Development of a CRISPR/Cas9-based therapy for Hutchinson-Gilford progeria syndrome

Abstract

CRISPR/Cas9-based therapies hold considerable promise for the treatment of genetic diseases. Among these, Hutchinson-Gilford progeria syndrome, caused by a point mutation in the LMNA gene, stands out as a potential candidate. Here, we explore the efficacy of a CRISPR/Cas9-based approach that reverts several alterations in Hutchinson-Gilford progeria syndrome cells and mice by introducing frameshift mutations in the LMNA gene.

Extended Data 1. Representative capillary electrophoresis-based fragment analysis of sgRNA-control- and sgRNA-LCS1-transduced Lmna^G609G/G609G mouse embryonic fibroblasts (n = 3 independent infections and MEF lines).

Red line and orange peaks correspond to size standards.

Extended Data 2. Representative capillary electrophoresis-based fragment analysis of sgRNA-control- and sgRNA-LCS1-transduced LMNA^G608G/+ human fibroblasts (n = 3 independent infections).

Red line and orange peaks correspond to size standards.

Extended Data 3. Percentage of indels in Lmna^G609G/G609G sgRNA-LC2-transduced male and female mouse tissues.

Data are mean ± s.e.m. (n = 5 tissues per group, except in sgRNA-LCS2-transduced female liver where n = 4; two-tailed Student’s t-test).

Extended Data 4. RT–qPCR analysis of progerin and lamin C in tissues from Lmna^G609G/G609G sgRNA-control-transduced and Lmna^G609G/G609G sgRNA-LCS2-transduced mice.

Data are mean ± s.e.m. (n = 4 tissues per group, except sgRNA-control-transduced liver and heart where n = 5; two-tailed Student’s t-test).

Extended Data 5

Progerin immunohistochemistry in lung, kidney and aorta from WT, Lmna^G609G/G609G sgRNA-control-transduced and Lmna^G609G/G609G sgRNA-LCS2-transduced mice (lung and kidney, n = 5 for WT and sgRNA-control-transduced mice and n = 4 for sgRNA-LCS2-transduced mice; aorta, n = 2 for WT and n = 3 for sgRNA-control- and sgRNA-LCS2-transduced Lmna^G609G/G609G mice). Scale bar, 100 μm.

Extended Data 6

Kaplan–Meier survival plot of Lmna^G609G/G609G male and female mice transduced with sgRNA-control (n = 6 males; n = 4 females) or sgRNA-LCS2 (n = 4 males; n = 6 females).

Extended Data 7. Progression of body weight of male and female mice transduced with sgRNA-control or sgRNA-LCS2, expressed as percentage of weight at 9 weeks.

Mean values ± s.e.m. are shown (for males, initial n = 9 sgRNA-control-transduced mice and n = 8 sgRNA-LCS2-transduced mice; for females, initial n = 6 mice per group; two-tailed Student’s t-test). Vertical arrow indicates the time point (3.5 months) at which the cohort destined for histological studies was sacrificed.

Extended Data 8

Images of three sex- and age-matched mice transduced with the sgRNA-LCS2 compared to sgRNA-control-transduced animals.

Extended Data 9. H&E staining of gastric mucosa from WT, Lmna^G609G/G609G sgRNA-controltransduced and Lmna^G609G/G609G sgRNA-LCS2-transduced mice.

The graph shows atrophy quantification according to a pathological score as described in Methods. Data are mean ± s.e.m. (n = 5 for WT and sgRNA-control-transduced mice; n = 3 for sgRNA-LCS2-transduced mice).

Figure 1. CRISPR/Cas9 testing in HGPS cellular models.

(a) sgRNA-LCS1 directs Cas9 nuclease against exon 11 of LMNA gene upstream of the HGPS mutation, disrupting lamin A and progerin without altering lamin C. (b) Cropped Western blot of lamin A, progerin and lamin C from wild-type and Lmna^G609G/G609G mouse embryonic fibroblasts (MEFs) transduced with sgRNA-control or sgRNA-LCS1 (n=3 independent infections and MEF lines; two-tailed Student’s t-test). (c) Immunofluorescence analysis of progerin-positive nuclei and quantification of nuclear alterations by 4′,6-diamidino-2-phenylindole (DAPI) staining (n=3 independent infections and MEF lines; two-tailed Student’s t-test). Arrowheads indicate nuclear aberrations. (d) Cropped Western blot of lamin A, progerin and lamin C from wild-type and LMNA^G608G/+ human fibroblasts transduced with sgRNA-control or sgRNA-LCS1 (n=3 independent infections; two-tailed Student’s t-test). (e) Progerin immunofluorescence and analysis of nuclear aberrations by DAPI staining (n=3 independent infections; two-tailed Student’s t-test). Arrowheads indicate blebbings and invaginations. Bar plots represent mean ± SD and individual values are overlaid. Scale bars, 40 μm. Uncropped blots are available as Source Data.

Figure 2. CRISPR/Cas9 delivery and phenotype amelioration in Lmna^G609G/G609G mice.

(a) Intraperitoneal injection of AAV9 in P3 mice. (b) Percentage of in-frame and frameshift mutations at the Lmna target region in liver, heart, muscle and lung. Data are mean ± SEM (n=10 tissues per group, except n=9 sgRNA-LCS2-transduced liver; two-tailed Student’s t-test for total indels). (c) Alignment of the most common indels in sgRNA-LCS2-transduced mice. Blue, target sequence; red, PAM sequence. (d) Progerin immunohistochemistry of liver, heart and muscle from wild-type and Lmna^G609G/G609G sgRNA-control-transduced or sgRNA-LCS2-transduced mice. Data are mean ± SD (n=5 wild-type and sgRNA-control-transduced mice; n=4 sgRNA-LCS2-transduced mice; two-tailed Student’s t-test). Insets, digital magnification of a selected area. (e) Kaplan-Meier survival plot of sgRNA-control- versus sgRNA-LCS2-transduced Lmna^G609G/G609G mice (n=10 mice per group; two-sided Log-rank test). (f) Progression of body weight of mice transduced with sgRNA-control or sgRNA-LCS2, expressed as percentage of weight at 9 weeks. Vertical arrow, time point (3.5 months) at which the cohort destined for histological studies (4-5 mice per group) was sacrificed. Mean values ± SEM are represented (initial n=15 sgRNA-control-transduced mice; n=14 sgRNA-LCS2-transduced mice; two-tailed Student’s t-test). (g) Representative image of Lmna^G609G/G609G sgRNA-control-transduced, sgRNA-LCS2-transduced and wild-type female mice at 3.5 months of age. (h) Glycemia in wild-type (males n=5; females n=5), sgRNA-control-transduced (males n=6; females n=4) and sgRNA-LCS2-transduced Lmna^G609G/G609G mice (males, n=5; females, n=5). Data are represented by box plots and whiskers are minimum to maximum values (two-tailed Student’s t-test). (i) TUNEL assay in kidney of 3.5-month-old mice. Data are mean ± SD (n=5 wild-type and sgRNA-control-transduced mice; n=4 sgRNA-LCS2-transduced mice; two-tailed Student’s t-test). (j) Gomori staining in 3.5-month-old mouse tissues showing moderate perivascular and interstitial fibrosis in heart and quadriceps muscle of Lmna^G609G/G609G mice (blue areas). Data are mean ± SD (n=5 wild-type and sgRNA-control-transduced mice; n=4 sgRNA-LCS2-transduced mice; two-tailed Student’s t-test). Scale bars, 100 μm (d, i, j).