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. To develop and test novel therapeutic strategies to treat HGPS, we have taken a diverse approach to target progerin at the protein, RNA, and DNA levels facilitated by generation of a transgenic mouse model that carries a human LMNA gene harboring the classic mutation (c.1824C>T, G608G), which exhibits progressive loss of smooth muscle cells in the media of large vessels. Although single-copy transgenic mice (LMNAG/+) die at 15-19 months of age, double-copy mice (LMNAG/G) show a more severe phenotype, with death at 6-7 months of age. 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, we demonstrated that the rapamycin analogue everolimus induced a modest increase in lifespan of homozygous G608G mice. We also demonstrated that everolimus has a beneficial effect on fibroblasts from atypical laminopathy patients atypical Werners syndrome and Emery-Dreifuss muscular dystrophy. Currently a trial of everolimus in children with progeria is underway at Boston Childrens Hospital. In an alternative genetic strategy to test the effect of mTOR inhibition in our mouse model mice carrying an mTOR hypomorphic allele (mTOR+/) were bred into the G608G transgenic mouse line resulting in mTOR+/LMNAG/G offspring exhibiting in a 30% increase in lifespan versus mTOR+/+LMNAG/G littermates. Furthermore, increased activation of the downstream effector S6 protein kinase (S6K) in mTOR+/+LMNAG/G MEFs relative to wild-type was normalized in mTOR+/LMNAG/G, consistent with lifespan extension in S6K-null mice, adding further support for pharmacological inhibition of this pathway in HGPS. A manuscript detailing these findings is currently in preparation. Given that most children with HGPS are currently taking farnesyltransferase inhibitors (FTIs), a second trial in heterozygous G608G mice treated with both an FTI (lonafarnib) and everolimus was initiated to determine if the combination of drugs improves their vascular phenotype. This study is nearing completion. At the RNA level, we are investigating a targeted gene therapeutic approach using antisense oligonucleotides to block aberrant splicing of LMNA at the cryptic splice site. In collaboration with Sarepta Therapeutics, we have demonstrated that a proprietary peptide-conjugated phosophorodiamidate morpholino oligonucleotide (PPMO) 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. We are now proceeding with pharmacokinetic and pharmacodynamic studies, as well as toxicology in anticipation of an eventual IND application to the FDA. In close collaboration with the laboratory of Dr. David Liu at the Broad Institute we are also investigating the most fundamental option for a cure by employing DNA base editing to correct the LMNA C1824T mutation. Dr. Liu and colleagues have engineered a family of base editors (ABEmax), composed of a fusion protein of catalytically impaired Cas9 and a laboratory-evolved adenine deaminase optimized for nuclear localization and expression, which is capable of precisely changing a single nucleotide rather than making a double-stranded break. In vivo delivery of this gene editing system 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. We are now engaged in an effort to assess therapeutic efficacy of these approaches by comparing retro-orbital (RO) injection at perinatal day 3 (P3) to RO at 2-weeks administration. 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 knock-in mouse model (Lmna G609G/G609G), we are defining the molecular and cellular alterations that occur in HGPS bone cell populations. Structural and mechanical analyses of murine long bones indicated increased fragility due to reduced cortical and trabecular bone mass in 2-month homozygous and 8-month heterozygous mice versus wild-type mice. Histologic examination of murine tissues revealed increased TRAcP-positive bone resorbing osteoclasts on trabecular bone surfaces, consistent with increased inflammatory cytokine-induced bone turnover. Furthermore, cultured Lmna G609G/G609G osteoblast precursors exhibited defective differentiation characterized by development of an adipogenic, rather than osteogenic, phenotype. These studies suggest that bone dysplasia in HGPS patients may be analogous to progressive bone deterioration that occurs in aging-related osteoporosis. Comprehensive understanding of how progerin drives HGPS phenotypes requires identification of protein interactions with 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 developed a novel biotin proximity-based labeling approach 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 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. Finally, expanding our interest in the cellular effects of premature and normal aging, we assessed the level of somatic cell DNA mutation accumulation in 44 aging related genes in normal, HGPS and Xeroderma Pigmentosa (XPA/C) fibroblast cell lines in early versus late passage. We identified specific mutations that indicate signs of selection during in vitro aging. We published these findings in Aging Cell.
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