.2021 Mar;27(3):536-545.

doi: 10.1038/s41591-021-01274-0. Epub 2021 Mar 11.

A targeted antisense therapeutic approach for Hutchinson-Gilford progeria syndrome

Michael R Erdos^#¹, Wayne A Cabral^#¹, Urraca L Tavarez¹, Kan Cao², Jelena Gvozdenovic-Jeremic¹, Narisu Narisu¹, Patricia M Zerfas³, Stacy Crumley⁴, Yoseph Boku¹, Gunnar Hanson⁴, Dan V Mourich⁵, Ryszard Kole^{4 6}, Michael A Eckhaus³, Leslie B Gordon^{7 8}, Francis S Collins⁹

Affiliations

¹ Molecular Genetics Section, Medical Genomics and Metabolic Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA.
² Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, USA.
³ Diagnostic and Research Services Branch, Division of Veterinary Resources, Office of Research Services, National Institutes of Health, Bethesda, MD, USA.
⁴ Sarepta Therapeutics, Inc., Cambridge, MA, USA.
⁵ Department of Veterinary Health Science, Oregon State University, Corvallis, OR, USA.
⁶ Department of Pharmacology, University of North Carolina, Chapel Hill, NC, USA.
⁷ Department of Pediatrics, Hasbro Children's Hospital and Warren Alpert Medical School of Brown University, Providence, RI, USA.
⁸ Department of Anesthesiology, Preoperative and Pain Medicine, Boston Children's Hospital, Boston, MA, USA.
⁹ Molecular Genetics Section, Medical Genomics and Metabolic Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA. collinsf@mail.nih.gov.

^# Contributed equally.

PMID:33707773
PMCID: PMC10158310
DOI: 10.1038/s41591-021-01274-0

A targeted antisense therapeutic approach for Hutchinson-Gilford progeria syndrome

Michael R Erdos et al. Nat Med.2021 Mar.

.2021 Mar;27(3):536-545.

doi: 10.1038/s41591-021-01274-0. Epub 2021 Mar 11.

Authors

Affiliations

¹ Molecular Genetics Section, Medical Genomics and Metabolic Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA.
² Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, USA.
³ Diagnostic and Research Services Branch, Division of Veterinary Resources, Office of Research Services, National Institutes of Health, Bethesda, MD, USA.
⁴ Sarepta Therapeutics, Inc., Cambridge, MA, USA.
⁵ Department of Veterinary Health Science, Oregon State University, Corvallis, OR, USA.
⁶ Department of Pharmacology, University of North Carolina, Chapel Hill, NC, USA.
⁷ Department of Pediatrics, Hasbro Children's Hospital and Warren Alpert Medical School of Brown University, Providence, RI, USA.
⁸ Department of Anesthesiology, Preoperative and Pain Medicine, Boston Children's Hospital, Boston, MA, USA.
⁹ Molecular Genetics Section, Medical Genomics and Metabolic Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA. collinsf@mail.nih.gov.

^# Contributed equally.

PMID:33707773
PMCID: PMC10158310
DOI: 10.1038/s41591-021-01274-0

Abstract

Hutchinson-Gilford progeria syndrome (HGPS) is a rare accelerated aging disorder characterized by premature death from myocardial infarction or stroke. It is caused by de novo single-nucleotide mutations in the LMNA gene that activate a cryptic splice donor site, resulting in the production of a toxic form of lamin A, which is termed progerin. Here we present a potential genetic therapeutic strategy that utilizes antisense peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) to block pathogenic splicing of mutant transcripts. Of several candidates, PPMO SRP-2001 provided the most significant decrease in progerin transcripts in patient fibroblasts. Intravenous delivery of SRP-2001 to a transgenic mouse model of HGPS produced significant reduction of progerin transcripts in the aorta, a particularly critical target tissue in HGPS. Long-term continuous treatment with SRP-2001 yielded a 61.6% increase in lifespan and rescue of vascular smooth muscle cell loss in large arteries. These results provide a rationale for proceeding to human trials.

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Conflict of interest statement

Competing interests

The authors declare no competing interests.

Figures

**Extended Data Fig. 1 |. PPMO structure.**
SRP-2001 is a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) comprising a cell-penetrating peptide (CPP) covalently linked to a phosphorodiamidate morpholino oligomer (PMO). The PMO contains 25 morpholino subunits each bearing a nucleobase forming the sequence 5’-GAGGAGATGGGTCCACCCACCTGGG-3’. The conjugated peptide comprises the amino acid sequence R₆G, where R is arginine and G is glycine. The glycine residue covalently links the CPP to the PMO by an amide bound to the 3’ end of the PMO. Mass spectrometric characterization was performed to confirm the structure and sequence of SRP-2001.

**Extended Data Fig. 2 |. Quantitative PCR screening of candidate PPMOs for targeted reduction of progerin in proband fibroblasts.**
Quantitation ofLMNA (A),*Progerin* (P), andLMNC (C) expression by ddPCR analysis of Classic HGPS (*LMNA* c.1824C > T) fibroblasts treated with 6uM candidate PPMOs.a, Transcript levels relative toTFRC.b, Each isoforms’ fraction relative to allLMNA transcripts.c, Each isoforms’ transcript level relative toLMNC transcripts. The greatest reduction of progerin transcripts in culture was achieved with the centrally located morpholino (SRP-2001). A,*LMNA*; P,*Progerin*; C,*LMNC*; WT, oligo complementary to normalLMNA sequence; CTRL, scrambled control; Values and error bars represent mean ± SD determined from triplicate reactions of biological triplicates. * p < 0.05, ** p < 0.01, *** p < 0.001 versus transcripts or protein from cells receiving saline (NT).

**Extended Data Fig. 3 |. Western immunoblot screening of candidate PPMOs for targeted reduction of progerin in proband fibroblasts.**
Quantitation of LMNA (A), Progerin (P), and LMNC (C) protein from immunoblots of lysates from Classic HGPS (*LMNA* c.1824C > T) fibroblasts treated with 6uM candidate PPMOs. Data was derived from the analysis shown in Fig. 1d and is expressed as isoform levels relative to ACTB (a), the isoforms’ fraction relative to allLMNA gene products (b), and the isoforms’ level relative to LMNC protein (c). The greatest reduction of progerin protein in culture was achieved with the centrally located morpholino (SRP-2001). A, LMNA; P, progerin; C, LMNC; WT, oligo complementary to normalLMNA sequence; CTRL, scrambled control; Values and error bars represent mean ± SD determined from biological triplicates shown in Fig. 1d. * p < 0.05, ** p < 0.01, *** p < 0.001 versus transcripts or protein from cells receiving saline NT).

**Extended Data Fig. 4 |. Quantitative PCR screening of SRP-2001 for targeted reduction of progerin in Classic and Non-classic HGPS cell lines.**
a, Transcript copy number in normal control (NL Control), Classic HGPS (c.1824C>T), and Non-classic HGPS (c.1822G>A, c.1968+1G>A, c.1968+2T>C, c.1968+5G>C) fibroblasts following 2 weeks of treatment with SRP-2001.b, IndividualLMNA transcripts expressed as a fraction of all totalLMNA-derived transcripts in each cell line.c, Full-lengthLMNA and progerin transcript levels relative toLMNC transcripts. A,*LMNA*; P,*progerin*; C,*LMNC*; Values and error bars represent mean ± SD determined from triplicate reactions of biological triplicates. * p < 0.05; ** p < 0.01; *** p < 0.001 vs same transcript in cells receiving saline only (NT).

**Extended Data Fig. 5 |. Reduction of progerin protein by SRP-2001 in Classic and Non-classic HGPS cell lines.**
a, Western immunoblots of normal control (NL Control), Classic HGPS (c.1824C>T), and Non-classic HGPS (c.1822G>A, c.1968+1G>A, c.1968+2T>C, c.1968+5G>C) fibroblasts following 2 weeks of treatment with SRP-2001.b, Quantitation ofLMNA gene products relative to ACTB.c, Quantitation of LMNA, progerin and LMNC isoforms expressed as a fraction of all totalLMNA-derived gene products in each cell line.d, Full-length LMNA and progerin protein levels relative to LMNC. A, LMNA; P, progerin; C, LMNC; Graphs represent mean ± SD determined from biological triplicates shown in panel A. * p < 0.05; ** p < 0.01; *** p < 0.001 vs same protein isoform in cells receiving saline only (NT).

**Extended Data Fig. 6 |. SRP-2001 reduces expression of transgene-derived progerin in murine aortas.**
a, Quantitative PCR analysis of transgene transcript levels in aortas ofLMNA^G/G mice receiving long-term treatment with saline (vehicle) or 60 mg/kg SRP-2001. Transcripts are quantitated relative toHprt expression, as a fraction of all transgene-derivedLMNA transcripts or relative to transgene-derivedLMNC transcripts. Data are presented as mean values ± SD determined from 12 independent samples per treatment group (n = 6 males, 6 females).b, Western immunoblots of A-type lamins and beta actin (ACTB) immunoprecipitated from aorta tissue.c, Quantitation of A-type lamins from immunoblots expressed relative to beta actin (ACTB), as a fraction of all A-type lamins and relative to LMNC. Data are presented as mean values ± SD determined from 12 independent samples per treatment group (n = 6 males, 6 females), shown in panel b. A, LMNA; P, progerin; C, LMNC; * p < 0.05; ** p < 0.01; *** p < 0.001 vs same transcript or protein in mice receiving saline only (vehicle).

**Extended Data Fig. 7 |. SRP-2001 reduces expression of transgene-derived progerin in murine hearts.**
a, Quantitative PCR analysis of transgene transcript levels in hearts ofLMNA^G/G mice receiving long-term treatment with saline (vehicle) or 60 mg/kg SRP-2001. Transcripts are quantitated relative toHprt expression, as a fraction of all transgene-derivedLMNA transcripts or relative to transgene-derivedLMNC transcripts. Data are presented as mean values ± SD determined from 12 independent samples per treatment group (n = 6 males, 6 females).b, Western immunoblots of A-type lamins and smooth muscle actin (SMA) immunoprecipitated from heart tissue.c, Quantitation of A-type lamins from immunoblots expressed relative to smooth muscle actin (SMA), as a fraction of all A-type lamins and relative to LMNC. Data are presented as mean values ± SD determined from 12 independent samples per treatment group (n = 6 males, 6 females), shown in panel b. A, LMNA; P, progerin; C, LMNC; * p < 0.05; ** p < 0.01; *** p < 0.001 vs same transcript or protein in mice receiving saline only (vehicle).

**Extended Data Fig. 8 |. SRP-2001 reduces expression of transgene-derived progerin in murine liver.**
a, Quantitative PCR analysis of transgene transcripts in liver tissue ofLMNA^G/G mice receiving long-term treatment with saline (vehicle) or 60 mg/kg SRP-2001. Expression ofLMNA (A), progerin (P) andLMNC (C) is quantitated relative toHprt expression, as a fraction of all transgene-derivedLMNA transcripts or relative to transgene-derivedLMNC transcripts. Data are presented as mean values ± SD determined from 12 independent samples per treatment group (n = 6 males, 6 females).b, Western immunoblots of A-type lamins and beta actin (ACTB) immunoprecipitated from liver homogenates.c, Quantitation of A-type lamins from immunoblots expressed relative to beta actin (ACTB), as a fraction of all A-type lamins and relative to LMNC. Data are presented as mean values ± SD determined from 12 independent samples per treatment group (n = 6 males, 6 females), shown in panel b. * p < 0.05; ** p < 0.01; *** p < 0.001 vs same transcript or protein in mice receiving saline only (vehicle).

**Extended Data Fig. 9 |. SRP-2001 reduces progerin transcripts in multiple mesenchyme-derived murine tissues.**
Quantitative PCR analysis of transgene transcripts ina, quadriceps,b, femoral bone, andc, kidneys ofLMNA^G/G mice receiving long-term treatment with saline (vehicle) or 60 mg/kg SRP-2001. Expression ofLMNA (A), progerin (P) andLMNC (C) is quantitated relative toHprt expression, as a fraction of all transgene-derivedLMNA transcripts or relative to transgene-derivedLMNC transcripts. Data are presented as mean values ± SD determined from 12 independent samples per treatment group (n = 6 males, 6 females). * p < 0.05; ** p < 0.01; *** p < 0.001 vs same transcript or protein in mice receiving saline only (vehicle).

**Extended Data Fig. 10 |. Histologic analysis of kidneys shows minimal toxicity due to long-term treatment with SRP-2001.**
Representative images of haemotoxylin and eosin (HE) staining of sectioned kidneys from treated (n = 12) and untreated (n = 12) mice. Mice receiving long-term treatment with SRP-2001 developed irregular cortical surfaces (ic), degenerate tubules (dt) and basophilic granules (bg) in tubular epithelial cells. Two sections per tissue sample were analyzed.

**Fig. 1 |. Design and testing of a PPMO-based strategy for blocking the production of progerin.**
a, Right, NormalLMNA transcript processing. Left: aberrantLMNA splicing. The HGPS G608G mutation position is indicated by the arrow at the junction of the retained exon 11 sequence (dark gray) and the 150-nt portion of exon 11 that is deleted (light gray) by splicing at the cryptic splice site. The antisense PPMO binds transcripts containing the mutation, thereby blocking cryptic splice site recognition by the spliceosome.b, Tiling of 11 alternatively designed PPMOs around the cryptic splice site generated by theLMNA G608G (c.1824C>T) mutation. The two controls included an oligonucleotide complementary to the normalLMNA sequence (wtLMNA, WT) and an oligo predicted to lack complementarity to any sequence in the human genome (scrambled control, CTRL).c, RT–qPCR results, normalized relative toTFRC RNA, of proband-derived fibroblasts cultured in the presence of 6 μM of alternative PPMOs for two weeks. Averages and s.d. were determined from triplicate reactions of biological triplicates.d, Immunoblots of triplicate lysates from proband-derived fibroblasts cultured in the presence of 6 μM of alternative PPMOs for two weeks.e, Quantitation of lamin A, progerin and lamin C proteins from western blot analysis, normalized relative to β-actin (ACTB). Values and error bars represent the mean ± s.d. of triplicate cultures. A, lamin A; P, progerin; C, lamin C; *P < 0.05, **P < 0.01,***P < 0.001 versus transcripts or protein from cells receiving saline.

**Fig. 2 |. Effect of SRP-2001 on cellular proliferation and B-type lamin protein expression in the fibroblasts of a patient with HGPS.**
a, Treatment of three independent patients with classic HGPS and sex-matched parental control fibroblast cell lines with SRP-2001 improves proliferative capacity in culture. Cells received either no treatment, saline (vehicle) or 6 μM of SRP-2001, performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle control.b, Western blot analysis of A- and B-type lamins showing partial rescue of B-type lamins, particularly LMNB2, in HGPS fibroblasts treated with SRP-2001. LMNB1 and LMNB2 levels increased 80 (P = 0.11) and 100% (P < 0.05), respectively in HGPS fibroblasts at 12 μM of SRP-2001 versus cells receiving no treatment. Values and error bars represent the mean ± s.d. of triplicate cultures.

**Fig. 3 |. Determination of SRP-2001 efficacy in culture.**
a–f, Fibroblasts derived from a proband with classic HGPS (a–c) and mice carrying two copies of the human mutantLMNA transgene (*LMNA*^G/G) (d–f) were treated for two weeks with SRP-2001 and analyzed forLMNA expression by qPCR and western immunoblot. For transcript quantitation, the averages and s.d. were determined from triplicate reactions of biological triplicates. The values for protein analyses were determined from three independent cultures. The EC₅₀ value was estimated at 3 μM based on intracellular progerin transcript (a) and protein (b,c) levels in HGPS cells and progerin transcript (d) and protein (e,f) levels in transgenic mouse cells. *P < 0.05; **P < 0.01; ***P < 0.001 versus the same transcripts or proteins in cells receiving saline only.

**Fig. 4 |. Efficient in vivo delivery of PPMO to murine vascular cells and tissues.**
a, The EGFP-654 splicing assay demonstrates efficient in vivo delivery of PPMOs to murine vascular cells and tissues. The schematic of the EGFP-654 reporter system illustrates that the mutation at nucleotide 654 of the intron results in partial intronic retention in spliced mRNA and prevention of proper translation of EGFP. The targeted PPMO blocks the aberrant splice site and restores proper splicing allowing for EGFP expression.b, Immunohistochemical analysis of sectioned ascending aortas after intravenous injection of EGFP-654 PPMO into reporter mice demonstrates restored expression of EGFP in VSMCs. Saline served as the negative control. Two mice (one male, one female) were injected in a single experiment and eight sections per aorta were analyzed.c–e, Single-dose pharmacodynamic analysis of SRP-2001 was performed on female and male mice expressing two copies of the humanLMNA transgene harboring the classic HGPS mutation, G608G (*LMNA*^G/G). The efficacy of intravenous drug delivery was determined by comparing progerin transcripts and PPMO levels in the aorta (c), heart (d) and kidney (e) after single intravenous doses of 60 mg kg⁻¹. Values and error bars represent the mean ± s.d. forn = 6 per treatment group.

**Fig. 5 |. Long-term treatment of HGPS mice with SRP-2001 significantly extends their lifespan.**
*LMNA*^G/G mice received twice weekly treatments via intravenous tail injection at 60 mg kg⁻¹ beginning at 2 weeks of age.a, Left: 7.5-month-oldLMNA^G/G mouse treated with SRP-2001. Right: 7.5-month-oldLMNA^G/G mouse receiving saline (vehicle).b, Growth curves ofLMNA^G/G mice treated with SRP-2001 or saline (vehicle) compared to C57BL/6 WT mice. Values and error bars represent the mean ± s.d. forn = 6 mice per treatment group.c, Kaplan–Meier plots for saline-treated controls (black line,n = 12) and SRP-2001-treated mice (blue line,n = 12) illustrate the 61.6% increase in lifespan of the treated group, from a median of 30.5 weeks to a median of 49.5 weeks (P < 0.0001).

**Fig. 6 |. Partial rescue of vascular tissue degradation in mice treated with SRP-2001.**
End point analysis of vascular tissues ofLMNA^G/G mice treated twice weekly until the end point at an average of 30.5 weeks with saline (vehicle) compared to the end point at an average of 49.5 weeks with 60 mg kg⁻¹ of SRP-2001.a, Representative images from Movat’s pentachrome-stained ascending aorta sections illustrate VSMC loss, elastin degradation, thickened medial layer and proteoglycan accumulation observed inLMNA^G/G mice receiving saline only. The aortas of mice treated with SRP-2001 contained less proteoglycan (blue) and retained VSMCs (red). H&E stain of the ascending aorta showing VSMC nuclei (blue) presence in SRP-2001-treated mice nearer the lumen of the tunica media. For each sample and stain, a total of four sections were imaged for analysis.b, Left: end point VSMC density within the tunica media of the ascending aortas from mice receiving PPMO or vehicle (saline) only. Right: adventitial area of ascending aortas at the end point.c, SRP-2001 reduces expression of progerin in vivo as determined by qPCR analysis ofLMNA transgene expression in vascular tissue from mice receiving saline (vehicle) or SRP-2001. Values and error bars represent the mean ± s.d. forn = 12 mice per treatment group (6 males, 6 females); *P < 0.05, **P < 0.01.

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Comment in

Splice-inhibition therapy targets progeria.
Revêchon G, Whisenant D, Eriksson M.Revêchon G, et al.Nat Med. 2021 Mar;27(3):377-379. doi: 10.1038/s41591-021-01267-z.Nat Med. 2021.PMID:33707774No abstract available.
Antisense approach slows progeria.
Villanueva MT.Villanueva MT.Nat Rev Drug Discov. 2021 May;20(5):343. doi: 10.1038/d41573-021-00056-0.Nat Rev Drug Discov. 2021.PMID:33772219No abstract available.

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1. Eriksson M et al. Recurrent de novo point mutations in lamin A cause Hutchinson–Gilford progeria syndrome. Nature 423, 293–298 (2003). - PMC - PubMed

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A targeted antisense therapeutic approach for Hutchinson-Gilford progeria syndrome

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A targeted antisense therapeutic approach for Hutchinson-Gilford progeria syndrome

Authors

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Abstract

Conflict of interest statement

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Comment in

References

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Full Text Sources

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