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SU The Hutchinson Gilford Progeria Syndrome a Disease Question

Brief Communication
https://doi.org/10.1038/s41591-018-0338-6
Development of a CRISPR/Cas9-based therapy
for Hutchinson–Gilford progeria syndrome
Olaya Santiago-Fernández1, Fernando G. Osorio1, Víctor Quesada 1,2, Francisco Rodríguez1,
Sammy Basso1, Daniel Maeso1, Loïc Rolas3, Anna Barkaway3, Sussan Nourshargh3, Alicia R. Folgueras1,
José M. P. Freije 1,2* and Carlos López-Otín 1,2*
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.
Hutchinson–Gilford progeria syndrome (HGPS) is a rare disease
characterized by aging-like manifestations emerging in childhood1.
Most cases (80–90%) result from a de novo point mutation in the
LMNA gene—encoding the nuclear lamins A and C—which activates a cryptic splice site in exon 11 (c.1824C >​  T; p.Gly608Gly)2,3.
This event leads to the expression of progerin, a truncated lamin A
variant with an internal deletion of 50 amino acids, which remains
farnesylated, inducing morphological and functional alterations of
the nuclear envelope4. A mouse model—LmnaG609G/G609G—recapitulating the mutation and many of the clinical features of these children5,
confirmed that HGPS is caused by progerin accumulation and not
by the loss of normal lamin A5,6. Several approaches against this syndrome were tested in preclinical models7, including farnesyltransferase inhibitors, which provided clinical benefits to HGPS patients8,9.
CRISPR/Cas9 gene-editing tools constitute promising alternatives for diseases such as Duchenne muscular dystrophy10, metabolopathies11 and deafness12. This system involves a Cas9 endonuclease
directed by a single-guide RNA (sgRNA) that recognizes its target
region, plus a protospacer-adjacent motif (PAM). The nuclease generates double-strand breaks in the DNA, repair of which through
non-homologous end-joining produces insertions and deletions
(indels)13. The finding that mosaic mice with both normal and
prelamin A-producing progeroid cells have a completely normal
phenotype14 indicates that a partial reduction in the accumulation
of farnesylated lamin A products could be sufficient for an important phenotype relief.
On this basis, we developed a CRISPR/Cas9-based strategy
against HGPS aimed at blocking the accumulation of lamin A and
progerin. The LMNA gene encodes lamin C (exons 1–10) and lamin
A (exons 1–12) through alternative splicing and polyadenylation.
Since lamin A appears to be dispensable5,6, our strategy would disrupt the last part of the LMNA gene, impeding lamin A/progerin
production without affecting lamin C. We first designed an sgRNA
(sgRNA-LCS1) with the 5′​-NGG PAM sequence of Streptococcus
pyogenes Cas9 to target LMNA exon 11 upstream of the HGPS mutation, in a region conserved across both humans and mice (Fig. 1a).
To test the efficacy of this approach, we cloned sgRNA-LCS1
or sgRNA-control in a lentiviral vector containing S. pyogenes
Cas9 (lentiCRISPRv2) and used these to transduce Lmna+/+ and
LmnaG609G/G609G murine fibroblasts. As a result, indels of variable
length were produced in sgRNA-LCS1-transduced cells, as assessed
by capillary electrophoresis-based fragment analysis (Extended
Data Fig. 1). Immunoblot analysis showed a significant decrease
in the accumulation of progerin and lamin A, while lamin C levels
were not affected (Fig. 1b). Likewise, immunofluorescence analysis demonstrated that numbers of progerin-positive nuclei were
reduced by 74% in sgRNA-LCS1-transduced cells compared to
sgRNA-control-transduced cells (Fig. 1c). Accordingly, we found a
65% decrease in the number of nuclear alterations in LmnaG609G/G609G
cells transduced with sgRNA-LCS1compared to sgRNA-controltransduced cells (Fig. 1c).
To test this system in human cells, we infected LMNAG608G/+
fibroblasts from HGPS patients and LMNA+/+ fibroblasts with
these lentiviral vectors. Similar to mouse fibroblasts, we observed
different indels in the DNA (Extended Data Fig. 2), a decrease in
progerin and lamin A by immunoblot (Fig. 1d), an 83% decrease
in progerin-positive nuclei and a 39% reduction in the number of
aberrant nuclei in sgRNA-LCS1- versus sgRNA-control-transduced
HGPS cells (Fig. 1e).
We next tested, in vivo, this editing approach using LmnaG609G/G609G
mice as an HGPS animal model. We chose an adeno-associated virus
9 (AAV9) delivery vector due to its safety and broad tissue tropism.
Given the packaging limit of these viruses (approximately 5 kb),
we used Staphylococcus aureus Cas9 nuclease15 and designed a new
sgRNA against the same region in exon 11 with the 5′​-NNGRRT
PAM sequence (sgRNA-LCS2). After packaging the vectors, with
either sgRNA-LCS2 or the sgRNA-control, we injected intraperitoneally 2 ×​  1011 AAV9 genome copies in P3 LmnaG609G/G609G mice
(Fig. 2a). To assess editing efficiency, we performed Illumina
sequencing of the target region in DNA from AAV9 target organs—
liver, heart, muscle and lung—of injected mice. Notably, Lmna contained indels in 13.6 ±​ 2.6% of the genome copies in liver, 5.3 ±​  1.0%
in heart, 4.1 ±​ 0.6% in muscle and 1.1 ±​ 0.2% in lung (Fig. 2b,c;
Extended Data Fig. 3; Supplementary Tables 1–4). Given the modest fraction of cells edited in vivo, the global decrease in progerin
messenger (RNA) was too low for reliable detection by quantitative reverse transcription polymerase chain reaction (RT–qPCR)
(Extended Data Fig. 4). However, immunohistochemical analysis
revealed a significant reduction in progerin-positive nuclei in liver,
heart and skeletal muscle from sgRNA-LCS2-transduced mice
1
Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología del Principado de Asturias, Universidad
de Oviedo, Oviedo, Spain. 2Centro de Investigación Biomédica en Red de Cáncer, Madrid, Spain. 3William Harvey Research Institute, Barts and The London
School of Medicine and Dentistry, Queen Mary University of London, London, UK. *e-mail: jmpf@uniovi.es; clo@uniovi.es
Nature Medicine | http://www.nature.com/naturemedicine
Brief Communication
NAtuRE MEDICInE
a
SD PolyA Lamin C
CTR
LCS1
Exon 11
C >T (G608G)
LCS1
Lamin A
Progerin
Lamin C
β-Actin
4
4
3
2
1
0
37
P = 0.0084
5 P = 0.7002 P = 0.9539
3
2
1
CTR LCS1
CTR LCS1
+/+
G609G/G609G
Progerin-positive
nuclei (%)
sgRNA-LCS1
80
60
40
20
P = 0.0018
40
30
20
10
LCS1
CTR
β-Actin
37
sgRNA-CTR
Progerin
sgRNA-LCS1
LMNAG608G/+
e
4
3
2
1
0
P = 0.0014
8
4
Lamin C/β-actin
kD
75
3
2
1
0
P = 0.9216 P = 0.3023
6
4
2
0
CTR LCS1
CTR LCS1
+/+
G608G/+
DAPI
LCS1
G609G/G609G
CTR LCS1 CTR LCS1
+/+
P = 0.0006
G608G/+
P = 0.0070
40
100
Nuclear defects (%)
Lamin A
Progerin
Lamin C
LCS1
5
5
Progerin/β-actin
CTR
P = 0.0370
Progerin-positive
nuclei (%)
LCS1
Lamin A/β-actin
CTR
G609G/G609G
0
CTR
6
1
50
0
LMNAG608G/+
2
+/+
P = 0.0008
100
LMNA+/+
3
CTR LCS1 CTR LCS1
G609G/G609G
d
4
0
0
DAPI
sgRNA-CTR
Progerin
LmnaG609G/G609G
kD
75
Exon 12
P = 0.0015
5
PolyA Lamin A
Nuclear defects (%)
c
6
LmnaG609G/G609G
CTR
SA
Lamin C/β-actin
Lmna+/+
Exon 10
SD
Progerin/β-actin
b
Exon 9
Lamin C
Lamin A
Progerin
Lamin A/β-actin
Exon 8
SD
SA
80
60
40
20
0
CTR
LCS1
G608G/+
30
20
10
0
CTR
LCS1
G608G/+
Fig. 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 immunoblot of lamin A, progerin and lamin C from WT and LmnaG609G/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 immunoblot of
lamin A, progerin and lamin C from WT and LMNAG608G/+ 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 ±​s.d. and individual values are overlaid. Scale bars, 40 µ​m.
Uncropped blots are available as Source data.
Nature Medicine | http://www.nature.com/naturemedicine
Brief Communication
NAtuRE MEDICInE
sgRNA-LCS2:
3′
P ​ 10. In the remaining cases,
normality was assumed based on previous data and we performed two-tailed
Student’s t-test to study the statistical significance. For survival comparisons we
used the log-rank test, and differences in maximum lifespan were calculated using
the one-tailed Fisher exact test comparing the number of live sgRNA-control- and
sgRNA-LCS2-transduced mice at the age corresponding to the 80th percentile of
lifespan in the joint survival distribution. We used Microsoft Excel or GraphPad
Brief Communication
Prism software for the analysis, and significant differences were considered when
*P ƫ-ŴāìŴaƫæąā°āÕìçŷ»ƗšŨŢŤƹaƚõƗ-ŦŠŨ-ŸÕçLMNA, the gene
āÓ°āÃç»ìÁÃûçą»âð÷â°æÕçƗaÓÕûæąā°āÕìç»°ąûÃûWGæÕûŴûõâÕ»ÕçÎāÓ°āõ÷ìÁą»Ãû
progerin, a toxic protein that induces rapid ageing and shortens the lifespan of children
đÕāÓõ÷ìÎÃ÷Õ°āì°õõ÷ìĖÕæ°āÃâėšŤėð÷ûš–ŤƗÁÃçÕçú°ûÃÃÁÕāì÷ûŷ”ûŸ»ìçĐÃ÷ā
ā°÷ÎÃāÃÁƫaº°ûÃõ°Õ÷ûāì-ƫº°ûÃõ°Õ÷ûđÕāÓæÕçÕæ°âºėŴõ÷ìÁą»āû°çÁđÕāÓìąā
÷ÃöąÕ÷ÕçÎÁìąºâÃŴûā÷°çÁ Gº÷ðàûì÷Áìçì÷ GāÃæõâ°āÃû5,ŦƗ2Ã÷ÃđÃÁÃû»÷ÕºÃāÓÃ
ąûÃìÍ°ç”āìÁÕ÷ûāâė»ì÷÷ûāāÓÃõ°āÓìÎÃçÕ»2-U[æąā°āÕìçÕ绹âāą÷ÃÁȻº÷ìºâ°ûāû
ÁÃ÷ÕĐÃÁÍ÷ìæ»ÓÕâÁ÷ÃçđÕāÓõ÷ìÎÃ÷Õ°°çÁÕç°æìąûÃæìÁÃâìÍ2-U[ƗBÃçāÕĐÕ÷°âÁÃâÕĐÃ÷ė
ìÍāÓÔāìȻº÷ìºâ°ûāûÍ÷ìæ»ÓÕâÁ÷ÃçđÕāÓ2-U[÷ÃûąâāÃÁÕçŨŧŵũšƂ»ì÷÷ûāÕìçìÍāÓÃ
õ°āÓìÎÃçÕ»°ââÃâÃƘæÕāÕΰāÕìçìÍWGæÕûŴûõâÕ»ÕçÎƘ÷ÃÁą»ÃÁâÃĐÃâûìÍõ÷ìÎÃ÷Õç°çÁ
»ì÷÷ûāÕìçìÍçą»âð÷°ºçì÷æ°âÕāÕÃûƗeçºÕ°ûÃÁìȺŴā°÷ÎÃā G°çÁWGÃÁÕāÕçÎ
°ç°âėûÕûÁÕÁçìāÁÃāûāìȺŴā°÷ÎÃāÃÁÕāÕçÎÕçā÷ðāÃÁõ°āÕÃçāŴÁÃ÷ÕĐÃÁȻº÷ìºâ°ûāûƗ4ç
transgenic mice that are homozygous for the human LMNA»ƗšŨŢŤƹa°ââÃâÃƘ°ûÕçÎâÃ
÷Ãā÷ìŴì÷ºÕā°âÕçÞûāÕìçìÍ°ÁÃçìŴ°ûûì»Õ°āÃÁĐÕ÷ąûũŷpũŸÃç»ìÁÕçÎāÓÔ÷ÃûąâāÃÁ
Õçûąºûā°çāÕ°âƘÁą÷°ºâûì÷÷ûāÕìçìÍāÓÃõ°āÓìÎÃçÕ»æąā°āÕìçŷ°÷ìąçÁŢŠŵŦŠƂ°»÷ìûû
Đ°÷Õìąûì÷ΰçûûÕĖæìçāÓû°ÍāÃ÷ÕçÞûāÕìçŸƘ÷Ãûāì÷°āÕìçìÍçì÷æ°âWGûõâÕ»ÕçΰçÁ
÷ÃÁą»āÕìçìÍõ÷ìÎÃ÷Õçõ÷ìāÃÕçâÃĐÃâûƗ4çĐÕĐ캰ûÃÃÁÕāÕçÎ÷Ãû»ąÃÁāÓÃĐ°û»ąâ°÷
pathology of the mice, preserving vascular smooth muscle cell counts and preventing
°ÁĐÃçāÕāÕ°âȻº÷ìûÕûƗûÕçÎâÃÕçÞûāÕìçìÍ”ŴÃĖõ÷ÃûûÕçÎpũ°āõìûāç°ā°âÁ°ėšŤ
Õæõ÷ìĐÃÁĐÕā°âÕāė°çÁÎ÷ðāâėÃĖāÃçÁÃÁāÓÃæÃÁÕ°çâÕÍÃûõ°çìÍāÓÃæÕ»ÃÍ÷ìæŢšťāìťšŠ
Á°ėûƗaÓÃûÃȻçÁÕçÎûÁÃæìçûā÷°āÃāÓÃõìāÃçāÕ°âìÍÕçĐÕĐ캰ûÃÃÁÕāÕçΰû°õìûûÕºâÃ
ā÷ðāæÃçāÍì÷2-U[°çÁìāÓÃ÷ÎÃçÃāÕ»ÁÕûðûÃûºėÁÕ÷ûāâė»ì÷÷ûāÕçÎāÓÃÕ÷÷ììā»°ąûÃƗ
2ąā»ÓÕçûìçŵ-ÕâÍì÷Áõ÷ìÎÃ÷Õ°ûėçÁ÷ìæÃŷ2-U[ì÷õ÷ìÎÃ÷Õ°ŸÕû°÷°÷Ã
genetic disease characterized by accelerated ageingŤƗ4çìĐÃ÷ũŠƂìÍ
õ°āÕÃçāûđÕāÓ2-U[ƘāÓÃÁÕûðûÃÕû»°ąûÃÁºė°ûÕçÎâÃÁÃçìĐìõìÕçāæąā°āÕìçŷ»ƗšŨŢŤƹaƚõƗ-ŦŠŨ-ŸÕçāÓÃâ°æÕçŷLMNA) geneš,ŢƗaÓÕûæąā°āÕìçõìāÃçāÕ°āÃû°»÷ėõāÕ»ûõâÕ»ÃûÕāÃÕçÃĖìçššƘâðÁÕçÎāì°æÕûŴûõâÕ»ÕçÎ
ÃĐÃçāāÓ°ā÷ÃûąâāûÕçāÓÃâìûûìÍťŠ°æÕçì°»ÕÁûÍ÷ìæāÓÃâ°æÕçõ÷ìāÃÕçš,Ţ
ŷ,ÕÎƗš°ŸƗaÓÕûā÷ąç»°āÃÁõ÷ìāÃÕçƘđÓÕ»ÓÕûàçìđç°ûõ÷ìÎÃ÷ÕçƘâ°»àû°õ÷ìāÃìâėāÕ»»âÃ°Đ°ÎÃûÕāÃÍì÷|FU[a”ŢŤƘđÓÕ»Ó»âðĐÃûāÓÃÍ°÷çÃûėâ°āÃÁāÃ÷æÕçąûìÍđÕâÁŴāėõÃõ÷ÃŴâ°æÕçšƗU÷ìÎÃ÷Õçõ÷ìāÃÕçÕæõ°Õ÷ûçą»âð÷ûā÷ą»āą÷Ã
and function, culminating in premature senescence and cell deathŧƗaÓÃ
pathogenic mutation is dominant-negative, so a single copy of the allele
is sufficient to cause progeria3Ɨ°÷ÁÕìĐ°û»ąâ°÷ÁÕûðûÃŶ»Ó°÷°»āÃ÷ÕĜÃÁºė
õ÷Ãæ°āą÷ðāÓÃ÷ìû»âÃ÷ìûÕûƘâìûûìÍĐ°û»ąâ°÷ûæììāÓæąû»âûÃââûŷp[FûŸ
°çÁĐ°û»ąâ°÷ûāÕÍÍÃçÕçÎŶÕûāÓÃõ÷ÃÁìæÕç°çā»°ąûÃìÍÁðāÓÕç»ÓÕâÁ÷Ãç
đÕāÓõ÷ìÎÃ÷Õ°ƘđÓìÓ°Đðç°ĐÃ÷°ÎÃâÕÍÃûõ°çìÍ°õõ÷ìĖÕæ°āÃâėšŤėð÷û3,ŧ–ššƗ
âāÓìąÎÓûā÷°āÃÎÕÃûÍì÷ā÷ðāÕçÎõ÷ìÎÃ÷Õ°Ƙûą»Ó°ûÎâ캰âÕçÓÕºÕāÕìçìÍ
protein farnesylation3,šŢ,šţ, offer benefits to patients, no approach has
yet been reported to directly reverse the mutation that causes HGPSšŤ–šŧƗ
aÓÃÁìæÕç°çāŴçÃΰāÕĐÃÍąç»āÕìçìÍõ÷ìÎÃ÷ÕçõìûÃû»Ó°ââÃçÎÃûÍì÷āÓÃ
treatment of HGPS by gene augmentation or gene disruption strateÎÕÃûƗLĐÃ÷ÃĖõ÷ÃûûÕìçìÍđÕâÁŴāėõÃLMNA does not rescue cellular phenotypesšŨƗâāÓìąÎÓW4[UWŵ°ûũŴæÃÁÕ°āÃÁÎÃçÃāÕ»ÁÕû÷ąõāÕìçìÍ
the pathogenic allele has been reported to improve phenotypes in
mouse models of progeriašť–šŧ, the resulting diversity of uncharacterized
Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA. 2Department of Chemistry and Chemical Biology, Harvard University,
Cambridge, MA, USA. 3Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA. 4National Human Genome Research Institute, National Institutes of Health, Bethesda, MD,
USA. 5Division of Cardiovascular Medicine, Vanderbilt University Medical Center, Nashville, TN, USA. 6Department of Cell Biology and Molecular Genetics, University of Maryland, College Park,
MD, USA. 7Department of Biostatistics, Vanderbilt University Medical Center, Nashville, TN, USA. 8Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA.
9
Therapeutic Innovation Center, Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA. 10Hasbro Children’s Hospital, Alpert Medical School of
Brown University, Providence, RI, USA. 11Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA. 12Present address: Kronos Bio Inc., Cambridge, MA, USA. 13These authors
contributed equally: Luke W. Koblan, Michael R. Erdos. ✉e-mail: francis.collins3@nih.gov; jonathan.d.brown@vumc.org; drliu@fas.harvard.edu
1
608 | Nature | Vol 589 | 28 January 2021
e
Untreated HGADFN167
ABE7.10max-VRQR, 10 d
ABE7.10max-VRQR, 20 d
Unaffected parent
7
6
5
4
3
*
2
****
****
1
0
LMNA
Lamin A/C
Progerin
Progerin
LMNC
7
6
Untreated HGADFN188
ABE7.10max-VRQR, 10 d
ABE7.10max-VRQR, 20 d
Unaffected parent
d
5
4
3
Nuclei
40
20
0
10 d 20 d
Untreated
ABE7.10maxVRQR
HGADFN188
C
T
80
60
40
20
0
10 d 20 d
Untreated
ABE7.10maxVRQR
Lamin A
Progerin
Lamin C
****
****
1
0
**
*
2
60
Base identity at c.1824 (%)
¥ &$* $*&&¥
80
100
HGADFN188
+ ABE
Normalized gene expression
c
¥ $* $*&&¥
Normalized gene expression
¥&$**7***7¥&$* *7¥
Progerin
LMNAΔ150 mRNA
Exon 12
HGADFN167
100
HGADFN188
Intron
b
HGADFN167
+ ABE
Base editing by
ABE7.10max-VRQR
Exon 11
Lamin A
¥ &$**7***&¥ &$* $*&&¥
HGADFN167
LMNA mRNA
Exon 12
¥ $* $*&&¥
Unaffected
parent
Intron
Base identity at c.1824 (%)
Exon 11
LMNA
Progerin
LMNC
Histone
H3
Merge
Unaffected
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HGADFN167
f
80
Morphologically abnormal
nuclei (%)
a
¥&$**7***&¥&$* *7¥
60
40
****
20
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na
ff
pa ect
re ed
nt
H
G
AD
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7
H
G
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+ N1
AB 67
E
0
HGADFN167
+ ABE
Fig. 1 | ABE-mediated correction of the LMNA c.1824 C>T mutation in
fibroblasts derived from patients with progeria. aƘaÓÃLMNA»ƗšŨŢŤƹa
æąā°āÕìçõìāÃçāÕ°āÃû°»÷ėõāÕ»ûõâÕ»ÃûÕāÃÕçÃĖìçššìÍāÓÃLMNA gene, resulting
Õç°çÕçŴÍ÷°æÃÁÃâÃāÕìçìÍšťŠçāŷLMNA‘šťŠŸ°çÁõ÷ìÁą»āÕìçìÍāÓÃõ°āÓìÎÃçÕ»
õ÷ìÎÃ÷Õçõ÷ìāÃÕçƗb, LMNA»ƗšŨŢŤçą»âÃìāÕÁÃÕÁÃçāÕāėÕçąçā÷ðāÃÁ
õ°āÕÃçāŴÁÃ÷ÕĐÃÁ2- ,GšŦŧ°çÁ2- ,GšŨŨ»Ãââû°çÁÕç»ÃââûšŠì÷ŢŠÁ°ėû
°ÍāÃ÷ā÷ðāæÃçāđÕāÓ”ŧƗšŠæ°ĖŴpWVWâÃçāÕĐÕ÷ąûƗ °ā°°÷ÃæðçƴûƗÁƗìÍÍÕĐÃ
āûÓçÕ»°â÷ÃõâÕ»°āÃûƗcƘVą°çāÕÍÕ»°āÕìçºėÁÕÎÕā°âÁ÷ìõâÃāUWŷÁÁUWŸìÍLMNA,
progerin and LMNC (a normal alternative splice form) transcripts in untreated
õ°āÕÃçāŴÁÃ÷ÕĐÃÁ»ÃââûƘ»ÃââûšŠì÷ŢŠÁ°ėû°ÍāÃ÷ā÷ðāæÃçāđÕāÓ”âÃçāÕĐÕ÷ąû°çÁ
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post-mitotic cell types and in a wide array of organismsŦƗ”ûąûð
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mutation in fibroblasts derived from children with HGPS and in a mouse
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notable improvement in vascular disease compared to saline-injected
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Nature | Vol 589 | 28 January 2021 | 609
80
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has been reported to be minimal or undetectableŦƗaìÕÁÃçāÕÍė»°çÁÕÁ°āðûŴÁÃõÃçÁÃçāìÍÍŴā°÷ÎÃā GÃÁÕāÕçÎûÕāÃû°ûûì»Õ°āÃÁđÕāÓāÓÃ
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LMNAŴā°÷ÎÃāÕçÎûÎWGŷ”ĖāÃçÁÃÁ °ā°,ÕÎƗŢŸƗqÃõÃ÷Íì÷æÃÁā°÷ÎÃāÃÁ
ûÃöąÃç»ÕçÎìÍÎÃçìæÕ» G°āāÓÃāìõţŢ»°çÁÕÁ°āÃìÍÍŴā°÷ÎÃāâì»Õ
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°ÍāÃ÷”âÃçāÕĐÕ÷ąûā÷°çûÁą»āÕìçƗqÃìºûÃ÷ĐÃÁçìÁÃāûā°ºâÃìÍÍŴā°÷ÎÃā
GÃÁÕāÕçÎŷŠƗšƂì÷âÃûûŸ°āāÓÃţŢāÃûāÃÁ»°çÁÕÁ°āÃìÍÍŴā°÷ÎÃāâì»ÕÕç
ÃÕāÓÃ÷»ÃâââÕçÃƘÁÃûõÕāÃŨŧŵũšƂìçŴā°÷ÎÃāÃÁÕāÕçÎŷ,ÕÎƗŢ°ŸƗ
aì°ûûÃûûìÍÍŴā°÷ÎÃāWGÃÁÕāÕçÎƘđÃõÃ÷Íì÷æÃÁā÷°çû»÷ÕõāìæÃŴđÕÁÃ
WGûÃöąÃç»ÕçÎŷWGŴûÃöŸìç”ŴâÃçāÕĐÕ÷ąûŴā÷ðāÃÁì÷ąçā÷ðāÃÁ
2- ,GšŦŧ °çÁ 2- ,GšŨŨ »ÃââûƘ æðûą÷ÕçÎ āÓà Í÷ÃöąÃç»ė ìÍ
°ÁÃçÕçÃŴāìŴÕçìûÕçÃWGÁðæÕç°āÕìçƘđÓÕ»Óç°āą÷°ââėì»»ą÷ûāÓ÷ìąÎÓout the transcriptome from endogenous cellular deaminasesŢŧƗaÓÃ
on-target nucleotide within the LMNA transcript was efficiently (more
āÓ°çŨŠƂŸ»ì÷÷ûāÃÁÍ÷ìæeāìÕç”Ŵā÷ðāÃÁ»Ãââûŷ,ÕÎƗŢºŸƗaÓðĐÃ÷°ÎÃÍ÷ÃöąÃç»ė°çÁÁÕûā÷ÕºąāÕìçìÍŴāìŴ4»ìçĐÃ÷ûÕìçÕçāÓÃā÷°çû»÷ÕõāìæÃìÍ”Ŵā÷ðāÃÁ»ÃââûđÃ÷ÃûÕæÕâ°÷āìāÓìûÃìÍąçā÷ðāÃÁ»Ãââûŷ,ÕÎƗŢ»,
“ĖāÃçÁÃÁ °ā°,ÕÎƗšÓŸƗGìā°ºâėƘ”ā÷ðāæÃçāìÍ»ÃââûÁÃ÷ÕĐÃÁÍ÷ìæ
patients with HGPS restored the transcriptome to a state resembling
Off-target editing analysis in patient cells
°çìçÕ»°â”ŧƗšŠÃÁÕāì÷û»°çÕçÁą»Ã°ûŴÁÃõÃçÁÃçāìÍÍŴā°÷ÎÃā
GÃÁÕāÕçΰçÁā÷°çûÕÃçāƘâìđŴâÃĐÃâ°ûŴÕçÁÃõÃçÁÃçāìÍÍŴā°÷ÎÃā
610 | Nature | Vol 589 | 28 January 2021
a
ABE-AAV9 injection
P3 retro-orbital
P14 retro-orbital
P14 intraperitoneal
ITR Promoter
WT TadA
deaminase
Homozygous
LMNA c.1824 C>T
(progeria) mice
32-aa Evolved TadA 32-aa Cas9 nickase-VRQR-N Intein-N Terminator ITR
linker deaminase linker
ITR Promoter Intein-C
ITR
C (corrected) DNA at c.1824 (%)
b
P3 retro-orbital injection
80
70
60
50
40
30
20
10
0
ddPCR count per 50 ng RNA
P14 retro-orbital injection
6 weeks
(n = 4)
6 months
(n = 12)
**
*
Heart
c
ddPCR count per 50 ng RNA
Terminator LMNA C>T sgRNA ITR
Cas9 nickase-VRQR-C
10,000
Quad
***
**
Liver
Aorta
Bone
**
2,000
6,000
**
***
Heart
Quad
Liver
Aorta
Bone
Liver, females (n = 6 saline, n = 6 ABE-AAV9)
P14 saline
P14 ABE-AAV9 WT
Lamin A
Progerin
Lamin C
Actin
6,000
0
6 weeks
(n = 5)
6 months
(n = 12)
d
Liver
(n = 12)
8,000
4,000
80
70
60
50
40
30
20
10
0
LMNA
Saline ABEAAV9
****
Progerin
Saline ABEAAV9
Heart
(n = 12)
Liver, males (n = 6 saline, n = 6 ABE-AAV9)
P14 saline
P14 ABE-AAV9 WT
Lamin A
Progerin
Lamin C
Actin
Heart, females (n = 6 saline, n = 6 ABE-AAV9)
P14 saline
P14 ABE-AAV9 WT
Lamin A
Progerin
Lamin C
Actin
4,000
**
2,000
0
Heart, males (n = 6 saline, n = 6 ABE-AAV9)
LMNA
Saline ABEAAV9
Progerin
Saline ABEAAV9
Lamin A
Progerin
Lamin C
Actin
P14 saline
P14 ABE-AAV9 WT
Fig. 3 | DNA, RNA and protein levels after a single in vivo injection of
ABE-expressing AAV9 in a mouse model of human progeria. aƘ ą°âpũ
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LMNAŴā°÷ÎÃāÕçÎûÎWGđÃ÷ÃÕçÞûāÃÁÕçāìõ÷ìÎÃ÷Õ°æÕ»ÃƗUţ÷Ãā÷ìŴì÷ºÕā°â
ÕçÞûāÕìçûŷťƶšŠšŠĐÎìÍð»ÓpƘšƶšŠššĐÎāìā°âŸƘUšŤ÷Ãā÷ìŴì÷ºÕā°âÕçÞûāÕìçû
ŷťƶšŠššĐÎìÍð»ÓpƘšƶšŠšŢĐÎāìā°âŸì÷UšŤÕçā÷°õÃ÷ÕāìçðâÕçÞûāÕìçû
ŷťƶšŠššĐÎìÍð»ÓpƘšƶšŠšŢĐÎāìā°âŸđÃ÷ðÁæÕçÕûāÃ÷ÃÁƗ4aWƘÕçĐÃ÷āÃÁ
āÃ÷æÕç°â÷ÃõðāûƗbƘ GŴÃÁÕāÕçÎÃÍÍÕ»ÕÃç»ÕÃûÍì÷»ì÷÷ûāÕçÎLMNA»ƗšŨŢŤÍ÷ìæ
aŷõ°āÓìÎÃçÕ»ŸāìŷđÕâÁāėõßÕçŦŴđÃÃàŴìâÁì÷ŦŴæìçāÓŴìâÁæÕ»ÃāÓ°āđÃ÷Ã
÷Ãā÷ìŴì÷ºÕā°ââėÕçÞûāÃÁđÕāÓ”ŴÃĖõ÷ÃûûÕçÎpũŷ”ŴpũŸ°āUţŷâÃÍāŸì÷
UšŤŷ÷ÕÎÓāŸƗ”ÁÕāÕçÎÃÍÍÕ»ÕÃç»ÕÃûÕçUšŤÕçā÷°õÃ÷ÕāìçðââėÕçÞûāÃÁæջð÷ÃûÓìđç
Õç”ĖāÃçÁÃÁ °ā°,ÕÎƗţ°ƗcƘÁÁUW»ìąçāûÍì÷āÓÃWGā÷°çû»÷Õõā°ºąçÁ°ç»ÃìÍ
human LMNA (grey bars) and progerin (red bars) in the liver and heart of mice
ŷŦæìçāÓûìâÁŸāÓ°āđÃ÷Ã÷Ãā÷ìŴì÷ºÕā°ââėÕçÞûāÃÁđÕāÓû°âÕçÃì÷”Ŵpũ°āUšŤƗ
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â°æÕçƘõ÷ìÎÃ÷Õç°çÁâ°æÕçõ÷ìāÃÕçûÕçāÓÃâÕĐÃ÷°çÁÓð÷āìÍæÕ»ÃāÓ°āđÃ÷Ã
÷Ãā÷ìŴì÷ºÕā°ââėÕçÞûāÃÁđÕāÓû°âÕçÃì÷”Ŵpũ°āUšŤƗ”°»Óâ°çÃûÓìđûāÕûûąÃ
Í÷ìæ°ÁÕÍÍÃ÷ÃçāæìąûÃƗƇqaƈÕçÁÕ»°āÃû°ťŧBƠŦæìąûÃāÓ°āâ°»àûāÓÃ
ā÷°çûÎÃçÃƘûÓìđÕçÎāÓ°āāÓðçāÕºìÁėÕûûõûÕÍÕ»āìÓąæ°çâ°æÕçõ÷ìāÃÕçûƗ[ÃÃ
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çąæºÃ÷ûìͺÕìâìÎÕ»°â÷ÃõâÕ»°āÃûƗƓPƸŠƗŠťƘƓƓPƸŠƗŠšƘƓƓƓPƸŠƗŠŠšƘ
ƓƓƓƓPƸŠƗŠŠŠšºė[āąÁÃçāƈûąçõ°Õ÷ÃÁāđìŴûÕÁÃÁtŴāÃûāƗ
āÓ°āìÍ»ÃââûÍ÷ìæ°çąç°ÍÍûāÃÁõ°÷Ãçāŷ,ÕÎƗŢÁƘßƗaÓÃûÃ÷Ãûąâāû»ìâlectively show that treating cells with the LMNAŴā°÷ÎÃāÕçÎûÎWG°çÁ
“æ°ĖŴpWVWÁÕÁçìā÷ÃûąâāÕçÁÃāûāÃÁìÍÍŴā°÷ÎÃā Gì÷WGÃÁÕāÕçÎ
ąûÕçÎāÓðºìĐðç°âėûÕûæÃāÓìÁûƘÁÃûõÕāÃÓÕÎÓâÃĐÃâûìÍìçŴā°÷ÎÃāÃÁÕāÕçÎƗ
In vivo ABE delivery in mice with progeria
“ç»ìą÷°ÎÃÁºėāÓÃûÃÍÕçÁÕçÎûƘđðõõâÕÃÁº°ûÃÃÁÕāÕçÎÕçĐÕĐìāì»ì÷÷ûā
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transgene that includes the complete human LMNA»ƗšŨŢŤƹa°ââÃâÃ
ŷťŧBƠŦŴāÎŷBFGƓ-ŦŠŨ-Ÿ2âçûƠ>Ƙõ÷ÃĐÕìąûâėąûÃÁ°ûÓÃāÃ÷ìĜėÎìąû
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ìÍ2-U[Õç»âąÁÕçÎp[FÁÃÍûāûƘÓ°Õ÷âìûûƘâ°»àìÍûąº»ąā°çÃìąûÍ°āƘ
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°ÁÃçìŴ°ûûì»Õ°āÃÁĐÕ÷ąûŷpŸŢŨ, a delivery modality that is approved
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trans-splicing inteins to reconstitute the full-length base editor in cells
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tropism, clinical validation and ability to transduce progeria-relevant
tissues including heart and muscleţŠ,ţšƗ
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āÕûûąÃûŷ,ÕÎƗ3Ƙ”ĖāÃçÁÃÁ °ā°,ÕÎƗţ°ŵ»ŸƗ
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injections generally achieved higher base-editing efficiencies than P3
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in the older miceŢŨ,ţŢ,33ƗaìÎÃāÓÃ÷ƘāÓÃûÃÁ°ā°÷ÃĐðâāÓ°ā°ûÕçÎâÃÕçĐÕĐì
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Long-term ABE treatment of progeria mice
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đÃ÷ÃÃąāÓ°çÕĜÃÁ°çÁ°ç°âėûÃÁÍì÷ Gº°ûÃŴÃÁÕāÕçÎÃÍÍÕ»ÕÃç»ėƘLMNA
WGûõâÕ»ÕçÎƘâÃĐÃâûìÍÓąæ°çõ÷ìÎÃ÷Õç°çÁâ°æÕçõ÷ìāÃÕçûƘ°çÁāÕûûąÃÓÕûāìâìÎėƗqÃõâ°»ÃÁāÓÃ÷Ãæ°ÕçÕçÎŢŤUţŴÕçÞûāÃÁæջðçÁŢŤ
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ûāąÁėāì°ûûÃûûâÕÍÃûõ°çƗ
DNA, RNA and protein analysis at six months
ç°âėûÕûìÍāÓà Gº°ûÃŴÃÁÕāÕçÎìąā»ìæÃûÕçûÕĖŴæìçāÓŴìâÁæÕ»Ã
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ìāÓ»ìÓì÷āûûÓìđÃÁÕç»÷ðûÃûÕç GŴÃÁÕāÕçÎÃÍÍÕ»ÕÃç»ėÕçûÃĐÃ÷°â
āÕûûąÃû»ìæõ°÷ÃÁāìāÓÃûÕĖŴđÃÃàāÕæÃõìÕçāŷ,ÕÎƗ3bŸƗ,ì÷ÃĖ°æõâÃƘ
ÃÁÕāÕçÎÕçāÓðì÷ā°÷ìûÃÍ÷ìæŤƗťƴŢƗťƂā욊ƴţƗŤƂÕçUţŴÕçÞûāÃÁæÕ»ÃƘ
°çÁÍ÷ìæšŧƴťƗŢƂāìŢţƴŨƗšƂÕçUšŤŴā÷ðāÃÁæÕ»ÃƗFìÁÃûāÃÁÕāÕçÎđ°û
ìºûÃ÷ĐÃÁÕçāÓÃâąçÎƘûàÕçƘĐÕû»Ã÷°âÍ°ā°çÁđÓÕāðÁÕõìûÃāÕûûąÃŷqaŸƘ
Nature | Vol 589 | 28 January 2021 | 611
Article
a
b
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*
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WT
Adventitial area (mm2)
0.2 0.4 0.6 0.8 1.0
Saline
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0
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1,000 2,000 3,000 4,000
P14 injection
WT
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0.2 0.4 0.6 0.8 1.0
Saline
****
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WT
ABE-AAV9
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c
Fraction survival
0.25
100
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ABE AAV9
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0.50
0
Saline-injected ABE-AAV-injected
(6 months old) (6 months old)
Lamin A/C
+ DAPI
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0
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(6 months old)
Males
Saline
ABE AAV9
1.00
Fraction survival
Untreated
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H&E
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200
Age (d)
300
400
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100
200
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Age (d)
đÓÃ÷ðûÃÁÕāÕçÎđ°ûæÕçÕæ°âÕçāÓÃàÕÁçÃė°çÁûõâÃÃçŷ”ĖāÃçÁÃÁ °ā°
,ÕÎƗţÁŸƗėûā°çÁÃ÷pŦũŠÃÁÕāÕçΰçÁÕçÁÃâûĐ°÷ÕÃÁºėāÕûûąÃºąāđÃ÷Ã
generally observed at low frequencies compared to on-target editing
ŷ”ĖāÃçÁÃÁ °ā°,ÕÎƗţÃƘÍŸƗaÓÃûÃ÷ÃûąâāûûąÎÎÃûāāÓ°āº°ûÃÃÁÕāÕçÎæ°ė
»ìçāÕçąÃÕçĐÕĐìÍ÷ìæûÕĖđÃÃàûāìûÕĖæìçāÓûìÍ°ÎÃƘ»ìçûÕûāÃçāđÕāÓāÓÃ
àçìđçõÃ÷ûÕûāÃç»ÃìÍpÕçæ°ææ°âûţŤ,35, or that edited cells may have
a survival advantage over uncorrected cells in some organs, increasing
āÓÃõ÷ÃĐ°âÃç»ÃìÍÃÁÕāÃÁ°ââÃâÃûìĐÃ÷āÕæÃƗ
“ÁÕāÕçÎÃÍÍÕ»ÕÃç»ÕÃû°āûÕĖæìçāÓûÕçæìûāāÕûûąÃû÷Ãæ°ÕçÃÁÓÕÎÓÃ÷
ÕçāÓÃUšŤŴÕçÞûāÃÁ»ìÓì÷āāÓ°çāÓÃUţŴÕçÞûāÃÁ»ìÓì÷āƘÕç»âąÁÕçκė
ŢƗŢŴÍìâÁÕçāÓðì÷ā°ƘŢƗšŴÍìâÁÕçûàÃâÃā°âæąû»âÃƘšƗŧŴÍìâÁÕçºìçðçÁ
šƗŤŴÍìâÁÕçâąçÎŷ”ĖāÃçÁÃÁ °ā°,ÕÎƗţÁŸƗaÓÃûÃ÷ÃûąâāûÕçÁÕ»°āÃāÓ°ā
ÕçÞûāÕçÎæÕ»ÃđÕāÓšŠšŢāìā°âĐΰāUšŤ÷ÃûąâāûÕçÓÕÎÓÃ÷âÃĐÃâûìÍLMNA
»ì÷÷ûāÕìçŦæìçāÓû°ÍāÃ÷ā÷ðāæÃçāƘ»ìæõ°÷ÃÁāìÕçÞûāÕçÎæÕ»ÃđÕāÓ
šŠššāìā°âĐΰāUţƗ
GÃĖāƘđÃöą°çāÕÍÕÃÁāÓÃÃÍÍûāûìÍÕçĐÕĐì”ā÷ðāæÃçāìçā÷°çû»÷Õõā°ºąçÁ°ç»Ã°çÁõ÷ìāÃÕçâÃĐÃâûÍì÷õ÷ìÎÃ÷Õç°çÁÓąæ°çâ°æÕçÕç
ûÕĖŴæìçāÓŴìâÁæÕ»ÃƗ°ûÃÃÁÕāÕçÎâÃÁāìÁû÷ðûÃûÕçõ÷ìÎÃ÷Õçā÷°çû»÷Õõā
°ºąçÁ°ç»Ãŷ,ÕÎƗ3cƘ”ĖāÃçÁÃÁ °ā°,ÕÎƗŤŸāÓ°āđÃ÷ÃûìæÃāÕæÃûâ°÷ÎÃ÷
āÓ°ç G»ì÷÷ûāÕìçâÃĐÃâûƚÍì÷ÃĖ°æõâÃƘÕçUšŤqađÃìºûÃ÷ĐÃÁ°
ţšƂ÷ÃÁą»āÕìçÕçāÓÃâÃĐÃâûìÍõ÷ìÎÃ÷ÕçæWGÁÃûõÕāÃìçâėŤƗŠƴţƗŦƂ
G»ì÷÷ûāÕìçƗaÓÃûÃÍÕçÁÕçÎûûąÎÎÃûāāÓ°ā»ì÷÷ûāÃÁ»Ãââûæ°ėºÃ
more transcriptionally active than uncorrected cells or that cells with
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612 | Nature | Vol 589 | 28 January 2021
400
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Fig. 4 | Aortic histopathology and lifespan of
progeria mice after a single injection of ABE-AAV9.
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adventitia in mice that were retro-orbitally injected
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PƸŠƗŠŠŠšŸƗe, Kaplan–Meier curve for mice that were
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ƓƓƓƓPƸŠƗŠŠŠšºė[āąÁÃçāƈûąçõ°Õ÷ÃÁāđìŴûÕÁÃÁt-test (b)
or Mantel–Cox test (d, eŸƗ
600
Finally, we noted increases in the abundance of correctly spliced LMNA
ā÷°çû»÷Õõāû°æìçΔŴā÷ðāÃÁæÕ»ÃÕç°Đ°÷ÕÃāėìÍāÕûûąÃû»ìæõ°÷ÃÁ
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corrected cells may be translationally more active than uncorrected
»ÃââûƗaìÎÃāÓÃ÷ƘāÓÃûÃÍÕçÁÕçÎûÕçÁÕ»°āÃāÓ°āÕçĐÕĐì”ŴæÃÁÕ°āÃÁ»ì÷÷ûtion of the pathogenic human LMNA»ƗšŨŢŤƹa°ââÃâÃÕçæջû°ç÷ÃÁą»Ã
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ABE treatment improves vascular pathology
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contribute to aortic stiffening and the impairment of cardiac functionŧ–ššƗqÃìºûÃ÷ĐÃÁāÓÃûÃÓ°ââæ°÷àĐ°û»ąâ°÷Íðāą÷ÃûìÍõ÷ìÎÃ÷Õ°
in saline-injected control miceŢŢŷ,ÕÎƗŤ°ŸƗì÷ā°ûÍ÷ìæUţŴÕçÞûāÃÁ
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÷Ãû»ąÃÁºìāÓ°ì÷āÕ»p[F»ìąçāûŷššŴÍìâÁÕç»÷ðûß°çÁ°ÁĐÃçāÕāÕ°â
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āÓ°āUšŤ”Ŵā÷ðāÃÁæÕ»ÃđÃ÷ÃÕçÁÕûāÕçÎąÕûÓ°ºâÃÍ÷ìæđÕâÁŴāėõÃæÕ»Ã
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with progeriaŧ,ššƗaÓÃÕæõ÷ìĐÃæÃçāûÕç°ì÷āÕ»õ°āÓìâìÎėÕç”Ŵā÷ðāÃÁ
mice prompted us to examine the protein levels of human progerin
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Óąæ°çâ°æÕçƠ°çÁÓąæ°çõ÷ìÎÃ÷Õçŷ,ÕÎƗŤ»Ƙ”ĖāÃçÁÃÁ °ā°,ÕÎƗŨŸƗ
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āÓ°ā Gº°ûÃÃÁÕāÕçÎìÍ°÷ìąçÁŢťƂÕçāÓðì÷ā°÷Ãû»ąÃûāđìàÃėĐ°û»ąâ°÷ÁÃÍûāûìÍõ÷ìÎÃ÷Õ°Ŷp[Fâìûû°çÁõÃ÷Õ°ÁĐÃçāÕāÕ°âÍÕº÷ìûÕûŵđÓÕâÃ
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of patients with progeriaŤƗ”ā÷ðāæÃçāæìÁÃûāâė÷Ãû»ąÃÁāÓÃâìûûìÍ
āÓÃÓėõìÁÃ÷æ°âÍ°āâ°ėÃ÷ÕçºìāÓUţŴ°çÁUšŤŴÕçÞûāÃÁæÕ»Ã÷Ãâ°āÕĐÃāì
û°âÕçÃŴÕçÞûāÃÁ»ìçā÷ìâûŷ”ĖāÃçÁÃÁ °ā°,ÕÎûƗũƘšŠŸƗìāÓ”Ŵā÷ðāÃÁ
and saline-injected mice exhibited moderate dermal hypoplasia comõ°÷ÃÁāìđÕâÁŴāėõÃťŧBƠŦæÕ»Ãŷ”ĖāÃçÁÃÁ °ā°,ÕÎûƗũƘšŠŸƗ
ABE treatment extends progeria mouse lifespan
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the potential origins of the liver tumours, we performed whole-genome
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