AAV Engineering Identifies a Species Barrier That Highlights a Portal to the Brain
Authors: Luk H. Vandenberghe
Journal: Molecular Therapy (2019)

In this issue of Molecular Therapy, Hordeaux et al.1Hordeaux J. Yuan Y. Clark P.M. Wang Q. Martino R.A. Sims J.J. Bell P. Raymond A. Stanford W.L. Wilson J.M. The GPI-Linked Protein LY6A Drives AAV-PHP.B Transport across the Blood-Brain Barrier.Mol. Ther. 2019; 27 (this issue): 912-921Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar present a new chapter in the fascinating story on gene transfer across the blood-brain barrier (BBB). Few in our field—and beyond—would have predicted the twists and turns in the story of this unexpected biology with transformative therapeutic implications. The tale starts in 2008 with a series of observations in which a 25 nm proteinaceous adeno-associated virus (AAV) particle at a high dose could transduce targets in the peripheral and CNS via systemic routes of administration in mice2Foust K.D. Nurre E. Montgomery C.L. Hernandez A. Chan C.M. Kaspar B.K. Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes.Nat. Biotechnol. 2009; 27: 59-65Crossref PubMed Scopus (949) Google Scholar, 3Duque S. Joussemet B. Riviere C. Marais T. Dubreil L. Douar A.M. Fyfe J. Moullier P. Colle M.A. Barkats M. Intravenous administration of self-complementary AAV9 enables transgene delivery to adult motor neurons.Mol. Ther. 2009; 17: 1187-1196Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar and larger animals.3Duque S. Joussemet B. Riviere C. Marais T. Dubreil L. Douar A.M. Fyfe J. Moullier P. Colle M.A. Barkats M. Intravenous administration of self-complementary AAV9 enables transgene delivery to adult motor neurons.Mol. Ther. 2009; 17: 1187-1196Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 4Bevan A.K. Duque S. Foust K.D. Morales P.R. Braun L. Schmelzer L. Chan C.M. McCrate M. Chicoine L.G. Coley B.D. et al.Systemic gene delivery in large species for targeting spinal cord, brain, and peripheral tissues for pediatric disorders.Mol. Ther. 2011; 19: 1971-1980Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar Moreover, the efficiencies of gene transfer supported remarkable levels of correction of disease models, most notably in spinal muscular atrophy (SMA).3Duque S. Joussemet B. Riviere C. Marais T. Dubreil L. Douar A.M. Fyfe J. Moullier P. Colle M.A. Barkats M. Intravenous administration of self-complementary AAV9 enables transgene delivery to adult motor neurons.Mol. Ther. 2009; 17: 1187-1196Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 5Foust K.D. Wang X. McGovern V.L. Braun L. Bevan A.K. Haidet A.M. Le T.T. Morales P.R. Rich M.M. Burghes A.H. Kaspar B.K. Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN.Nat. Biotechnol. 2010; 28: 271-274Crossref PubMed Scopus (591) Google Scholar A decade later, these findings culminated in a successful Ph1/2 study for SMA type 1 and the likelihood of a drug approval for this otherwise fatal disorder.6Mendell J.R. Al-Zaidy S. Shell R. Arnold W.D. Rodino-Klapac L.R. Prior T.W. Lowes L. Alfano L. Berry K. Church K. et al.Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy.N. Engl. J. Med. 2017; 377: 1713-1722Crossref PubMed Scopus (1185) Google Scholar This initial academic success was translated commercially by Avexis (now acquired by Novartis), and this SMA program is currently being reviewed for drug approval by the US Food and Drug Administration (FDA). These findings and developments have energized a field of vector discovery to further improve on this unique ability that allows gene transfer to the CNS and peripheral nervous system via a non-invasive injection route. That potential was reached in 2016 when Deverman et al.7Deverman B.E. Pravdo P.L. Simpson B.P. Kumar S.R. Chan K.Y. Banerjee A. Wu W.L. Yang B. Huber N. Pasca S.P. Gradinaru V. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain.Nat. Biotechnol. 2016; 34: 204-209Crossref PubMed Scopus (530) Google Scholar identified an AAV variant that leapfrogged AAV9 in its ability to traverse the BBB. AAV-PHP.B was selected from a diverse library of AAV9 variants that incorporated random 7-mer peptides in a known permissive insertion site of the rigid icosahedral AAV capsid. Using a system called Cre recombination-based AAV-targeted evolution (CREATE), developed in the Gradinaru lab at Caltech, Deverman was able to enrich those AAVs that landed in glial fibrillary acidic protein (GFAP)-positive astrocytes in the brains of mice that were injected intravenously with the library. The clever design of CREATE gave them a handle on specificity and sensitivity: the in vivo selection was performed in GFAP-transgenic C57BL/6 mice and the CREATE-library of vectors enabled selective amplification of Cre-recombined AAV genomes with high sensitivity in astrocytes in the CNS. After several rounds of purifying selection, PHP.B arose as a dominant species. Validation studies confirmed the efficiency of transduction by AAV-PHP.B following intravenous administration to be between one and two orders of magnitude greater than AAV9. PHP.B has since been widely adopted as a research tool in neuroscience. The AAV9 variant was also quickly considered as a therapeutic vector system and licensed to various gene therapy companies, and its remarkable improvements in efficiency compared to AAV9 in mice suggested a tantalizing set of opportunities to unlock targets in neurological and neuromuscular disorders, to reduce dose requirements to achieve therapeutic levels of transduction in the CNS, and to minimize dose-related toxicities. The authors continued on this successful track and have since generated other variants with improved properties.8Chan K.Y. Jang M.J. Yoo B.B. Greenbaum A. Ravi N. Wu W.L. Sánchez-Guardado L. Lois C. Mazmanian S.K. Deverman B.E. Gradinaru V. Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems.Nat. Neurosci. 2017; 20: 1172-1179Crossref PubMed Scopus (543) Google Scholar The next twist in this already remarkable story was unfortunately more sobering. In a Molecular Therapy report last year, Hordeaux et al.9Hordeaux J. Wang Q. Katz N. Buza E.L. Bell P. Wilson J.M. The Neurotropic Properties of AAV-PHP.B Are Limited to C57BL/6J Mice.Mol. Ther. 2018; 26: 664-668Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar presented data that the BBB advantage of PHP.B over AAV9 did not translate to a non-human primate model or to another mouse strain, BALB/c. Concerns about species barriers had been raised in the past for other AAVs, but such barriers had not been observed in such a dramatic and narrow manner. In this issue of Molecular Therapy, Hordeaux et al. 1Hordeaux J. Yuan Y. Clark P.M. Wang Q. Martino R.A. Sims J.J. Bell P. Raymond A. Stanford W.L. Wilson J.M. The GPI-Linked Protein LY6A Drives AAV-PHP.B Transport across the Blood-Brain Barrier.Mol. Ther. 2019; 27 (this issue): 912-921Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar present the next surprising chapter in the story of AAV and BBB, following up on their initial observation demonstrating a species barrier. In an elegant study that combines both old and new school genetics, the authors were able to segregate the enhanced PHP.B BBB-crossing phenotype to the C57BL/6 Ly6a (also known as Sca-1) haplotype. Confirmatory studies in Ly6a knockout (KO) mice, the mapping of the phenotype to a small set of small nucleotide polymorphisms (SNPs) in the BALB/c Ly6a, and the observation that AAV-PHP.B's CNS infectivity was not affected following an intracerebroventricular injection of BALB/c mice gave further credence to its specific role in BBB transport of this AAV variant. Remarkably, the 18-kDa mouse glycosyl phosphatidylinositol-anchored cell surface protein is well-known for its role in hematopoiesis and its utility as a marker of stem cells and progenitors, but not for its role in BBB transport. So where does this tale of unexpected biology leave us after all its twists and turns? For starters, AAV-PHP.B was, is, and remains a powerful research tool for neuroscientists, and the current study really defines its utility to strains of mice with the C57BL/6 Ly6a haplotype. Second, the development of AAV-PHP.B by Deverman et al.7Deverman B.E. Pravdo P.L. Simpson B.P. Kumar S.R. Chan K.Y. Banerjee A. Wu W.L. Yang B. Huber N. Pasca S.P. Gradinaru V. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain.Nat. Biotechnol. 2016; 34: 204-209Crossref PubMed Scopus (530) Google Scholar illustrates the potential of AAV engineering and the dynamic range in activity or potency that can be obtained. It puts the bar quite high for the rest of us working in a dense AAV discovery space. It also highlights how methodological sophistication, such as CREATE, may be required to identify AAVs with a specific transduction performance, such as PHP.B. Third, the consecutive studies by Hordeaux et al.1Hordeaux J. Yuan Y. Clark P.M. Wang Q. Martino R.A. Sims J.J. Bell P. Raymond A. Stanford W.L. Wilson J.M. The GPI-Linked Protein LY6A Drives AAV-PHP.B Transport across the Blood-Brain Barrier.Mol. Ther. 2019; 27 (this issue): 912-921Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 9Hordeaux J. Wang Q. Katz N. Buza E.L. Bell P. Wilson J.M. The Neurotropic Properties of AAV-PHP.B Are Limited to C57BL/6J Mice.Mol. Ther. 2018; 26: 664-668Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar underline the limits of vector engineering, particularly those using methods of directed evolution such as the one that generated AAV-PHP.B. These potent selective screens are subject to the genetic biases of the model upon which they are used and, hence, the output reagents require significant scrutiny and validation before consideration for translation. The exquisite sensitivity of CREATE and other such systems might inadvertently heighten the concern that model-specific biases are introduced. Future studies should reveal where the verdict lands on this glass-half-full, glass-half-empty assessment of the utility of vector engineering approaches such as directed evolution. Lastly, the fact that we can tie a mechanism and a host factor to explain aspects of AAVs biology and tropism in vivo is a remarkable step forward. While this study may present findings on a vector with relatively limited therapeutic utility given the species restriction, the implication of Ly6a in BBB transport is novel and opens the door to use it and its functional homologs as a target portal to the brain for AAV and other therapies in humans. L.H.V. is a co-inventor of AAV9, which was licensed to Avexis (now Novartis), and other AAV technologies; holds equity in Akouos; and consults for a number of AAV gene therapy, biotechnology, and pharmaceutical entities. His laboratory receives sponsored research from Lonza Houston, Selecta Biosciences, Solid Biosciences, and Oxford Biomedica. The GPI-Linked Protein LY6A Drives AAV-PHP.B Transport across the Blood-Brain BarrierHordeaux et al.Molecular TherapyFebruary 19, 2019In BriefThis study unravels a new transport mechanism across the blood-brain barrier and identifies LY6A (SCA-1) as the receptor for adeno-associated virus (AAV)-PHP.B and AAV-PHP.eB capsids. Importantly, LY6A is not expressed in several strains of mice and is non-existent in primates, limiting the usefulness of those capsids to nonclinical applications. Full-Text PDF Open Archive