Progerin, an Aberrant Spliced form of Lamin A, is a Potential Therapeutic Target for HGPS

1. Introduction

Hutchinson–Gilford progeria syndrome (HGPS; OMIM#176670) was first reported more than 100 years ago by Hutchinson and Gilford in 1886 and 1897, respectively [1]. The condition was designated as a premature aging syndrome by Gilford based on the fact that the symptoms associated with aging are similar to those of older people in general, including a lack of subcutaneous fat, hair loss, joint contractures, a progressive cardiovascular disease similar to atherosclerosis, and death from heart attacks and strokes in childhood. As patients typically live in their teens or early 20s and usually die before their reproductive age, this syndrome is not inherited. Diagnosis is not possible at birth but can be done within the first 6 months of age, although prominent and noticeable symptoms may be observed after the age of 2 years [2,3,4,5,6,7,8]. This fatal pediatric disease remained a medical mystery until genetic mapping revealed that 90% of patients have a de novo point mutation in the LMNA gene that replaces cytosine with thymine [9,10].

Nuclear membrane proteins (lamins A and C), encoded by the LMNA gene, are structural components of the nuclear lamina, a network of proteins inside the nuclear membrane that determines the shape and size of the nucleus [11,12]. LMNA produces four proteins as a result of alternative splicing: lamin A and lamin C as major products, lamin C2 and lamin A delta 10 as minor products. Lamin A and C are similar through the first 566 amino acids (encoded by exons 1-10) but deviate at the carboxyl terminus [3,13,14]. Prelamin A, but not lamin C, contains a CaaX motif at its C-terminus and undergoes farnesylation and methylation (Figure 1). Lamin A is synthesized as a precursor (prelamin A) and matures through four sequential post-translational processing steps [15]. First, FTase adds a 15-carbon farnesyl moiety to the carboxyl-terminal cysteine. Second, the Zmpste24 endopeptidase cleaves the last three amino acids of prelamin A prenyl protein-specific methyltransferase. Finally, the endopeptidase removes carboxyl-terminal 15 amino acids from the protein, resulting in the release of mature lamin A.

HGPS belongs to a group of diseases called laminopathies, in which, mutations across the LMNA gene result in a wide range of overlapping disorders [16]. Genetic mapping of the genome from patients elucidated that a sporadic, autosomal dominant de novo point mutation c.1824C>T (p.G608G) (NM_170707.3) in exon 11 of the human LMNA gene mediates abnormal alternative splicing [9,10], which produces an abnormal variant protein called progerin, which is responsible for this accelerated aging disease [17,18,19] (Figure 1).

The current review describes the discovery of progerin as a causative agent of HGPS and provides evidence of its deleterious effects when expressed intracellularly, and the benefits of inhibiting its expression. Additionally, it introduces efforts to develop therapies and clinical trials for HGPS.

2. Puzzling Observation in HGPS: The Absence of Primary Neurological Disease

Extensive studies have been conducted to examine mutations in the LMNA gene encoding prelamin A and lamin C, which result in distinct muscular dystrophy, cardiomyopathy, partial lipodystrophy, and progeroid syndromes. These laminopathies mostly affect mesenchymal tissues (e.g., the myocardium, skeletal muscle, adipose tissue, fibrous tissue, and bone tissues). However, one confusing observation in patients with HGPS is that they generally show fundamental and dramatic premature aging but do not exhibit any noticeable cognitive damage. For many years, it has been puzzling that patients with HGPS do not have any primary neurological disease. However, recent research has confirmed a lack of lamin A expression, the major isoform of LMNA, in HGPS patient-driven induced pluripotent stem cells (iPSCs) [20,23,30,31]. In most tissues, the amounts of lamins A and C are approximately equal; however, the brain mostly generates lamin C and very little lamin A [20,21]. Immunohistochemistry has indicated that lamin C is expressed at high levels in the neurons and glia of the brain, but the expression of prelamin A and lamin A is restricted to the vascular endothelial cells [21]. Further studies have shown that the expression of prelamin A in the brain is downregulated by miR-9, a microRNA highly expressed in the brain that binds to a single site in the 3′ untranslated region of prelamin A [20,21,23]. The ectopic expression of miR-9 in fibroblasts or HeLa cells decreases the levels of prelamin A and lamin A proteins but does not influence lamin C expression [21]. In lamin A-only knock-in mice, where there is no alternative splicing and the output of all genes is directed to the prelamin A transcript, high levels of lamin A are found in the peripheral tissues, but very little lamin A is found in the brain [21]. Likewise, a knock-in mouse was created to direct the production of LMNA towards the progerin transcript. In this model, high levels of progerin were expressed in peripheral tissues, while minimal levels were observed in the brain [21], demonstrating that the unique expression pattern of lamin A/lamin C in the brain is not the result of alternative splicing. This regulation of lamin A in the brain provides us a scope to research further about improperly processed progerin and toxic lamin A. Children with HGPS have aging-like phenotypes in many tissues but lack common features of physiological aging in the central nervous system (CNS), such as senile dementia. Therefore, progerin accumulation in cells is considered a pathology-inducing factor.

Additionally, several studies have highlighted the important role of nuclear lamins in the CNS, indicating that type B lamins, lamins B1/B2, play an important role in neuronal migration in the developing brain [25,26,27]. Duplication of the LMNB1 gene encoding lamin B1 has been shown to cause autosomal dominant leukodystrophy (ADLD) [28,29]. More recently, both LMNB1 deficiency and overexpression have been reported to inhibit proliferation, but only LMNB1 overexpression induces senescence, which is prevented by telomerase expression or p53 inactivation. A concomitant decrease in lamin A/C levels aggravates this phenotype. These findings show that changes in the expression of LMNB1 inhibit proliferation and are potentially relevant for understanding the molecular pathophysiology of ADLD [112].

We also questioned whether the post-translational processing steps of prelamin A are essential for targeting the protein to the nuclear envelope [24]. Mice that could directly produce mature lamin A without going through the usual prelamin A synthesis and processing steps were created. However, no detectable disease phenotype was observed in the mice and the nuclear membrane of mature lamin A appeared normal [24], suggesting that prelamin A processing is minimally important for the nuclear targeting of mature lamin A and is independent of lamin B in laboratory mice.

3. Increased Malfunction in the Phenotype by Intracellular Progerin Expression

Notably, the expression levels of progerin and lamins A and C (lamin A/C) were significantly reduced in iPSCs derived from patients with HGPS [20,23,30,31]. Moreover, these cells showed decreased patterns of cellular senescence markers, including nuclear deformation, histone H3 trimethyl Lys9 (H3K9-Me3), and senescence-associated β-gal (SA–β-gal). In contrast, HGPS cells differentiated from iPSCs start expressing progerin and lamin A/C and re-expressing senescence markers [30]. This implies that the expression of the LMNA gene is tightly regulated at an early developmental stage; therefore, progerin is expressed in differentiated HGPS cells and its expression is affected by its pathologic nature. Twenty years ago, Collins et al. showed that progerin is the main factor for inducing a premature aging phenotype in HGPS by the disruption of lamin-related functions ranging from the maintenance of nuclear shape to the regulation of gene expression and DNA replication [17].

A correlation between progerin levels and the severity of HGPS phenotypes has been reported. Progerin levels in HGPS fibroblasts increase with culture passage number [18,84,129]. Recently, Gordon et al. developed a plasma assay to assess the amount of progerin in response to progerin-targeted therapy and its correlation with patient survival. The extent of survival improvement was related to both the magnitude and duration of progerin reduction at low levels, demonstrating that plasma progerin is a biomarker of HGPS that enables short- and long-term assessment of progerin-targeted therapeutic efficacy through progerin reduction [101].

Over the last 20 years, several kinds of mice have been developed as animal models to investigate diverse aspects of HGPS as follows: the knock-out or transgenic mice affecting the whole body level are Zmpste24^−/− [113,114], transgenic G608G BAC [81], Lmna^G609G [74], Apo^−/− Lmna^G609G/G609G [42], Ldlr^−/−, and Lmna^G609G/G609G [115]; the mice affecting specific tissue or cells are Lmna^LCS/LCS SM22αCre [42], Apoe^−/− Lmna^LCS/LCS SM22αCre [42], LmnaLCS/LCS Tie2Cre [116,117], Lmna^f/f; TC [118], Prog-Tg [119]—see reference 120 for further detail. The limited number of patients with HGPS worldwide renders it difficult to conduct longitudinal studies and clinical trials, and there is also a scarcity of human samples available for ex vivo analyses. Therefore, the use of animal models and their derived cell lines has contributed to the understanding of progeria phenotypes and is particularly important for developing therapeutic reagents for the disease. The current section aims to examine how progerin affects the phenotypes of various cell types and mouse models by compiling research carried out by various experts.

Blood vascular diseases are the predominant cause of death in classical HGPS [32]. As childhood progresses, other symptoms appear including hair loss, joint stiffness, body fat loss, osteoporosis, and other aspects of physiological aging. The most clinically relevant aspect of HGPS is the hardening of the arteries (atherosclerosis), which leads to premature death from cardiovascular disease or stroke at an average age of approximately 15 years [33]. In HGPS, atherosclerosis is accompanied by pathological changes in the aortic wall including severe vascular smooth muscle cell (VSMC) depletion in the media, extracellular matrix deposition, calcification, and early thickening of the aortic wall [34,35,36]. Skin tissue sections from patients with HGPS have shown that progerin accumulates primarily in the nucleus of vascular cells, suggesting that its accumulation has a direct association with vascular diseases in progeria [18]. Similarly, several reports have suggested that progerin in vascular muscles could accelerate atherosclerosis by inducing endoplasmic reticulum (ER) stress, DNA damage, wound healing impairment, mislocalization of a myocardin-related transcription factor, and replication stress [37,38,39,40,41]. Recently, Hamczyk, et al. (2018) produced the first mouse model (Apoe^–/–Lmna^LCS/LCSSM22αCre) with progerin-induced atherosclerosis acceleration expressing progerin specifically in VSMC and demonstrated that restricting progerin expression to VSMCs is sufficient to accelerate atherosclerosis, trigger plaque vulnerability, and reduce lifespan [42]. Moreover, they succeeded in developing CRISPR-Cas9 technology to generate HGPSrev mice (Lmna^{HGPSrev/HGPSrev}), engineered to express progerin throughout the body while lacking lamin A and allowing progerin suppression and restoration of lamin A in a temporal and cell type-specific manner upon Cre recombinase activation. Regardless of the broad expression of progerin and its pathological effects in several organs, restricting its suppression to VSMCs and cardiomyocytes is adequate to ameliorate vascular diseases and extend the lifespan of mouse models [43].

Progerin accumulation is associated with fat tissue disorders [105,106,108] and its expression decreases the capacity for adipocyte differentiation in both iPSCs and human mesenchymal stem cells (hMSCs) derived from patients with HGPS [65,107]. Mateos, et al. [44] executed quantitative proteomics to study the effect of progerin accumulation in a preadipocyte cell line, 3T3L1 cell. They reported that progerin accumulation in adipocytes contributed to the generation of reactive oxygen species and premature aging features, establishing a relationship between mitochondrial malfunction and proteostasis failure in HGPS [44].

Patients with HGPS exhibit unique skeletal dysplasia with bone morphological abnormalities and short stature. The HGPS mouse model (homozygous transgenic G608G BAC) also shows a similar bone structure pattern [109,110,111]. The cartilage abnormalities observed in this HGPS mouse model were similar to those observed in age-matched WT controls, including the premature loss of glycosaminoglycans and decreased cartilage thickness and volume. These alterations may mimic degenerative joint diseases prevalent in the elderly [110]. Zmpste24^−/− HGPS and progeria mouse model showed the development of kyphosis and spontaneous bone fractures in multiple locations [130]. More recently, the Lmna^G609G/G609G mouse model exhibited joint immobility and skeletal deformities in the vertebral column and skull [111].

Several studies have examined the relationship between progerin expression and inflammatory responses [45,46]. Endothelial cells expressing progerin recapitulate some characteristics of aging-associated cell dysfunction, including pro-inflammatory features, oxidative stress, DNA damage, increased expression of cell cycle arrest proteins, and cellular senescence [45]. Using HGPS fibroblasts, it was shown that progerin-induced replication stress causes genomic instability by stalling the replication fork and nuclease-mediated degradation, along with upregulation of the cGAS/STING cytosolic DNA sensing pathway and activation of a robust STAT1-regulated interferon (IFN)-like inflammatory response [46]. Hamczyk et al. (2018) also showed that exogenously expressed progerin increased inflammation [42]. A significant correlation was observed between chronic inflammation and ZMPSTE24 levels. Additionally, patients with cardiovascular diseases showed an abnormal lamin A/C expression associated with progerin levels [136]. The pro-atherogenic role of progerin in HGPS-related early atherosclerosis was proposed by Bidault et al. [45]. They found that progerin overexpression increased the expression of pro-inflammatory cytokines IL-6 and IL-1β, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), enhancing inflammation along with oxidative stress [45]. González-Dominguez, et al. (2021) recently suggested that progerin was responsible for the activation of the NLRP3 inflammasome [137], which is a multiprotein complex having an intracellular sensor comprising NLRP3 itself, the adapter protein ASC, and the catalytic subunit (Caspase 1) which cleaves pro IL-1β to the mature IL-1β. Furthermore, it was revealed that the activation was associated with alterations in nuclear morphology, indicating its relation to the induction of IL-1β by progerin [45,137].

The arrangement of chromatin in the nucleus is crucial for regulating many aspects of nuclear function and protecting nuclear integrity. Nuclear envelope (NE) is an essential factor in the dimensional scattering of these chromosomes. It binds to and ties a wide range of chromatin domains by interacting with the nuclear lamina and other associated proteins. Progerin sequesters NRF2 at the NE site, causing a subnuclear localization mismatch that impairs NRF2 transcriptional activity and consequently increases chronic oxidative stress [47]. Progerin also induces an altered 3D telomere organization between telomeres and the nuclear lamina, and an altered telomeric chromatin state [48]. Overexpression of telomerase reverse transcriptase (TERT) enhances the proliferative capability of HGPS fibroblasts and repairs progerin-induced DNA damage [49,50]. Chojnowski, et al. [51] suggested a controllable cellular model of progeria and showed that exogenous TERT prevents proliferation deficiency, DNA damage, lamin B1 reduction, and gene expression differences induced by progerin, suggesting that progerin disturbs the interaction between LAP2α and telomeres. However, TERT could not restore defects in nuclear morphology or altered H3K27me3 deposition [51]. Other reports have indicated that progerin expression is sufficient to cause heterochromatin loss by inducing DNA damage [52,53].

ER stress can be induced by the disruption of cellular energy levels, such as redox status or Ca²⁺ concentration, leading to the accumulation and aggregation of unfolded proteins [54,55]. Progerin expression disrupts calcium homeostasis and whole-body energy consumption. A mouse model (CAG-Progerin+ and MCK-Cre+) was developed to investigate the role of human progerin in sarcoplasmic reticulum function [56]. The study demonstrated that conditional overexpression of progerin in muscle tissue is sufficient to provoke premature death and impair the regulatory control of the expression of thermogenesis-related genes.

It has been found that the abnormal expression of progerin could aggravate the defenestration of liver sinusoidal endothelial cells (LSEC) during liver fibrosis, whereas the knockdown of progerin expression could attenuate premature liver senescence [57,58]. It has been proposed that in response to DNA damage, the binding between LC3 and UBC9-sumoylated lamin A/C enables autophagy to specifically mediate the disruption of lamin A/C and leaky nuclear DNA, suggesting that in response to cancer-promoting stresses such as DNA damage, autophagy breaks down nuclear material to prevent tumor formation [59]. Another study has shown that progerin is involved in nucleophagy [60]. It was indicated that deficient nucleophagy due to progerin expression caused oxidative stress presumably due to the decomposition of basal lamin B1. Furthermore, acetylation of nuclear LC3 was responsible for the unusual deposition of progerin, whereas its deacetylation promoted progerin removal, thus suggesting a potentially novel approach to maintain the LSEC phenotype [61].

Progerin-mediated functions at the protein level and their relationship with miRNA expression have also been described in recent studies [122,123,126]. A structure-based study has revealed that unfarnesylated progerin can form a disulfide bond with an Ig-like domain in the nuclear lamina. The Alphafold2-assisted docking structure showed that disulfide bond formation was promoted by a weak interaction between the groove of the Ig-like domain and the unfarnesylated C-terminal tail region of progerin, providing molecular insights into the abnormal interactions caused by progerin [122]. The evaluation of miRNA expression profiles in HGPS and normal fibroblasts revealed an enriched set of overexpressed miRNAs belonging to the 14q32.2-14q32.3 miRNA cluster and showed that inducing their overexpression in normal fibroblasts reduced cell proliferation and increased senescence, whereas inhibiting them in HGPS fibroblasts alleviated proliferation defects, senescence, and reduced progerin accumulation [123]. Progerin overexpression induced notable changes in miRNA expression and confirmed that has-miR-59 (miR-59) was markedly upregulated in cells from patients with HGPS and in multiple tissues of an HGPS mouse model (Lmna^G609G/G609G) [126].

In this report, we describe the relationship between progerin and cellular homeostasis, and how its role in inducing premature aging in most cells. Several groups have examined progerin as a biomarker of aging and indicated that it may be one of the few known biological indicators that initiate the aging process at a certain age [62,63,64,65]. Recently, fibroblasts cultured from older individuals were shown to have nuclear features similar to HGPS cells [62]. Notably, although these cells expressed progerin mRNA transcripts at barely detectable levels [62], they contained several abnormal nuclei that were clearly positive for progerin-specific antibodies after prolonged culture [66,68], indicating that progerin can be expressed in normal cells. To further investigate the biological relationship between progerin expression and aging in normal humans, Djabali et al. examined skin biopsies from 150 unaffected individuals. They found that similar splicing events occurred in vivo at low levels in the skin of individuals of all ages [69]. Although the mRNA expression level of progerin is low, it accumulates with age in a subpopulation of skin fibroblasts and terminally differentiated keratinocytes [36,69], suggesting that research on HGPS may improve our knowledge of physiological aging.

Here, we discuss the pathologies caused by progerin and its possible link to aging in normal individuals without mutations in the LMNA gene. Considerable evidence reveals that a reduction in progerin in cells leads to a reduction in pathology. In the following two sections, we discuss various attempts to directly or indirectly target progerin to achieve therapeutic benefits for HGPS and address the challenges of translating them into clinical trials.

4. Targeting and Inhibiting Progerin at mRNA and DNA Level

Cellular progerin levels are correlated with the severity of premature aging phenotype. Approximately 20 years ago, specific knockdown of progerin mRNA by RNA interference was shown to alleviate cellular aging features [67,70]. Short hairpin RNA constructs were designed to target progerin mRNA and mutate pre-spliced LMNA mRNAs with 1824 C->T mutations. The expression of shRNA using lentiviruses significantly decreased progerin expression, and in turn led to the improvement of abnormal nuclear morphology, recovery of proliferative potential, and a reduction in senescent cells [67,70]. These findings justify the potential use of gene therapy for HGPS treatment.

Antisense oligonucleotide (ASO)-based therapies are promising strategies for treating various diseases by blocking the expression of the target gene at the mRNA level through binding, followed by inactivation of the specific RNA by steric blockade or by promoting RNA degradation to specific sites on the mRNA [71,72]. The first ASO treatment in HGPS was tested in vitro by transfecting fibroblasts from patients with HGPS with an ASO targeting the progerin mRNA sequence. This approach effectively reduced progerin expression and ameliorated progerin-induced phenotypes [73]. In subsequent studies using several types of ASOs, similar results were obtained with fibroblasts from patients with HGPS [74,75,76] and from HGPS-like patients containing non-classical LMNA mutations that can induce progerin expression [77]. However, these studies revealed that progerin-targeting ASOs also decrease endogenous lamin A levels, raising concerns about the probable risk of lamin A depletion in vivo. However, mice lacking lamin A but maintaining lamin C expression showed no apparent phenotypes when compared to wild-type controls [74,78], encouraging researchers to test the potential of ASOs to reduce progerin levels in vivo. Another study showed that aortic progerin levels were reduced by approximately 50% in Lmna^G609G/G609G mice treated with ASOs selected through an in vitro approach that targeted a 70-nucleotide region located upstream of the mutation which causes HGPS. This treatment alleviated the HGPS-associated vascular phenotype by reducing the loss of SMCs and early fibrosis; however, no longevity data have been reported [75]. Two recent studies demonstrated the promising efficacy of ASO-dependent inhibition of progerin expression in homozygous transgenic G608G BAC mice [79,80], an HGPS mouse model that encodes human progerin and lamin A/C in addition to endogenous mouse lamin A/C [81]. The main advantage of this model over the Lmna^G609G/G609G mice is that the candidate ASOs have greater translational potential because they target the human (not mouse) LMNA gene. The top candidates were selected based on their ability to reduce progerin and lamin A levels without reducing lamin C expression both in vitro and in vivo. Weekly treatment of asymptomatic two- to six-days to a week-old BAC mice with selected ASOs significantly reduced progerin mRNA levels in many tissues; however, the reduction in progerin protein levels was less pronounced [79,80], much like the results obtained in Lmna^G609G/G609G mice [74], raising concerns about the high stability of protein levels and the need for inhibitors that efficiently reduce progerin expression. In two studies, ASO treatment was shown to increase the lifespan of BAC mice by 35-60% [79,80]; however, in one study, it was shown to partially prevent SMC loss and early thickening of the ascending aorta, two key features of the HGPS vascular phenotype [80]. The use of oligonucleotides to manipulate protein production has become an important therapeutic strategy for treating genetic diseases and cancer; however, the development of chemically modified nucleic acids, bio-conjugation to escort the moiety, and formulation of nanoparticle carriers are required for the delivery of oligonucleotide drugs [82]. Although these technological advances have led to the clinical approval of several ASO drugs, efficient and targeted delivery remains a major challenge for HGPS, in which the causative protein is expressed throughout the body and progressively affects tissues and organs.

Although ASOs have proven useful in mouse models (transgenic human G608G BAC mice) of HGPS, these models require continuous administration and do not eliminate the cause of the disease. Additionally, the treated animals died prematurely from HGPS. Considering the currently available information, adenine base editors (ABEs) appear to be more advantageous than CRISPR/Cas9 approaches because they do not induce double-stranded DNA breaks, inhibit lamin A, or efficiently correct mutations that cause HGPS. Furthermore, it prevents HGPS-associated vascular features and extends lifespan more than any other tested treatment [83]. Recently, transient expression of an ABE and single-guide RNA using MS2 bacteriophage-lentivirus chimeric particles corrected the mutation in 20.8-24.1% of skin cells in an HGPS mouse model (human tetop-LAG608G minigene), indicating that it could be a good approach for future gene-editing therapies [127]. However, two major challenges limit their application: moderate editing efficiency and off-target mutagenesis of DNA and RNA. Further pre-clinical studies are required to improve the safety and effectiveness of ABE-based therapies by enhancing their efficiency, optimizing vectors, fine-tuning doses, and defining the optimal treatment duration to achieve the best outcomes for patients before moving to clinical trials.