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, scleroderma 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. 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 LMNA G/G increases the lifespan of HGPS mice by 62% compared to vehicle only. 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 in our mouse model, 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 1.48e14 viral genomes per kg in 2-week-old LMNA G/G mice (P14) resulted in a 2.4-fold increase in median lifespan. The use of a dual vector base editor requires high total viral dose. For both greater efficacy and reduced toxicity, it would be advantageous to achieve delivery with a single vector. By deleting the unnecessary vector sequence, minimizing the promoter, and re-engineering the cas9 coding sequence, the Liu lab has constructed three different single-vector adenine base editors that retain similar base editing efficiencies in cell culture as ABEmax7.10. A preliminary trial administrating the AAV9 single-vector adenine base editors to LMNA G/G mice as well as the ABEmax7.10 standard, demonstrated comparative in vivo DNA editing capacity and beneficial effect on blocking progerin splicing. Single-vector ABEs showed comparable on-target gene editing efficacies on LMNA c.1824T>C to dual-vector ABE and additional “bystander edits” on nearby thymines, producing missense changes mainly on c.1820T>C (p.V607A), c.1832T>C (p.I611T), and c.1834 T>C (p.S612P). Thus, it will be necessary to determine if these “bystander edits” alter the structure and/or function of LMNA before proceeding to clinical trials. We developed two in vitro approaches to determine the effects of these bystander edits, which rely on either ectopic or endogenous expression of the LMNA variants being investigated. We have generated stable-transfected immortalized HEK-293 and HeLa clonal cell lines that express LMNA cDNA containing the bystander edits in the context of the normal LMNA sequence. Characterization of these cell lines has demonstrated no obvious alterations of cell proliferation and nuclear structure. To investigate the effects of bystander edits in a clinically relevant cell type in HGPS, we initiated an in vitro approach using iPSC-derived vascular smooth muscle cells (VSMCs). HGPS-iPSCs carrying heterozygous G608G mutation in the LMNA gene were transduced with single-vector lentiviral constructs and achieved on-target 1824C/T gene correction (> 93%) along with anticipated bystander edits. We performed VSMC differentiation and completed characterization with specific markers (SMA, Calponin, and Myh11) indicating functioning contractile VSMCs with high efficiencies (>95%). The analyses of cellular phenotypes showed significant improvement of cell proliferation and restoration of normal nuclear shape in VSMCs, with or without I611T editing, compared to untreated HGPS VSMCs. The VSMCs with p.V607A edit demonstrated rescued nuclear blebbing but compromised cell proliferation. The S612P bystander edit was not evaluated in VSMCs because the frequency is too low for effective clonal capture. In collaboration with Kan Cao’s group at the University of Maryland a pro-angiogenic growth factor, termed Angiopoietin-2 (Ang2) was found to be downregulated in HGPS endothelial cells and HGPS mouse vascular tissue, resulting in the loss of Ang2/Tie2/AKT-mediated transcriptional regulation of genes involved in endothelial cell survival, proliferation, migration, chemotaxis, and tube formation. Treatment with exogenous Ang2 reversed progerin-induced dysfunction by improving vasculogenesis, migration, survival, nitric oxide production and the paracrine secretory profile of HGPS endothelial cells, suggesting that Ang2 may hold therapeutic potential to treat the vascular disease that is the leading cause of death in HGPS patients. In collaboration with the laboratory of Jay Humphrey at Yale, we generated echocardiographic results indicating diastolic dysfunction in the left ventricle of G/G mice contributing to cardiac strain common to HGPS patients. We identified significant upregulation of two known heart failure genes, Nppa and Nppb, in middle- and late-stage G/G mice compared to age-matched WT mice. Elevated plasma NT-proBNP peptide, a standard biomarker monitoring heart failure, was significantly reduced in ABE-treated G/G mice compared to nontreated mice, indicating improved heart function corresponding to reduced expression of toxic progerin upon gene correction in heart (average 26%). We are currently analyzing heart histology focusing on cardiomyocytes, interstitial fibrosis, cardiac vessel structure and adventitia changes upon ABE treatment. Taking advantage of bone as a surrogate system for testing tissue structural and functional characteristics, we have continued to investigate pathomechanisms of the HGPS bone phenotype. Our recent collaborative studies have focused on dysregulation of the two distinct processes of bone formation (i.e., endochondral and intramembranous ossification). We have demonstrated that craniofacial abnormalities in the LMNAG608G/G608G transgenic mouse model recapitulate the clinical features of HGPS in humans. These alterations of relative cranial size and structure most likely stem from defects in pluripotent progenitor cells responsible for processes of remodeling and ossification within the cranial sutures. In addition, a detailed histologic and histomorphometric analysis showed that long bones of the LmnaG609G/G609G knock-in mouse model present a distinct chondrodysplasia evidenced by abnormal development of long bone growth plates. The observed thinning of growth plates may be associated with increased resorption by TRAcP-rich chondroclasts at the osteochondrous junction, suggesting that low bone mineral density (BMD) in HGPS is due to decreased bone volume rather than low mineralization of the bone material. We are completing a follow-up analysis of our dual vector base editor approach, where treatment with ABEmax7.10 attained 22% transgene correction in murine bone, and which is sufficient to partially rescue the phenotype as demonstrated by improved structural and mechanical bone parameters, reversal of the loss of specific bone cell populations, and normalization of gene transcriptional programs and intracellular signaling pathways critical for tissue homeostasis.