Generation and characterization of a novel knockin minipig model of Hutchinson-Gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disorder for which no cure exists. The disease is characterized by premature aging and inevitable death in adolescence due to cardiovascular complications. Most HGPS patients carry a heterozygous de novo LMNA c.1824C > T mutation, which provokes the expression of a dominant-negative mutant protein called progerin. Therapies proven effective in HGPS-like mouse models have yielded only modest benefit in HGPS clinical trials. To overcome the gap between HGPS mouse models and patients, we have generated by CRISPR-Cas9 gene editing the first large animal model for HGPS, a knockin heterozygous LMNA c.1824C > T Yucatan minipig. Like HGPS patients, HGPS minipigs endogenously co-express progerin and normal lamin A/C, and exhibit severe growth retardation, lipodystrophy, skin and bone alterations, cardiovascular disease, and die around puberty. Remarkably, the HGPS minipigs recapitulate critical cardiovascular alterations seen in patients, such as left ventricular diastolic dysfunction, altered cardiac electrical activity, and loss of vascular smooth muscle cells. Our analysis also revealed reduced myocardial perfusion due to microvascular damage and myocardial interstitial fibrosis, previously undescribed readouts potentially useful for monitoring disease progression in patients. The HGPS minipigs provide an appropriate preclinical model in which to test human-size interventional devices and optimize candidate therapies before advancing to clinical trials, thus accelerating the development of effective applications for HGPS patients.


CRISPR/Cas9 pLMNA sgRNAs
The human codon-optimized Cas9 (gift from George Church, Addgene plasmid #41815) and sgRNAs were expressed from separate plasmids. CRISPR/Cas9 sgRNAs targeting the porcine LMNA gene were designed using the online sgRNA design tool Zifit (https://crispr-cas9.com/96/zifit-targeter-crispr-cas9/). Three individual sgRNAs were designed to span the pLMNA region comprising the targeted nucleotide, cloned into plasmids, and validated before gene editing of porcine fibroblasts 1 . For each sgRNA construct, two pLMNAspecific complementary oligonucleotides were denatured and slowly annealed before ligation of the annealed oligonucleotides to a sgRNA scaffold plasmid (pFUS-U6-sgRNA) based on BsaI assembly 1 . The ligation mixture was used to transform XL-2 Blue ultracompetent bacterial cells (Agilent Technologies, cat. no. 200150). The resulting bacterial cell clones were screened by PCR, and positive sgRNA clones were validated by DNA sequencing of isolated plasmid DNA. Oligonucleotides and target sites used for generation of the sgRNA vectors are listed in Supplemental Table S1.

pLMNA-specific C-check vector
The three generated sgRNAs were validated using a previously developed single strand annealing (SSA)directed, dual fluorescent surrogate reporter system called C-check. This vector comprises two expression cassettes: a truncated EGFP expression cassette for detection of double strand break (DSB)-induced SSA events and an AsRED expression cassette for measuring transfection efficiency and for normalization 1 . The EGFP cassette is interrupted by one or a series of target sites for the sgRNAs of interest. Upon sgRNAinduced DSB in this target region, the C-check reporter construct will express AsRED and EGFP if repaired by SSA, whereas only AsRED will be expressed if no DSB is induced or repair occurs by non-homologous end joining. The pLMNA-specific C-check vector was constructed as described 1 . Briefly, two complementary oligonucleotides comprising the sgRNA target site(s) were annealed and cloned into the BsaI-digested Ccheck vector. The ligated plasmid was used for bacterial cloning in XL-2 Blue Ultracompetent cells 1 . The resulting bacterial cell clones were PCR screened, and positive C-check clones were validated by DNA sequencing of isolated plasmid DNA. The oligonucleotides used to generate the pLMNA-specific C-check vector comprising overlapping sgRNA1, sgRNA2, and sgRNA3 target sites are listed in Supplementary  Table S1.

Flow cytometry evaluation of pLMNA gRNA efficiency
The efficiency of the three generated sgRNAs was evaluated by transfection into HEK293T cells and flow cytometry of the transfected cells 1 . The cells were seeded the day before transfection into 6-well plates (3 x10 5 cells/well). The next day, cells were co-transfected with one of the three sgRNAs plasmids (75, 150, and 300 ng) together with the hCas9 plasmid (75, 150, and 300 ng), the pLMNA specific C-check plasmid (300 ng), and stuffer plasmid DNA (to adjust the amount of total DNA to 1 µg) using X-tremeGENE 9 DNA transfection reagent (Sigma-Aldrich). As controls, cells were transfected with only the C-check or hCas9 plasmid, or with sgRNA plasmid together with the C-check vector. At 48 hours post transfection, the cells were harvested by trypsinization and analyzed by flow cytometry in a BD FACSARIA III sorter (BD Bioscience) (FACS Core Facility, Dept. of Biomedicine, AU) to quantify the efficiency (EGFP expression) of the individual sgRNAs in transfected (AsRED + ) cells.

pLMNA c.1824C>T donor plasmid
A donor plasmid comprising the homologous pLMNA sequence including the c.1824C>T mutation was constructed by 2-fusion PCR. Two PCRs with overlapping sequences were run, each using a Platinum Pfx DNA polymerase, genomic DNA isolated from a WT male Yucatan minipig as template DNA, and primer sets including a primer containing the C>T mutation. The two resulting overlapping c.1824C>T-containing amplicons were used as a template (100 ng of each amplicon per 25 µl PCR reaction) in a 2-fusion PCR resulting in a 378-bp product comprising a 236-bp left homology arm (LHA) and a 141-bp right homology arm (RHA) flanking the pLMNA C>T mutation. The HGPS-causing LMNA c.1824C>T mutation results in aberrant splicing of exon 11 and exon 12. To mimic this, and to avoid eventual additional interference with the intended splicing of the gene-edited c.1824C>T pLMNA allele, the PAM site located downstream of the sgRNA1 recognition site in the donor pLMNA sequence was left intact. The 2-fusion PCR was performed using Platinum Pfx polymerase and the following PCR protocol: 94C/2´; 35x (94C/20´´-62C/30´´-68C/30´´), 68C/7 min. Fusion products were cloned using a Zero Blunt TOPO PCR cloning kit (Invitrogen, cat. no. 450245) and the ligation mixture used for transformation of XL-2 Blue Ultracompetent cells. The resulting bacterial colonies were PCR screened before plasmid DNA isolation from positive bacterial cell clones. Finally, the pLMNA c.1824C>T plasmid was verified by DNA sequencing before being used as a donor plasmid for gene editing of porcine fibroblasts. Primers used to generate the pLMNA c.1824C>T plasmid are listed in Supplementary Table S1.

Transfection of primary porcine fibroblasts and expansion of gene-edited cells
Primary fibroblasts from newborn male Yucatan piglets were seeded onto a gelatin-coated 10 cm cell culture dish one day before transfection (1.5  10 6 cells). Before transfection, the culture medium was changed and supplemented with bFGF (5 ng/µL). The cells were then co-transfected with the sgRNA1 vector (500 ng), hCas9 vector (1500 ng), the pLMNA c.1824C>T donor construct (2500 ng), and EGFP-N3 plasmid (1000 ng, Clontech) using Lipofectamine LTX with Plus Reagent (Thermo Fischer Scientific, cat. no. 15338100). The transfected cells were harvested by trypsinization 48 hours post transfection and analyzed by FACS (FACS Core Facility, Dept. of Biomedicine, AU) to enrich for gene-edited cells based on EGFP expression. Propidium-iodide-negative EGFP-expressing cells were sorted using a BD FACSARIA III sorter and were seeded in 7 gelatin-coated 96-well plates (3 cells per well). Single cell clones were subsequently expanded by culturing in 15% DMEM supplemented with 5 ng/ml bFGF. At subconfluency, the cell clones were trypsinized, and 1/2 of the resulting cell suspension was transferred to 96-well PCR plates for lysis and PCR screening. The remaining half of the cell suspension was cultured in gelatin-coated 96-well cell culture plates for further expansion and freezing at early passages of PCR-validated gene-edited cell clones before their use as nuclear donor cells for SCNT.

PCR screening and DNA sequencing of gene edited donor cells
EGFP + cells in 96-well PCR plates were centrifuged and re-suspended in 25 L lysis buffer (50 mM KCl, 1.5 mM MgCl2, 10 mM Tris-Cl, pH 8.5, 0.5% Nonidet P40, 0.5% Tween, 400 g/ml Proteinase K) 2 . The cells were lysed (65C for 30 mins) followed by proteinase K inactivation (95C for 10 mins), and 1 L lysate was used as template for allele-specific PCR screening. A forward primer (5´-GCCTCTCAAGCCCTGTCACC-3´) and an allele-specific reverse primer (5´-GAGCCAGAGGAGATGGATCCA-3´) discriminating between the WT and gene-edited pLMNA alleles were used with the following PCR conditions: 94C/3´, 35x (94C/30´´-70C/30´´-72C/20´´), 72C/7´. Negative controls for the PCR screen were WT Yucatan minipig genomic DNA and water. Cell clones found to be positive for the pLMNA c.1824C>T mutation by the PCR described above were further analyzed by two PCR protocols to validate the fidelity of the 5´-and 3´-gene-targeting events. These PCR protocols used Platinum Pfx DNA polymerase and primer sets consisting of primers located outside and internally in the targeted region, respectively (Supplementary Table S2). Successfully gene-edited cell clones (positive for all three PCRs) were further verified by DNA sequencing of the amplicons resulting from both the 5´-and the 3´-gene-targeting PCRs prior to being selected as donor cells for hand-made cloning.

Off-target analysis of gene edited cell clones
The CRISPR RGEN Cas-OFFinder algorithm was used to identify potential off-target sites for the employed sgRNA1. Allowing for up to 3 mismatches between the sgRNA and genomic sequence, 9 genomic regions, in addition to the targeted pLMNA target gene, were identified on chromosomes 1, 5, 6, 8, 11, and 13. Two potential off-target sites reside in annotated genes on chromosome 6 (USB1 and PRKCZ). Except for one locus on chromosome 5, which proved difficult to amplify, all potential off-target regions were amplified by standard PCR using genomic DNA from WT or gene-edited Yucatan cells clones and a Platinum Pfx DNA polymerase. The resulting amplicons were subjected to DNA sequencing to verify if off-target activity had occurred.
In addition, the gene-edited cell clones were analyzed for potential unwanted random integration of the plasmid constructs used for co-transfection (sgRNA, hCas9, EGFP-N3, and the donor plasmid). These analyses were performed by standard PCR using genomic DNA from either WT or gene-edited cells and primer sets specific for the individual plasmids used for transfection. Primers used for off-target and random integration analyses are shown in Supplementary Table S2.

Genotyping of cloned gene edited piglets
Genotyping of the cloned pLMNA c.1824C>T gene-edited minipigs was performed by standard PCR using the allele-specific PCR and the 3´-gene-targeting PCRs mentioned above (for primer sequences, see Supplementary Table S2). Gene editing of the cloned piglets was also verified by DNA sequencing of amplicons resulting from a 5´+ 3´-gene targeting PCR essentially combining the 5´-and 3´-gene targeting PCRs but using Platinum Pfx DNA polymerase and primers flanking the entire targeted DNA region (for primer sequences, see Supplementary Table S2).

Handmade cloning, embryo culture and transfer, pregnancy period, and delivery of piglets
Handmade cloning was performed as described 3 . Cumulus-oocyte complexes harvested from slaughterhousederived ovaries were in-vitro matured and treated to remove cumulus cells and partially remove zonae pellucidae. The oocytes were bisected manually, and the chromatin-free cytoplasts were collected. Each cytoplast was first attached to one knockin HGPS fibroblast cell before fusion (BTX microslide 0.5 mm fusion chamber, model 450; BTX San Diego, US). After 1 h incubation, each cytoplast-fibroblast pair was fused with an additional cytoplast to create the reconstructed embryo. All reconstructed embryos were then incubated in culture medium for 5-6 days, when blastocysts and morulae were selected based on morphology. Two pools of cloned transgenic embryos were prepared, and respectively 92, 66 and 70 blastocysts/morulae were transferred surgically into three recipient landrace surrogate sows 4 . Pregnancy was diagnosed in all sows by ultrasonography after approximately 25 days. Farrowing was hormonally initiated at day 114 by intra-muscular prostaglandin injection. The sows farrowed a total of 19 piglets (15, 3 and 1), of which 10 were still alive after two weeks (Supplementary Fig. S2b).

Epididymal sperm extraction
Immediately after death, the testes with attached epididymes and vas deferens of 3 HGPS boars were placed in a portable cooler at 18-20ºC and transported for processing to the Physiology and Biotechnology of Reproduction in Swine Laboratory at the INIA. Epididymal samples were collected following a previously reported flushing technique 5 . The cauda epididymis and ductus deferens were isolated from the rest of the epididymis by making a cut near the junction of the corpus and the proximal cauda. For epididimal fluid collection, the vas deferens cannulated with a blunted 21G needle, and epididimal fluid was collected by retrograde washing from the vas deferens and epididymis tail using a syringe loaded with 1 mL Beltsville thawing solution (BTS) extender. The vas deferens and the cauda region were perfused with the extender, followed by air injection, until all the contents were removed from the cauda epididymis. Samples from each testicle were collected in 10 ml sterile plastic tubes and cryopreserved for storage in liquid nitrogen.

Surgical intrauterine insemination of wild-type gilts
Two gilts (approximate 5.5 months old and weighing 90-95 kg) were induced to ovulate with sequenced injection of eCG (1500 IU, Foligon; Intervet International BV, Boxmeer, Holland) followed by hCG (750 IU Veterin Corion; Divasa Farmavic, Barcelona, Spain) 72 h after the first injection 6 . The expected time of ovulation was 38-42 h after hCG injection 7 . Pigs were kept in individual pens, and after a 24 h fast, they were pre-medicated with an intramuscular injection of morphine (0.2 mg/kg), metadomidine (0.2 mg/kg), and ketamine (10 mg/kg). Anesthesia was maintained with isofluorane (ISOFLO, Esteve 2-3% O2) 8 . Animals were placed in lateral recumbency and under CO2 pneumoperitoneum of 8-10 mmHg. Paralumbar laparoendoscopic single-site surgery (LESS) was carried out using a monoport device (GelPOINT Advanced Access Platform, Applied Medical, Rancho Santa Margarita, California, USA). After rapid visualization of the abdominal organs, non-traumatic laparoscopy forceps were used to grasp the ad-ovarian end of the uterine horn towards them on port opening. Pneumoperitoneum and the monoport cap were then removed to allow gentle manipulation of the reproductive organs 9 . Frozen semen samples were thawed at 37º for 30 seconds and diluted in tempered BTS 10 . Semen doses were deposited into the oviduct with a blunt needle 11 . The gilts were kept under the usual farm conditions, and pregnancy status was assessed by ultrasonography 25-28 days after insemination (100 FALCO-VET scan, Esaote Espana S.A., Barcelona, Spain).

Progerin expression
Total mRNA was extracted in Tri-Reagent solution (Invitrogen AM9738), quantified in a NanoDrop spectrophotometer (Wilmington), and retrotranscribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). PCR to detect progerin mRNA was performed with a forward primer (5´-GCAACAAGTCCAATGAGGACCA-3´), a reverse primer (5´-CATGATGCTGCAGTTCTGGGGGCTCTGGAC-3´), 100-200 ng of cDNA, and 1 U/ml of Taq DNA polymerase (Biotools) in a c1000 Thermal Cycler (Bio-Rad). The 482-bp PCR product was purified after agarose gel separation and subjected to DNA sequencing. The sequence was analyzed with Chromas software.

Histology and immunofluorescence
Tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Serial 4 μm sections were stained with hematoxylin-eosin (H&E) and Masson trichrome (MT). Antigen retrieval was performed with 10 mM sodium citrate buffer (pH 6). Samples were blocked for 1 h at room temperature (RT) with 100 mM glycine and permeabilized for 1 h at RT in PBS containing 0.3% Triton X-100 (Sigma 9002-93-1), 5% normal goat serum (Jackson ImmunoResearch), and 5% bovine serum albumin (BSA, Sigma). Sections were then incubated overnight at 4°C with anti-CD31 (DIA-310 DIANOVA, 1:50), anti-N-cadherin (33-3900, ThermoScientific, 1:100) and anti-connexin 43 (Abnova PAB12759, 1:100). After washing, samples were incubated with corresponding secondary antibodies (goat anti-rabbit Alexa Fluor 647, Invitrogen A21245), a nucleic acid stain Hoechst 33342 (Sigma B2261) and anti-α-smooth muscle actin-Cy3 (Sigma SMA-Cy3 C6198) for 2 h at RT and then mounted in Fluoromount G imaging medium (Affymetrix eBioscience). Images were acquired with LSM 700 and 880 Carl Zeiss confocal microscopes. Stained sections were scanned with a NanoZoomer-RS scanner (Hamamatsu), and images were exported with NDP.view2 software and analyzed with ImageJ Fiji software by two researchers blinded to genotype. Collagen content in myocardial tissue was quantified by deconvolution of MT-stained images as the mean % of 7 areas distributed along the LV and 6 areas along the septum (470 m 2 free of vessels). Coronary artery number and degeneration were analyzed in areas of 120 mm 2 in septum and 170 mm 2 in LV in MT-stained images of transverse heart sections. Connexin 43 and N-cadherin localization in heart tissue was quantified as the mean of three 236 µm 2 regions per minipig using ImageJ JACoP plugin 12 .  (a) Representative photographs of similar-aged HGPS and WT minipigs, illustrating the small size and lean body of HGPS minipigs. The WT minipig in this picture is a castrated male 1 month younger than the HGPS minipig. The graph shows the body-mass index of minipigs at ages between 4.3 and 5.5 months (n=6 WT; n=8 HGPS).
(b-d) HGPS minipigs recapitulate many of the external features of human HGPS, including alopecia, wrinkled and dry skin with patchy pigmentation, and sclerodermia (b); prominent eyes with short eyelids, sculpted nose, thin lips, and prominent teeth (c); as well as thin limbs with severe joint stiffness and abnormal hooves (d) (finger and toe alterations occur in HGPS patients).