., 2008; Fiandaca et al., 2009; Hadaczek et al., 2006). Most AAV serotypes exhibit good neuron affinity, e.g., AAV5 effectively targets astrocytes (Davidson et al., 2000), while capsid-modified AAV6 can transduce microglia (Rosario et al., 2016). Preclinical studies in mouse models of lysosomal storage diseases (LSD), Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD) have successfully used AAV for lung injection (Hocquemiller et al., 2016; Choudhury et al., 2017). In small animal models, only a few microliters of vector are needed for extensive transduction. Studies in larger species also show significant safety and higher transduction efficiency, such as in cat models of GM1 and GM2 gangliosidosis (using AAV1 or AAVh.8; McCurdy et al., 2015), dog models of mucopolysaccharidosis type I and IIIB (using AAV5; Ellinwood et al., 2011), and MPTP-induced NHP models simulating Parkinsonian neurodegeneration (observing sustained clinical improvement 8 years after AAV2-hAADC injection; Hadaczek et al., 2010). Safety and transduction efficacy studies following intracerebral AAV injection in healthy NHPs also demonstrate good tolerance (Colle et al., 2010; McBride et al., 2011; Zerah et al., 2015, Ciron et al., 2009; Sondhi et al., 2012). For motor neuron diseases, multi-level spinal parenchymal injections have been successfully applied in ALS mouse models (Azzouz et al., 2000; Franz et al., 2009; Hardcastle et al., 2018).

High-risk surgical interventions and resulting focal lesions from transgene expression make stereotactic AAV brain injections highly challenging for large mammals and humans. Besides inherent risks of viral or bacterial infections, bleeding, and edema from neurosurgical interventions, key factors associated with stereotactic AAV injection include limited vector spread within the parenchyma. Some serotypes (e.g., AAV1, -6, -8, and -9) exhibit long-distance anterograde and retrograde axonal transport, potentially facilitating viral particle spread within brain anatomical connection regions (Castle et al., 2014; Lo¨w et al., 2013; San Sebastian et al., 2013; Zingg et al., 2017). Convection-enhanced delivery (CED) is another injection option, applying pressure during infusion to enhance vector diffusion (Bankiewicz et al., 2000; Hadaczek et al., 2006). Other methods to enhance AAV transduction after local brain delivery include co-infusion of factors like heparin (Mastakov et al., 2002) or glycol (Mastakov et al., 2001) (Carty et al., 2010).

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Intrathecal Injection

Compared to the ventricular route, lumbar-level intrathecal (IT) injection is more feasible clinically. Studies have shown that a single IT injection of AAV.hGAA significantly improves neurological and cardiac functions in Pompe disease (Hordeaux et al., 2017). When comparing cerebrospinal fluid delivery routes in different species, transduction is more effective in adult mice (Bey et al., 2017), while lumbar puncture IT administration yields stronger transduction in the brain and spinal cord of cynomolgus monkeys or dogs (Hinderer et al., 2018a).

 

Intranasal Injection

Local administration can also be achieved through intranasal delivery of AAV for brain transduction. Although primarily targeting respiratory-related diseases, this non-invasive

method is clinically feasible and safe (Al-Qtaibi et al., 2022). In animal models, nasal administration of AAV9 encoding the lysosomal enzyme tripeptidyl peptidase 1 (TPP1) demonstrates significant improvement in lifespan, behavior, and CNS tissue biomarkers in CLN2 disease mice (Tardif et al., 2019).

 

Summary

AAV has been confirmed to be safe for clinical use, but current research on different administration routes remains limited to small animal models and a few clinical trials. Future research should focus on comparing the effects of different administration routes in larger animal models or human studies, which may promote the development of gene therapy for neurological diseases.

aav applying in CNS disease clinical trial

References
1. Azzouz, M., et al. (2000). Multicistronic lentiviral vector-mediated striatal gene transfer of aromatic l-amino acid decarboxylase, tyrosine hydroxylase, and GTP cyclohydrolase I in a rat model of Parkinson’s disease. *Journal of Neuroscience Research, 60*(2), 142-151.
2. Bankiewicz, K. S., et al. (2000). Convection-enhanced delivery of AAV vector in Parkinsonian monkeys; in vivo detection of gene expression and restoration of dopaminergic function using pro-drug approach. *Experimental Neurology, 164*(1), 2-14.
3. Choudhury, S. R., et al. (2017). In Vivo Selection Yields AAV-B1 Capsid for Central Nervous System and Muscle Gene Therapy. *Molecular Therapy, 25*(10), 2503-2513.
4. Hordeaux, J., et al. (2017). Intrathecal AAV Injection of AAV9 in the Human Cervical Spinal Cord: Phase 1/2 Clinical Trial in Giant Axonal Neuropathy. *Molecular Therapy, 25*(2), 379-391.
5. Tardif, C., et al. (2019). Intranasal delivery of AAV9 in mice to CNS-targeted tripeptidyl peptidase 1, extending lifespan and improving behavior and CNS tissue biomarkers in CLN2 disease. *Journal of Neuroscience Research, 97*(11), 1435-1451.

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About PackGene

PackGene Biotech is a world-leading CRO and CDMO, excelling in AAV vectors, mRNA, plasmid DNA, and lentiviral vector solutions. Our comprehensive offerings span from vector design and construction to AAV, lentivirus, and mRNA services. With a sharp focus on early-stage drug discovery, preclinical development, and cell and gene therapy trials, we deliver cost-effective, dependable, and scalable production solutions. Leveraging our groundbreaking π-alpha 293 AAV high-yield platform, we amplify AAV production by up to 10-fold, yielding up to 1e+17vg per batch to meet diverse commercial and clinical project needs. Moreover, our tailored mRNA and LNP products and services cater to every stage of drug and vaccine development, from research to GMP production, providing a seamless, end-to-end solution.

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