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

National Human Genome Research Institute

Investigators

Linked publications & trials

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. 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. 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 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 1012 viral genomes in 2-week-old LMNAG/G mice (P14) resulted in a 2.4-fold increase in median lifespan. To support a potential IND application with the FDA we are conducting a study comparing initiation of DNA editing at one month and at four months followed by evaluation of DNA editing at seven months. The use of a dual vector base editor requires high total viral load. For both higher efficacy and reduced toxicity it would be much better to be able 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 four different single-vector adenine base editors that retain similar base editing efficiencies in cell culture as ABEmax7.10. We have initiated 6-week preliminary trial administrating the AAV9 single-vector adenine base editors to LMNA G/G mice as well as the ABEmax7.10 standard, to examine the comparative in vivo DNA editing capacity and beneficial effect on blocking progerin splicing. A biomarker that can be used to assess response to therapeutic interventions is critically needed for HGPS future trials. We are investigating cell free DNA (cfDNA) released by senescing cells as a potential biomarker for HGPS. Though initial assays quantified cfDNA concentration using a High Sensitivity DNA bioanalyzer chip, we developed a much more sensitive ddPCR assay for LINE ORF1 and SINE B1 retrotransposable elements, allowing serial sampling of mice with as little as 5uL of plasma. We were able to show for murine HGPS models that cfDNA levels correspond to age and severity of disease progression. Validation of cfDNA levels as a clinical biomarker for therapeutic response was achieved by demonstrating quantitative reductions in plasma LINE and SINE copy number from mice treated in vivo with gene editing, where cfDNA levels mirrored the partial phenotypic rescue. In collaboration with the laboratory of Jay Humphrey at Yale, we have expanded the exploration of cardiac phenotypes in LMNA G/G progeria mice focusing on heart function assessment and gene expression profiling. Echocardiographic study showed an elevated E/E ratio in G/G mice compared to WT mice, indicating diastolic dysfunction in the left ventricle. Applying snRNA-seq to cardiac muscle from left ventricle, the major cell types in WT and G/G mouse heart were identified including cardiomyocytes, endothelial cells, fibroblasts, macrophages, and pericytes. When focusing on the myocytes, 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. Overexpression of these genes was validated by RNA in situ hybridization. These results, together with our previous findings, provide solid evidence of major primary cardiac abnormalities in the G/G mouse model of HGPS. Progressive bone dysplasia is one of the defining phenotypic features that we have sought to further characterize utilizing mouse models of HGPS. Our studies using these mouse models have demonstrated that osteoblast precursors develop an adipogenic phenotype and gene expression profile in vitro and in vivo that is associated with an inflammatory cytokine profile. Additional analysis of HGPS murine bone cell populations has revealed a low bone turnover condition that is not only due to reduced osteoblast function, but also to decreased bone resorption associated with upregulation of the osteoclastic inhibitory factor OPG. Furthermore, the HGPS bone phenotype can now be more accurately described as an osteo-chondrodysplasia in which long bone growth plate defects and altered cartilage content can be attributed to abnormal chondrocyte maturation and survival. The work describing the tissue and cellular phenotype of HGPS knock-in mouse bone was published this year in Aging Cell. We have also continued to investigate the efficacy of AAV9-mediated delivery of an adenine base editor (ABE) to correct the mutation in vivo using bone as a surrogate system for improved tissue structure and function. Treatment with ABE was shown to improve the ability of HGPS murine osteoblasts to deposit and mineralize extracellular matrix in vitro by as much as 40%. Partial rescue of the HGPS murine bone phenotype was demonstrated, including improved physical and mechanical bone parameters, with normalization of expression of mesenchymal stem cell, osteoblast and osteoclast markers.