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: [email protected]; [email protected]
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Brief Communication
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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.
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Brief Communication
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sgRNA-LCS2:
3′
P < 0.0001
P = 0.0031
5′
5′
3′
5′
3′
5′
75
P = 0.0026
50
25
0
g
0
50
100
3′
G609G
LCS2
C
T
LC R
S
C 2
T
LC R
S
C 2
T
LC R
S
C 2
T
LC R
S2
Liver Heart Muscle Lung
Lmna+/+
LmnaG609G/G609G CTR
i
LmnaG609G/G609G LCS2
250
h
WT
Week 12: P = 0.0028
Week 13: P = 0.0063
****
G609G CTR
G609G LCS2
0 2 10 12 14 16 18 20 22 24 26 28 30
Age (weeks)
250
200
150
100
50
0
Males
P = 0.0433
P = 0.0005
WT CTR LCS2
Lmna+/+
LmnaG609G/G609G CTR
Kidney
j
WT CTR LCS2
LmnaG609G/G609G
Lmna+/+
LmnaG609G/G609G CTR
P = 0.0008
60
40
20
0
WT
WT CTR LCS2
LmnaG609G/G609G LCS2
P = 0.0177
2
1
0
G609G/G609G
Lmna
WT
P = 0.0361
LmnaG609G/G609G
Area with
fibrosis (%)
2.0
WT CTR LCS2
CTR LCS2
LmnaG609G/G609G
Muscle
Progerin-positive
nuclei (%)
Muscle
P = 0.0076
100
80
60
40
20
0
CTR LCS2
LmnaG609G/G609G
3
Heart
Progerin-positive
nuclei (%)
Progerin-positive
nuclei (%)
Liver
Heart
100
80
60
40
20
0
P = 0.0134
LmnaG609G/G609G LCS2
WT CTR LCS2
LmnaG609G/G609G
P = 0.0007
Females
P = 0.0206
LmnaG609G/G609G
P < 0.0001
100
80
60
40
20
0
250
200
150
100
50
0
TUNEL-positive cells (%)
d
200
Time (days)
G609G
CTR
0
AAV9
150
110
100
90
80
70
60
10
0
Area with
fibrosis (%)
P = 0.0002
G609G CTR
G609G LCS2
Percentage of initial
weight
In-frame
Frameshift
20
10
f
100
Blood glucose
(mg dl–1)
e
P < 0.0001
Blood glucose
(mg dl–1)
Total genomes (%)
30
Survival (%)
b
1.5
1.0
0.5
0.0
WT
CTR LCS2
LmnaG609G/G609G
Fig. 2 | CRISPR/Cas9 delivery and phenotype amelioration in LmnaG609G/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 ±​ s.e.m. (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 WT and LmnaG609G/G609G sgRNA-controltransduced or sgRNA-LCS2-transduced mice. Data are mean ±​ s.d. (n =​5 WT 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-LCS2transduced LmnaG609G/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 ±​s.e.m. 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 LmnaG609G/G609G sgRNA-control-transduced, sgRNA-LCS2-transduced and WT female mice at
3.5 months of age. h, Glycemia in WT (males n =​ 5; females n =​5), sgRNA-control-transduced (males n =​ 6; females n =​4) and sgRNA-LCS2-transduced
LmnaG609G/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 ±​ s.d. (n =​5 WT and sgRNA-control-transduced mice; n =​ 4 sgRNALCS2-transduced mice; two-tailed Student’s t-test). j, Gomori staining in 3.5-month-old LmnaG609G/G609Gmouse tissues showing moderate perivascular and
interstitial fibrosis in heart and quadriceps muscle (blue areas). Data are mean ±​ s.d. (n =​5 WT and sgRNA-control-transduced mice; n =​ 4 sgRNA-LCS2transduced mice; two-tailed Student’s t-test). Scale bars, 100 μ​m (d, i, j).
compared to sgRNA-control-transduced animals (Fig. 2d), which
concurred with the DNA sequencing results. In lung, kidney and
aorta, no reduction in the number of progerin-positive nuclei
was observed, possibly due to the lower tropism of AAV9 in these
organs (Extended Data Fig. 5). Given the importance of vascular
alterations in HGPS, the lack of noticeable direct effects on the aorta
is a setback of the approach tested. Nevertheless, vascular pathologies characteristic of HGPS such as atherosclerosis are strongly
influenced by systemic factors. Therefore, a reliable assessment of
potential vascular benefits will require the use of susceptible mouse
models carrying additional genetic alterations, such as Apoe or Ldlr
inactivation.
Importantly, progerin reduction in AAV9-sgRNA-LCS2transduced mice was translated into an increase in their median
survival of 33.5 days, from 127 to 160.5 days, compared to the
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sgRNA-control-transduced cohort, which represents a 26.4% lifespan increase (Fig. 2e; Extended Data Fig. 6). Mean survival was
extended from 128.1 days (s.d. 15.73; 95% confidence interval
116.8–139.4) to 167.4 days (s.d. 30.41; 95% confidence interval
145.6–189.2). Likewise, maximum survival was extended from
151 to 212 days (P = 0.0163; one-tailed Fisher exact test) (Fig. 2e).
Phenotypically, sgRNA-LCS2-transduced LmnaG609G/G609G mice
presented a healthier appearance, with retarded loss of grooming,
slightly improved body weight and increased blood glucose levels,
partially rescuing the hypoglycemia characteristic of these mice
(Fig. 2f–h; Extended Data Figs. 7 and 8). Likewise, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis
revealed that this group presented significantly fewer apoptotic cells
in the kidney (Fig. 2i). Since progerin reduction was not detected
in this organ, this suggests an effect dependent on systemic factors.
Brief Communication
We also observed a slight decrease in gastric mucosa atrophy
(Extended Data Fig. 9) and reduced focal and perivascular fibrosis in
heart and quadriceps muscle in sgRNA-LCS2-transduced compared
to sgRNA-control-transduced mice, in accordance with the higher
mobility of the former group (Fig. 2j; Supplementary Video 1).
Antisense oligonucleotides blocking the aberrant splicing of
LMNA transcripts have been proposed for the treatment of progeria5,16. However, a longer-term therapy would be desirable. Here,
we present a CRISPR/Cas9-based permanent genome-editing
approach that targets LMNA exon 11, specifically interfering with
lamin A/progerin expression. Because the Cas9 endonuclease is not
directed specifically against the p.Gly608Gly point mutation, this
strategy could also be applicable to laminopathies caused by other
LMNA mutations17. However, although lamin A is dispensable in
cells and mice5,6, the consequences of abrogating its expression in
humans remain unexplored. Therefore, alternative CRISPR/Cas9based systems, such as base editors18, also need to be tested. The
coexistence of progeroid and normal cells at a ratio of approximately
50/50 in mosaic mice resulted in a completely normal phenotype
and lifespan14. In the current work, although the editing efficiency
is lower, extensive phenotype amelioration and lifespan extension
were obtained. The extent to which the modest editing efficiency
attained is due to low delivery efficacy could not be determined reliably, as the packaging limit of the vector precluded the inclusion of
a suitable reporter. Interestingly, the concurrent study by Beyret and
colleagues19 describes another successful CRISPR/Cas9-based treatment of the LmnaG609G/G609G mouse model of HGPS. Nevertheless,
further research on putative off-target events and adverse effects of
the Cas9 nuclease will be needed to ensure the safety of this intervention. Regardless of these limitations, these two studies show the
preclinical efficacy of genome editing in a mouse model of progeria and pave the way for using CRISPR/Cas9 in HGPS and other
currently incurable systemic diseases.
Online content
Any methods, additional references, Nature Research reporting
summaries, source data, statements of data availability and associated accession codes are available at https://doi.org/10.1038/
s41591-018-0338-6.
Received: 22 January 2018; Accepted: 18 December 2018;
Published: xx xx xxxx
References
1. Hennekam, R. C. Am. J. Med. Genet. A. 140, 2603–2624 (2006).
2. De Sandre-Giovannoli, A. et al. Science 300, 2055 (2003).
3. Eriksson, M. et al. Nature 423, 293–298 (2003).
NAtuRE MEDICInE
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
Goldman, R.D. et al. Proc. Natl Acad. Sci. USA 101, 8963–8968 (2004).
Osorio, F. G. et al. Sci. Transl. Med. 3, 106ra107 (2011).
Fong, L. G. et al. J. Clin. Invest. 116, 743–752 (2006).
Gordon, L. B., Rothman, F. G., Lopez-Otin, C. & Misteli, T. Cell 156,
400–407 (2014).
Gordon, L. B. et al. Circulation 130, 27–34 (2014).
Gordon, L. B. et al. JAMA 319, 1687–1695 (2018).
Amoasii, L. et al. Sci. Transl. Med. 9, eaan8081 (2017).
Yang, Y. et al. Nat. Biotechnol. 34, 334–338 (2016).
Gao, X. et al. Nature 553, 217–221 (2018).
Doudna, J. A. & Charpentier, E. Science 346, 1258096 (2014).
de la Rosa, J. et al. Nat. Commun. 4, 2268 (2013).
Ran, F. A. et al. Nature 520, 186–191 (2015).
Scaffidi, P. & Misteli, T. Nat. Med. 11, 440–445 (2005).
Bar, D. Z. et al. J. Med. Genet. 54, 212–216 (2017).
Gaudelli, N. M. et al. Nature 551, 464–471 (2017).
Beyret, E. et al. Nat. Med. https://doi.org/10.1038/s41591-019-0343-4 (2019).
Acknowledgements
We thank G. Velasco, R. Villa-Bellosta, C. Bárcena, A.P. Ugalde and X.M. Caravia for
helpful comments and advice, and R. Feijoo, A. Moyano, D.A. Puente and S.A. Miranda
for excellent technical assistance. We also acknowledge the generous support by
J.I. Cabrera and Associazione Italiana Progeria Sammy Basso and the contribution of
Dr. Matthew Golding to the generation of the anti-murine progerin antibody.
The Instituto Universitario de Oncología del Principado de Asturias is supported by
Fundación Bancaria Caja de Ahorros de Asturias. J.M.P.F. is supported by Ministerio de
Economía y Competitividad (MINECO/FEDER: No. SAF2015-64157-R) and Gobierno
del Principado de Asturias. C.L.-O. is supported by grants from the European Research
Council (ERC-2016-ADG, DeAge), Ministerio de Economía y Competitividad (MINECO/
FEDER: Nos. SAF2014-52413-R and SAF2017-87655-R), Instituto de Salud Carlos III
(RTICC) and Progeria Research Foundation (No. PRF2016-66). O.S.-F. is recipient of
an FPU fellowship. A.R.F. is recipient of a Ramón y Cajal fellowship. The generation of
progerin antibody was funded by the Wellcome Trust (No. 098291/Z/12/Z to S.N.).
Author contributions
F.G.O., J.M.P.F. and C.L.-O. conceived and designed experiments. O.S.-F., F.G.O., V.Q.,
F.R., S.B., D.M. and A.R.F. performed experiments and analyzed data. L.R., A.B. and S.N.
provided reagents. O.S.-F., F.G.O., A.R.F., J.M.P.F., and C.L.-O. wrote the manuscript. All
authors revised the manuscript.
Competing interests
The authors declare no competing interests.
Additional information
Extended data is available for this paper at https://doi.org/10.1038/s41591-018-0338-6.
Supplementary information is available for this paper at https://doi.org/10.1038/
s41591-018-0338-6.
Reprints and permissions information is available at www.nature.com/reprints.
Correspondence and requests for materials should be addressed to J.M.P.F. or C.L.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in
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© The Author(s), under exclusive licence to Springer Nature America, Inc. 2019
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Methods
Plasmids and sgRNA cloning. All sgRNA-LCS were designed to target
exon 11 of the LMNA gene using the Benchling CRISPR Design tool. For
infections involving human and mouse fibroblasts, we used the lentiviral
vector lentiCRISPRv2 (Addgene) in which we cloned the sgRNA-control
(5′​-GGAGACGGGATACCGTCTCT-3′​) or the sgRNA-LCS1
(5′​-AGCGCAGGTTGTACTCAGCG-3′​). For AAV injection, we cloned the
sgRNA-control or sgRNA-LCS2 (5′​-GTGCAGCGGCTCGGGGGACCCCG-3′​)
in the pX601-AAV vector from Addgene, containing the Cas9 nuclease
from S. aureus.
Virus production. pX601-sgRNA-control or pX601-sgRNA-LCS2 plasmids
were packaged as AAV serotype 9 by The Viral Vector Production Unit of the
Universitat Autònoma de Barcelona (Barcelona, Spain), followed by polyethylene
glycol precipitation and iodixanol gradient purification. Aliquots of 2 ×​  1011
genome copies in 60 μ​l of phosphate buffered saline (PBS)-MK were prepared for
the injections.
Animal experiments. All animal experiments were performed in accordance
with institutional guidelines and were approved by the Committee of Animal
Experimentation of University of Oviedo (Oviedo, Spain). For AAV injection, P3
LmnaG609G/G609G mice were injected intraperitoneally with either sgRNA-control- or
sgRNA-LCS2-containing vectors. All gene-editing and phenotypic analyses were
performed at 3.5 months of age. To analyze blood glucose, animals were starved
overnight and glucose levels were measured with an Accu-Check glucometer
(Roche Diagnostics) using blood from the tail vein.
Histological analysis and TUNEL staining. Tissues were collected in 4%
paraformaldehyde in PBS and embedded in paraffin. Hematoxylin and eosin
(H&E) staining was performed on stomach tissue, and atrophy of the gastric
mucosa was blindly evaluated by a pathologist on three different sections per
mouse, establishing a pathological score (0, normal; 1, mild; 2, moderate;
3, severe athrophy). TUNEL staining in mouse kidneys was done according to the
manufacturer’s instructions (In-Situ Cell Death Detection Kit, TMR red, Roche).
To determine the number of TUNEL-positive nuclei, ten random fields per mouse
were blindly analyzed using ImageJ. H&E with Gomori’s trichrome staining was
performed in heart and quadriceps muscle, and five random fields per tissue
were quantified with a FIJI plugin provided by A. M. Nistal (Servicios CientíficoTécnicos, Universidad de Oviedo).
Illumina sequencing and bioinformatic analysis. MiSeq DNA sequencing was
performed by Macrogen, using the Illumina 300bpPE. To prepare the library, DNA
was isolated from liver, heart, muscle and lung from mice transduced with sgRNAcontrol- or sgRNA-LCS2-encoding AAVs. Next, we amplified the target region of
the Cas9 nuclease with the Pfu DNA polymerase (Promega) adding the Illumina
adapters by two PCRs: N­GS­1_­fwd: A­CA­CT­CT­TT­CC­CT­AC­AC­GA­CG­CT­CT­TC­
CG­AT­CT­NN­NN­TGTGACACTGGAGGCAGAAG and N­GS­1_­rev: G­TG­AC­TG­
GA­GT­TC­AG­AC­GT­GT­GC­TC­TT­CC­GA­TC­TC­AAGTCCCCATCACTTGGTT
for the first PCR, and NGS2_fwd: A­ATGATACGGCGACCACCGAGAT
CTACACTCTTTCCCTACACGACGCTCTTCCGATCT and NGS2_rev:
CAAGCAGAAGACGGCATACGAGATXXXXXXGTGACTGGAGTTC for the
second PCR. The N represents random bases and the X the sequence used for the
index. Genomic reads in FASTQ format were aligned to the GRCm38.p6 assembly
of the mouse genome using BWA v. 0.7.5a-r405 (ref. 20). Then, reads spanning the
genomic region putatively affected by the CRISPR/Cas9 action (chr3: 8848255588482615) were extracted with Samtools v. 1.3.1 (ref. 21) and analyzed using inhouse Perl scripts. Briefly, these scripts isolate the part of each read spanning the
chosen region, highlight small insertions/deletions and output a count of each
regional sequence. We then analyzed the percentage of the sequences
showing regional differences in sgRNA-control- and sgRNA-LCS2-transduced
mouse samples.
Capillary electrophoresis-based fragment analysis. We performed PCR
amplification of the target region with the forward oligonucleotide labeled
with 6FAM fluorophore in the 5′​position, facilitating fragment analysis of the
resulting products by capillary electrophoresis. For human cells, we used the
oligonucleotides HsLMNA_Fwd: [6FAM] GCACAGAACCACACCTTCCT and
HsLMNA_Rev: TGACCAGATTGTCCCCGAAG, while for mouse cells we used
MmLmna_Fwd: [6FAM] GTCCCCATCACTTGGTTGTC and MmLmna_Rev:
TGACTAGGTTGTCCCCGAAG.
Cell culture, transfection and viral transduction. We maintained HEK-293T
cell cultures in Dulbecco’s modified Eagle’s medium supplemented with 10%
fetal bovine serum, 1% penicillin-streptomycin-l-glutamine and 1% antibioticantimycotic (Gibco) at 37 °C in 5% CO2. In the case of human and mouse
fibroblasts, ×​1 non-essential amino acids, 10 mM HEPES buffer, 100 μ​M
2-mercaptoethanol and ×​1 sodium pyruvate (Gibco) were also added to the
previous medium and 15% fetal bovine serum was used. For lentiviral infection,
HEK-293T cells were transfected with lentiCRISPRv2 vector together with
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second-generation packaging plasmids using Lipofectamine reagent (Life
Technologies). Supernatants were filtered through 0.45 μ​m polyethersulfone filters
to collect the viral particles, and added at 1:3 dilution to previously seeded human
and mouse fibroblasts supplemented with 0.8 μ​g ml–1 of polybrene (Millipore).
Selection with puromycin (2 μ​g ml–1) was performed 2 days after infection, and the
editing efficiency and nuclear aberrations were quantified one week later.
RNA preparation and RT–qPCR. Collected cells or tissues were homogenized
in TRIzol reagent (Life Technologies) and RNA was extracted with the
RNeasy Mini kit following the manufacturer’s instructions (QIAGEN). cDNA
was synthesized with the QuantiTect Reverse Transcription kit (QIAGEN)
using 1 μ​g of total RNA, and then RT–qPCR analysis of mouse tissues
was performed. For progerin analysis, TaqMan PCR Universal Mastermix
(Applied Biosystems) and the following oligonucleotides and probe were used:
MmProgerin_fwd (5′​-TGAGTACAACCTGCGCTCAC-3′​), MmProgerin_rev
(5′​-TGGCAGGTCCCAGATTACAT-3′​) and MmProgerin_probe
(5′​-CGGGAGCCCAGAGCTCCCAGAA-3′​); using a β​-actin (Applied
Biosystems) as endogenous control. For lamin C analysis, we used SYBR green
PCR Universal Mastermix (Applied Biosystems) and the oligonucleotides
Lmnc_fwd (5′​-CGACGAGGATGGAGAAGAGC-3′​) and Lmnc_rev
(5′​-AGACTTTGGCATGGAGGTGG-3′​) for lamin C; or Actb_Fwd
(5′​-CTGAGGAGCACCCTGTGCT-3′​) and Actb_Rev
(5′​-GTTGAAGGTCTCAAACATGATCTG-3′​) for β​-actin as
endogenous control.
Protein isolation and immunoblot analysis. Cells were washed with ×​1 PBS
and homogenized in RIPA lysis buffer containing 100 mM Tris pH 7.4, 150 mM
NaCl, 10 mM EDTA pH 8.0, 1% sodium deoxycholate, 1% Triton X-100 and
0.1% sodium dodecyl sulfate (SDS), supplemented with protease inhibitor
cocktail (Complete, EDTA-free, Roche) and phosphatase inhibitors (PhosSTOP,
Roche). Protein concentration was determined with the Pierce BCA Protein
Assay Kit, and 30 µ​g per lane were loaded onto 8% SDS–polyacrylamide gels.
Gels were then transferred to nitrocellulose membranes, blocked with 5%
nonfat dry milk in TBS-T buffer (20 mM Tris pH 7.4, 150 mM NaCl and 0.05%
Tween 20) and incubated overnight at 4 °C with primary antibodies: 1:500
mouse monoclonal anti-lamin A/C (MANLAC1, provided by G. Morris) for
experiments involving mouse cells, 1:1,000 rabbit polyclonal anti-lamin A/C
(sc-20681, Santa Cruz Biotechnology) for experiments involving human cells or
1:10,000 anti-β​-actin (AC-15, Sigma) as an endogenous control. Finally, blots
were incubated with 1:10,000 goat anti-mouse (Jackson ImmunoResearch) or
1:3,000 goat anti-rabbit horseradish peroxidase (HRP) (Cell Signaling) in 1.5%
nonfat dry milk in TBS-T and washed with TBS-T. Immunoreactive bands
were developed with Immobilon Western chemiluminescent HRP substrate
(Millipore) in a LAS-3000 Imaging System (Fujifilm). Bands were quantified
using ImageJ.
Immunofluorescence, immunohistochemistry and nuclear morphology
analysis. For immunofluorescence assays, cells were fixed in 4% paraformaldehyde
solution, rinsed in PBS and permeabilized with 0.5% Triton X-100. Afterwards,
they were blocked with 15% goat serum solution and incubated overnight at 4 °C
with a rabbit polyclonal anti-progerin antibody in PBS (1:200). Next, slides were
washed with TBS-T and incubated with 1:500 anti-rabbit secondary antibody
Alexa Fluor 488 (Life Technologies). Nuclei were stained with 4′​,6-diamidino-2phenylindole (DAPI, Invitrogen). In the case of immunohistochemical analysis,
tissues were fixed in 4% paraformaldehyde solution and incubated with Target
Retrieval Solution at 95 °C for 20 min, Peroxidase Blocking Solution for 5 min
and Protein Block Serum Free (all from Dako) for 20 min before incubation with
the anti-progerin primary antibody (1:300 dilution) for 1 h. An HRP-conjugated
polyclonal anti-rabbit was applied for 30 min and then 3,3′​-diaminobenzidine for
10 min. Tissues were counterstained with hematoxylin (Dako) and visualized by
light microscopy. Rabbit anti-progerin polyclonal antibody was generated using
peptide immunogens and standard immunization procedures (S. Nourshargh et al.,
manuscript in preparation). The specificity of the antibody was confirmed by
nuclear staining of LmnaG609G/G609G mouse-derived fibroblasts, which was negative
in the case of WT cells. To determine the percentage of progerin-positive cells and
nuclei with aberrations, five random fields per culture or tissue sample were blindly
analyzed. For tissue samples, in each field a pre-established grid was used and five
random areas were quantified.
Statistical analysis. Animals of the same age were used for comparisons between
mice groups, and no statistical method was used to predict sample size. For
statistical analysis of differences between mouse cohorts, normality was assessed
using the Shapiro–Wilk test in those cases where n >​ 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 < 0.05, **P < 0.01, ***P < 0.001.
Reporting Summary. Further information on research design is available in the
Life Sciences Reporting Summary attached to this paper.
Code availability
The in-house Perl scripts used for gene edition analysis can be freely downloaded at
https://github.com/vqf/genotypes.
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Data availability
The MiSeq data were deposited in the NCBI SRA (no. PRJNA505974). Other data
from this study are available from the corresponding authors. Any materials that
can be shared will be released via a Material Transfer Agreement.
References
20. Li, H. & Durbin, R. Bioinformatics 26, 589–595 (2010).
21. Li, H. et al. Bioinformatics 25, 2078–2079 (2009).
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Extended Data Fig. 1 | Representative capillary electrophoresis-based fragment analysis of sgRNA-control- and sgRNA-LCS1-transduced LmnaG609G/G609G
mouse embryonic fibroblasts (n = 3 independent infections and MEF lines). Red line and orange peaks correspond to size standards.
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Extended Data Fig. 2 | Representative capillary electrophoresis-based fragment analysis of sgRNA-control- and sgRNA-LCS1-transduced LMNAG608G/+
human fibroblasts (n = 3 independent infections). Red line and orange peaks correspond to size standards.
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Extended Data Fig. 3 | Percentage of indels in LmnaG609G/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).
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Brief Communication
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Extended Data Fig. 4 | RT–qPCR analysis of progerin and lamin C in tissues from LmnaG609G/G609G sgRNA-control-transduced and LmnaG609G/G609G sgRNALCS2-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).
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Extended Data Fig. 5 | Progerin immunohistochemistry in lung, kidney and aorta from WT, LmnaG609G/G609G sgRNA-control-transduced and
LmnaG609G/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 LmnaG609G/G609G mice). Scale bar, 100 μ​m.
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Extended Data Fig. 6 | Kaplan–Meier survival plot of LmnaG609G/G609G male and female mice transduced with sgRNA-control (n =​ 6 males; n =​ 4 females)
or sgRNA-LCS2 (n =​ 4 males; n =​ 6 females).
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Extended Data Fig. 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.
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Extended Data Fig. 8 | Images of three sex- and age-matched mice transduced with the sgRNA-LCS2 compared to sgRNA-control-transduced animals.
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Extended Data Fig. 9 | H&E staining of gastric mucosa from WT, LmnaG609G/G609G sgRNA-control-transduced and LmnaG609G/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).
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Life Sciences Reporting Summary
Nature Research wishes to improve the reproducibility of the work that we publish. This form is intended for publication with all accepted life
science papers and provides structure for consistency and transparency in reporting. Every life science submission will use this form; some list
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Experimental design
1. Sample size
Describe how sample size was determined.
Sample size was determined based on previous experiments of our laboratory, carried out
with the same animal model under identical environmental conditions.
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Jose M.P. Freije
Corresponding author(s): Carlos López-Otín
2. Data exclusions
Describe any data exclusions.
Two AAV9-treated mice (one from the control cohort and another one from the sgRNA-LCS2
group) were excluded from the survival plot due to perinatal death. This event could be
explained by an early manipulation and a higher frailty of progeroid mice. The analysis of
indel mutations in the liver of a LCS2-transduced mouse was also excluded due to a low
number of reads in NGS (only 24 reads compared to more than 100,000 in the rest of
analyzed samples). These exclusion criteria had not been pre-established.
3. Replication
Describe the measures taken to verify the reproducibility
of the experimental findings.
Three independent infections were done for in vitro experiments. All attempts at replication
were successful.
4. Randomization
Describe how samples/organisms/participants were
allocated into experimental groups.
Allocation of both mice and cell cultures to each group was random.
5. Blinding
Describe whether the investigators were blinded to
group allocation during data collection and/or analysis.
Nuclear abnormalities and progerin analysis in cultures were quantified by investigators who
were blinded to the identity of the analyzed cells. In the same way, histological analysis
(Gomori staining, TUNEL assay and progerin immunohistochemistry) were also performed by
investigators blinded to group identity.
Note: all in vivo studies must report how sample size was determined and whether blinding and randomization were used.
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1
6. Statistical parameters
n/a Confirmed
The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement (animals, litters, cultures, etc.)
A description of how samples were collected, noting whether measurements were taken from distinct samples or whether the same
sample was measured repeatedly
A statement indicating how many times each experiment was replicated
The statistical test(s) used and whether they are one- or two-sided
Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of any assumptions or corrections, such as an adjustment for multiple comparisons
Test values indicating whether an effect is present
Provide confidence intervals or give results of significance tests (e.g. P values) as exact values whenever appropriate and with effect sizes noted.
A clear description of statistics including central tendency (e.g. median, mean) and variation (e.g. standard deviation, interquartile range)
Clearly defined error bars in all relevant figure captions (with explicit mention of central tendency and variation)
See the web collection on statistics for biologists for further resources and guidance.
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For all figures and tables that use statistical methods, confirm that the following items are present in relevant figure legends (or in the
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Software
Policy information about availability of computer code
7. Software
Describe the software used to analyze the data in this
study.
-Microsoft Excel v. 15.21.1 and GraphPad Prism v. 6.0.2 were used for the statistical analysis.
-BWA v. 0.7.5a-r405 and Samtools v. 1.3.1 were used for MiSeq analysis.
-ImageJ v. 1.48v was used for Western blot and TUNEL analysis.
-FIJI v. 1.52i was used for Gomori quantification.
For manuscripts utilizing custom algorithms or software that are central to the paper but not yet described in the published literature, software must be made
available to editors and reviewers upon request. We strongly encourage code deposition in a community repository (e.g. GitHub). Nature Methods guidance for
providing algorithms and software for publication provides further information on this topic.
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Materials and reagents
Policy information about availability of materials
8. Materials availability
Indicate whether there are restrictions on availability of
unique materials or if these materials are only available
for distribution by a third party.
Unique materials used are available from the authors upon Material Transfer Agreement
signature. AAVs were obtained from The Viral Vector Production Unit (UPV) of the Universitat
Autònoma de Barcelona (Barcelona, Spain).
9. Antibodies
Describe the antibodies used and how they were validated The primary antibodies used were:
for use in the system under study (i.e. assay and species). -Mouse monoclonal anti-lamin A/C (MANLAC1) was provided by Prof. Glenn Morris (Wolfson
The secondary antibodies used were:
-Goat anti-mouse IgG HRP-linked antibody from Jackson ImmunoResearch (cat: 115-035-062;
lot:121006) diluted 1:10,000.
-Goat anti-rabbit IgG HRP-linked antibody from Cell Signaling (cat: 7074S; lot: 27) diluted
1:3,000.
-Goat anti-rabbit IgG – H&L 488 from Alexa Fluor (cat: A11034; lot: 1670152) diluted 1:500.
November 2017
Centre for Inherited Neuromuscular Disease, UK) and used at a dilution 1:500. For validation,
cultured mouse fibroblast cell extracts were used.
-Rabbit polyclonal anti-lamin A/C (H-110; cat: sc-20681) from Santa Cruz Biotechnology was
used at a dilution 1:1,000. According to the websites of the manufacturer, this antibody
reacts against Lamin A/C from mouse, rat and human origin.
-Mouse monoclonal anti-beta-actin (AC-15; cat: A5441; lot: 014M4759) was pursached from
Sigma and used at a dilution 1:10,000. According to the websites of the manufacturer, reacts
against guinea pig, canine, Hirudo medicinalis, feline, pig, carp, mouse, chicken, rabbit, sheep,
rat, human and bovine orthologs.
-The anti-progerin polyclonal antibody was generated using peptide immunogens and
standard immunization procedures (S. Nourshargh et al., manuscript in preparation).
1:200-1:300 dilutions were used and its specificity was confirmed by nuclear staining of
LmnaG609G/G609G mice-derived fibroblasts, which was negative in the case of wild-type
cells.
2
`
a. State the source of each eukaryotic cell line used.
HEK-293T cells are from ATCC. Controls and progeroid mouse fibroblasts cultures were
established in our laboratory from control and mutant mice. Human control and HGPS
fibroblasts are from Coriell.
b. Describe the method of cell line authentication used.
The identity of control and progeroid fibroblasts was confirmed by Western blot of lamin A/C.
PCR-based microsatellite characterization of HEK-293T cells was performed at the University
of Oviedo.
c. Report whether the cell lines were tested for
mycoplasma contamination.
The cell lines were not tested for mycoplasma contamination.
d. If any of the cell lines used are listed in the database
of commonly misidentified cell lines maintained by
ICLAC, provide a scientific rationale for their use.
HEK-293T cells are widely used for infection experiments. The identity of HEK-293T was
assessed by PCR-based microsatellite characterization.
Animals and human research participants
Policy information about studies involving animals; when reporting animal research, follow the ARRIVE guidelines
11. Description of research animals
Provide all relevant details on animals and/or
animal-derived materials used in the study.
Progeria LmnaG609G/G609G mouse model (Lmna tm1.1Otin) was used in a C57BL/6N
background. Both males and females were used for the study. Samples from wild-type and
LmnaG609G/G609G sgRNA-transduced mice were collected at the age of 3.5 months.
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10. Eukaryotic cell lines
Policy information about studies involving human research participants
12. Description of human research participants
Describe the covariate-relevant population
characteristics of the human research participants.
The study did not involve human research participants.
November 2017
3
Article
In vivo base editing rescues Hutchinson–
Gilford progeria syndrome in mice
https://doi.org/10.1038/s41586-020-03086-7
Received: 9 June 2020
Accepted: 30 November 2020
Published online: 6 January 2021
Luke W. Koblan1,2,3,13, Michael R. Erdos4,13, Christopher Wilson1,2,3, Wayne A. Cabral4,
Jonathan M. Levy1,2,3, Zheng-Mei Xiong4, Urraca L. Tavarez4, Lindsay M. Davison5,
Yantenew G. Gete6, Xiaojing Mao6, Gregory A. Newby1,2,3, Sean P. Doherty5, Narisu Narisu4,
Quanhu Sheng7, Chad Krilow4, Charles Y. Lin8,9,12, Leslie B. Gordon10,11, Kan Cao6,
Francis S. Collins4 ✉, Jonathan D. Brown5 ✉ & David R. Liu1,2,3 ✉
Check for updates
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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: [email protected]; [email protected]; [email protected]
1
608 | Nature | Vol 589 | 28 January 2021
e
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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
»ÃââûÍ÷ìæ°çąç°ÍÍûāÃÁõ°÷ÃçāƗ-ÃçÃÃĖõ÷ÃûûÕìçâÃĐÃâûđÃ÷Ãçì÷æ°âÕĜÃÁāì
transferrin receptor (TFRCŸÃĖõ÷ÃûûÕìçâÃĐÃâûƗ °ā°Í÷ìæāÓÃąç°ÍÍûāÃÁõ°÷Ãçā
°÷ÃûÓìđçÕçºìāÓÎ÷°õÓûÍì÷»ìæõ°÷ÕûìçƗ °ā°°÷ÃæðçƴûƗÁƗìÍāÓ÷ÃÃ
āûÓçÕ»°â÷ÃõâÕ»°āÃûƗdƘqÃûāÃ÷çºâìāìÍąç°ÍÍûāÃÁõ°÷Ãçā»ÃââûƘąçā÷ðāÃÁ
õ°āÕÃçāŴÁÃ÷ÕĐÃÁ»Ãââûì÷õ°āÕÃçāŴÁÃ÷ÕĐÃÁ»ÃââûŢŠÁ°ėû°ÍāÃ÷”âÃçāÕĐÕ÷°â
ā÷ðāæÃçāąûÕçÎāÓÃ>LBŢ°çāÕºìÁėûõûÕÍÕ»Íì÷Óąæ°çâ°æÕçƘõ÷ìÎÃ÷Õç°çÁ
â°æÕçƗìæõâÃāúâìāûđÕāÓæìâûąâ°÷đÃÕÎÓāæ°÷àÃ÷û°÷Ã°Đ°Õâ°ºâÃÕç
[ąõõâÃæÃçā°÷ė,ÕÎƗšƗÁÁÕāÕìç°âÕçÁÃõÃçÁÃçāºÕìâìÎÕ»°â÷ÃõâÕ»°āÃû°÷Ã
õ÷ìĐÕÁÃÁÕç”ĖāÃçÁÃÁ °ā°,ÕÎƗšƗeƘGą»âð÷æì÷õÓìâìÎėìÍąç°ÍÍûāÃÁõ°÷Ãçā
»ÃââûƘąçā÷ðāÃÁ2- ,GšŦŧ»Ãââûì÷2- ,GšŦŧ»ÃââûŢŠÁ°ėû°ÍāÃ÷ā÷ðāæÃçā
đÕāÓ”ŴÃĖõ÷ÃûûÕçÎâÃçāÕĐÕ÷ąûƘûā°ÕçÃÁđÕāÓ°â°æÕçŴŴûõûÕÍÕ»°çāÕºìÁėƘ°
õ÷ìÎÃ÷ÕçŴûõûÕÍÕ»°çāÕºìÁėì÷ U4Ɨ[»°âú°÷ûƘŢŠŁæƗÁÁÕāÕìç°â÷ÃõâÕ»°āÃû
đÃ÷ÃçìāõÃ÷Íì÷æÃÁƗf, Frequency of morphologically abnormal nuclei in
samples of cells shown in eƗ °ā°°÷ÃæðçƴûƗÁƗÍ÷ìæāÓ÷ÃûìąçāûìÍ
independent images from the experiment in eƗƓPƸŠƗŠťƘƓƓPƸŠƗŠšƘƓƓƓPƸŠƗŠŠšƘ
ƓƓƓƓPƸŠƗŠŠŠšºė[āąÁÃçāƈûąçõ°Õ÷ÃÁāđìŴûÕÁÃÁtŴāÃûāƗ
insertion and deletion (indel) products at the target locus together
đÕāÓāÓÃ÷ÕûàìÍÁÕû÷ąõāÕçÎāÓÃđÕâÁŴāėõÃLMNA allele, which differs only
at a single base pair from the pathogenic allelešũ,ŢŠ, pose challenges
āì»âÕçÕ»°âā÷°çûâ°āÕìçìÍÎÃçÃÁÕû÷ąõāÕìçûā÷°āÃÎÕÃûāìā÷ðāõ÷ìÎÃ÷Õ°Ɨ
°ûÃÃÁÕāì÷û°÷ÃÎÃçìæÃÃÁÕāÕçΰÎÃçāûāÓ°āÁÕ÷ûāâė»ìçĐÃ÷āā°÷ÎÃāÃÁº°ûÃõ°Õ÷ûđÕāÓìąāæ°àÕçÎÁìąºâÃŴûā÷°çÁ Gº÷ðàûŦƗėāìûÕçÃ
º°ûÃÃÁÕāì÷ûŷ”ûŸŢš»ìçĐÃ÷āƫ-āìaƫƘđÓÃ÷ðû°ÁÃçÕçú°ûÃÃÁÕāì÷û
ŷ”ûŸ5»ìçĐÃ÷āƫaāì-ƫƗ°ûÃÃÁÕāì÷ûÍąç»āÕìçÕçæ°çėæÕāìāÕ»°çÁ
post-mitotic cell types and in a wide array of organismsŦƗ”ûąûð
laboratory-evolved deoxyadenosine deaminase to convert adenine to
ÕçìûÕçÃŷđÓÕ»Óº°ûÃõ°Õ÷ûâÕàÃÎą°çÕçßđÕāÓÕç°ûæ°ââđÕçÁìđìÍ°÷ìąçÁ
Ťŵťçą»âÃìāÕÁÃû°ā°°ûŴõ÷ìāÃÕçŴûõûÕÍÕÃÁâì»ąûƘ°çÁÕçÁą»ÃāÓûÃââ
āì÷Ãõâ°»ÃāÓûìæõâÃæÃçā°÷ėāÓėæÕçÃđÕāÓ»ėāìûÕçúėçÕ»àÕçΰçÁ
stimulating repair of the non-edited strand5,ŦƗ
2Ã÷ÃđÃ÷Ãõì÷ā”ŴæÃÁÕ°āÃÁ»ì÷÷ûāÕìçìÍāÓÃLMNA»ƗšŨŢŤƹa
mutation in fibroblasts derived from children with HGPS and in a mouse
model in which mice contain two genomically integrated copies of the
human LMNA»ƗšŨŢŤƹaõ÷ìÎÃ÷Õ°°ââÃâÃŢŢƗ4绹âāą÷ÃÁõ°āÕÃçāŴÁÃ÷ÕĐÃÁ
»ÃââûƘđÃìºûÃ÷ĐÃÁÃÍÍÕ»ÕÃçāŷ°÷ìąçÁũŠƂŸÎÃçìæÕ» G»ì÷÷ûāÕìçāÓ°ā
ameliorates pathogenic mis-splicing of the LMNA transcript, reduces
the abundance of progerin protein and restores normal nuclear morõÓìâìÎėƗqÓÃçÁÃâÕĐÃ÷ÃÁÕçāì°æìąûÃæìÁÃâìÍÓąæ°çõ÷ìÎÃ÷Õ°ºė
single retro-orbital injection of therapeutically relevant doses of
pũÃç»ìÁÕçÎāÓÔ°çÁûÕçÎâÃŴÎąÕÁÃWGŷûÎWGŸƘāÓÔ»ì÷rected the LMNA»ƗšŨŢŤƹa°ââÃâÃÕçĐ°÷ÕìąûāÕûûąÃû°āāÓà GƘWG
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notable improvement in vascular disease compared to saline-injected
»ìçā÷ìâûƘđÕāÓ°ì÷āÕ»p[F»ìąçāû°çÁ°ÁĐÃçāÕāÕ°âÍÕº÷ìûÕûÕçÁÕûāÕçguishable from those of wild-type mice, as well as reduced numbers of
õ÷ìÎÃ÷ÕçŴõìûÕāÕĐÃp[Fû°çÁÕç»÷ðûÃÁçąæºÃ÷ûìÍâ°æÕçì÷â°æÕç
ŷâ°æÕçƠŸŴõìûÕāÕĐÃp[FûƗaÓÃæÃÁÕ°çâÕÍÃûõ°çìÍ”Ŵā÷ðāÃÁæÕ»Ã
đÕāÓõ÷ìÎÃ÷Õ°đ°ûąõāìŢƗŤŴÍìâÁâìçÎÃ÷āÓ°çāÓ°āìÍû°âÕçÃŴÕçÞûāÃÁ»ìçā÷ìâûƗaÓÃûÃÍÕçÁÕçÎûûąÎÎÃûā°õìāÃçāÕ°âāÓÃ÷°õÃąāÕ»ûā÷°āÃÎėÍì÷2-U[
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ABE corrects the HGPS mutation in patient cells
aìõìûÕāÕìçāÓÃõ°āÓìÎÃçÕ»LMNA»ƗšŨŢŤƹaæąā°āÕìçđÕāÓÕçāÓð»āÕĐÕāėđÕçÁìđìÍ°ç”5ŷõìûÕāÕìçûŤŵŧƘđÓÃ÷ÃāÓÃõ÷ìāìûõ°»Ã÷Ŵ°ÁÞ°»Ãçā
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Nature | Vol 589 | 28 January 2021 | 609
80
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changes after treating fibroblasts derived from patients with progeria
with ABEmax-VRQR. aƘ GûÃöąÃç»ÕçÎÍì÷āÓÃāìõţŢ4WB”ŴûÃöŴÕÁÃçāÕÍÕÃÁŢŦ
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»ÃââûƘąçā÷ðāÃÁõ°āÕÃçāŴÁÃ÷ÕĐÃÁ»Ãââû°çÁõ°āÕÃçāŴÁÃ÷ÕĐÃÁ»ÃââûšŠì÷ŢŠÁ°ėû
°ÍāÃ÷ā÷ðāæÃçāđÕāÓ”ŴÃĖõ÷ÃûûÕçÎâÃçāÕĐÕ÷ąûƗ °ā°°÷ÃæðçƴûƗÁƗìÍāÓ÷ÃÃ
āûÓçÕ»°â÷ÃõâÕ»°āÃûƗd, Heat map of zŴû»ì÷ÃûÍì÷āÓÃāìõšŠŠÁÕÍÍÃ÷ÃçāÕ°ââė
expressed genes between unaffected control fibroblasts (obtained from the
ì÷ÕÃââÃââ°çàŷì÷ÕÃâ⟰çÁÍ÷ìæ°õ÷ÃĐÕìąûâėõąºâÕûÓÃÁÁ°ā°ûÃāŤŧŷF°āÃìûŸƚ
FÃāÓìÁûŸ°çÁąçā÷ðāÃÁì÷âÃçāÕĐÕ÷°âŴ”Ŵā÷ðāÃÁõ°āÕÃçāŴÁÃ÷ÕĐÃÁ»ÃââûƗ
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ÁÕÍÍÃ÷ÃçāÕ°ââėÃĖõ÷ÃûûÃÁÎÃçÃûÕçđÕâÁŴāėõÃŷqaŸ»ÃââûŷF°āÃìûŤŧ, Coriell and
ąç°ÍÍûāÃÁõ°÷ÃçāŸƘÁÕûðûÃÁ»Ãââûŷąçā÷ðāÃÁ2- ,GšŦŧ°çÁ2- ,GšŨŨŸ
°çÁā÷ðāÃÁ»ÃââûŷâÃçāÕĐÕ÷°âŴ”Ŵā÷ðāÃÁ2- ,GšŦŧ°çÁ2- ,GšŨŨ°āšŠ°çÁ
ŢŠÁ°ėûŸƗÓðāæ°õìÍâìÎ ŢŴā÷°çûÍì÷æÃÁ, WĐ°âąÃûÍì÷āÓÃûÚũÎÃçÃûÃāûÕû
shown, with overexpressed gene sets in red and underexpressed gene sets in
ºâąÃƗUâ°ûƗæÃæƗƘõâ°ûæ°æÃæº÷°çÃƚ÷ÃÎƗƘ÷ÃÎąâ°āÕìçƗ
°āšŠÁ°ėûƘ°çÁŨŧƂ°çÁũšƂ»ì÷÷ûāÕìç°āŢŠÁ°ėûƘ÷ÃûõûāÕĐÃâėŷ,ÕÎƗšºŸƗ
âìđÍ÷ÃöąÃç»ėŷšƗšŵŢƗŢƂŸìͺėûā°çÁÃ÷ÃÁÕāÕçÎđ°ûìºûÃ÷ĐÃÁ°āāÓÃƫa°ā
õ÷ìāìûõ°»Ã÷õìûÕāÕì皊ƘđÓÕ»Ó÷ÃûąâāûÕçpŦũŠŷ”ĖāÃçÁÃÁ °ā°,ÕÎƗš°ŸƗ
4çÁÃâÍ÷ÃöąÃç»ÕÃûđÃ÷ÃæÕçÕæ°âŷŠƗšťƂì÷âìđÃ÷ŸÍì÷ºìāÓ»ÃâââÕçÃûŷ”ĖāÃçÁÃÁ
°ā°,ÕÎƗšºŸƗaÓÃûÃ÷ÃûąâāûÕçÁÕ»°āÃāÓ°ā°ç”»°çÃÍÍÕ»ÕÃçāâė»ì÷÷ûāāÓÃ
2-U[æąā°āÕìçāìđÕâÁāėõÃđÕāÓÍÃđÃÁÕāÕçκėŴõ÷ìÁą»āû°āāÓÃā°÷ÎÃāâì»ąûƗ
Consistent with genomic correction of LMNA»ƗšŨŢŤƹaƘđÃìºûÃ÷ĐÃÁ
°çŨƗšŴÍìâÁ°çÁ°ŤƗŤŴÍìâÁ÷ÃÁą»āÕìçÕçāÓÃâÃĐÃâûìÍæÕûŴûõâÕ»ÃÁLMNA
æWGÕç”ŴâÃçāÕĐÕ÷ąûŴā÷°çûÁą»ÃÁ2- ,GšŦŧ°çÁ2- ,GšŨŨ»ÃââûƘ
÷ÃûõûāÕĐÃâėƘŢŠÁ°ėû°ÍāÃ÷ā÷ðāæÃçāƘ»ìæõ°÷ÃÁāìąçā÷ðāÃÁ»Ãââû
ŷ,ÕÎƗš»ŸƗ”ā÷ðāæÃçā°âûì÷ÃÁą»ÃÁāÓÃâÃĐÃâûìÍõ÷ìÎÃ÷Õçõ÷ìāÃÕçºė
ŦƗšŴ°çÁšťŴÍìâÁƘ÷ÃûõûāÕĐÃâėƘ÷Ãâ°āÕĐÃāìąçā÷ðāÃÁ»ÃââûƘ°çÁæìÁÃûāâė
Õç»÷ðûÃÁâ°æÕç°ºąçÁ°ç»Ãŷ,ÕÎƗšÁŸƗGą»âð÷æì÷õÓìâìÎėÕæõ÷ìĐÃÁ
Õç”Ŵā÷ðāÃÁ»ÃââûƘđÓÕ»ÓÓ°ÁšƗŨŴÍìâÁÍÃđÃ÷°ºçì÷æ°âçą»âÃÕ»ìæõ°÷ÃÁāìąçā÷ðāÃÁ»Ãââûŷ,ÕÎƗšÃƘÍƘ”ĖāÃçÁÃÁ °ā°,ÕÎƗš»ŵßƗaìÎÃāÓÃ÷Ƙ
these results show that base editing to correct the LMNA»ƗšŨŢŤƹa
mutation in cells derived from patients with HGPS rescues the molecuâ°÷°çÁõÓÃçìāėõÕ»»ìçûÃöąÃç»ÃûìÍāÓÃæąā°āÕìçƗ
WGÃÁÕāÕçÎŦƗ°ûŴÕçÁÃõÃçÁÃçāìÍÍŴā°÷ÎÃā GÃÁÕāÕçκė”ŧƗšŠ
has been reported to be minimal or undetectableŦƗaìÕÁÃçāÕÍė»°çÁÕÁ°āðûŴÁÃõÃçÁÃçāìÍÍŴā°÷ÎÃā GÃÁÕāÕçÎûÕāÃû°ûûì»Õ°āÃÁđÕāÓāÓÃ
ûÎWG°çÁ°ûũŴpWVWĐ°÷Õ°çāąûÃÁāì»ì÷÷ûāāÓÃ2-U[æąā°āÕìçƘ
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LMNAŴā°÷ÎÃāÕçÎûÎWGŷ”ĖāÃçÁÃÁ °ā°,ÕÎƗŢŸƗqÃõÃ÷Íì÷æÃÁā°÷ÎÃāÃÁ
ûÃöąÃç»ÕçÎìÍÎÃçìæÕ» G°āāÓÃāìõţŢ»°çÁÕÁ°āÃìÍÍŴā°÷ÎÃāâì»Õ
ÕÁÃçāÕÍÕÃÁºė4WB”ŴûÃöÕç2- ,GšŦŧ°çÁ2- ,GšŨŨ»ÃââûŢŠÁ°ėû
°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
Ãç»ìÁÕçÎûõâÕāŴÕçāÃÕç”ŧƗšŠæ°ĖŴpWVWº°ûÃÃÁÕāì÷Ó°âĐÃûŢŨ and the
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
[ÃÔĖāÃçÁÃÁ °ā°,ÕÎƗŤÍì÷°ÁÁÕāÕìç°âÁ°ā°ƗdƘqÃûāÃ÷çºâìā°ç°âėûÕûìÍÓąæ°ç
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
“ĖāÃçÁÃÁ °ā°,ÕÎƗťÍì÷°ÁÁÕāÕìç°âÁ°ā°Ɨ °ā°°÷ÃæðçƴûƗÁƗÍì÷āÓÃÕçÁÕ»°āÃÁ
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
°æìąûÃæìÁÃâìÍõ÷ìÎÃ÷Õ°ƗqÃąûÃÁťŧBƠŦæÕ»ÃÓìæìĜėÎìąûÍì÷°
transgene that includes the complete human LMNA»ƗšŨŢŤƹa°ââÃâÃ
ŷťŧBƠŦŴāÎŷBFGƓ-ŦŠŨ-Ÿ2âçûƠ>Ƙõ÷ÃĐÕìąûâėąûÃÁ°ûÓÃāÃ÷ìĜėÎìąû
miceŢŢŸƚāÓÃûÃÓìæìĜėÎìąûæÕ»ÃÓÃ÷ðÍāÃ÷°÷Ã÷ÃÍÃ÷÷ÃÁāì°ûƇõ÷ìÎÃ÷Õ°
æÕ»ÃƈƗUÓÃçìāėõÕ»°ââėƘāÓÕûæìÁÃâ÷û°õÕāąâ°āÃûÓ°ââæ°÷àûėæõāìæû
ìÍ2-U[Õç»âąÁÕçÎp[FÁÃÍûāûƘÓ°Õ÷âìûûƘâ°»àìÍûąº»ąā°çÃìąûÍ°āƘ
æąû»ąâìûàÃâÃā°â°ºçì÷æ°âÕāÕÃû°çÁûÓì÷āÃçÃÁâÕÍÃûõ°ç3,Ť,ŢŢƗ-ÕĐÃçāÓÃ
ÁÕĐÃ÷ûÃāÕûûąÃû°ÍÍûāÃÁºėõ÷ìÎÃ÷Õ°ƘđÃûìąÎÓāûėûāÃæÕ»ÕçĐÕĐìÁÃâÕĐÃ÷ė
ìÍāÓÔ°çÁûÎWG»Ó°÷°»āÃ÷ÕĜÃÁ°ºìĐÃƗ
qÃ÷ûÃçāâėÁÃĐÃâìõÃÁ°ûā÷°āÃÎėÍì÷º°ûÃÃÁÕāì÷ÁÃâÕĐÃ÷ėÕçĐÕĐìąûÕçÎ
°ÁÃçìŴ°ûûì»Õ°āÃÁĐÕ÷ąûŷpŸŢŨ, a delivery modality that is approved
ºėāÓÃe[,ììÁ°çÁ ÷ąÎÁæÕçÕûā÷°āÕìçŷ, ŸƗaÓÕû°õõ÷ì°»ÓąûÃû
trans-splicing inteins to reconstitute the full-length base editor in cells
Í÷ìæ°õ°Õ÷ìÍpûƘð»ÓÃĖõ÷ÃûûÕçÎìçÃÓ°âÍìÍāÓú°ûÃÃÁÕāì÷ŢŨ,Ţũ
ŷ,ÕÎƗ3aŸƗqðÁ°õāÃÁāÓÕûûėûāÃæāìÁÃâÕĐÃ÷”æ°ĖŴpWVW°çÁāÓÃLMNA
»ƗšŨŢŤŴā°÷ÎÃāÕçÎûÎWGƗqûÓìûÃāÓÃpũ»°õûÕÁÍì÷Õāûº÷ì°ÁāÕûûąÃ
tropism, clinical validation and ability to transduce progeria-relevant
tissues including heart and muscleţŠ,ţšƗ
aì»ìæõ°÷ÃāÓÃÃÍÍûāûìÍÕçÞûāÕìç÷ìąāðçÁāÕæÕçÎìçÕçĐÕĐìÃÁÕāing, we performed retro-orbital injection of P3 (3-day-old) mice (nƷŤŸ
°çÁUšŤŷŢŴđÃÃàŴìâÁŸæÕ»ÃŷnƷťŸƘ°çÁÕçā÷°õÃ÷ÕāìçðâÕçÞûāÕìçìÍUšŤ
mice (nƷťŸƗUţÕçÞûāÕìçûąûÃÁťƶšŠšŠĐÕ÷°âÎÃçìæÃûŷĐΟìÍð»Óp
Íì÷°āìā°âìÍšƶšŠššĐÎõÃ÷æìąûÃƗìāÓUšŤÕçÞûāÕìçûąûÃÁťƶšŠššĐÎ
ìÍð»ÓpÍì÷°āìā°âÕçÞûāÕìçìÍšƶšŠšŢĐÎõÃ÷æìąûÃƗFÕ»ÃđÃ÷Ã
ÃąāÓ°çÕĜÃÁ°āāÓðÎÃìÍûÕĖđÃÃàû°çÁÃÁÕāÕçÎđ°ûÃĐ°âą°āÃÁÕçĐ°÷Õìąû
āÕûûąÃûŷ,ÕÎƗ3Ƙ”ĖāÃçÁÃÁ °ā°,ÕÎƗţ°ŵ»ŸƗ
āûÕĖđÃÃàûìÍ°ÎÃƘUšŤ÷Ãā÷ìŴì÷ºÕā°âÕçÞûāÕìç÷ÃûąâāÃÁÕçāÓÃÓÕÎÓest editing efficiencies in aorta and bone among the tested injection
÷ìąāÃûŷ,ÕÎƗ3bƘ”ĖāÃçÁÃÁ °ā°,ÕÎƗţ°ŸƗ”ÁÕāÕçÎÃÍÍÕ»ÕÃç»ÕÃûÕçºąâàÓð÷ā
āÕûûąÃŷÃĖ»âąÁÕçΰì÷ā°ŸđÃ÷ÃûÕæÕâ°÷Íì÷āÓÃāÓ÷ÃÃÕçÞûāÕìç÷ìąāÃûƗUšŤ
injections generally achieved higher base-editing efficiencies than P3
ÕçÞûāÕìçûƘõìûûÕºâėìđÕçÎāìāÓÃāÃçÍìâÁÓÕÎÓÃ÷ÁìûÃìÍpāÓ°ā»ìąâÁ
ºÃÕçÞûāÃÁÕçāìUšŤæÕ»Ãì÷Õç»÷ðûÃÁÃĖõ÷ÃûûÕìçìÍpũ÷ûÃõāì÷û
in the older miceŢŨ,ţŢ,33ƗaìÎÃāÓÃ÷ƘāÓÃûÃÁ°ā°÷ÃĐðâāÓ°ā°ûÕçÎâÃÕçĐÕĐì
ÕçÞûāÕìçìÍ”ŴÃç»ìÁÕçÎp÷ÃûąâāûÕçæìÁÃûāāìÓÕÎÓâÃĐÃâûìÍ»ì÷÷ûāÕìçŷšŠŵŦŠƂŸìÍāÓû°ąû°āÕĐÃLMNAõìÕçāæąā°āÕìçÕçĐ°÷Õìąûì÷ΰçûƗ
Long-term ABE treatment of progeria mice
qÃõÃ÷Íì÷æÃÁâìçÎŴāÃ÷æûāąÁÕÃûÕçæÕ»ÃąûÕçκìāÓUţ°çÁUšŤ
÷Ãā÷ìŴì÷ºÕā°â p ÕçÞûāÕìçû āì °ûûÃûû āÓà ÷Ãâ°āÕìçûÓÕõ ºÃāđÃÃç
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āÕûûąÃûûą»Ó°ûāÓðì÷ā°ƘÕçđÓÕ»ÓÃÁÕāÕçÎâÃĐÃâûÕçUšŤŴÕçÞûāÃÁæÕ»Ã
đÃ÷ÃţƗŨŴÍìâÁÓÕÎÓÃ÷÷Ãâ°āÕĐÃāìUţŴÕçÞûāÃÁæջðāûÕĖđÃÃàûìÍ°ÎÃƗ
qÃ÷Ãā÷ìŴì÷ºÕā°ââėÕçÞûāÃÁŢŤõ÷ìÎÃ÷Õ°æջðāUţđÕāÓšŠšš total vg of
Áą°âpũƘ°çÁŢŤæջðāUšŤđÕāÓšŠšŢāìā°âĐΰûºÃÍì÷ÃƗû»ìçā÷ìâûƘ
ŢŤæջðāUţ°çÁŢŤæջðāUšŤđÃ÷ÃÕçÞûāÃÁ÷Ãā÷ìŴì÷ºÕā°ââėđÕāÓ
û°âÕçÃƗââÎ÷ìąõû»ìçā°ÕçÃÁÃöą°âçąæºÃ÷ûìÍæ°âðçÁÍÃæ°âÃæÕ»ÃƗā
ŦæìçāÓûìÍ°ÎÃƘđÓÃçąçā÷ðāÃÁõ÷ìÎÃ÷Õ°æÕ»ÃāėõÕ»°ââėûÓìđõÓÃçìāėõÕ»ÁûâÕçúąā°÷ÃçìāėÃā°āāÓÃÃçÁìÍāÓÃÕ÷âÕÍÃûõ°çƘŢŤUţŴÕçÞûāÃÁ
æջðçÁŢŤUšŤŴÕçÞûāÃÁæÕ»ÃŷÓ°âÍpũŴā÷ðāÃÁƘÓ°âÍû°âÕçûìçā÷ìâûŸ
đÃ÷ÃÃąāÓ°çÕĜÃÁ°çÁ°ç°âėûÃÁÍì÷ Gº°ûÃŴÃÁÕāÕçÎÃÍÍÕ»ÕÃç»ėƘLMNA
WGûõâÕ»ÕçÎƘâÃĐÃâûìÍÓąæ°çõ÷ìÎÃ÷Õç°çÁâ°æÕçõ÷ìāÃÕçûƘ°çÁāÕûûąÃÓÕûāìâìÎėƗqÃõâ°»ÃÁāÓÃ÷Ãæ°ÕçÕçÎŢŤUţŴÕçÞûāÃÁæջðçÁŢŤ
UšŤŴÕçÞûāÃÁæÕ»ÃŷÓ°âÍpũŴā÷ðāÃÁƘÓ°âÍû°âÕçûìçā÷ìâûŸÕç°âìçÎÃĐÕāė
ûāąÁėāì°ûûÃûûâÕÍÃûõ°çƗ
DNA, RNA and protein analysis at six months
ç°âėûÕûìÍāÓà Gº°ûÃŴÃÁÕāÕçÎìąā»ìæÃûÕçûÕĖŴæìçāÓŴìâÁæÕ»Ã
÷ÃĐðâÃÁçìā°ºâÃÁÕÍÍÃ÷Ãç»ÃûºÃāđÃÃçāÓÃUţŴ°çÁUšŤŴÕçÞûāÃÁ»ìÓì÷āûƗ
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
Males
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WT
Adventitial area (mm2)
0.2 0.4 0.6 0.8 1.0
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1,000 2,000 3,000 4,000
P14 injection
WT
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WT
ABE-AAV9
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c
Fraction survival
0.25
100
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(6 months old) (6 months old)
Lamin A/C
+ DAPI
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0.75
0
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(6 months old)
Males
Saline
ABE AAV9
1.00
Fraction survival
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H&E
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d
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200
Age (d)
300
400
0.75
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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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Meier curve for mice that were retro-orbitally injected
with saline (nƷšŢŸì÷”ŴpũŷnƷššŸ°āUţƗFÃÁÕ°ç
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PƸŠƗŠŠŠšŸƗe, Kaplan–Meier curve for mice that were
retro-orbitally injected with saline (nƷšŢŸì÷”Ŵpũ
(nƷšŠŸ°āUšŤƗFÃÁÕ°çâÕÍÃûõ°çûƙû°âÕçÃŴÕçÞûāÃÁæÕ»ÃƘ
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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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Óð÷āÃĖ»âąÁÕçΰì÷ā°ŷŨŦƴũƗšƂ÷ÃÁą»āÕì矰çÁ°ì÷ā°ŷŤũƴšũƂ÷ÃÁą»āÕì矻ìæõ°÷ÃÁāìû°âÕçÃŴÕçÞûāÃÁ»ìçā÷ìâûŷ,ÕÎƗ3dƘ”ĖāÃçÁÃÁ °ā°
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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
õ÷ìÎÃ÷ÕçWG°çÁõ÷ìāÃÕçâÃĐÃâûÕçûÃĐÃ÷°â»âÕçÕ»°ââė÷ÃâÃĐ°çāāÕûûąÃûƗ
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°
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mice prompted us to examine the protein levels of human progerin
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of patients with progeriaŤƗ”ā÷ðāæÃçāæìÁÃûāâė÷Ãû»ąÃÁāÓÃâìûûìÍ
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and saline-injected mice exhibited moderate dermal hypoplasia comõ°÷ÃÁāìđÕâÁŴāėõÃťŧBƠŦæÕ»Ãŷ”ĖāÃçÁÃÁ °ā°,ÕÎûƗũƘšŠŸƗ
ABE treatment extends progeria mouse lifespan
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revealed gastrointestinal necrosis in one, liver tumours in five and no
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the potential origins of the liver tumours, we performed whole-genome
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