CRISPR-Cas9-Mediated In Vivo Gene Integration at the Albumin Locus Recovers Hemostasis in Neonatal and Adult Hemophilia B Mice

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 loaded by vectors could induce high rates of specific site genome editing and correct disease-causing mutations. However, most monogenic genetic diseases such as hemophilia are caused by different mutations dispersed in one gene, instead of an accordant mutation. Vectors developed for correcting specific mutations may not be suited to different mutations at other positions. Site-specific gene addition provides an ideal solution for long-term, stable gene therapy. We have demonstrated SaCas9-mediated homology-directed factor IX (FIX) in situ targeting for sustained treatment of hemophilia B. In this study, we tested a more efficient dual adeno-associated virus (AAV) strategy with lower vector dose for liver-directed genome editing that enables CRISPR-Cas9-mediated site-specific integration of therapeutic transgene within the albumin gene, and we aimed to develop a more universal gene-targeting approach. We successfully achieved coagulation function in newborn and adult hemophilia B mice by a single injection of dual AAV vectors. FIX levels in treated mice persisted even after a two-thirds partial hepatectomy, indicating stable gene integration. Our results suggest that this CRISPR-Cas9-mediated site-specific gene integration in hepatocytes could transform into a common clinical therapeutic method for hemophilia B and other genetic diseases.

Keywords: AAV; CRISPR-Cas9; albumin; hemophilia B; targeted integration.

Graphical abstract

Figure 1

In Vivo Genome Editing of Albumin Locus in Mouse Liver by AAV.CRISPR-Cas9 (A) Schematic illustrating albumin targeting strategy. (B) In vitro validation of sgRNAs targeted to mAlb1 in the H2.35 mouse cell line by transient transfection and Surveyor nuclease assays. sgRNA4 showed the highest efficiency in inducing indels in the targeted loci and was therefore chosen for subsequent studies. Arrows denote Surveyor nuclease cleaved fragments of the Alb PCR products. Results were replicated in two independent experiments. (C) Dual AAV vector system for liver-directed and SpCas9-mediated gene insertion. The AAV8.sgRNA.donor vector encoded a promoterless hFIX cassette containing exons 2–8 of the hFIX gene flanked by a SA signal and a poly(A) (PA) sequence.

Figure 2

Efficient Restoration of hFIX Expression in Hemophilia B Mice Treated by Dual AAV8 Strategy (A) Plasma hFIX was measured by ELISA following tail vein injections of 8-week-old male hemophilia B mice with the AAV8.SpCas9 (9 × 10¹⁰ GC/mouse) and AAV8.sgRNA.donor (4.5 × 10¹¹ GC/mouse) (n = 5). Untargeted hemophilia B mice received AAV8.SpCas9 (9 × 10¹⁰ GC/mouse) and AAV8.control.donor (4.5 × 10¹¹ GC/mouse) (n = 5). Untreated hemophilia B mice (n = 5) were included as control. (B) hFIX activity in adult-treated hemophilia B mouse plasma. (C) Plasma hFIX was measured by ELISA following temporal vein injections of newborn male hemophilia B mice with the AAV8.SpCas9 (3 × 10¹⁰ GC/mouse) and AAV8.sgRNA.donor (1.5 × 10¹¹ GC/mouse) (n = 5). All targeted mice were subjected to two-thirds partial hepatectomy (PH) 14 weeks after vector treatment. Untargeted hemophilia B mice received AAV8.SpCas9 (3 × 10¹⁰ GC/mouse) and AAV8.control.donor (1.5 × 10¹¹ GC/mouse) (n = 6). Three untargeted mice were sacrificed at week 14 for analyses. Untreated hemophilia B mice (n = 3) were included as control. (D) hFIX activity in hemophilia B mouse plasma after neonatal vector treatment. (E) Immunofluorescence staining with antibodies against hFIX (red) with 4′,6-diamidino-2-phenylindole (DAPI) nuclear counterstain (blue) on liver sections, which were treated as adults collected at 16 weeks after injection (left). Scale bars, 100 μm. Quantification of gene expression was based on the percentage of area on liver sections expressing hFIX by immunostaining (right). (F) Immunofluorescence staining with antibodies against hFIX with DAPI nuclear counterstain on liver sections, which were treated as newborns collected at 14 and 48 weeks after injection (left). Scale bars, 100 μm. Quantification of gene expression was based on the percentage of area on liver sections expressing hFIX by immunostaining (right). Error bars represent mean ± SEM. Dunnett’s test. n.s., not significant.

Figure 3

Indel and HDR-Mediated Gene Integration Efficiency and Transgene Expression Analyses Liver mRNA and DNA were isolated from hemophilia B mice at 14, 16, and 48 weeks after treatment with dual gene integration vectors or untargeted vectors. DNA and mRNA from untreated hemophilia B mice served as control. Indel analysis on the mAlb locus was analyzed by NGS on liver DNA. HDR-mediated gene integration was analyzed by LM-PCR following digestion with XbaI on liver DNA. The pooled PCR amplicons were performed by NGS. Chimeric m-hFIX mRNA levels in liver were measured by qPCR using primers spanning the junction of mAlb and hFIX cDNA. (A) Indel frequency in mice treated as adults. (B) Indel frequency in mice treated as neonates. (C) HDR frequency in mice treated as adults. (D) HDR frequency in mice treated as neonates. (E) Quantification of chimeric m-hFIX mRNA levels in the liver treated as adults and neonates. Symbols represent individual mice. Means ± SEM are shown. Dunnett’s test. n.s., not significant.

Figure 4

In Vivo Off-target Validation on Liver DNA Samples (A) NGS data of off-target sites for liver DNA samples isolated from adult mice 16 weeks after vector treatment. Cumulative indel mutations within the protospacer region were plotted for each off-target locus in the targeted group (n = 5) compared to untreated mice (n = 3). (B) NGS data of off-target sites for liver DNA samples isolated from newborn mice 14 weeks after vector treatment. Cumulative indel mutations within the protospacer region were plotted for each off-target locus in the targeted group (n = 5) compared to untreated mice (n = 3). Means ± SEM are shown.

Figure 5

Liver Function Tests and Toxicity Examination in Adult and Newborn Animals Treated with Dual AAV8 Vectors (A) Histological analysis by hematoxylin and eosin stain on adult livers harvested 16 weeks following dual vector treatment. Scale bar, 100 μm. (B and C) Liver transaminase (B, ALT; C, AST) levels in untreated hemophilia B mice (n = 3) or 16 weeks following targeted vector (n = 5) and untargeted vector (n = 5) treatment. (D) Quantification of SpCas9 mRNA levels in liver isolated from adult mice 16 weeks after vector treatment by qPCR. (E) Quantification of SpCas9 vector genome in liver from adult treatment group by qPCR. (F) Western blot analysis. Liver lysates were prepared from adult untreated or hemophilia B mice treated with the dual AAV vectors for detection of Alb protein. (G) Quantification of Alb mRNA in the liver by qPCR. (H) Histological analysis by hematoxylin and eosin stain on newborn livers harvested 14 weeks following dual vector treatment. Scale bar, 100 μm. (I and J) ALT and AST levels in untreated hemophilia B mice (n = 5) or newbron mice 14 weeks after targeted vector (n = 5) and untargeted vector (n = 5) treatment. (K) Quantification of SpCas9 mRNA levels in liver isolated from newborn mice 14 and 48 weeks after vector treatment by qPCR. (L) Quantification of SpCas9 vector genome in liver from newborn treatment group by qPCR. (M) Western blot analysis. Liver lysates were prepared from untreated or newborn hemophilia B mice treated with the dual AAV vectors for detection of Alb protein. (N) Quantification of Alb mRNA in the liver by qPCR in newborn treatment group. Means ± SEM are shown. Dunnett’s test. ∗p < 0.05, ∗∗p < 0.01. n.s., not significant.