Hutchinson-Gilford Progeria syndrome--a model for the genetics of aging.

National Human Genome Research Institute

Investigators

Linked publications, trials & patents

Abstract

Hutchinson-Gilford progeria syndrome (HGPS) is the most dramatic human syndrome of premature aging. Although children with this rare condition appear normal at birth, they develop bone dysplasia, growth deficiency, sclerotic dermis and atherosclerotic lesions within the vasculature, leading to mortality from heart attacks or strokes at an average age of 14.6 years. Our laboratory discovered that most cases of HGPS are caused by a rare single nucleotide mutation (c.1824C>T, p.G608G) that does not alter the coding sequence, but instead activates a cryptic splice donor within exon 11 of LMNA. The resulting protein product, termed progerin, lacks a 50-residue region required for processing, leading to incorporation of a permanently farnesylated truncated protein within the nuclear lamina that acts in a dominant negative manner to disrupt nuclear scaffold structure, chromosome segregation and distribution of histone chromatin marks. As part of the process of developing a model for testing alternative therapeutic approaches to treat HGPS in the context of the human gene, transcript and protein sequence, we have completed a detailed characterization of the phenotype of homozygous G608G mice. At the tissue level, progerin transcript and protein expression levels in multiple tissues have been correlated with the loss of subcutaneous fat, progressive kyphosis, osteopenia and degeneration of vascular tissue involving complete loss of smooth muscle cells and fibrotic expansion of vascular adventitia. Since the mTor inhibitor rapamycin extends lifespan in wild-type mice and improves the phenotype of HGPS fibroblasts by activating autophagic turnover of progerin aggregates, an alternative genetic strategy to test the effect of mTOR inhibition was initiated by breeding mice carrying an Mtor hypomorphic allele (Mtor+/) into the G608G transgenic line resulting in Mtor+/LMNAG/G offspring. Our studies have indicated that genetic reduction of Mtor results in a 30% increase in lifespan versus Mtor+/+LMNAG/G littermates. However our analyses also indicated that, although mTOR signaling is hyperactivated at an early age, it is suppressed in vivo as pathology develops in aging mice. A manuscript describing these findings is in press in Aging Cell. In collaboration with Sarepta Therapeutics, we have demonstrated that a proprietary peptide-conjugated phosophorodiamidate morpholino oligonucleotide (PPMO), SRP-2001, targeted to the G608G mutation site inhibited aberrant splicing in vitro nearly 100%, achieves efficient in vivo delivery to aortic vascular smooth muscle cells in mice by intravenous and subcutaneous injection, and in a preclinical trial in LMNAG/G increases the lifespan of HGPS mice by 62% compared to vehicle only. This study was published in Nature Medicine. To facilitate treatment of progeria patients, we are collaborating with the Progeria Research Foundation and Pace Analytical Life Sciences to develop and characterize a subcutaneous injection formulation of SRP-2001 in anticipation of an IND application to the FDA. In close collaboration with the laboratory of Dr. David Liu we are also investigating the most fundamental option for a cure by employing DNA base editing to correct the LMNA C1824T mutation. In vivo delivery of this gene editing system using dual split-intein AAV9 delivery vectors produced base correction of 59% in liver, 32% in heart muscle, 17% in aorta and 16% in skeletal muscle, resulting in significant reduction of progerin mRNA and protein levels. Targeted PCR amplification/high throughput sequencing indicated no editing events above genomic background. Retro-orbital (RO) injection of 1012 viral genomes in 2-week old LMNAG/G mice (P14) resulted in the reduction of progerin transcript levels and more significant reduction of progerin protein levels in cardiovascular tissues at 6 months of age, apparently as the result of positive selection of corrected cells. Histological analysis of P14-injected mice revealed striking improvements in VSMC counts, 11-fold higher than saline controls, statistically indistinguishable from wild type C57BL/6 mice. The P14 longevity cohort survived 510 days compared to 215 days for untreated control mice , representing a 2.4-fold increase in median lifespan. The manuscript describing this study was published in Nature. In collaboration with the Progeria Research Foundation and Beam Therapeutics we are preparing for IND application with the FDA. In addition, we are characterizing the most recent generation of more efficient DNA editors targeted to the LMNA C1824T mutation. The LMNA gene is one of most frequently mutated genes related to dilated cardiomyopathy (DCM) among 50+ genes involved. To investigate myocardial defects in HGPS that potentially contribute to premature death, we are employing cell biology, tissue pathology, transmission electron microscopy (TEM), and single cell transcriptomics of cardiomyocytes (CMs) from LMNAG/G mice. Progerin-expressing binucleated CM nuclei fail to separate the nuclei completely, suggesting a compromised mitotic process consistent with other HGPS cell types. TEM analysis of LMNAG/G mice reveals fragmented and disorganized sarcomere Z-lines, and histopathological staining indicates increased extracellular matrix deposition in small coronary arteries and partial loss of the characteristic striated structure in the myocardium. Although mortality is due to myocardial infarction or stroke due to rapidly progressive atherosclerosis, cells and tissues derived from common mesenchymal progenitors are particularly susceptible to the pathology that results from progerin accumulation. While progressive bone dysplasia is one of the defining phenotypic features of HGPS, several studies suggest epidemiologic and biologic links between aging-related cardiovascular disease and osteoporosis. Using a different knock-in mouse model from Carlos Lopez-Otin (LmnaG609G/G609G), we are defining the phenotypic, molecular, and cellular alterations that occur in HGPS bone cell populations. We found significantly reduced structural and physical parameters by CT analysis and mechanical testing. These alterations are associated with shortening of the long bones resulting in rhizomelia, accompanied by disorganization and increased TUNEL staining of growth plate chondrocytes, which is a marker of DNA damage and apoptosis. Furthermore, our hypothesis that progerin expression dysregulates tissue remodeling was verified by biochemical and histologic analyses, showing increased osteoclastic activity in murine serum and tissue, and consistent with the preponderance of immature collagen crosslinks in bone from these mice. Building on our previous findings that intracellular signaling pathways critical to the differentiation and functioning of bone cell populations are altered in HGPS, we have generated preliminary data suggesting that dysregulation of Akt-Mtor signaling may be responsible for inhibition of the Unfolded Protein Response through a previously unrecognized direct interaction with the IRE1-XBP1 branch of this pathway. We have studied the effects of gene editing on bone structure and function, and have demonstrated that the level of in vivo transgene editing (14-22%) in bone tissue is sufficient to restore the reduced structural parameters in LMNAG/G mice to wild-type values. A manuscript describing the full characterization of the bone phenotype in these mice is currently in preparation.