Abstract
RNA interference (RNAi) for spinocerebellar ataxia type 1 can prevent and reverse behavioral deficits and neuropathological readouts in mouse models, with safety and benefit lasting over many months. The RNAi trigger, expressed from adeno-associated virus vectors (AAV.miS1), also corrected misregulated microRNAs (miRNA) such as miR150. Subsequently, we showed that the delivery method was scalable, and that AAV.miS1 was safe in short-term pilot nonhuman primate (NHP) studies. To advance the technology to patients, investigational new drug (IND)-enabling studies in NHPs were initiated. After AAV.miS1 delivery to deep cerebellar nuclei, we unexpectedly observed cerebellar toxicity. Both small-RNA-seq and studies using AAVs devoid of miRNAs showed that this was not a result of saturation of the endogenous miRNA processing machinery. RNA-seq together with sequencing of the AAV product showed that, despite limited amounts of cross-packaged material, there was substantial inverted terminal repeat (ITR) promoter activity that correlated with neuropathologies. ITR promoter activity was reduced by altering the miS1 expression context. The surprising contrast between our rodent and NHP findings highlight the need for extended safety studies in multiple species when assessing new therapeutics for human application.
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Data availability
Sequencing datasets generated as a part of this manuscript can be accessed using NCBI Gene Expression Omnibus (GEO) at accession number: GSE182666. The following individual files in GSE182666are associated with the indicated figure: Fig. 3, GSM5534318, GSM5534319, GSM5534320, GSM5534321, GSM5534322, GSM5534323; Fig. 4, GSM5534296, GSM5534297, GSM5534298, GSM5534299, GSM5534300, GSM5534301, GSM5534302, GSM5534303, GSM5534304, GSM5534305; Fig. 5, GSM5534306, GSM5534307, GSM5534308, GSM5534309, GSM5534310, GSM5534311, GSM5534312, GSM5534313, GSM5534314, GSM5534315, GSM5534316, GSM5534317; Extended Data Fig. 5, GSM5534324, GSM5534325, GSM5534326, GSM5534327, GSM5534328, GSM5534329. Raw data are available as Supplementary Data for graphs shown in Figs. 1c,f, 2i–n and 5c and Extended Data Figs. 2c, 3b–d, 4b–e,g and 6b,d–f. The original uncropped gel is given in the Supplementary Data for Extended Data Fig. 3a. The following public datasets used: Ensemble, Rhesus macaque genome (Mmul10), http://ftp.ensembl.org/pub/release-104/fasta/macaca_mulatta/dna/Macaca_mulatta.Mmul_10.dna.toplevel.fa.gz for Figs. 3–5 and Extended Data Fig. 5; Ensembl, (GRCh38), http://ftp.ensembl.org/pub/release-104/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.toplevel.fa.gz for Fig. 4 and Extended Data Figs. 4 and 5. All vectors presented in this work are available on request with approval from the CHOP Office of Technology Transfer. Source data are provided with this paper.
Code availability
The software tools generated as a part of this study are archived at https://github.com/DavidsonLabCHOP/Keiser_NatMed_2021.
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Acknowledgements
This work was funded by the NIH NS094355 (M.K., P.G.-A., T.J.L., B.L.D.), NIH T32 HG009495 (P.T.R.) and the Children’s Hospital of Philadelphia Research Institute.
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Contributions
M.S.K. designed the study, performed experiments, evaluated the data and wrote and edited the paper. P.T.R. designed, performed and analyzed all small RNA and RNA sequencing and wrote and edited the paper. C.M.Y. designed, performed and analyzed the nanopore sequencing and edited the paper. E.M.C. designed constructs, performed rodent work and edited the paper. G.R.S. and A.L.M. assisted with all NHP and rodent work. J.M.S. read and assessed surgical MRIs. Y.H.C. performed antibody analyses and prepared test article to keep study staff blinded. R.L.W. read and assessed the in-life imaging studies. E.R. graded histopathology and microscopic image acquisition. T.J.L. developed the neurosurgical approaches. P.G.-A. designed the study, evaluated data and edited the manuscript. B.L.D. designed the study, evaluated the data and wrote and edited the manuscript.
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Competing interests
B.L.D. is a founder of Spark Therapeutics and Spirovant Sciences. She serves an advisory role and/or receives sponsored research support for her laboratory from Roche, NBIR, Homology Medicines, Triplet Therapeutics, Resilience, Intellia Therapeutics, Spirovant Sciences, Panorama Medicines and Voyager Therapeutics. P.G.-A. has consulted for Eisai Therapeutics, Spark Therapeutics and NeuExcell Therapeutics. The remaining authors declare no competing interests.
Additional information
Peer review information Nature Medicine thanks H.T. Orr, L. Naldini and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Jerome Staal was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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Extended data
Extended Data Fig. 1 Immunohistochemistry and lesion scores in AAV.miS1 treated animals.
Representative images from vehicle- (a) and AAV.miS1-injected (b) sagittal cerebellar sections (10 µm thick) immunostained for glial fibrillary acidic protein (GFAP). N = 4 or 6 animals per group. Granule cell layer (GCL) and molecular layers (ML) are identified. Scale bar = 100 µm. c, Tissue lesion scoring parameters and associated heat chart (See Extended Data Table 2). d, Quantitation and statistics of lesion scores. Each dot represents a single animal. Data are represented as mean ± SEM (N = 2, 3, 4 or 7 animals per group), significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison post hoc for each location (** P(3E12vg) = 0.0052; **P(1E13vg) = 0.0079 relative to vehicle). e-f, Representative images (N = 10 independent 20X objective fields) from cerebellar sections immunostained for Calbindin in vehicle- (e; N = 4 animals) and AAV.miS1-injected (f; N = 6 animals) used to quantify Purkinje cell counts throughout the cerebellum. Scale bar = 300 µm.
Extended Data Fig. 2 Non-coding stuffer sequence does not exhibit promoter activity in HEK293 cells.
a, Cartoon map of AAV.miS1 proviral plasmid. Bracketed regions below indicate locations of unexpected RNA-seq reads. Bracketed regions above represent the full or partial stuffer regions tested for activity. b, Construct series designed to test promoter activity of sequences near the 3’ ITR. Proviral plasmid segments were cloned upstream Renilla luciferase coding sequence. c, Renilla luciferase activity in HEK293 cells at 24 hrs post-transfection. Activity was normalized to Firefly luciferase activity encoded within the same plasmid.
Extended Data Fig. 3 Probe based ddPCR assays.
a, Assays were designed to quantify cross-packaged sequence upstream of the left ITR (5’ backbone (BB)), downstream of the right ITR (3’ BB), or within the cargo (stuffer sequence) of AAV.miS1, to reflect transcripts identified by RNA-seq (See Fig. 4). b, Copies of vector cargo (stuffer sequence) and cross-packaged sequence measured from punches taken from the intermediate lobule of NHPs. c, Cargo, 5‘- and 3’- transcript levels isolated from the superficial medial punches of animals injected with high dose AAV.miS1. d, Mock RT cDNA for the cargo sequence (stuffer for AAV.miS1 and EF1α for AAV.IntmiS1) was quantified in parallel to confirm that expression was not arising from contaminating vector DNA. All data are represented as mean ± SEM (N = 3 animals per group).
Extended Data Fig. 4 Testing of AAV.IntmiS1.
AAV1.IntmiS1 was injected bilaterally into B05 SCA1 mice cerebella (N = 4 or 6) and tissues collected 3 weeks later. a, Representative agarose gels of cDNAs following PCR using primers flanking the miRNA-containing intron (N = 3 biological replicates). b-d, mRNA levels of miS1 (***P = 0.0003; ****P < 0.0001), (b) hATXN1L (**P = 0.001; ****P < 0.0001), (c) and hATXN1 (**P = 0.0060; ****P, 0.0001), (d) in mice cerebella following AAV.IntmiS1 injection at the indicated doses. All data are represented as mean ± SD (N = 4 and 6 animals per treatment group), with significance determined by one-way ANOVA followed by Dunnett’s multiple comparisons post hoc. e) Relative expression of miS1 as quantified by stem-loop qPCR in WT mice injected bilaterally with AAV.miS1 or AAV.IntmiS1 into the striatum. Data are represented as mean ± SD, (N = 3 animals per group). f) Cartoons of the packaged AAV genome and plasmid sequences for assessing transcript levels by ddPCR. Green arrowheads depict the probe locations for assessing transcripts arising from the 5’ ITR (5’ backbone (BB)), the 3’ ITR (3’ BB), or the EF1α promoter of AAV.IntmiS1. g, ddPCR quantification of RNA isolated from striatum of mice injected with AAV.miS1 or AAV.IntmiS1. Data are represented as mean ± SEM (N = 3 animals per group).
Extended Data Fig. 5 AAV.IntmiS1 does not cause small RNA dysregulation.
a, Differential expression analysis on small RNASeq data obtained from medial deep cerebellar tissue punches from AAV.IntmiS1 and empty capsid treated NHPs. b,c, Heatmaps of the top 30 most highly detected (b) or 11 specific neuronal miRNAs (c). * denotes 28 day in-life animal.
Extended Data Fig. 6 Study 2 histological and molecular readouts.
a, Quantitation of lesion scores from cerebellar sections of NHPs injected with the indicated AAVs (Empty, miSCA7, Stuffer, IntmiS1). Each dot represents a single animal. b, Human ATXN1L levels normalized to endogenous GAPDH as assessed by RT-qPCR. c, Total read counts of human ATXN1L by RNA-seq. d, miS1 levels in AAV.IntmiS1 treated animals, normalized to endogenous U6 RNA and relative to empty capsid treated animals. e, Ionized calcium binding adapter molecule 1 (IBA1) mRNA levels. f, Glial fibrillary acidic protein (GFAP) mRNA levels. Data are represented as mean ± SEM (N = 3 animals per group). There was no significant difference as measured by two-way ANOVA followed by a Dunnett’s multiple comparisons post hoc.
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Keiser, M.S., Ranum, P.T., Yrigollen, C.M. et al. Toxicity after AAV delivery of RNAi expression constructs into nonhuman primate brain. Nat Med 27, 1982–1989 (2021). https://doi.org/10.1038/s41591-021-01522-3
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DOI: https://doi.org/10.1038/s41591-021-01522-3
