Hutchinson-Gilford progeria syndrome (HGPS) is the most dramatic human syndrome of premature aging. Children with this rare condition appear normal at birth, but by age 2 they have stopped growing, lost their hair, and shown skin changes and loss of subcutaneous tissue that resemble the ravages of old age. Untreated, they rarely live past adolescence, dying at an average age of 14.6 years due to advanced cardiovascular disease (heart attack and stroke). Our laboratory discovered that 90% of cases of HGPS harbor a de novo point mutation in codon 608 of the LMNA gene. This mutation, denoted G608G, generates an exonic cryptic splice donor site, producing an mRNA with an internal deletion of 150 nt. This is translated into a mutant form of the lamin A protein (referred to as progerin) that lacks 50 amino acids near the C-terminus. Normal pre-lamin A is post-translationally processed by the addition of a farnesyl group at the C-terminus, followed by cleavage of 18 C-terminal residues by the enzyme ZMPSTE24 to produce mature lamin A. Progerin lacks the recognition site for this final cleavage, and so remains permanently farnesylated. We have shown that progerin acts in a dominant negative manner to disrupt the structure of the nuclear scaffold, interfere with proper chromosome segregation, and alter the distribution of various histone chromatin marks. Based on encouraging results with farnesyltransferase inhibitors (FTIs) in cell culture and our mouse model, clinical trials of FTIs in children with HGPS have been conducted and demonstrated benefit to the vascular system, with reduction in peripheral vascular resistance in most of the children treated. This treatment is not a cure, however, and so the search for other therapeutic options continues. Our lab has developed a bacterial artificial chromosome (BAC) transgenic mouse model for HGPS that carries the human LMNA gene with the G608G mutation. Animals carrying the G608G transgene show progressive loss of smooth muscle cells in the media of large vessels. In contrast to single-copy transgenic mice that die at 15-19 months of age, homozygotes (double copy) for the transgene show a considerably more severe phenotype, with death at 6-7 months of age. We have used this mouse model to investigate the potential therapeutic benefit of the rapamycin analog, everolimus, alone or in combination with FTIs. Rapamycin has been demonstrated to extend lifespan in wild-type mice, and improves the phenotype of HGPS fibroblasts by activating autophagy and increasing turnover of progerin aggregates. We conducted a small-scale drug trial in homozygous G608G mice to see if there might be therapeutic benefit from everolimus, and found a modest improvement in lifespan. We will soon be completing a second trial on heterozygous G608G mice that are being treated with a combination of an FTI (lonafarnib) and everolimus, to determine if the drugs improve their vascular phenotype. We also published a paper this year showing that everolimus has a beneficial effect on fibroblasts from patients with other LMNA mutations, including those that cause atypical Werners syndrome and Emery-Dreifuss muscular dystrophy. Based on the promising results from cell culture studies, and the encouraging safety profile of everolimus from long experience in organ transplantation, a Phase 1 trial of everolimus in children with progeria has been completed at Boston Childrens Hospital, and Phase 2 is actively enrolling. We are also investigating a more precisely targeted gene therapeutic approach to treating HGPS patients using antisense oligonucleotide strategies to inhibit abnormal splicing at the cryptic G608G splice site. In collaboration with Sarepta Therapeutics, we previously tested several phosophorodiamidate morpholino oligonucleotides (PMOs) in HGPS patient fibroblasts, two of which achieved significant reduction of progerin mRNA and protein. The best candidate PMO was subsequently synthesized by Sarepta Therapeutics as a proprietary peptide-conjugated phosophorodiamidate morpholino oligonucleotide (PPMO) for further testing. Having demonstrated that intravenous and subcutaneous injection of PPMOs achieved excellent delivery to vascular smooth muscle cells of the mouse aorta using a GFP reporter system, we have now demonstrated in a preclinical trial that twice weekly administration of PPMO by IV tail vein injection increases the lifespan of HGPS mice by 62% compared to mice treated with vehicle only. We are now proceeding with pharmacokinetic and pharmacodynamic studies, as well as toxicology in anticipation of an eventual IND application to the FDA. Although mortality is due to myocardial infarction or stroke as a result of rapidly progressive atherosclerosis, HGPS patients also exhibit alopecia, bone and joint abnormalities, and subcutaneous fat loss. Thus cells and tissues derived from common mesenchymal progenitors are particularly susceptible to the pathology that results from the accumulation of progerin. While progressive bone dysplasia is one of the defining phenotypic features of HGPS, several studies have also suggested epidemiologic and biologic links between aging-related cardiovascular disease and osteoporosis. Using a knock-in mouse model (LmnaG609G/G609G), we have initiated studies to characterize the molecular and cellular alterations that occur in HGPS bone cell populations. We are interested in knowing what would happen to normal cells or organisms if progerin production was completely prevented. To pursue this, we have created a mouse knock-in model that alters the sequence of mouse exon 11 to block the use of the cryptic splice site, without changing the encoded amino acid sequence. The knock-in heterozygotes and homozygotes are viable, and quantitative RNA measurements have confirmed that homozygotes make no progerin. Although progerin expression has been observed in human ductus arteriosus and is thought to play a role in the normal closure of this vessel, no defects in ductus arteriosus closure were observed in this model. Furthermore, extensive histological analyses of the vessels and organs have not revealed any differences between the knock-in homozygotes and wild type mice, and the homozygotes show no enhancement in longevity . Cell culture studies of this progerin-free mouse strain are under way and potentially might reveal what role progerin plays in natural aging. Comprehensive understanding of how progerin drives HGPS phenotypes requires the identification of the protein interactors of lamin A/C and progerin. The high order insoluble structure of the nuclear envelope makes this challenging, even in cell culture, and has not been previously attempted in human tissues. We have developed a novel biotin proximity-based labeling approach and used it to identify lamin A/C and progerin interactors both in cell culture and directly from human tissue. We have identified instances where tissue culture did not faithfully replicate the interactome of primary tissues, and we have found multiple tissue-specific lamin A/C interactors. We have also identified proteins enriched or depleted in the presence of progerin. We have optimized the method for quantitative comparative proteomics of primary human samples and are generating data identifying known and novel nuclear envelope proteins, including tissue specific proteins. We have also identified, in an unbiased manner, changes to the composition of HGPS primary fibroblasts. We are working on additional variants of this method and applying it to primary tissues of old and young, wild type, and HGPS models.
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