AAV-CNS matters turn from gray to white
Authors: Aaron Mitchell-Dick, Aravind Asokan
Journal: Molecular Therapy (2021)

Adeno-associated virus (AAV)-mediated gene therapies for central nervous system (CNS) disorders are experiencing a period of rapid development. A string of recent clinical trial outcomes for rare and complex CNS disorders complemented by preclinical studies in animal models continue to highlight both the prospects and challenges facing therapeutic gene transfer to the brain.1Hudry E. Vandenberghe L.H. Therapeutic AAV Gene Transfer to the Nervous System: A Clinical Reality.Neuron. 2019; 101: 839-862Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 2Rafii M.S. et al.Adeno-Associated Viral Vector (Serotype 2)–Nerve Growth Factor for Patients With Alzheimer Disease: A Randomized Clinical Trial.JAMA Neurol. 2018; 75: 834-841Crossref PubMed Scopus (83) Google Scholar, 3Deverman B.E. Ravina B.M. Bankiewicz K.S. Paul S.M. Sah D.W.Y. Gene therapy for neurological disorders: progress and prospects.Nat. Rev. Drug Discov. 2018; 17: 641-659Crossref PubMed Scopus (2) Google Scholar Notably, regional disparities in AAV vector spread within the brain parenchyma and transgene expression have spurred efforts in two key areas: optimizing physical methods of CNS dosing and better characterizing AAV biology in the brain.4Castle M.J. et al.Postmortem Analysis in a Clinical Trial of AAV2-NGF Gene Therapy for Alzheimer's Disease Identifies a Need for Improved Vector Delivery.Hum. Gene Ther. 2020; 31: 415-422Crossref PubMed Scopus (24) Google Scholar In this issue of Molecular Therapy, Heller et al.5Heller G.J. Marshall M.S. Issa Y. Marshall J.N. Nguyen D. Rue E. Pathmasiri K.C. Domowicz M.S. van Breeman R.B. Tai L.M. et al.Waning efficacy in a long-term AAV-mediated gene therapy study in the murine model of Krabbe disease.Mol Ther. 2021; 29 (this issue): 1883-1902Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar highlight a third temporal component that shines a spotlight on the interface of CNS disease and AAV biology. Krabbe disease (KD), also known as globoid cell leukodystrophy, affects newborn children, often leading to death within the first few years of life.6Wenger D.A. Rafi M.A. Luzi P. Datto J. Costantino-Ceccarini E. Krabbe Disease: Genetic Aspects and Progress toward Therapy.Mol. Genet. Metab. 2000; 70: 1-9Crossref PubMed Scopus (157) Google Scholar As KD is caused by loss-of-function mutations in the lysosomal enzyme galactosylceramidase (GALC), several groups have published promising results showing that AAV-delivered GALC significantly improves behavior and pathology in Twitcher mice, a murine model for KD.7Marshall M.S. et al.Long-Term Improvement of Neurological Signs and Metabolic Dysfunction in a Mouse Model of Krabbe Disease after Global Gene Therapy.Mol. Ther. 2018; 26: 874-889Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 8Karumuthil-Melethil S. et al.Intrathecal administration of AAV/GALC vectors in 10-11-day-old twitcher mice improves survival and is enhanced by bone marrow transplant.J. Neurosci. Res. 2016; 94: 1138-1151Crossref PubMed Scopus (30) Google Scholar, 9Rafi M.A. Rao H.Z. Luzi P. Wenger D.A. Long-term Improvements in Lifespan and Pathology in CNS and PNS After BMT Plus One Intravenous Injection of AAVrh10-GALC in Twitcher Mice.Mol Ther. 2015; 23: 1681-1690Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar These results have sparked optimism for treating KD via AAV gene therapy. In a recent long-term study, the Heller group found that AAV9-GALC-treated Twitcher mice showed little to no signs of disease for most of their significantly lengthened lifespans.7Marshall M.S. et al.Long-Term Improvement of Neurological Signs and Metabolic Dysfunction in a Mouse Model of Krabbe Disease after Global Gene Therapy.Mol. Ther. 2018; 26: 874-889Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar However, a wide range of lifespans were found to result from the treatment, suggesting differences in underlying factors affecting long-term survival. Upon deeper investigation, Heller et al.5Heller G.J. Marshall M.S. Issa Y. Marshall J.N. Nguyen D. Rue E. Pathmasiri K.C. Domowicz M.S. van Breeman R.B. Tai L.M. et al.Waning efficacy in a long-term AAV-mediated gene therapy study in the murine model of Krabbe disease.Mol Ther. 2021; 29 (this issue): 1883-1902Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar found that late-onset focal lesions of demyelination develop in white matter following AAV9-GALC treatment. Interestingly, the lesions developed in areas where, at earlier time points, the investigators observed healthy tissue with robust GALC expression, suggesting waning efficacy. Several groups have investigated whether Twitcher mice can be cured of disease using AAV-mediated GALC expression in the brain. Delivery of AAVrh10-GALC simultaneously via intravenous, intracerebellar, and intracerebroventricular injection in postnatal day 2 (P2) mice resulted in significantly extended lifespans up to 8 months. This approach, which requires three simultaneous injections, was further refined to a single AAVrh10-GALC injection at P10 and resulted in longer lifespans, although the lifespan was only extended by 1 month following injections.10Rafi M.A. et al.Intravenous injection of AAVrh10-GALC after the neonatal period in twitcher mice results in significant expression in the central and peripheral nervous systems and improvement of clinical features.Mol. Genet. Metab. 2015; 114: 459-466Crossref PubMed Scopus (32) Google Scholar Combining bone marrow transplant (BMT) with a single injection of AAVrh10 around P10 further improved lifespan, with many treated Twitcher mice living longer than 150 days. A similar strategy with multiple AAV serotypes led to the conclusion that AAV9-GALC delivered intrathecally in combination with BMT greatly increased the lifespan of Twitcher mice.8Karumuthil-Melethil S. et al.Intrathecal administration of AAV/GALC vectors in 10-11-day-old twitcher mice improves survival and is enhanced by bone marrow transplant.J. Neurosci. Res. 2016; 94: 1138-1151Crossref PubMed Scopus (30) Google Scholar Most recently, the Heller group demonstrated that intracranial delivery of AAV9-GALC or high-dose intravenous delivery in the first day after birth significantly improved average lifespan beyond 250 days and was not statistically different from a cohort receiving AAV9-GALC+BMT (via intravenous delivery of non-myeloablated syngenic bone marrow cells) at P1–P2.7Marshall M.S. et al.Long-Term Improvement of Neurological Signs and Metabolic Dysfunction in a Mouse Model of Krabbe Disease after Global Gene Therapy.Mol. Ther. 2018; 26: 874-889Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar These results demonstrate remarkable promise, yet posed a major question: what might be the underlying causes driving heterogeneity in long-term survival of Twitcher mice following AAV-GALC treatment? In this follow up study, Heller et al.5Heller G.J. Marshall M.S. Issa Y. Marshall J.N. Nguyen D. Rue E. Pathmasiri K.C. Domowicz M.S. van Breeman R.B. Tai L.M. et al.Waning efficacy in a long-term AAV-mediated gene therapy study in the murine model of Krabbe disease.Mol Ther. 2021; 29 (this issue): 1883-1902Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar tackled this challenging question and reported that waning efficacy can be attributed to psychosine accumulation and decreased GALC activity in specific demyelinated areas of the CNS following treatment. Notably, the peripheral nervous system (PNS)/spinal cord and other areas of the CNS do not exhibit these focal lesions or deficits at later time points. Rather than gray matter, focal demyelination is observed in the CNS white matter, accompanied by surrounding gliotic scarring, local inflammation, and accumulating Iba1+ microglia. In addition, extravasation of serum factors and fibrinogen into focal lesions appears to recruit microglia, resulting in axonal damage. The authors go on to show that dilution of AAV episomes due to proliferation of oligodendrocyte precursor cells (OPCs) is associated with these focal lesions. These latter findings turn our attention to an underappreciated area in CNS gene therapy, i.e., gliogenesis. Astrocytes are known to proliferate in response to injury, concomitantly with the process of astrogliosis.11Shimada I.S. LeComte M.D. Granger J.C. Quinlan N.J. Spees J.L. Self-renewal and differentiation of reactive astrocyte-derived neural stem/progenitor cells isolated from the cortical peri-infarct area after stroke.J. Neurosci. 2012; 32: 7926-7940Crossref PubMed Scopus (101) Google Scholar Both OPCs and astrocytes display robust proliferative potential post-specification.12Richardson W.D. Young K.M. Tripathi R.B. McKenzie I. NG2-glia as multipotent neural stem cells: fact or fantasy?.Neuron. 2011; 70: 661-673Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar While still unclear, NG2+ glia can potentially serve as OPCs and retain the ability to proliferate in response to injury.12Richardson W.D. Young K.M. Tripathi R.B. McKenzie I. NG2-glia as multipotent neural stem cells: fact or fantasy?.Neuron. 2011; 70: 661-673Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar Further, myelination, which follows synaptic pruning and gliogenesis, has been shown to occur robustly during postnatal development, and myelin turnover requirements may be greatest at sites of high neural activity.13Gibson E.M. et al.Neuronal Activity Promotes Oligodendrogenesis and Adaptive Myelination in the Mammalian Brain.Science. 2014; 344: 1252304Crossref PubMed Scopus (721) Google Scholar Linking these aspects of CNS biology to that of AAV genome persistence, it is plausible that proliferation of OPCs early in development, in adulthood in areas of frequent myelin remodeling, or both could account for the loss of AAV-mediated GALC activity and re-emergence of KD phenotypes over time. Importantly, the authors conclude that, since other areas of the PNS/CNS in AAV9-GALC treated Twitcher mice remain healthy, the emergence of focal demyelinated lesions is not likely to arise due to AAV transgene silencing, but rather from local myelin turnover, generation of new oligodendrocytes, and dilution of AAV episomes on a per-cell basis. The exact reason behind the focal nature of these lesions is unclear, although the authors propose that such might be seeded by accelerated glial proliferation in regions undergoing myelin remodeling. Taken together, the findings from the Heller et al.5Heller G.J. Marshall M.S. Issa Y. Marshall J.N. Nguyen D. Rue E. Pathmasiri K.C. Domowicz M.S. van Breeman R.B. Tai L.M. et al.Waning efficacy in a long-term AAV-mediated gene therapy study in the murine model of Krabbe disease.Mol Ther. 2021; 29 (this issue): 1883-1902Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar study investigating long-term effects of AAV gene therapy in a mouse model for KD highlight a previously under-appreciated temporal component in CNS gene transfer. Envisioning successful gene therapy in KD invariably includes gene therapy very early after birth. Ongoing efforts will undoubtedly help improve CNS coverage through AAV gene transfer. However, we may discover potential trade-offs to early treatment arising from proliferative events and resulting in AAV genome dilution. These observations in the Twitcher mouse model have important implications toward clinical trial design and assessment of different endpoints. The implications also extend beyond KD and, in general, underscore the need to assess long-term AAV-mediated gene expression in the brain. Overall, as we continue to evaluate the potential of AAV-mediated gene therapy for CNS disorders, the importance of pursuing a multidimensional approach that hinges at the interface of physical delivery methods, AAV vectorology, and CNS biology is clear.