Single-dose CRISPR-Cas9 therapy extends lifespan of mice with Hutchinson-Gilford progeria syndrome

Abstract

Hutchinson-Gilford progeria syndrome (HGPS) is a rare lethal genetic disorder characterized by symptoms reminiscent of accelerated aging. The major underlying genetic cause is a substitution mutation in the gene coding for lamin A, causing the production of a toxic isoform called progerin. Here we show that reduction of lamin A/progerin by a single-dose systemic administration of adeno-associated virus-delivered CRISPR-Cas9 components suppresses HGPS in a mouse model.

Extended Data Fig. 1|. Immunoblots and RNA sequencing of Lmna gRNA-treated fibroblasts of adult HGPS mice.

(a) Immunoblots of individual (g1 or g2) or multiplex (g1&2) gRNA-treated fibroblasts derived from adult HGPS (Pro); Cas9 mice. Fibroblasts derived from heterozygous (Pro/+) and homozygous (Pro/Pro) HGPS mice on heterozygous Cas9 (Cas9/+) background were treated with lentivirally-delivered gRNAs. No Cas9 (Pro/+; +/+) sample and mock-treatments were used as negative controls. Middle panels are longer exposures. aTubulin and pActin were used as loading controls, (b) Relative intensities of Lamin A, Progerin and Lamin C bands on the immunoblots. The intensity of each band was normalized by the pActin intensity and compared with the normalized mock control, (c) Custom tracks of the RNA sequences identified with high- throughput sequencing. Magnified region corresponds to the squared area in blue. The red line denotes the truncated region in HGPS (Pro). Shown are the RNA samples derived from mouse fibroblasts of the indicated genotypes and treatments: + denotes wild type. Mock treatments and heterozygous HGPS mice without Cas9 (Pro/+; +/+) were used as negative controls. The arrows are not drawn to scale, (d) Quantification of the RNA sequencing analyses shows reduced transcription of the exons 11 and 12 in the Cas9-positive cells treated with the gRNAs relative to the mock-treated or Cas9-negative cells. Figures S1a–d are representatives of two independent replicates.

Extended Data Fig. 2|. Fluorescent images of Lmna gRNA injected mice.

(a) Whole body fluorescent images of a P4 (4dpp) mouse injected at PO with a 1:1 mix of gRNA1 and gRNA2 viral preps (each 1.29 × 1011 gc per viral prep), (b) Same organs as in Fig. 1c under individually adjusted exposure levels relative to their negative controls. The organs were harvested from a 6dpp mouse, 5 days post injection (DPI) at P1 (P1–5DPI). The inset corresponds to the squared area. Ctrl: No injection. Scale bar, 1.5 mm, except for Int 2mm. (c) Expression of the mCherry reporter in the adult (5.5 months post injection of a 1:1 mix of gRNA 1 and 2 viral preps) (1.3 X 1011 gc per viral prep). Figures S2a–c are representative replicates of at least two independent experiments. The numbers on the upper right corner denote the exposure time. Scale bar, 2.5 mm. Stom, Pane, Int, Sp, Kid, Br, Liv, Mscl: stomach, pancreas, intestine, spleen, kidney, brain, liver, muscle.

Extended Data Fig. 3|. Genomic DNA and protein analyses of Lmna gRNA treated mice.

(a) Relative intensities of Lamin A, Progerin and Lamin C bands on the immunoblot in Figure 1e. The intensity of each band was normalized by the aTubulin intensity and compared with the normalized mock control, (b) Immunoblot of adult liver lysate from g1&2-treated (5 X 10¹¹ gc per viral prep) heterozygous HGPS (Pro/+) on hemizygous or homozygous Cas9 background shows single copy of Cas9 is sufficient. Figures S3a and S3b were representative replicate of two independent experiments with similar results. Figures S3a and S3b are representative replicates of two independent experiments with similar results.(c-e) Deep sequencing was performed on liver and heart DNA to measure the level of in vivo indel formation by each gRNA. (c) Scheme of the deep sequencing strategy. gLmna-1 and gLmna-2 target sites are shown by red arrows. ¥ denotes the HGPS mutation. PCR amplicons of deep sequencing are indicated by black curved arrows. The locations of the primers (Lmna F and Lmna R) used for the first round of the nested PCR are shown by green arrows. Liver (d) and heart (e) DNA samples were collected from 2.5 months old Cas9 mice that had been injected at PO with either of the gRNA viruses (gLmna-1 or gLmna-2) (1.5 × 10¹¹ gc viral prep), or with no virus. The gRNA treatments caused indels in their corresponding target sites and not in the target site of the other gRNA. No indels were detected in the “no injection” controls. Each treatment was evaluated in two independent in vivo experiments.

Extended Data Fig. 4|. Physiological analyses of Lmna gRNA treated mice.

(a) Histological analysis of the aortic arch of untreated and gRNA-treated (g1&2) HGPS; Cas9 mice that are 19 weeks old injected at PO with a 1:1 mix of gRNA1 and gRNA2 viral preps. The black bracket shows the medial layer of the aortic arch. Scale bar: 100 pm. The bar graph on the right shows the number of nuclei in the medial layer (n = 21 for each group), (b) Electrocardiographic (ECG) analysis of untreated and gRNA-treated (g1&2) HGPS; Cas9 mice (Untreated: n = 8; g1&2: n = 9). Heart rate is shown as beats per minute (bpm). Data are presented as mean ± SEM. P-values were calculated by unpaired Student’s t-test in a) and b). (c) Body weight of the mice used for the grip strength test. Males and females are on the left and right, respectively. Cyan, red and green bars denote wild-type, negative control and treated mice. For each box plot, first and third quartiles denote 25th and 75th percentile and the black line represent the median. The whiskers represent the upper and lower limits of the data. Significance was determined by one-way ANOVA with Bonferroni correction.

Extended Data Fig. 5|. Gastro-intestinal phenotype in the treated HGPS mice.

(a) Prevalence of Megacolon/ Megacaecum incidences among homozygous HGPS mice treated with gLmnal and 2 (g1&2) versus the negative controls. Asymp: Asymptomatic (no obvious gastrointestinal phenotype), G.l: gastrointestinal phenotype characterized with Megacolon/Megacaecum and inability to defecate. (b) Body weight progression of a representative g1&2-treated homozygous mouse that acutely died after 28 weeks, (c) Necropsy of a g1&2-treated homozygous mouse exhibiting Megacolon/Megacaecum (left and bottom photographs) unlike the negative control (right photograph).

Figure 1:. Targeted disruption of lamin A and progerin by CRISPR/Cas9.

a, Scheme of lamin A/progerin targeting by CRISPR/Cas9. gLmna-1 targets upstream of the farnesylation site, gLmna-2 recognizes mutation and wildtype site (Ұ). SA/D: Splice Activator/Donor site. b, The in vivo gene therapy scheme. AAV9-mCherry-gLmna was injected into 0-to-2-day-old mice (P0–2). Upper panel shows the mCherry signal 4 days post-injection (DPI) into a P0 mouse (P0–4DPI) versus the PBS-injected control (lower panel). c, Expression of the mCherry reporter in different organs at 6dpp (days postpartum, P1–5DPI). The numbers at the lower right corners denote the exposure time in seconds. Scale bar, 2 mm. d, Efficiency of the lamin A/progerin targeting. When the two gRNAs act simultaneously, the region between them is deleted and the re-ligated ends can be detected by PCR. Black arrows denote the location of the primers. Tfrc: positive control for PCR. Mock: A gRNA with no target site on the genome. e, Immunoblots of adult organ lysates from heterozygous HGPS (Pro/+) mice on homozygous Cas9 (C9/C9) background treated with g1&2 versus mock-treated to evaluate lamin A/C/progerin levels. Middle panel corresponds to the uppermost blot under different exposures. α Tubulin: loading control (Fig. 1b–e are representative replicates of at least two independent experiments). f, Gross morphology of the mice. Shown are 16.4 weeks (wk) old mock-treated wild-type and homozygous (homo) female siblings next to 19.4 weeks old heterozygous (hetero) and g1&2-treated homozygous female siblings (Also displayed in the videos 1 and 2). The difference in the morphology is although the mock-treated control is 3 weeks younger. g, Body weight comparison between 9 and 17 weeks with the males depicted on the left, and the females on the right. For each box plot, first and third quartiles denote 25^th and 75^th percentile and the black line represent the median. The whiskers represent the upper and lower limits of the data. Significance was determined by one-way ANOVA with Bonferroni correction. Liv, TT, Hrt, Lng, Kid, Stom, Msl, Int, Sp: liver, tail-tip, heart, lung, kidney, stomach, muscle, intestine, spleen.

Figure 2:. CRISPR/Cas9-induced reduction of lamin A/progerin extends the health and lifespan of the HGPS mice.

a, Body weight and b, survival rate through time. Magenta line denotes untreated homozygous HGPS (Progeria) mice analyzed in the paper of A. Ocampo et al. The mice depicted in blue and red lines are a cross between HGPS and Cas9 transgenic mice (Progeria/Cas9 mice). They are homozygous for HGPS and carry at least one copy of the Cas9 transgene (hemizygous or homozygous). Blue line denotes untreated mice. Except for green and orange diamonds on the blue line in (b) denote mock-treated and gRNA-only negative controls, respectively. Red line denotes g1&2-treated mice. The sample size (n) indicates the initial number of mice at week 9 in Fig. 2a and 2b. Shown are mean values ± standard deviations. Multiple t-tests and log rank (Mantel Cox) test were used for body weight and survival curves, respectively. Although introduction of Cas9 alone seems to increase the survival rate, this may result from the different strain backgrounds following crossing with Cas9 mice. c, Histological analyses of the stomach (upper panel) and skin (lower panel) derived from 19 weeks old g1&2-treated versus mock-treated homozygous mice next to mock-treated wild-type (Wt). The arrows and asterisks denote epidermis of the skin and parietal cells of the stomach, respectively. Ұ: dermal fat. The figures at the bottom are the enlarged views of the epidermal regions pointed by the arrows. The images are representative replicates of two independent experiments with similar results. Scale bar, 100 μm. d, Measurement of the epidermal thickness (n = 15 sections per group from 3 biologically independent samples). Shown are mean, SEM and statistical significance were calculated by unpaired student’s t-test. e, Grip strength test. Left and right panels depict forelimb and hindlimb grip strength of adult mice (16–18 weeks old), respectively. Each estimate corresponds to 25 reads derived from 5 animals, measured 5 times. For each box plot, first and third quartiles denote 25^th and 75^th percentile and the black line represent the median. The whiskers represent the upper and lower limits of the data. Significance was determined by one-way ANOVA with Bonferroni correction. f, Representative graph of the running wheel activities. Shown are the activities of untreated or mock-treated versus g1&2-treated homozygous mice. All treated mice were 17 weeks old and control mice were 4 weeks younger. g, Quantitation of the running wheel activities. Mice tested were between 10–18 weeks old. Blue bars denote wild-type mice (n=5), red and green bars denote control (n=5) and g1&g2-treated (n=6) homozygous HGPS mice, respectively. Shown are mean values ± standard deviations and significance according to unpaired t-test.