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 de novo 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. 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 increases the lifespan of HGPS mice by 62% compared to vehicle only. 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 developing 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. Retro-orbital (RO) injection of 1012 viral genomes in 2-week old HGPS mice (P14) resulted in a 2.4-fold increase in median lifespan. In collaboration with the Progeria Research Foundation and Beam Therapeutics we have partnered with Columbus Childrens Foundation and AskBio to develop the next generation AAV therapeutic delivery vehicle and 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. 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 progeria 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 progeria 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. To investigate pathology at the cellular level we are collaborating with the Allen Institute to employ LMNB-GFP reporter iPSC line containing the LMNA G608G mutation, differentiated to cardiomyocytes to examine changes in developmental stages as well as differences in chromatin structure, gene expression and protein function. Successful differentiation of two independent iPSC lines of each G608G and WT iPSCs into CMs has demonstrated spontaneous contraction in all 4 lines. The progerin-specific nuclear blebbing phenotype was not obvious at this immature phase at <15 days post differentiation. Single nucleus transcriptome analysis of CMs isolated from the progeria mice compared to WT mice revealed dysregulation of key genes involved in the calcium signaling pathway critical for heart muscle excitation-contraction coupling. To complement the cellular and tissue analysis we are examining cardiac function in vivo -- in particular left ventricular systolic and diastolic function as well as cardiac strain, using diagnostic ultrasound at the NIH Mouse Imaging Facility. Progressive bone dysplasia is one of the defining phenotypic features that we have sought to further characterize utilizing both knock-in and transgenic mouse models of HGPS. In HGPS mouse bone tissue and differentiating osteoblast cultures abnormal ATP levels result in hyperactivation of AMPK causing suppression of mTORC1 to initiate autophagic removal of ER membrane localized IRE1. In a more direct role, suppression of mTORC1 results in hyperactivation of AKT which, through a direct physical interaction with IRE1, inhibits its role in splicing and activation of the XBP1 transcriptional regulator of the intracellular response to stress that occurs during differentiation. While chemical inhibition or genetic reduction of mTOR further inhibits IRE1 activity, inhibition of Akt partially rescues the reduced IRE1 activity in response to acute and chronic stress in vitro. These data point to a specific pathomechanism that can be targeted as a potential therapeutic approach to restore normal bone development in HGPS. Additional studies using this mouse model have demonstrated that osteoblast precursors take on adipogenic phenotype in vitro and in vivo that is associated with abnormal histone chromatin marks, and therefore dysregulated osteoblast-specific gene expression. Our work describing the tissue and cellular phenotype of HGPS knock-in mouse bone is currently under review. We have also continued to investigate effects of the CRISPR gene-editing therapeutic approach described above on the mouse skeletal system. While single-copy transgenic mice exhibit a relatively normal phenotype at 6 months of age, double copy littermates have developed compromised bone structural and mechanical parameters compared to wild-type mice, including reduced BMD, bone volume and cortical thickness that results in significantly decreased stiffness and load to fracture. We have studied the effects of gene editing on bone structure and function in these mice and have achieved, depending on the age and route of administration, 10-22% base correction in bone tissue. Notably, this level of editing in bone is sufficient to restore the reduced mechanical, but not structural, parameters in double copy mice to wild-type values suggesting that changes in material properties are largely responsible for improvement in bone quality following base editor treatment. In addition to identifying clusters of corrected bone cells near the periosteal surface of cortical bone by immunostaining, rescue of tissue function in treated mice was associated with normalized expression of specific markers of bone cell populations, including mesenchymal stem cells, osteoblasts, osteocytes and osteoclasts. In parallel studies utilizing primary osteoblasts derived from double copy mice we have found that the same level of correction obtained in vivo results in 40% reduction of progerin transcripts, 13% reduction in progerin protein, and a 10% increase in mineral deposition in vitro. A manuscript describing the full characterization of the bone phenotype and the efficacy of base editing in transgenic mice is currently in preparation.