Modulation of LMNA splicing as a strategy to treat prelamin A diseases
John M. Lee, … , Stephen G. Young, Loren G. Fong
John M. Lee, … , Stephen G. Young, Loren G. Fong
Published March 21, 2016
Citation Information: J Clin Invest. 2016;126(4):1592-1602. https://doi.org/10.1172/JCI85908.
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Research Article Aging Therapeutics

Modulation of LMNA splicing as a strategy to treat prelamin A diseases

Abstract

The alternatively spliced products of LMNA, lamin C and prelamin A (the precursor to lamin A), are produced in similar amounts in most tissues and have largely redundant functions. This redundancy suggests that diseases, such as Hutchinson-Gilford progeria syndrome (HGPS), that are caused by prelamin A–specific mutations could be treated by shifting the output of LMNA more toward lamin C. Here, we investigated mechanisms that regulate LMNA mRNA alternative splicing and assessed the feasibility of reducing prelamin A expression in vivo. We identified an exon 11 antisense oligonucleotide (ASO) that increased lamin C production at the expense of prelamin A when transfected into mouse and human fibroblasts. The same ASO also reduced the expression of progerin, the mutant prelamin A protein in HGPS, in fibroblasts derived from patients with HGPS. Mechanistic studies revealed that the exon 11 sequences contain binding sites for serine/arginine-rich splicing factor 2 (SRSF2), and SRSF2 knockdown lowered lamin A production in cells and in murine tissues. Moreover, administration of the exon 11 ASO reduced lamin A expression in wild-type mice and progerin expression in an HGPS mouse model. Together, these studies identify ASO-mediated reduction of prelamin A as a potential strategy to treat prelamin A–specific diseases.

Authors

John M. Lee, Chika Nobumori, Yiping Tu, Catherine Choi, Shao H. Yang, Hea-Jin Jung, Timothy A. Vickers, Frank Rigo, C. Frank Bennett, Stephen G. Young, Loren G. Fong

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Figure 2

ASOs modulate LMNA expression.

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ASOs modulate LMNA expression. (A) ASOs modulate LMNA alternative splici...
(A) ASOs modulate LMNA alternative splicing in human cells. Triplicate wells of wild-type human fibroblasts (clone CRL-2429) were transfected with ASO E11-26, E11-28, E11-31, E11-33, E11-36, E11-38, and E11-41. After 3 days, prelamin A and lamin C mRNA levels were measured by qRT-PCR. The data are expressed relative to cells treated with a scrambled ASO (Con) (set at a value of 1.0). The mean ± SD is shown. All yielded statistically significant differences compared with the control (P < 0.01, t test). (B) Modulating LMNA alternative splicing with ASO E11-31 in multiple cell types. Human skin fibroblasts (SKF; clone AG2492), HepG2 cells, HeLa cells, osteoblasts (OsteoB), chondrocytes (Chond), and smooth muscle cells (SMC) were transfected with ASO E11-31 or transfection reagent alone. After 2 days, prelamin A and lamin C transcript levels were measured by qRT-PCR and expressed relative to the nontemplate control (set at a value of 1.0). (C) Decreasing lamin A protein levels with ASO E11-31 in several human cell types. The same cell types described in B were transfected with ASO E11-31 or transfection reagent alone. After 3 days, the expression of lamin A and C was measured by Western blotting. Actin levels were measured as a loading control. Lamin protein expression was quantified and expressed relative to the nontemplate control (set at a value of 1.0). (D) ASO E11-31 decreases progerin levels in HGPS cells. Duplicate wells of HGPS cells were transfected once with ASO E11-31. Cells were harvested 1–5 days later, and the expression of lamin A, lamin C, and progerin was measured by Western blotting. Actin levels were measured as a loading control. The data are expressed relative to cells treated with a scrambled ASO (set at a value of 1.0).