A Highly Efficacious PS Gene Editing System Corrects Metabolic and Neurological Complications of Mucopolysaccharidosis Type I

Abstract

Our previous study delivered zinc finger nucleases to treat mice with mucopolysaccharidosis type I (MPS I), resulting in a phase I/II clinical trial (ClinicalTrials.gov: NCT02702115). However, in the clinical trial, the efficacy needs to be improved due to the low transgene expression level. To this end, we designed a proprietary system (PS) gene editing approach with CRISPR to insert a promoterless α-l-iduronidase (IDUA) cDNA sequence into the albumin locus of hepatocytes. In this study, adeno-associated virus 8 (AAV8) vectors delivering the PS gene editing system were injected into neonatal and adult MPS I mice. IDUA enzyme activity in the brain significantly increased, while storage levels were normalized. Neurobehavioral tests showed that treated mice had better memory and learning ability. Also, histological analysis showed efficacy reflected by the absence of foam cells in the liver and vacuolation in neuronal cells. No vector-associated toxicity or increased tumorigenesis risk was observed. Moreover, no off-target effects were detected through the unbiased genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) analysis. In summary, these results showed the safety and efficacy of the PS in treating MPS I and paved the way for clinical studies. Additionally, as a therapeutic platform, the PS has the potential to treat other lysosomal diseases.

Copyright © 2020 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Figures

Graphical abstract

Figure 1

Molecular Mechanism of the PS Gene Editing Approach First, Cas9 nuclease creates a double-strand break at the target locus. Through homology-directed repair, the splicing acceptor (SA) and human IDUA cDNA (IDUA), and the poly(A) (PA) sequence, are integrated. Through non-homologous end joining, the whole AAV sequence is integrated. Through the interaction between the splicing donor (SD) and SA, the fusion transcript is the same, which includes exon 1 of the albumin gene and the IDUA sequence. Exon 1 primarily encodes a signal peptide, which will be cleaved thereafter. The mature proteins are only the therapeutic IDUA proteins. ITR, inverted terminal repeat; HA, homology arm.

Figure 2

The Efficacy of PS822 in Neonatal MPS I Mice (A) Blood samples were collected from treated neonatal mice and controls monthly. Plasma IDUA enzyme activity increased significantly throughout 10 months. (B) Kaplan-Meier analysis showed the improved survival rate of treated neonatal mice. Three mice from the high-dose group were euthanized at 1 month post-dosing for early assessment and thus were not included in the Kaplan-Meier survival analysis. p 

Figure 3

Enzyme Activities and GAG Levels in Neonatal MPS I Mice after Treatment with PS822 (A and B) Enzyme activities (A) increased significantly and GAG storage levels (B) decreased significantly in tissues, including the brain of treated neonatal mice at 11 months post-dosing. Mean ± SEM. ∗p 

Figure 4

Histology and IHC Analysis of Neonatal MPS I Mice after Treatment with PS822 (A–C) Columns show tissues from normal mice (A), untreated MPS I mice (B), and treated MPS I mice (C). The upper panel shows H&E staining of liver. Untreated MPS I mice have several foam cells (the hallmark of MPS I) present but these are absent in the treated MPS I and normal mice. The middle panel shows H&E staining of cerebellum. The Purkinje neurons in untreated MPS I mice have cytoplasmic cellular vacuolation (arrows), which is absent in high-dose MPS I treated and normal mice. The lower panel shows IHC for IDUA. There is a subtle but slightly increased intensity of signal in the choroid plexus of treated MPS I mice, which is suggestive of the presence of enzyme in the brain. Also, occasional individual choroid plexus epithelial cells (arrows) are strongly immunopositive for IDUA.

Figure 5

Cutting Efficiency and Expression of Cas9, as well as Humoral Immune Response against IDUA Proteins (A) Indel percentage in the liver of treated neonatal mice (high dose) was analyzed through next-generation sequencing. Untreated MPS I mice were included as controls. (B) SaCas9 mRNA level in the liver was quantified through qRT-PCR. The liver from treated neonatal MPS I mice (high, middle, or low dose) at 11 months post-dosing is depicted; RNA was also extracted from three mice from the high-dose group at 1 month post-dosing. (C) ELISA was performed with plasma samples from treated neonatal MPS I mice (high, middle, or low dose) at 30, 90, and 270 days post-dosing. In addition, ELISA was also performed with plasma samples from treated adult MPS I mice at 7 and 30 days post-dosing. The dashed line indicates the antibody level in untreated control mice (n = 6). (D) Plasma albumin levels were measured by ELISA with samples from untreated MPS I mice, neonatal MPS I mice treated with a high dose and low dose at 1 and 10 months post-dosing, as well as adult MPS I mice treated with a middle dose at 1 month post-dosing. Mean ± SEM. ∗p 

Figure 6

The Cutting Efficiency and Specificity of PS822 in Human Hepatocytes (A) Plasmids encoding SpCas9 and three candidate gRNAs were transfected into human hepatocytes (Huh7 cell line), and the cleavage activity was measured through sequencing the target locus. Only gRNA3 showed significant cleavage, and it was selected as the candidate for further analyses. (B) The plasmid encoding SpCas9 and the donor plasmid encoding IDUA cDNA and gRNA3 were cotransfected into human hepatocytes (HepG2) cells. After 48 h, genomic DNA was extracted from collected cells, and nested PCR was performed. Cells without transfection and cells transfected with the donor plasmid only were used as controls. The gel image of the second round PCR is shown. Only cells transfected with both Cas9 and donor plasmids had the band at the expected size of 1,031 bp. (C) Cell pellets and supernatants were processed for IDUA enzyme assays. IDUA enzyme activities in cells transfected with Cas9 and donor plasmids had significantly higher IDUA enzyme activities in cell pellets and supernatants compared with untransfected cells and cells transfected with the donor plasmid only. (D) SpCas9 and gRNA3 ribonucleoprotein were cotransfected with double-strand oligonucleotide tag (dsTag) into Huh7 cells. Genomic DNA was extracted and used for library preparation. Deep sequencing was performed to search for the dsTag. (E) Only on-target cleavage was identified through GUIDE-seq.