Rapid evolution of blood-brain-barrier-penetrating AAV capsids by RNA-driven biopanning
2020; Cell Press; Volume: 20; Linguagem: Inglês
10.1016/j.omtm.2020.12.006
ISSN2329-0501
AutoresMathieu Nonnenmacher, Wei Wang, Matthew A. Child, Xiao‐Qin Ren, Carol Huang, Amy Ren, Jenna Tocci, Qingmin Chen, K Bittner, Katherine Tyson, Nilesh Pande, Charlotte Hiu-Yan Chung, Steven M. Paul, Jay Hou,
Tópico(s)Neurogenetic and Muscular Disorders Research
ResumoTherapeutic payload delivery to the central nervous system (CNS) remains a major challenge in gene therapy. Recent studies using function-driven evolution of adeno-associated virus (AAV) vectors have successfully identified engineered capsids with improved blood-brain barrier (BBB) penetration and CNS tropism in mouse. However, these strategies require transgenic animals and thus are limited to rodents. To address this issue, we developed a directed evolution approach based on recovery of capsid library RNA transcribed from CNS-restricted promoters. This RNA-driven screen platform, termed TRACER (Tropism Redirection of AAV by Cell-type-specific Expression of RNA), was tested in the mouse with AAV9 peptide display libraries and showed rapid emergence of dominant sequences. Ten individual variants were characterized and showed up to 400-fold higher brain transduction over AAV9 following systemic administration. Our results demonstrate that the TRACER platform allows rapid selection of AAV capsids with robust BBB penetration and CNS tropism in non-transgenic animals. Therapeutic payload delivery to the central nervous system (CNS) remains a major challenge in gene therapy. Recent studies using function-driven evolution of adeno-associated virus (AAV) vectors have successfully identified engineered capsids with improved blood-brain barrier (BBB) penetration and CNS tropism in mouse. However, these strategies require transgenic animals and thus are limited to rodents. To address this issue, we developed a directed evolution approach based on recovery of capsid library RNA transcribed from CNS-restricted promoters. This RNA-driven screen platform, termed TRACER (Tropism Redirection of AAV by Cell-type-specific Expression of RNA), was tested in the mouse with AAV9 peptide display libraries and showed rapid emergence of dominant sequences. Ten individual variants were characterized and showed up to 400-fold higher brain transduction over AAV9 following systemic administration. Our results demonstrate that the TRACER platform allows rapid selection of AAV capsids with robust BBB penetration and CNS tropism in non-transgenic animals. IntroductionClinical applications of gene therapy in the central nervous system (CNS) are currently limited by the poor transduction of brain and spinal cord by adeno-associated virus (AAV) and other viral vectors.1Deverman 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,2Gray S.J. Woodard K.T. Samulski R.J. Viral vectors and delivery strategies for CNS gene therapy.Ther. Deliv. 2010; 1: 517-534Crossref PubMed Scopus (79) Google Scholar The blood-brain barrier (BBB) represents a formidable obstacle for delivery of AAV into brain tissue following intravenous administration, and even the best-in-class natural BBB-penetrating serotypes, namely AAV9 and other clade F derivatives,3Mendell 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 (1122) Google Scholar,4Ellsworth J.L. Gingras J. Smith L.J. Rubin H. Seabrook T.A. Patel K. Zapata N. Olivieri K. O'Callaghan M. Chlipala E. et al.Clade F AAVHSCs cross the blood brain barrier and transduce the central nervous system in addition to peripheral tissues following intravenous administration in nonhuman primates.PLoS One. 2019; 14: e0225582Crossref PubMed Scopus (15) Google Scholar only allow limited brain distribution.1Deverman 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,5Wang D. Tai P.W.L. Gao G. Adeno-associated virus vector as a platform for gene therapy delivery.Nat. Rev. Drug Discov. 2019; 18: 358-378Crossref PubMed Scopus (664) Google Scholar,6Hocquemiller M. Giersch L. Audrain M. Parker S. Cartier N. Adeno-Associated Virus-Based Gene Therapy for CNS Diseases.Hum. Gene Ther. 2016; 27: 478-496Crossref PubMed Scopus (167) Google Scholar This challenge can be partially overcome by using local delivery routes, such as intraparenchymal injection,7Taymans J.-M. Vandenberghe L.H. Van den Haute C. Thiry I. Deroose C. Mortelmans L. et al.Comparative analysis of adeno-associated viral vector serotypes 1, 2, 5, 7, and 8 in mouse brain.Hum. Gene Ther. 2007; 18: 195-206https://doi.org/10.1089/hum.2006.178Crossref PubMed Scopus (233) Google Scholar,8Cearley C.N. Wolfe J.H. Transduction characteristics of adeno-associated virus vectors expressing cap serotypes 7, 8, 9, and Rh10 in the mouse brain.Mol. Ther. 2006; 13: 528-537Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar intrathecal infusion,9Hinderer C. Bell P. Katz N. Vite C.H. Louboutin J.P. Bote E. Yu H. Zhu Y. Casal M.L. Bagel J. et al.Evaluation of Intrathecal Routes of Administration for Adeno-Associated Viral Vectors in Large Animals.Hum. Gene Ther. 2018; 29: 15-24Crossref PubMed Scopus (62) Google Scholar or cisterna magna administration.10Hinderer C. Bell P. Vite C.H. Louboutin J.P. Grant R. Bote E. Yu H. Pukenas B. Hurst R. Wilson J.M. Widespread gene transfer in the central nervous system of cynomolgus macaques following delivery of AAV9 into the cisterna magna.Mol. Ther. Methods Clin. Dev. 2014; 1: 14051Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar However, these methods are invasive and only achieve limited distribution and transduction throughout the brain and spinal cord, short of therapeutically desired coverage. These shortcomings could be mitigated by engineered AAV capsids capable of efficiently crossing the BBB via intravascular delivery.High-throughput mutagenesis and directed evolution of AAV capsids were first described in 200311Perabo L. Büning H. Kofler D.M. Ried M.U. Girod A. Wendtner C.M. Enssle J. Hallek M. In vitro selection of viral vectors with modified tropism: the adeno-associated virus display.Mol. Ther. 2003; 8: 151-157Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar,12Müller O.J. Kaul F. Weitzman M.D. Pasqualini R. Arap W. Kleinschmidt J.A. Trepel M. Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors.Nat. Biotechnol. 2003; 21: 1040-1046Crossref PubMed Scopus (303) Google Scholar and are greatly facilitated by the simplicity of the viral genome organization, extensive knowledge of capsid structure,13Xie Q. Bu W. Bhatia S. Hare J. Somasundaram T. Azzi A. Chapman M.S. The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy.Proc. Natl. Acad. Sci. USA. 2002; 99: 10405-10410Crossref PubMed Scopus (456) Google Scholar and the natural propensity of wild-type AAV to assemble capsids with low mosaicism and high genome-capsid correlation.14Nonnenmacher M. van Bakel H. Hajjar R.J. Weber T. High capsid-genome correlation facilitates creation of AAV libraries for directed evolution.Mol. Ther. 2015; 23: 675-682Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 15Schmit P.F. Pacouret S. Zinn E. Telford E. Nicolaou F. Broucque F. Andres-Mateos E. Xiao R. Penaud-Budloo M. Bouzelha M. et al.Cross-Packaging and Capsid Mosaic Formation in Multiplexed AAV Libraries.Mol. Ther. Methods Clin. Dev. 2019; 17: 107-121Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 16Körbelin J. Hunger A. Alawi M. Sieber T. Binder M. Trepel M. Optimization of design and production strategies for novel adeno-associated viral display peptide libraries.Gene Ther. 2017; 24: 470-481Crossref PubMed Scopus (11) Google Scholar Early designs of AAV-directed evolution were strictly tailored for in vitro selection in cultured cells and used helper adenovirus coinfection to enrich transduction-competent variants.11Perabo L. Büning H. Kofler D.M. Ried M.U. Girod A. Wendtner C.M. Enssle J. Hallek M. In vitro selection of viral vectors with modified tropism: the adeno-associated virus display.Mol. Ther. 2003; 8: 151-157Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar,12Müller O.J. Kaul F. Weitzman M.D. Pasqualini R. Arap W. Kleinschmidt J.A. Trepel M. Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors.Nat. Biotechnol. 2003; 21: 1040-1046Crossref PubMed Scopus (303) Google Scholar,17Grimm D. Lee J.S. Wang L. Desai T. Akache B. Storm T.A. Kay M.A. In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses.J. Virol. 2008; 82: 5887-5911Crossref PubMed Scopus (426) Google Scholar,18Maheshri N. Koerber J.T. Kaspar B.K. Schaffer D.V. Directed evolution of adeno-associated virus yields enhanced gene delivery vectors.Nat. Biotechnol. 2006; 24: 198-204Crossref PubMed Scopus (391) Google Scholar Helper-dependent selection is not easily accomplished in vivo, and library selection in the mouse initially relied on indiscriminate PCR amplification of AAV genomes from the tissue of interest.19Michelfelder S. Kohlschütter J. Skorupa A. Pfennings S. Müller O. Kleinschmidt J.A. Trepel M. Successful expansion but not complete restriction of tropism of adeno-associated virus by in vivo biopanning of random virus display peptide libraries.PLoS ONE. 2009; 4: e5122Crossref PubMed Scopus (61) Google Scholar Although this approach proved successful to some degree, tremendous effort has been spent on developing new biopanning approaches to efficiently select true positives in vivo.20Körbelin J. Sieber T. Michelfelder S. Lunding L. Spies E. Hunger A. Alawi M. Rapti K. Indenbirken D. Müller O.J. et al.Pulmonary Targeting of Adeno-associated Viral Vectors by Next-generation Sequencing-guided Screening of Random Capsid Displayed Peptide Libraries.Mol. Ther. 2016; 24: 1050-1061Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar,21Körbelin J. Trepel M. How to Successfully Screen Random Adeno-Associated Virus Display Peptide Libraries In Vivo.Hum. Gene Ther. Methods. 2017; 28: 109-123Crossref PubMed Scopus (6) Google Scholar Over the past decade, functional AAV library screens based on cell-specific sorting,22Dalkara D. Byrne L.C. Klimczak R.R. Visel M. Yin L. Merigan W.H. Flannery J.G. Schaffer D.V. In vivo-directed evolution of a new adeno-associated virus for therapeutic outer retinal gene delivery from the vitreous.Sci. Transl. Med. 2013; 5: 189ra76Crossref PubMed Scopus (426) Google Scholar in vivo helper virus coinfection,23Lisowski L. Dane A.P. Chu K. Zhang Y. Cunningham S.C. Wilson E.M. Nygaard S. Grompe M. Alexander I.E. Kay M.A. Selection and evaluation of clinically relevant AAV variants in a xenograft liver model.Nature. 2014; 506: 382-386Crossref PubMed Scopus (287) Google Scholar or cell-specific Cre-lox selection24Ojala D.S. Sun S. Santiago-Ortiz J.L. Shapiro M.G. Romero P.A. Schaffer D.V. In Vivo Selection of a Computationally Designed SCHEMA AAV Library Yields a Novel Variant for Infection of Adult Neural Stem Cells in the SVZ.Mol. Ther. 2018; 26: 304-319Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 25Hanlon K.S. Meltzer J.C. Buzhdygan T. Cheng M.J. Sena-Esteves M. Bennett R.E. Sullivan T.P. Razmpour R. Gong Y. Ng C. et al.Selection of an Efficient AAV Vector for Robust CNS Transgene Expression.Mol. Ther. Methods Clin. Dev. 2019; 15: 320-332Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 26Deverman 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 (503) Google Scholar have identified improved capsid variants. In particular, two AAV9 variants, PHP.B and PHP.eB, showed an unprecedented ability to transduce C57BL/6 mouse brain via systemic injection.26Deverman 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 (503) Google Scholar,27Chan 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 (498) Google Scholar Follow-up studies, however, showed that these properties did not translate to other laboratory mouse strains or to non-human primates (NHPs).28Matsuzaki Y. Konno A. Mochizuki R. Shinohara Y. Nitta K. Okada Y. Hirai H. Intravenous administration of the adeno-associated virus-PHP.B capsid fails to upregulate transduction efficiency in the marmoset brain.Neurosci. Lett. 2018; 665: 182-188Crossref PubMed Scopus (88) Google Scholar, 29Hordeaux 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 (120) Google Scholar, 30Liguore W.A. Domire J.S. Button D. Wang Y. Dufour B.D. Srinivasan S. McBride J.L. AAV-PHP.B Administration Results in a Differential Pattern of CNS Biodistribution in Non-human Primates Compared with Mice.Mol. Ther. 2019; 27: 2018-2037Abstract Full Text Full Text PDF PubMed Scopus (53) Google ScholarImportantly, Cre-dependent AAV library screening methods24Ojala D.S. Sun S. Santiago-Ortiz J.L. Shapiro M.G. Romero P.A. Schaffer D.V. In Vivo Selection of a Computationally Designed SCHEMA AAV Library Yields a Novel Variant for Infection of Adult Neural Stem Cells in the SVZ.Mol. Ther. 2018; 26: 304-319Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 25Hanlon K.S. Meltzer J.C. Buzhdygan T. Cheng M.J. Sena-Esteves M. Bennett R.E. Sullivan T.P. Razmpour R. Gong Y. Ng C. et al.Selection of an Efficient AAV Vector for Robust CNS Transgene Expression.Mol. Ther. Methods Clin. Dev. 2019; 15: 320-332Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 26Deverman 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 (503) Google Scholar strictly rely on transgenic animals for specific recovery of transduction-competent variants, which precludes the use of clinically relevant animals such as NHPs. In this study, we describe TRACER (Tropism Redirection of AAV by Cell-type-specific Expression of RNA), an AAV evolution platform based on recovery of bulk capsid library RNA expressed in a cell-type-specific manner from non-transgenic animal tissue. We used TRACER in a directed evolution screen focused on mouse CNS and were able to isolate multiple capsid variants capable of widespread brain transduction via systemic administration. We demonstrate that TRACER is a highly effective AAV evolution platform, with potential applications in a broad range of tissues and non-transgenic animal species.ResultsDesign and construction of AAV libraries with cell-type-specific expression vectors and biopanningThe AAV genome is transcriptionally repressed in the absence of helper virus.31Stutika C. Gogol-Döring A. Botschen L. Mietzsch M. Weger S. Feldkamp M. Chen W. Heilbronn R. A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins.J. Virol. 2015; 90: 1278-1289Crossref PubMed Scopus (18) Google Scholar,32Mouw M.B. Pintel D.J. Adeno-associated virus RNAs appear in a temporal order and their splicing is stimulated during coinfection with adenovirus.J. Virol. 2000; 74: 9878-9888Crossref PubMed Scopus (31) Google Scholar In order to generate transcription-competent libraries for RNA-driven directed evolution, we inserted a non-AAV promoter upstream of the Cap gene, which would confer cell-type expression specificity while retaining the minimal regulatory elements essential for capsid protein expression and stoichiometry (Figure 1A). We determined that a minimal Rep fragment starting at nucleotide 1700 that contains the AAV P40 promoter and splice donor and acceptor sequences was sufficient for efficient virus production, albeit with a lower yield than wild-type AAV (Figure S1).We performed a series of in vitro experiments to test possible interference between the P40 promoter and the non-AAV promoters placed in tandem configuration. We tested three promoters in the context of AAV9 TRACER vectors: the ubiquitous cytomegalovirus (CMV) enhancer-chicken β-actin (CAG) promoter, the neuron-specific human synapsin 1 (SYN) promoter,33Schoch S. Cibelli G. Thiel G. Neuron-specific Gene Expression of Synapsin I.J. Biol. Chem. 1996; 271: 3317-3323Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar and the astrocyte-specific gfaABC1D (GFAP) promoter.34Lee Y. Messing A. Su M. Brenner M. GFAP promoter elements required for region-specific and astrocyte-specific expression.Glia. 2008; 56: 481-493Crossref PubMed Scopus (218) Google Scholar All constructs were used to produce AAV9 capsids, and the resulting virions were added to HEK293T cells or cultured primary mouse brain cells. As expected, RNA expression from SYN and GFAP promoters was strongly repressed in non-CNS HEK293T cells (170-fold and 400-fold lower than CAG, respectively), but not in primary mouse brain cells, where both SYN and GFAP showed a strength similar to CAG promoter (Figure 1B, left panel). A similar trend was observed with GFP vectors without P40 sequence (Figure 1B, right panel), indicating that the presence of the P40 element has little or no impact on the regulation of upstream tandem promoters used for biopanning.Since TRACER constructs lack a full-length functional REP reading frame, REP proteins were provided in trans by a separate plasmid during virus production. We first tested a Rep2 plasmid missing most of the Cap gene, but higher titers were obtained with a Rep2-Cap9Δ plasmid containing a CAP C terminus deletion and in-frame stop codons to eliminate VP1-3 translation (Figure S1).Capsid libraries were generated by inserting 7-mer randomized peptides between residues 588 and 589 in the hypervariable surface loop VIII35DiMattia M.A. Nam H.-J. Van Vliet K. Mitchell M. Bennett A. Gurda B.L. McKenna R. Olson N.H. Sinkovits R.S. Potter M. et al.Structural insight into the unique properties of adeno-associated virus serotype 9.J. Virol. 2012; 86: 6947-6958Crossref PubMed Scopus (129) Google Scholar of AAV9. Random peptides were N-terminally flanked by the original AAV9 residues AQ(587, 588) or by the PHP.eB-derived residues DG(587, 588) or DGT(587, 588, 589) (Figure 1C).27Chan 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 (498) Google Scholar To avoid the loss of variants resulting from bacterial transformation, library DNA assembled with SYN or GFAP vectors was amplified in vitro by rolling circle amplification (RCA) and protelomerase end cleavage joining (Figure S1).36Deneke J., Ziegelin, G. Lurz R. Lanka E. The protelomerase of temperate Escherichia coli phage N15 has cleaving-joining activity.Proc. Natl. Adac. Sci. U S A. 2000; 97: 7721-7726Crossref PubMed Scopus (74) Google Scholar This technique generated large amounts of transfection-ready DNA with a diversity beyond the capacity of our next-generation sequencing (NGS) analysis (>108 unique variants) and without obvious sequence bias. Viral libraries were produced in HEK293T cells using low-DNA-input conditions to minimize capsid mosaicism and cross-packaging14Nonnenmacher M. van Bakel H. Hajjar R.J. Weber T. High capsid-genome correlation facilitates creation of AAV libraries for directed evolution.Mol. Ther. 2015; 23: 675-682Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar and were administered intravenously to adult C57BL/6 mice (n = 6). Whole-brain RNA was isolated after 28 days and capsid library sequences were recovered by RT-PCR. Amplified pools were re-cloned into SYN or GFAP TRACER vectors for a second round of selection (Figure 1D). Abundant capsid amplicons were recovered from all brain samples regardless of the promoter driving the expression (Figure 1E), indicating that some variants achieve high transduction in the CNS, a tissue with one of the lowest AAV biodistributions.37Zincarelli C. Soltys S. Rengo G. Rabinowitz J.E. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection.Mol. Ther. 2008; 16: 1073-1080Abstract Full Text Full Text PDF PubMed Scopus (870) Google ScholarSelection of AAV9 capsid variants with enhanced BBB penetration and CNS transductionNGS analysis was performed after each step of in vivo selection to estimate variant diversity and enrichment (Figure 2A). The first round of selection eliminated approximately 95% of variants (from ≥10 million to 500,000 unique sequences), and the second round removed 60% of the remaining variants (from 500,000 to 200,000 unique sequences). Enrichment analysis performed after the second round of biopanning showed that hundreds of capsid variants displayed high enrichment scores compared to AAV9 (Figure 2B).Figure 2NGS-driven evolution of TRACER libraries in C57BL/6 miceShow full caption(A) TRACER workflow and library diversity through successive rounds of evolution and pooled synthesis. Values indicate the number of unique variants detected by NGS. (B) Enrichment analysis of P2 brain RNA. Enrichment score E indicates the relative RNA abundance of each variant (RP2) normalized to P1 virus stock (RP1v). Top 1,000 variants of SYN and GFAP libraries are depicted. (C and D) Fitness analysis of SYN-driven (C) and GFAP-driven (D) pool of 330 capsid candidates plus AAV9, PHP.B, and PHP.eB controls. Heatmaps represent relative RNA enrichment score in brain and spinal cord and DNA enrichment score in heart and liver. Values are normalized to AAV9 control. Numbered columns represent individual animals (n = 6). Values represent the average of two codon variants for each mutant and are ranked according the average of 6 brains. Ranking of control capsids is indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Bioinformatics analysis based on absolute read numbers, enrichment scores, cross-animal consistency, and collapsing of pseudo-variants from sequencing errors led us to select 330 capsid candidates with a favorable CNS enrichment profile (see Materials and methods). Phylogenetic analysis of this brain-enriched variant pool identified several conserved families of variants harboring 9-mer peptides with striking sequence similarities (Figure S2). The largest families clustered in three dominant groups: the most prominent (107 variants) shared the consensus motif DGTxxxxGW, a second group (68 variants) displayed the motif DGTxxxP(F/P)(K/R) reminiscent of the PHP.eB capsid and herein referred to as "PHP-like," and a third group (43 variants) displayed the motif DGTxxxLSS (Figure S2). Smaller groups displayed the motif (DG/AQ)xxxxYD(A/S) or AQxxxxxRW (8 and 9 variants, respectively). Of note, we also observed a cluster of 9 variants sharing the motif AQWxxxxGY, similar to the recently identified PHP.C2,38Ravindra Kumar S. Miles T.F. Chen X. Brown D. Dobreva T. Huang Q. Ding X. Luo Y. Einarsson P.H. Greenbaum A. et al.Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types.Nat. Methods. 2020; 17: 541-550Crossref PubMed Scopus (55) Google Scholar and also one single variant (AQFVVGQQY) closely related to the CNS-trophic AAV-F capsid (AQFVVGQSY).25Hanlon K.S. Meltzer J.C. Buzhdygan T. Cheng M.J. Sena-Esteves M. Bennett R.E. Sullivan T.P. Razmpour R. Gong Y. Ng C. et al.Selection of an Efficient AAV Vector for Robust CNS Transgene Expression.Mol. Ther. Methods Clin. Dev. 2019; 15: 320-332Abstract Full Text Full Text PDF PubMed Scopus (48) Google ScholarA synthetic library containing these 330 candidates plus AAV9, PHP.B, and PHP.eB, each encoded as two distinct codon versions (666 nucleotide variants total), was produced de novo using pooled primer synthesis as recently described38Ravindra Kumar S. Miles T.F. Chen X. Brown D. Dobreva T. Huang Q. Ding X. Luo Y. Einarsson P.H. Greenbaum A. et al.Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types.Nat. Methods. 2020; 17: 541-550Crossref PubMed Scopus (55) Google Scholar and cloned in SYN or GFAP vectors (Figure 2A). Each AAV pool was injected to C57BL/6 mice (n = 6), and NGS enrichment analysis was performed 28 days post-injection using RNA from the brain and spinal cord, as well as DNA from heart and liver tissues. Overall, 300 variants showed a brain transduction superior to AAV9, and 92 variants outperformed AAV9 by more than 10-fold (Figures 2C and 2D). By contrast, whereas AAV9 was among the lowest performers in the brain and spinal cord, it showed the highest score in the heart and liver, consistent with CNS cell-type-specific selection conferred by the TRACER platform used in this study (Figures 2C and 2D). The alternative codon versions of each variant showed highly correlated behavior in DNA, virus, and tissue samples, supporting the robustness of the assay (Figure S3). SYN- and GFAP-driven versions of each variant behaved very consistently at the DNA level but diverged at the RNA level (Figure S3), which was not unexpected, since the DNA enrichment is not subject to variations in promoter activity among cell types. Consistent enrichment scores were observed among animals injected with the SYN-driven pool, whereas the GFAP-driven library showed a higher inter-animal variability (Figures 2C and 2D). As expected, both PHP.eB- and PHP.B-positive controls showed a high CNS enrichment score in both screens (Figures 2C and 2D; Data S1). Taken together, the high inter-animal reproducibility, the strong codon variant correlation, and the precise calibration from internal controls allowed us to identify multiple capsids with improved CNS fitness with a very high degree of confidence. Strikingly, despite the absence of effort to select variants with low liver and heart tropism in our screen, the majority of CNS-trophic capsids showed substantial de-targeting from both tissues.Correlation of variant origin and genotype with in vivo and in vitro propertiesOur synthetic library of 330 brain-enriched variants contained 120 candidates originating from SYN-driven library evolution and 210 candidates from GFAP-driven evolution (Data S1). We asked if the capsid evolution path would favor variants with a bias toward neurons (SYN) or astrocytes (GFAP). When brain RNA enrichment data were stratified according to the library selection method, we observed a significant, albeit modest, transduction bias in favor of the cell type where the library screen was performed (Figure 3A). On average, capsids selected with the GFAP-TRACER library showed a 2-fold increased fitness for astrocyte versus neuron than capsids evolved with the SYN-TRACER library. This relatively modest cell-type specificity likely reflects the absence of negative selection in our screen, combined with the ability of most variants to transduce both neurons and astrocytes, at least to some extent.Figure 3Genotype-to-phenotype analysis of synthetic capsid pool from C57BL/6 CNS biopanningShow full caption(A) Comparative neuron and astrocyte fitness of the capsid variants originating from SYN- or GFAP-driven library biopanning (black and red circles, respectively). Each data point represents the average neuron (SYN-driven) and astrocyte (GFAP-driven) RNA enrichment score in i.v.-dosed C57BL/6 mice (n = 6), normalized to AAV9. Linear regression trendline of each population is indicated. p value indicates the statistical difference between the average GFAP-to-SYN score ratio of each subpopulation (unpaired t test). Frequency plots of peptides from SYN- and GFAP-evolved subpools are indicated on top. (B) Enrichment scores of each capsid sequence family in GFAP- (y axis) and SYN-driven RNA assays (x axis). The frequency plots and number of variants in each group are indicated. (C) Comparative brain RNA enrichment of 330 variants in C57BL/6 mice (n = 6) and BALB/c mice (n = 6) following i.v. injection. Color scale indicates the average RNA enrichment score normalized to AAV9. Variants are ranked by SYN-driven RNA enrichment score in C57BL/6 mice. (D) Comparative SYN-driven RNA enrichment score of distinct capsid families in C57BL/6 and BALB/c mice. The frequency plots and number of variants of each group are indicated. (E) Multiplexed binding assay of synthetic capsid pool to C57BL/6 mouse primary brain microvascular endothelial cells (BMVECs). Values indicate bound viral DNA enrichment score relative to AAV9. Ranking of reference PHP.eB, PHP.B, and AAV9 capsids is indicated. (F) Scatterplot presenting the correlation between virus binding to mouse BMVECs and C57BL/6 brain RNA enrichment scores. The PHP-like capsid variants are indicated by blue dots, all other variants by gray dots.View Large Image
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