How can we cure neurodegenerative disorders? In this article, we discuss the latest human gene therapy trials showing that delivering genes using the adeno-associated virus into the brain can effectively and durably correct the pathogenic mechanisms of neurodegeneration.
by Vanessa Lunardi
Across the globe, we are facing an unprecedented crisis: there are now more people older above age 64 than there are younger individuals below age five.1 Although population ageing has been widely recognised as a human success story and celebrated as a triumph in medical care, the dramatic increase in life expectancy did not come with a proportionate increase in quality of life for the elderly.2 In fact, there is surprisingly little evidence to suggest that older people today are experiencing their later years in better health than their parents.3 This is because, even with advancements in technology and medicine, we have yet to find a way to stave off sicknesses at old age.
One of the most common diseases affecting the elderly is neurodegenerative disorders. As its name suggests, neurodegenerative diseases stem from the progressive damage done to cells and nervous system connections that are essential for mobility, coordination, strength, sensation, and cognition.4 Some of the more common neurodegenerative diseases include Alzheimer’s, which affect one’s memory and mental function, and Parkinson’s, which debilitate movement and coordination, while others like Amyotrophic Lateral Sclerosis, Creutzfeldt-Jakob disease, and Huntington’s disease are rarer.
At present, researchers and clinicians have come up with a variety of intervention strategies in hopes of preventing or curing neurodegeneration. However, current therapies have not been able to address the underlying pathology and merely focus on providing symptomatic relief. This is, in large part, due to the obstructive blood-brain barrier that prevents therapeutic agents from reaching the brain.5
What is the Blood-Brain Barrier?
To many, the brain is arguably the most important organ in the human body, controlling and coordinating our thoughts, actions, and reactions. Considering its role, evolution has gone to great lengths to protect the brain from damage. Immediately, it would be reasonable to think of the most obvious protective gear of the brain – the skull. But apart from this thick bone, the brain is also protected by the cerebrospinal fluid and the blood-brain barrier.
True to its name, the blood-brain barrier acts as a structural and functional roadblock between the brain’s capillaries, the cells, and other components that make up the brain tissue. But more importantly, it is a key regulator of microorganism entry into the central nervous system as well as the brain’s microenvironment. To protect the brain from harm, the blood-brain barrier is highly selective in only allowing certain molecules to enter the central nervous system, restricting the passage of microorganisms, solutes, and most large molecules. This feature greatly benefits the brain in terms of protection. However, the high selectivity of the blood-brain barrier restricts the delivery of many pharmaceutical and therapeutic antibodies as well, thus complicating the administration of medications for neurodegenerative diseases. To achieve therapeutic parenchymal levels, clinicians would need to administer dangerously high dosages of medication, which can adversely cause significant side effects.
As an alternative, scientists have strategised to use direct intracerebral delivery of therapeutics. With this method, they can circumvent the blood-brain barrier and target the disease process with functional-anatomic specificity. For instance, therapeutic molecules like growth factors could be delivered directly into the brain parenchyma. Unfortunately, this strategy is still limited in clinical practice as it requires chronic re-administration of the therapeutic agent, thus calling the need for more effective and durable therapeutic interventions.5 One promising strategy of intracerebral drug delivery that we can look to is gene therapy, which has shown strong potential to mitigate these medical deficiencies.
Bridging the Gaps of the Blood-Brain Barrier With Gene Therapy
In the past few decades, gene therapy has rapidly emerged to become a powerful therapeutic strategy for a wide range of neurodegenerative disorders, including Alzheimer’s, Parkinson’s, and Huntington’s disease. Its popularity rose because it solved the long-standing problem of the impenetrable blood-brain barrier. But what makes gene therapy so special is its ability to induce sustained, and even permanent therapeutic effects for compartmentalised organs such as the central nervous system. Even some genetic targets that are usually refractory to traditional agents have shown potential to be manageable by gene therapy. Gene silencing and gene overexpression can be induced to manage gain-of-function mutations and loss-of-function mutations respectively.
In clinical practice, however, scientists first need to find a way to safely and effectively bring gene-editing tools to the site of interest before they can induce these potentially life-saving genetic changes. Based on past studies, it has been shown that both viral and non-viral vectors alike can successfully direct transgenes that express therapeutic proteins, antibodies, caspase/guide RNAs for gene editing, and microRNAs to diseased tissues in humans and animals. Intravenous injections of therapeutic agents have also demonstrated promising potential as an effective delivery route as it is non-invasive. Furthermore, therapeutic antibodies produced or delivered via intramuscular injections can potentially cross physiological barriers like the blood-brain barrier at clinically sufficient levels as well. But amongst these options, the adeno-associated viruses have become the most widely applied vector for neurodegenerative disorders.6
Adeno-Associated Virus as a Vector for Gene Therapy
At first glance, it may seem unsettling that a virus should be used to deliver supposedly life-saving medications. However, adenoviruses have become promising viral vectors because they offer several key features like safety and durability. Adenoviruses cannot insert their own gene into the host genome, thus giving them an excellent safety profile. They also demonstrate sustained expression in neurons, thereby making them potentially suitable vectors to help treat neurodegenerative diseases.
To ensure that the adenovirus can best perform its role as a delivery vehicle, scientists have taken a step further to determine which serotype amongst the different types of adenoviruses is best suited for gene therapy. This is because the serotype can heavily influence several important characteristics like biodistribution and tissue tropism that are needed for successful adeno-associated virus-based gene therapy.
According to one study using mice models, serotype 4 has shown a predilection to transfect ependymal cells, which constitute the epithelial lining of neuroblasts and the lateral ventricles, after administration near or into cerebral ventricles. Their findings concluded that serotype 4 can become a new tool to manipulate glial cells in the rostral migratory stream.7 In a more recent clinical trial, scientists have found that using serotype 2 for the intracerebral administration of the nerve growth factor, which is used to rescue and stimulate cholinergic neurons, is well-tolerated and demonstrates promising therapeutic effects on the cognitive decline in dementia related to Alzheimer’s disease.8 Most notably, serotype 9 and serotype rh.10 have demonstrated the ability to penetrate the blood-brain barrier, thus making them excellent candidates to advance treatments for neurodegenerative diseases.9,10
Clinical Implementations of Adeno-Associated Viruses
Neurodegenerative diseases are typically caused by genetic abnormalities and enhanced by environmental and epigenetic factors. Therefore, viral vector-based gene therapies are well-suited to reverse the lethal impacts of these inheritable diseases on a genetic level. In the case of Parkinson’s disease, an adeno-associated virus vector system has been implemented for gene therapy in several ways: increasing dopamine levels in target cells to make up for the loss of dopaminergic neurons,11 delivering α-synuclein expression vector to target the neural parenchyma,12 and transporting C3-ADP ribosyltransferase to facilitate axon regeneration.13,14
Another fatal neurodegenerative disease, known as amyotrophic lateral sclerosis, has also been a subject of interest for gene therapy. Amyotrophic lateral sclerosis is characterised by loss of motor neurons and can lead to progressive muscle weakness, paralysis, and death. Currently, treatment for this disease is still limited to the standard drug riluzole, which has only improved patient survival by a few months. Fortunately, one study using mice models has shown that injecting a single-stranded adeno-associated vector serotype 9 encoding an artificial microRNA against the human superoxide dismutase 1 gene can extend survival by 50 per cent and delay paralysis. 20 per cent of amyotrophic lateral sclerosis cases are attributed to mutations in the superoxide dismutase 1 gene. The experiment also showed a reduction of mutant superoxide dismutase 1 mRNA levels in upper and lower motor neurons and remarkable improvements in the numbers of spinal motor neurons, the extent of neuroinflammation, and diameter of ventral root axons.15
Besides neurodegenerative diseases, adeno-associated viruses have also been used as a vector to deliver medications to facilitate brain repair after ischemic injury. In 2020, scientists from the Pennsylvania State University and the University of Puerto Rico School of Medicine have reported the successful regeneration of neurons post-ischemic injury through NeuroD1-mediated adeno-associated virus-based gene therapy. They were able to regenerate one-third of the total neurons lost to ischemic injury and protect another one-third of injured neurons by converting astrocytes to neurons in situ, thus supporting neuronal recovery.16
Additionally, adeno-associated virus-mediated restricted expression of calcium channel-binding domain 3 in primary sensory neurons can also prevent the development of cutaneous mechanical hypersensitivity. Using a rat neuropathic pain model, a 2019 study revealed that delivering this application of adeno-associated virus vector is an effective molecular strategy to treat established neuropathic pain.17 But even with all these promising, clinical translations of approved gene therapy products, especially for neurodegenerative disorders, are still slow due to several limitations.
Addressing the Challenges of Adeno-Associated Virus-Based Therapies
Due to the pathogenic nature of the adenovirus, a large dose of systematically administered adeno-associated viruses will inevitably stimulate host immune responses, which can lead to anti-capsid and anti-transgene immunity with implications for transgene expression, treatment longevity, and patient safety. Therefore, clinicians have attempted to deliver lower doses directly into the central nervous system as an alternative. This method can induce high transgene expression without triggering apparent immune responses.
However, this targeted delivery of adeno-associated viruses into the central nervous system has been shown to induce neuroinflammatory responses instead. According to some findings from preclinical studies, scientists have reported signs of adeno-associated virus-associated neuroinflammation like dorsal root ganglion and spinal cord pathology with mononuclear cell infiltration. Neuroinflammation can lead to the breakdown of the blood-brain barrier and adversely exacerbate and accelerate the pathogenesis of neurodegenerative diseases like Parkinson’s, Alzheimer’s, and Multiple Sclerosis.18
To prevent the deleterious effects of neuroinflammation, researchers have been exploring various methods to better design and deliver the adeno-associated virus vector. For instance, many scientists have experimented on several routes of administration like direct intraparenchymal, intracerebroventricular, intra-cisterna magna, and intrathecal injections to deliver gene therapeutics to determine the least invasive and immunologically safest approach. They have also attempted to genetically modify the vector and/or transgene to minimise neuroinflammation and incorporate immunosuppressive strategies into clinical protocols.19
At present, researchers are still trying to identify new vectors, therapeutic targets, and reliable delivery routes for transgenes. By optimising transgene design, delivery, and vectors, it is hoped that new knowledge can be uncovered regarding the underlying mechanisms of the onset and progression of neurodegenerative diseases. But above all, as we gear towards precision medicine, experts hope that refinements in vector-based gene therapy can facilitate prompt diagnosis, targeted selection, and bring us closer to treating currently incurable diseases.
- Ritchie, H. (2019, May 23). The world population is changing: For the first time there are more people over 64 than children younger than 5. Our World in Data. https://ourworldindata.org/population-aged-65-outnumber-children
- Brown G. C. (2015). Living too long: the current focus of medical research on increasing the quantity, rather than the quality, of life is damaging our health and harming the economy. EMBO reports, 16(2), 137–141. https://doi.org/10.15252/embr.201439518
- World Health Organisation. (2018, February 5). Ageing and health. https://www.who.int/news-room/fact-sheets/detail/ageing-and-health
- O’Donnell Jr. Brain Institute. (n.d.). Neurodegenerative Disorders. UT Southwestern Medical Center. Retrieved September 20, 2021, from https://utswmed.org/conditions-treatments/neurodegenerative-disorders/
- Sudhakar, V., & Richardson, R. M. (2018). Gene Therapy for Neurodegenerative Diseases. Neurotherapeutics, 16(1), 166–175. https://doi.org/10.1007/s13311-018-00694-0
- Chen, W., Hu, Y., & Ju, D. (2020). Gene therapy for neurodegenerative disorders: advances, insights and prospects. Acta pharmaceutica Sinica. B, 10(8), 1347–1359. https://doi.org/10.1016/j.apsb.2020.01.015
- Liu, G., Martins, I. H., Chiorini, J. A., & Davidson, B. L. (2005). Adeno-associated virus type 4 (AAV4) targets ependyma and astrocytes in the subventricular zone and RMS. Gene Therapy, 12(20), 1503–1508. https://doi.org/10.1038/sj.gt.3302554
- Rafii, M. S., Tuszynski, M. H., Thomas, R. G., Barba, D., Brewer, J. B., Rissman, R. A., Siffert, J., & Aisen, P. S. (2018). Adeno-Associated Viral Vector (Serotype 2)–Nerve Growth Factor for Patients With Alzheimer Disease. JAMA Neurology, 75(7), 834. https://doi.org/10.1001/jamaneurol.2018.0233
- Duque, S., Joussemet, B., Riviere, C., Marais, T., Dubreil, L., Douar, A. M., Fyfe, J., Moullier, P., Colle, M. A., & Barkats, M. (2009). Intravenous Administration of Self-complementary AAV9 Enables Transgene Delivery to Adult Motor Neurons. Molecular Therapy, 17(7), 1187–1196. https://doi.org/10.1038/mt.2009.71
- Albright, B. H., Storey, C. M., Murlidharan, G., Castellanos Rivera, R. M., Berry, G. E., Madigan, V. J., & Asokan, A. (2018). Mapping the Structural Determinants Required for AAVrh.10 Transport across the Blood-Brain Barrier. Molecular Therapy, 26(2), 510–523. https://doi.org/10.1016/j.ymthe.2017.10.017
- Stoker, T. B., Torsney, K. M., & Barker, R. A. (2018). Emerging Treatment Approaches for Parkinson’s Disease. Frontiers in Neuroscience, 12. https://doi.org/10.3389/fnins.2018.00693
- Morabito, G., Giannelli, S. G., Ordazzo, G., Bido, S., Castoldi, V., Indrigo, M., Cabassi, T., Cattaneo, S., Luoni, M., Cancellieri, C., Sessa, A., Bacigaluppi, M., Taverna, S., Leocani, L., Lanciego, J. L., & Broccoli, V. (2017). AAV-PHP.B-Mediated Global-Scale Expression in the Mouse Nervous System Enables GBA1 Gene Therapy for Wide Protection from Synucleinopathy. Molecular Therapy, 25(12), 2727–2742. https://doi.org/10.1016/j.ymthe.2017.08.004
- Gutekunst, C. A., Tung, J. K., McDougal, M. E., & Gross, R. E. (2016). C3 transferase gene therapy for continuous conditional RhoA inhibition. Neuroscience, 339, 308–318. https://doi.org/10.1016/j.neuroscience.2016.10.022
- Mijanović, O., Branković, A., Borovjagin, A., Butnaru, D. V., Bezrukov, E. A., Sukhanov, R. B., Shpichka, A., Timashev, P., & Ulasov, I. (2020). Battling Neurodegenerative Diseases with Adeno-Associated Virus-Based Approaches. Viruses, 12(4), 460. https://doi.org/10.3390/v12040460
- Stoica, L., Todeasa, S. H., Cabrera, G. T., Salameh, J. S., ElMallah, M. K., Mueller, C., Brown, R. H., & Sena-Esteves, M. (2016). Adeno-associated virus-delivered artificial microRNA extends survival and delays paralysis in an amyotrophic lateral sclerosis mouse model. Annals of Neurology, 79(4), 687–700. https://doi.org/10.1002/ana.24618
- Chen, Y. C., Ma, N. X., Pei, Z. F., Wu, Z., Do-Monte, F. H., Keefe, S., Yellin, E., Chen, M. S., Yin, J. C., Lee, G., Minier-Toribio, A., Hu, Y., Bai, Y. T., Lee, K., Quirk, G. J., & Chen, G. (2020). A NeuroD1 AAV-Based Gene Therapy for Functional Brain Repair after Ischemic Injury through In Vivo Astrocyte-to-Neuron Conversion. Molecular Therapy, 28(1), 217–234. https://doi.org/10.1016/j.ymthe.2019.09.003
- Yu, H., Shin, S. M., Xiang, H., Chao, D., Cai, Y., Xu, H., Khanna, R., Pan, B., & Hogan, Q. H. (2019). AAV-encoded CaV2.2 peptide aptamer CBD3A6K for primary sensory neuron-targeted treatment of established neuropathic pain. Gene Therapy, 26(7–8), 308–323. https://doi.org/10.1038/s41434-019-0082-7
- Kempuraj, D., Thangavel, R., Selvakumar, G. P., Zaheer, S., Ahmed, M. E., Raikwar, S. P., Zahoor, H., Saeed, D., Natteru, P. A., Iyer, S., & Zaheer, A. (2017). Brain and Peripheral Atypical Inflammatory Mediators Potentiate Neuroinflammation and Neurodegeneration. Frontiers in Cellular Neuroscience, 11. https://doi.org/10.3389/fncel.2017.00216
- Perez, B. A., Shutterly, A., Chan, Y. K., Byrne, B. J., & Corti, M. (2020). Management of Neuroinflammatory Responses to AAV-Mediated Gene Therapies for Neurodegenerative Diseases. Brain Sciences, 10(2), 119. https://doi.org/10.3390/brainsci10020119