Targeted gene correction of laminopathy-associated LMNA mutations in patient-specific iPSCs

Abstract

Combination of stem cell-based approaches with gene-editing technologies represents an attractive strategy for studying human disease and developing therapies. However, gene-editing methodologies described to date for human cells suffer from technical limitations including limited target gene size, low targeting efficiency at transcriptionally inactive loci, and off-target genetic effects that could hamper broad clinical application. To address these limitations, and as a proof of principle, we focused on homologous recombination-based gene correction of multiple mutations on lamin A (LMNA), which are associated with various degenerative diseases. We show that helper-dependent adenoviral vectors (HDAdVs) provide a highly efficient and safe method for correcting mutations in large genomic regions in human induced pluripotent stem cells and can also be effective in adult human mesenchymal stem cells. This type of approach could be used to generate genotype-matched cell lines for disease modeling and drug discovery and potentially also in therapeutics.

Figure 1. Correction of HGPS-Associated LMNA Mutation in iPSCs with LMNA-c-HDAdV

(A) Schematic molecular representation of LMNA gene correction with LMNA-c-HDAdV. The primers for PCR are shown as arrows (P1, P2, P3, and P4). The probes for Southern analyses are shown as black bars (5′ probe and 3′ probe). HSVtk stands for herpes simplex virus thymidine kinase gene cassette used for negative selection; neo stands for neomycin-resistant gene cassette used for positive selection; CMV-β-gal indicates the β-gal expression cassette for determination of HDAdV titer; blue triangle, FRT site; red X, mutation sites in exon 11. (B) Schematic representation of the gene-correction approach employed in iPSCs with laminopathy-associated mutation(s). (C) Gene-targeting and gene-correction efficiencies at the LMNA locus in HGPS-iPSCs achieved by different infection conditions. N.D., not determined. (D) PCR analyses of HGPS-iPSCs and cHGPS-iPSCs via 5′ primer pair (P1 and P2; 13.9 kb) or 3′ primer pair (P3 and P4; 9.4 kb). M, DNA ladder. (E) Southern blot analyses of HGPS-iPSCs and cHGPS-iPSCs. The approximate molecular weights (kb) corresponding to the bands are indicated. (F) Sequencing results of C1824T mutation site in exon 11 of LMNA in BJ-iPSCs (wild-type), HGPS-iPSCs, and cHGPS-iPSCs. All iPSCs employed represent high-passage iPSCs (>30). See also Figure S1.

Figure 2. Abrogation of HGPS-Associated Phenotypes by Correction of LMNA in iPSCs

(A) Replicate error analysis indicating that cHGPS-iPSCs and HGPS fibroblasts were essentially genetically identical, whereas HGPS fibroblasts, H9 hESCs, and BJ-iPSCs came from unrelated individuals. (B) CNV calling via CNV Partition, identifying one region of duplication (CNV value = 3) at chr7:141322-162448, which was present in both the original HGPS fibroblasts and cHGPS-iPSCs. (C) Hierarchical clustering on RMA-normalized probe sets intensity values for HGPS- and AWS-iPSCs before and after gene correction, and their original fibroblast cell lines. (D) Hierarchical clustering on genome-wide DNA methylation profiles of the indicated cell lines. (E) Immunoblotting analysis of lamin A/C and progerin expression in the indicated cell lines with lamin A/C antibody. Tubulin was used as loading control. d-SMC, iPSC-derived SMC; d-fib, iPSC-derived fibroblast. (F) RT-PCR analysis of expression of full-length lamin A (top band) and truncated lamin A (progerin; bottom band) in hESC/iPSC-derived SMCs (passage 3) with the indicated primer pair. GAPDH was used as loading control. (G) Quantitative RT-PCR analysis of progerin expression in BJ-iPSC-, HGPS-iPSC-, or cHGPS-iPSC-derived SMCs at passage 3. **p < 0.01. (H) Senescence-associated (SA)-β-gal staining of SMCs derived from HGPS-iPSC or cHGPS-iPSC at passage 5. Correction of HGPS-iPSCs resulted in more than a 68% reduction on the derived SMCs undergoing senescence; *p < 0.05, **p < 0.01. (I) Immunostaining of lamin A in iPSC-derived fibroblasts at passage 15. Arrows denote dysmorphic nuclei. Dysmorphic nuclei were quantified and represented in the lower panels. **p < 0.01. Data are shown as mean ± SD; n = 3. Scale bar represents 20 μm. All iPSCs employed represent high-passage iPSCs (>30). See also Figure S1, Table S1, and Movie S1.

Figure 3. Editing the LMNA Locus in iPSCs and MSCs via LMNA-c-HDAdV

(A) Immunostaining of different pluripotency markers in AWS-iPSCs. Nuclei were stained with Hoechst 33342. Scale bars represent 20 μm. (B) H&E staining (left) and immunofluorescence (right) of three germ layer-specific markers (endoderm: AFP, FOXA2; mesoderm: ASMA, ASA; ectoderm: Tuj1, GFAP) in teratomas derived from AWS-iPSCs. Nuclei were stained with DAPI. (C) Gene-targeting and gene-correction efficiencies at the LMNA locus in AWS-iPSCs achieved by different infection conditions. N.D., not determined. (D) PCR analyses of AWS-iPSCs and gene-corrected AWS-iPSCs (cAWS-iPSCs) via 5′ primer pair (P1 and P2; 13.9 kb) or 3′ primer pair (P3 and P4; 9.4 kb). M, DNA ladder. (E) Southern blot analyses of AWS-iPSCs and cAWS-iPSCs. The approximate molecular weights (kb) corresponding to the bands are indicated. (F) Sequencing results of A1733T mutation site in exon 11 in BJ-iPSCs (wild-type), AWS-iPSCs, and cAWS-iPSCs. (G) Schematic molecular representation of mutation- and SNP-correction with LMNA-c-HDAdV in AWS-iPSCs. The mutation site (A1733T) and SNP sites 1 and 2 are located 0.3, 3.3, and 4.4 kb downstream of the neo-insertion site, respectively, on the same chromosome as the AWS mutation. (H) Sequencing results of SNP sites 1 and 2 downstream of exon 12 in cAWS-iPSCs with and without SNP correction. (I) Schematic illustration of the putative gene editing capacity of LMNA-c-HDAdV at LMNA locus. (J) Gene-targeting efficiency in OE-MSCs with LMNA-c-HDAdV achieved by different infection conditions. All iPSCs employed represent high-passage iPSCs (>30). See also Figure S1.