Novel assays and novel methods of treatment for Hutchinson-Gilford Progeria Syndrome

Abstract

Described herein is a newly developed immunoassay that can sensitively detect progerin, but not wild-type lamin A. Also described is the application of the newly developed immunoassay to novel methods for the diagnosis, prognosis, and treatment of Progeria.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 294,418, filed December 29, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] Technical Field The present disclosure relates to a newly developed immunoassay that can sensitively detect progerin, but not wild-type lamin A, and novel methods for the diagnosis, prognosis, and treatment of Progeria.

[0003] Government support acknowledged This invention was made in part with government support under Grant No. 1U01FD006886-01 awarded by the U.S. Food and Drug Administration. The Government has certain rights in this invention. [Background technology]

[0004] Hutchinson-Gilford progeria syndrome (HGPS) is an ultra-rare (prevalence 1 in 18-20 million) and uniformly fatal progeria syndrome that affects all sexes, ethnicities, and geographic regions. Morbidity includes growth retardation, generalized lipodystrophy, alopecia, osteogenesis imperfecta, and progressive arteriosclerosis, with death occurring at a mean age of 14.6 years, primarily due to heart failure.

[0005] HGPS is a sporadic autosomal dominant disease caused by mutations in LMNA that produce a toxic splice variant of the nuclear membrane protein lamin A called progerin (De Sandre-Giovannoli et al.; Eriksson et al.). 90% of cases are classical HGPS caused by a point mutation c.1824C>T, while the remaining 10% are non-classical HGPS caused by a single nucleotide mutation within the spliceosome recognition sequence in intron 11 (Gordon et al., 2018; Gordon et al., 2014). The progerin protein acts in a dominant-negative manner; it is the presence of progerin and the relative absence of a reduction in normal amounts of lamin A that results in disease (Scaffidi et al.). Supporting this, mice lacking lamin A display a normal phenotype (Fong et al.). The effect of progerin dosage is supported by mouse studies showing graded disease severity and shortened life span in heterozygous and homozygous progerin expressing mice, and extended life span in HGPS mice treated with progerin targeted therapy (Benedicto et al.; Cabral et al.). Case reports have reported low and high levels of progerin expressed in fibroblast cultures from patients with mild and severe progerin syndrome, respectively (Bar et al.; Hisama et al.).

[0006] Unlike lamin A, progerin lacks a cleavage site for zinc metalloproteases and therefore remains persistently farnesylated and is chronically incorporated as part of the nuclear lamina, where it accumulates and causes cytotoxicity. In vitro, mouse model, and human studies have demonstrated that inhibition of farnesylation delays and / or reverses the HGPS phenotype. Clinical trials in children with HGPS have demonstrated that treatment with the farnesyltransferase inhibitor (FTI), lonafarnib, resulted in cardiovascular, bone, hearing, and weight improvements (Gordon et al., 2012) and was associated with an estimated mean survival benefit of 2.5 years. Taking this evidence together, lonafarnib became the first FDA-approved drug for the treatment of HGPS.

[0007] Currently, there are no validated biomarkers for HGPS. Progerin is the disease-causing protein and therefore an optimal biomarker candidate. Progerin protein has been detected in human autopsy tissues and skin biopsies using immunofluorescence or ELISA methods, but these methods are not sensitive or quantitative enough for use in clinical trials, and tissue detection is not feasible for obtaining a range of patient samples. As limitations of immunoassay approaches have long been recognized, alternative methods of immunodetection using targeted mass spectrometry have been developed in recent years (Camafeita et al.). However, their clinical utility may also be limited. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Bar DZ, et al., J Med Genet.2017;54:212-216 [Non-Patent Document 2] Benedicto I et al., Cells.2021;10 [Non-Patent Document 3] Cabral WA et al., Aging Cell.2021:e13457 [Non-Patent Document 4] Camafeita E et al.,Int.J.of Molec.Sci.2022,3,11733 [Non-Patent Document 5] De Sandre-Giovannoli A et al., Progress in molecular and subcellular biology.2006;44:199-232 [Non-Patent Document 6] Eriksson M et al., Nature. 2003;423:293-297 [Non-Patent Document 7] Fong LG et al., The Journal of Clinical Investigation.2006;116:743-752 [Non-Patent Document 8] Gordon LB et al., PNAS.2012;109:16666-16671 [Non-Patent Document 9] Gordon LB et al., Circulation. 2014;130:27-34 [Non-Patent Document 10] Gordon LB et al., JAMA.2018;319:1687-1695 [Non-Patent Document 11] Hisama FM et al., American Journal of Medical Genetics Part A.2011;155A:3002-3006 [Non-Patent Document 12] Scaffidi P et al., Nature Medicine.2005;11:440-445 Summary of the Invention [Problem to be solved by the invention]

[0009] Thus, there is a continuing need for methods to detect progerin in a sensitive and specific manner in biological material that can be obtained longitudinally from children with HGPS to assess treatment efficacy. Ongoing studies (see online at grantome.com / grant / NIH / U01-FD006886-01) aim to validate progerin as a biomarker, but it has not previously been demonstrated that sensitive detection of progerin can be used for diagnostic or prognostic purposes or is sensitive enough to monitor patient changes in progerin in response to a given HGPS treatment. [Means for solving the problem]

[0010] overview Described herein is a method for prognosis of Hutchinson-Gilford Progeria Syndrome (HGPS) in a subject, comprising the steps of: providing a sample from the subject; quantifying the progerin concentration in the sample using a quantitative immunoassay, where the quantitative immunoassay detects progerin but not wild-type Lamin A protein; and comparing the progerin concentration in the sample to an HGPS positive control, where a progerin concentration in the sample that is less than the progerin concentration in the HGPS positive control indicates an increase in life expectancy in the subject.

[0011] In certain embodiments of the described methods, the sample is a liquid sample selected from plasma, serum, urine, and saliva.

[0012] In other specific embodiments, a quantitative immunoassay for use in the described methods comprises the following steps: contacting a sample with a capture antibody that binds to both wild-type lamin A and progerin to obtain a mixture of captured lamin A and captured progerin; separating the mixture of captured lamin A and captured progerin from the sample; contacting the mixture of captured lamin A and captured progerin with a progerin-specific detection antibody that contains a detectable label, thereby binding the detection antibody to the captured progerin but not to the captured lamin A; separating the detectable label from the mixture with captured lamin A; and quantifying the amount of progerin in the sample by detecting the detectable label.

[0013] In certain embodiments of the described methods, the control is from the same subject at an earlier time point.

[0014] In some embodiments, the lower limit of quantification of the quantitative immunoassay is 59 pg / ml. In other embodiments, the upper limit of quantification of the quantitative immunoassay is 30,000 pg / ml. In yet other embodiments, the range of quantification of the quantitative immunoassay is 59 pg / ml to 30,000 pg / ml.

[0015] In certain embodiments, the described methods include the further step of repeating the methods one or more times after the subject has been treated for HGPS, where a decrease, further decrease, or sustained decrease in progerin levels after treatment indicates an increase in life expectancy.

[0016] In certain embodiments, the reduction in progerin levels is between 35 and 62%.

[0017] In yet other embodiments, the greater the decrease in progerin levels, the greater the increase in life expectancy.

[0018] Also described herein is a method for determining the effectiveness of a treatment for Hutchinson-Gilford Progeria Syndrome (HGPS), the method comprising the steps of: providing a pre-treatment sample from the subject taken prior to administration of a therapeutic agent for HGPS; determining the progerin concentration in the pre-treatment sample by a quantitative immunoassay, where the quantitative immunoassay detects progerin but not wild-type Lamin A protein; providing a post-treatment sample from the subject taken during or after administration of a therapeutic agent for HGPS; determining the progerin concentration in the post-treatment sample by a quantitative immunoassay; and comparing the pre-treatment progerin concentration with the post-treatment progerin concentration, where a significant decrease in the progerin concentration in the post-treatment sample indicates that the treatment is effective.

[0019] In certain embodiments, the pre-treatment and post-treatment samples are liquid samples selected from plasma, serum, urine, and saliva.

[0020] In other embodiments, the method further includes providing one or more additional post-treatment samples taken from the subject at a time point after the initial post-treatment sample, where a continued or further decrease in progerin concentration indicates continued effectiveness of the treatment.

[0021] In certain embodiments, the quantitative immunoassay includes the steps of: contacting a sample with a capture antibody that binds both wild-type lamin A and progerin to obtain a mixture of captured lamin A and captured progerin; separating the mixture of captured lamin A and captured progerin from the sample; contacting the mixture of captured lamin A and captured progerin with a progerin-specific detection antibody that contains a detectable label, thereby binding the detection antibody to the captured progerin, but not the captured lamin A; separating the detectable label from the mixture with captured lamin A; and detecting the detectable label, thereby quantifying the amount of progerin in the sample.

[0022] In some embodiments, the lower limit of quantification of the quantitative immunoassay is 59 pg / ml. In other embodiments, the upper limit of quantification of the quantitative immunoassay is 30,000 pg / ml. In yet other embodiments, the range of quantification of the quantitative immunoassay is 59 pg / ml to 30,000 pg / ml.

[0023] In certain embodiments of the methods described, the treatment of HGPS includes a farnesyltransferase inhibitor, which in certain embodiments is lonafarnib.

[0024] Further described in the present specification is a method for treating Hutchinson-Gilford Progeria Syndrome (HGPS) in a subject, the method comprising the steps of providing a sample from the subject; quantifying the progerin concentration in the sample using a quantitative immunoassay, where the quantitative immunoassay detects progerin but not wild-type Lamin A protein; comparing the progerin concentration in the sample to an HGPS positive control, where the progerin concentration in the sample indicates that the subject has HGPS; and, if the subject has HGPS, administering to the subject a treatment for HGPS that reduces the progerin concentration.

[0025] In some embodiments, the lower limit of quantification of the quantitative immunoassay is 59 pg / ml. In other embodiments, the upper limit of quantification of the quantitative immunoassay is 30,000 pg / ml. In yet other embodiments, the range of quantification of the quantitative immunoassay is 59 pg / ml to 30,000 pg / ml.

[0026] In certain embodiments of the methods described, the treatment of HGPS includes a farnesyltransferase inhibitor, which in certain embodiments is lonafarnib.

[0027] The above and other objects, features, and advantages will become more apparent from the following detailed description which proceeds with reference to the accompanying drawings. [Brief description of the drawings]

[0028] [Figure 1] Figure 1 is a schematic diagram of the progerin-specific SMC immunoassay: 1. Lamin A / C capture antibody is bound to magnetic microparticles (circular) at the heavy chain non-specific region; 2. Progerin-containing solution is added to the well, and progerin is bound to the antibody binding site of the Lamin A / C capture antibody; 3. Fluorescently labeled progerin-specific detection antibody is added and bound to progerin; 4. Elution - Chemically separate progerin + Fl (fluorescently labeled) progerin-specific antibody from the magnetic microparticles; and 5. Single molecules are counted using laser detection of the fluorescent label (rectangle). [Diagram 2] Figure 2 shows the analytical performance of the progerin assay. Curve fits were generated using inverse interpolation of the progerin standard curve generated from 10 consecutive assay runs. A. Full range of quantitation. B. Lower limit of quantitation range. [Diagram 3] Figure 3 shows plasma progerin levels in non-HGPS and untreated clinical trial patients. Each symbol represents an individual patient's comparison of levels and age. The Y-axis is on a logarithmic scale. A. Non-HGPS patients (N=69) = triangles. B. Untreated HGPS patients (N=74) = circles. This is displayed with the non-HGPS patients shown in panel A who are in the same age range as the HGPS patients = triangles. [Figure 4A] Figure 4 shows plasma progerin levels in clinical trial patients. Mean ± SEM progerin compared with local study site visits and treatment time. Study visits to BCH occurred at various times after Visit 1 for the different studies. All Visit 1 patients were naive to treatment. A. ProLon1 (N=25), treated with Lonafarnib. [Figure 4B] B. Triple study patients (N=13) treated with triple therapy from baseline through Visit 5 who then switched to lonafarnib monotherapy (Visits 6, 7; N=10). [Figure 4C] C. ProLon2 lonafarnib monotherapy (N=26). [Figure 4D] D. Long-term continuation treatment (N=13). Upper and lower box ends represent interquartile (IQR) ranges for the 75th and 25th quartiles, respectively. Horizontal lines represent medians and x's within boxes represent means. Lower and upper whiskers represent Q1-1.5×IQR and Q3+1.5×IQR. *p<0.05; **p<0.01, ***p<0.001, ****p<0.0001; NS, p>0.05. Each visit is compared to the linked visit. [Diagram 5] Figure 5 shows that progerin levels are associated with survival. Using time-dependent joint modeling, risk of death gradually decreased with decreasing plasma progerin levels (p<0.0001). Change in risk of death (y-axis) versus all values ​​(x-axis) in the range of observed progerin reduction from 0 to 65,000 pg / mL. Solid lines are percent reduction in risk of death and dashed lines are 95% confidence intervals (CI). N=74 subjects (9 untreated with a single sample and 65 treated with untreated baseline and multiple on-therapy samples). [Figure 6] Figure 6 shows that progerin load is associated with survival (p=0.0008). Reduction in plasma progerin is associated with reduced risk of death. Time-dependent Cox model with progerin to predict survival, adjusted for age and sex. Change in risk of death (y-axis) versus all values ​​in the range of observed reduction in progerin from 0 to 65,000 pg / mL (x-axis). Solid lines are percent reduction in risk of death, dashed lines are 95% confidence intervals (CI). N=74 (9 untreated with a single sample, 65 treated with untreated baseline and multiple on-therapy samples). [Figure 7] Figure 7 shows that the degree of life expectancy increase is stepwise associated with the decline in plasma progerin and the duration of therapy. Using dynamic modeling based on cRMST, the increase in life expectancy (y-axis) is shown against the duration of therapy (x-axis) for three selected changes in plasma progerin (N=65 subjects with multiple samples at baseline and on therapy). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Detailed Description term Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. Furthermore, it should be understood that all base or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are provided for illustration purposes. Although methods and materials similar or equivalent to those described herein can be used to practice or test the present disclosure, suitable methods and materials are described below. The abbreviation "eg" comes from the Latin "exempli gratia," and is used herein to denote a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example." Consistent with the definition established in the United States, the term "comprise" means "include," and the terms "comprise essentially of" or "consisting essentially of" indicate that the active ingredients or steps of a described composition or method include only those ingredients or steps that are explicitly recited. Moreover, consistent with the definition established in the United States, a composition that "comprises" a given ingredient can, in other embodiments, "consist essentially of" that ingredient. Similarly, a method that "comprises" a given sequence of steps can, in other embodiments, "consist essentially of" the sequence of steps explicitly set forth.

[0030] In case of conflict, the present specification, including explanations of terms, will control.All materials, methods, and examples are illustrative and not intended to be limiting.

[0031] Prognostic Methods for HGPS The development of clinically relevant biological markers is essential to identify and implement effective treatments for children and young adults with HGPS. However, thus far, the identification of such biomarkers has been hampered by immunological methods that have not been sensitive or quantitative enough to be useful in clinical trials. Furthermore, detection of such biomarkers in tissues has not been possible due to the availability of consecutive patient samples. Because of the limitations of immunoassay approaches, alternatives to immunodetection have been developed using targeted mass spectrometry (Camafeita et al.), but their clinical utility may also be limited. The work described herein establishes that progerin is a quantifiable biomarker for HGPS and shows that progerin concentrations can be used to directly evaluate new treatments for this universally fatal disease. Furthermore, the work described also demonstrates a quantitative relationship of plasma progerin to patient survival, enabling for the first time a quantitative method to prognose patient survival.

[0032] Thus, a newly developed immunoassay is described herein that selectively quantifies progerin in liquid samples (e.g., plasma, urine, and saliva) that can be easily obtained from a subject. This developed immunoassay allows the development of a method for the prognosis of HGPS in a subject, which can be used both to determine the effectiveness of treatment for HGPS and to prognose the relative duration of patient survival.

[0033] To determine the effectiveness of a HGPS treatment, the described method includes providing a sample from a subject prior to receiving HGPS treatment; quantifying the progerin concentration in the pre-treatment sample using a quantitative immunoassay; providing a sample taken from the subject after the subject receives HGPS treatment; quantifying the progerin concentration in the post-treatment sample using a quantitative immunoassay; and comparing the progerin concentration in the post-treatment sample to the progerin concentration in the pre-treatment sample. Based on this comparison, if the progerin concentration in the post-treatment sample is significantly lower than the progerin concentration in the pre-treatment sample, this indicates the effectiveness of the treatment.

[0034] In certain embodiments, the described methods are used to quantify progerin levels in samples taken from a subject at a single time point after administration of a therapeutic agent. In other embodiments, the described methods can be used to quantify progerin in longitudinal samples taken from a subject at successive time points after the initial administration of a treatment, and in certain embodiments during continued administration of a treatment. In these methods of quantifying progerin levels over multiple time points, a given treatment is considered effective if the progerin levels are further reduced from the levels of progerin after the first measured time point, or if the reduced progerin levels are maintained over time.

[0035] As mentioned above, the described method embodiment can be used to determine the relative length of survival of HGPS patients. Such an embodiment includes providing a sample from a subject; quantifying the progerin concentration in the subject with a quantitative immunoassay; and comparing the progerin concentration in the sample with that of a control that represents a HGPS-positive subject. Based on this comparison, a lower progerin concentration in the subject than that of the control indicates an increased life expectancy of the subject.

[0036] Samples for use in the described methods include any sample that can be obtained directly or indirectly from a subject and from which cellular or extracellular proteins can be detected. Such samples include, but are not limited to, whole blood, plasma, serum, tears, mucus, saliva, urine, sputum, tissues, cells (such as fibroblasts, peripheral blood mononuclear cells, or muscle cells), organs, and / or extracts of tissues, cells (such as fibroblasts, peripheral blood mononuclear cells, or muscle cells), or organs. Samples are collected or obtained using methods well known to those skilled in the art. In certain embodiments, samples for use in the described methods include liquid samples such as plasma, serum, urine, or saliva. In yet other specific embodiments, the sample collected from a subject and provided for use in the described methods is a plasma sample.

[0037] In the described methods, "providing" a sample includes any method known in the art for collecting and processing a sample from a subject for use in an immunological method of detecting a protein. In certain embodiments, the sample is provided to the described immunoassay immediately after it is taken from the subject. In other embodiments, the sample is preserved, such as by freezing, for short-term and long-term storage of days, weeks, months, or years. Methods of collection and storage of protein-containing samples are standard and well known in the art. Similarly, in certain embodiments, progerin is detected using the described quantitative immunoassay without additional extensive sample pretreatment. Alternatively, the sample is sufficiently processed to allow detection of the protein by the immunoassay described herein. Such protein extraction and concentration procedures are not necessary for all samples, but can be performed as necessary before providing the sample to the described method to increase the signal detected.

[0038] The prognostic method described herein quantifies progerin by using a quantitative immunoassay that selectively detects progerin in a sample(s) taken from a subject, but not the wild-type (non-mutant) form of the protein, lamin A. The quantitative immunoassay must be sensitive enough to detect differences in progerin concentration in a sample from a comparison source (e.g., a historical control or an earlier time point from the subject) in the picogram / ml range. For example, the quantitative immunoassay should be able to detect differences of 10,000 pg / ml, 5,000 pg / ml, 4,000 pg / ml, 3,000 pg / ml, 2,000 pg / ml, 1,000 pg / ml or less. In a particular embodiment, the lower limit of quantification of the quantitative immunoassay is 59 pg / ml. In another embodiment, the upper limit of quantification of the quantitative immunoassay is 30,000 pg / ml.

[0039] Immunoassays are well known in the art, and it will be understood that any immunoassay that can be adapted to specifically distinguish between wild-type and mutant forms of a protein and has sufficient sensitivity to quantify a target protein on the picogram scale as described can be adapted for use in the described methods. Specific non-limiting examples of immunoassays that can be used in the described methods include radioimmunoassays (RIA), immunohistochemistry, immunofluorescence histochemistry with or without confocal microscopy, and enzyme-linked immunosorbent assays (ELISA) and related non-enzymatic techniques, as well as flow cytometry-related methodologies (see, for example, Cox et al., "Immunoassay Methods" in Assay Guidance Manual, eds., Markossian et al., Eli Lilly and Co., updated 2019). In certain embodiments, the immunoassay is a typical "sandwich assay" or a derivative thereof, in which an antigen of interest is first captured from a sample by an antibody and then detected by a second antibody that specifically recognizes the antigen and is conjugated to a detectable label.

[0040] The immunoassays used in the described methods use antibodies that specifically recognize lamin A and progerin. Such antibodies can be monoclonal or polyclonal. Particular examples of antibodies for use in the described methods include intact immunoglobulins and variants and portions thereof well known in the art, such as Fab' fragments, F(ab)'2 fragments, single chain Fv proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv").

[0041] Beyond the specific structure or molecular sequence, antibodies for use in the described methods should be capable of specifically binding to Lamin A and Progerin (capture antibodies) or specifically binding to Progerin but not Lamin A (detection antibodies). The terms "specifically bind" and "specific binding" refer to the ability of an antibody to bind to a target molecular species in preference to binding to other molecular species in the presence of the specific binding agent and the target molecular species. A specific binding agent specifically recognizes a target molecular species if it is capable of specifically binding to that target molecular species.

[0042] The amino acid sequences of lamin A and progerin are known in the art (available online under NCBI accession numbers NP_733821.1 (lamin A) and AAR29466.1 (progerin)). Lamin A is a 664 amino acid polypeptide and its truncated variant, progerin, is 614 amino acids. Thus, antibodies or fragments thereof used in the described methods that specifically recognize progerin but not lamin A are designed to recognize C-terminal amino acids that are present in progerin but not in lamin A. A specific, non-limiting example of such an antibody recognizes an epitope between amino acids 604 and 611 of progerin, spanning the deleted region of lamin A. Antibodies for use in the described methods are commercially available.

[0043] The detection antibody for use in the described method is conjugated to a detectable label, including any detectable compound or composition that can be directly or indirectly conjugated to an antibody that specifically recognizes progerin but does not recognize or bind to lamin A. Specific non-limiting examples of detectable labels for use in the described method include radioisotopes, enzyme substrates, cofactors, ligands, chemiluminescent or fluorescent agents, haptens, enzymes. Methods for detecting such labels are similarly standard.

[0044] In certain embodiments, the described quantitative immunoassay is a sandwich-type immunoassay comprising the following steps: (a) contacting a sample with a capture antibody that binds to both wild-type lamin A and progerin to obtain a mixture of captured lamin A and captured progerin; (b) separating the mixture of captured lamin A and captured progerin from the sample; (c) contacting the mixture of captured lamin A and captured progerin with a progerin-specific detection antibody comprising a detectable label, whereby the captured progerin binds to the detection antibody but the captured lamin A does not; (d) separating the detectable label from the mixture with captured lamin A; and (e) detecting the detectable label, thereby quantifying the amount of progerin in the sample.

[0045] As in step (b) of the above method, various methods are known in the art for separating a given target protein from a complex mixture. In certain embodiments, the capture antibody is attached to a solid substrate, such as, but not limited to, magnetic particles, polystyrene beads (which may also be coated with affinity tags), wells of a culture dish, and the like. By using such a substrate, the target protein (Lamin A or Progerin in this example) will bind to the substrate if it is specifically recognized and bound by the capture antibody. The captured antibody-protein-substrate can then be separated from the remaining sample components by standard methods and according to the nature of the substrate. For example, in certain embodiments where the capture antibody is coated on a magnetic particle, the captured antibody-protein-substrate complex can be immobilized by a magnetic field, while the remainder of the sample can be washed away under conditions that are sufficiently stringent to prevent non-specific binding of the protein to the antibody. In another exemplary embodiment, the capture antibody is affixed to the bottom of a culture plate, such as a 96-well culture plate, thereby allowing the captured antibody-protein complex to remain on the plate, thereby allowing the remainder of the sample to be washed away.

[0046] Similarly, various methods of separating the bound detectable label (step (d)) after the detection antibody binds to the captured antibody-protein complex (step (c) above) and excess detection antibody is washed away under conditions that limit non-specific detection antibody binding are possible within the scope of the described method. In certain embodiments, the moiety separated from the detection antibody-progerin-capture antibody complex is the detection antibody-progerin subcomplex (similar to separation step 4 depicted in FIG. 1). In other embodiments, it is only the antibody with the label that is removed, while in still other embodiments, it is only the label that is separated from the bound complex. In certain embodiments, the label is separated by enzymatic means that cleave one or more portions of the polypeptide complex (whether as antibody-progerin, as part of the antibody, or away from the antibody). In other embodiments, it is separated by non-enzymatic means that release the label from the complex (whether as part of the antibody or as the antibody-progerin complex).

[0047] In determining the effectiveness of a treatment for HGPS, the subject's progerin concentration is determined before and after administration of the therapeutic agent. In certain embodiments, the effectiveness is determined at a single time point after administration of the therapeutic agent. In other certain embodiments, the effectiveness is determined at multiple time points after or during administration of the therapeutic agent. For example, samples can be taken from the subject over weeks, months, or even years after administration of the therapeutic agent or during the continuation of treatment. In certain embodiments, samples are taken from the subject every 2, 4, 6, 8, 10, 12 months and every increment therebetween after or during administration of the therapeutic agent. In another embodiment, samples are taken every six months or yearly during or after the end of treatment.

[0048] The efficacy of a treatment for HGPS is indicated by the described methods detecting a significant decrease in the concentration of HGPS after or during administration of the therapeutic agent. A significant decrease can be, but need not be, a statistically significant decrease in the present methods. A significant decrease in progerin concentration indicative of efficacy of a treatment includes a decrease of up to 10%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or more from the detected pre-administration progerin concentration. In certain embodiments, the decrease in progerin concentration is determined to be at least 35%-65% lower than the pre-administration progerin concentration.

[0049] In those embodiments in which samples are taken from the subject at multiple time points after an initial administration of a treatment to determine progerin concentrations, the efficacy of the treatment can be determined by either a further decrease in progerin concentrations as described, or by maintenance of the decreased progerin concentrations determined from samples taken at the first time point after administration of the therapeutic agent. In such embodiments, it will be understood that any further decrease in progerin concentrations detected may be less than the initial decrease. Such further decrease may be as little as 1% to 10%, but may be greater as described above.

[0050] The method for determining the effectiveness of a treatment for HGPS can be used to monitor the effectiveness of any treatment that is expected to alter the expression or accumulation of progerin in a subject. Potential treatments for HGPS have been developed or are currently being developed with various mechanisms of action aimed at altering the amount of progerin (reviewed by Macicior et al.), including gene editing (Koblan et al.), RNA-based therapy (Erdos et al., and Puttaraju et al.), small molecules that target the interaction with lamin A, the rate of autophagy, and post-translational processing of progerin protein. In certain embodiments, the method monitors the effectiveness of administering a farnesyltransferase inhibitor, such as lonafarnib, to a subject with HGPS.

[0051] Certain embodiments of the disclosed method are directed to prognosis of relative life expectancy in subjects with HGPS. As previously mentioned, lower progerin concentrations in samples from subjects correlate with increased life expectancy. The described prognostic method compares the progerin concentration in a sample from a subject to an HGPS positive control. As is generally understood in the art, a control is a reference standard. Thus, the HGPS positive control can be a known or previously determined progerin concentration of a subject, or can be derived from a historical average value. In a particular example, the HGPS positive control is a determined progerin concentration of a sample from the same subject (from whom the sample of the method is provided) at an earlier time in life, or before receiving HGPS treatment. In another example, the HGPS positive control is a progerin concentration determined from one or more other known (non-subject) HGPS positive individuals. In a particular embodiment, the HGPS positive control is the average concentration of progerin determined in samples from HGPS positive subjects, as recorded as the historical baseline of HGPS patients.

[0052] Accurate determination of progerin in a clinical setting is an important aspect for effective diagnosis, prognosis, and treatment of HGPS (see, e.g., Benedicto et al.; Cabral et al.). Using the described immunoassay, it can be shown for the first time that a greater reduction in progerin concentration in a subject compared to a HGPS positive control is a quantitative indication of an increase in relative life expectancy. As with the methods for determining therapeutic efficacy described above, the relative reduction does not have to be statistically significant, but as described above, the reduction from the control can be up to 10%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or more. In certain embodiments, the reduction in progerin concentration is determined to be at least 35%-65% lower than the control. The expected life expectancy is increased.

[0053] As with the methods for determining the effectiveness of HGPS treatment, in certain embodiments, the prognostic methods described herein can be employed at a single time point after a subject (patient) is diagnosed. In other embodiments, the methods described herein can be used to monitor the progress of a patient and the changes in progerin concentration over time. Such monitoring can use multiple time points, for example, months, years, or more (and any time in between) after diagnosis and initiation of treatment can be suitable time points to measure the concentration of progerin in a sample from a patient.

[0054] Methods for the Treatment of HGPS In the present disclosure, progerin is established as a biomarker for HGPS. It has been shown that patients diagnosed with HGPS not only have significantly elevated progerin concentrations, but also that patients who have undergone certain treatments for HGPS have significantly decreased progerin concentrations, and that decreased progerin concentrations correlate with increased life expectancy. Thus, described herein is a method of treating a subject with HGPS. In such a method, a sample is provided from a subject who may be suspected of having HGPS or who may not be suspected of having HGPS. The progerin concentration in the sample is determined as described above and compared to a HGPS positive control, which may be a progerin concentration from one or more individuals known to have HGPS. If the progerin concentration in the sample indicates that the subject has HGPS (e.g., because the progerin concentration is similar to, equal to, or exceeds the progerin concentration of the HGPS positive control), the subject is provided with a treatment for HGPS that reduces the progerin concentration.

[0055] As mentioned above, multiple treatments for HGPS are under development to alter progerin levels using a wide range of different mechanisms of action. Any of the described treatments could be used in this manner if proven. The only treatment approved to date is the use of the farnesyltransferase inhibitor lonafarnib.

[0056] Farnesyltransferase inhibitors (FTIs) are a class of compounds that inhibit the ability of farnesyltransferase to transfer a farnesyl group from farnesyl pyrophosphate to a given target protein. FTIs can be used to inhibit the addition of farnesyl to progerin, thereby preventing the abnormal binding of progerin or prelamin A to the inner nuclear envelope. FTIs can be generally divided into three groups: (1) tetrapeptides that contain or mimic the CAAX motif (Brown et al., Proc. Natl. Acad. Sci. USA 89:8313-8316, 1992; Reiss et al., Proc. Natl. Acad. Sci. USA 88:732-736, 1991; Goldstein et al., J. Biol. Chem. 266:15575-15578, 1991); (2) analogues of farnesyl pyrophosphate (FPP) (Gibbs et al., J. Biol. Chem 268:7617-7620, 1993); and (3) inhibitors with structures that resemble neither tetrapeptides nor FPP (Liu et al., J. Antibiot. 45:454-457, 1992; Miura et al., FEBS Lett. 318:88-90, 1993; Omura et al., J. Antibiot. 46:222-228, 1993; Van Der Pyl et al., J. Antibiot. 45:1802-1805, 1992). The latter category of inhibitors is generally less active than the first two categories. As an example, the FTI lonafarnib (Zokinvy®) is a non-peptidomimetic FTI, and FTI-277 is a peptidomimetic FTI. Another non-limiting example of an FTI for use in the methods described herein includes R115777 (tipifarnib, Zarnestra®). The development and chemistry of FTIs is well documented and known to those skilled in the art.To date, lonafarnib is the only FTI approved for use in the treatment of HGPS, but it is believed that all categories of FTIs can be used in the methods provided herein; and selecting a particular FTI is within the skill of one of ordinary skill in the art. In some embodiments, it is beneficial to select an inhibitor compound that is more selective for farnesyltransferase compared to geranylgeranyltransferase I. In other embodiments, it may be beneficial to select an inhibitor compound that is dually selective in that it inhibits both FTase and GGTase I. Considerations for determining the selectivity criteria of an FTI include, but are not limited to, the potential for lower toxicity with an FTase-specific FTI versus a dual-specific FTI, although both efficacy and toxicity may vary depending on the particular compound and the particular patient. As one of ordinary skill in the art would recognize, other considerations may also apply, such as pharmacological and medical considerations.

[0057] In certain instances, FTIs such as lonafarnib are administered at 115 mg / m 2 ~150mg / m 2 / day, for example, in the range of about 115 mg / m 2 , 120 mg / m 2 , 125 mg / m 2 , 130 mg / m 2 , 135 mg / m 2 , 140 mg / m 2 , 145 mg / m 2 , 150 mg / m 2 and the like. A person of ordinary skill in the art will recognize that other dosage ranges typical of FTIs are encompassed by the present disclosure.

[0058] The treatments for use in the methods described herein are generally included in a pharma- ceutically acceptable carrier and / or pharmaceutical excipient. The term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapy is administered.

[0059] Examples of pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. Certain treatments may also contain small amounts of wetting agents, emulsifying agents, or pH buffering agents, as needed. Treatments for use in the methods described herein can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like. Oral formulations can include standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.

[0060] Other pharmaceutical composition embodiments are prepared using conventional pharma- ceutically acceptable counterions, as would be known to one of skill in the art.

[0061] Therapeutic preparations will contain a therapeutically effective amount of at least one active ingredient, preferably in purified form, together with a suitable amount of carrier so as to permit proper administration to the patient. The formulation should suit the mode of administration.

[0062] The combination therapy of the present disclosure can be formulated according to routine procedures as a pharmaceutical composition suitable for intravenous administration to humans.In general, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.If necessary, the composition can also contain a solubilizing agent and a local anesthetic such as lidocaine to ease the pain at the injection site.

[0063] The ingredients in various embodiments are supplied in unit dosage form, either separately or mixed together, for example, in solid, semi-solid and liquid dosage forms such as tablets, pills, powders, liquid solutions or suspensions, or as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. When one or more of the indicated medications are administered by infusion, they can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.

[0064] The specific dosage level and frequency of administration for any particular subject may vary and will depend on a variety of factors, such as the activity of the particular compound, the metabolic stability and duration of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, and the severity of the condition of the recipient being treated.

[0065] Therapeutic compounds and compositions of the present disclosure may be administered at about the same dose throughout the treatment period, in an escalating dose regimen, or in a loading dose regimen (e.g., a loading dose is about 2-5 times the maintenance dose). In some embodiments, the dose is modified over the course of treatment based on the condition of the subject being treated, the severity of the disease or condition, the apparent response to therapy, and / or other factors as determined by one of skill in the art. In some embodiments, chronic treatment with the drug is contemplated.

[0066] In some embodiments, sustained local release of a pharmaceutical preparation containing a therapeutically effective amount of a therapeutic compound or composition may be beneficial. Sustained release formulations are known to those skilled in the art. As an example, sustained local release can be achieved using polymers such as bis(p-carboxyphenoxy)propane-sebacic acid or suspensions of lecithin.

[0067] The following examples are provided to illustrate particular features and / or embodiments and should not be construed as limiting the disclosure to the particular features or embodiments described. EXAMPLES

[0068] Example 1: Methods Single Molecule Counting (SMC™) Progerin Immunoassay We have launched an SMC ultrasensitive immunoassay for plasma progerin detection. Overall, the assay employs a sandwich immunoassay format with an anti-lamin A capture antibody coated on magnetic microparticles and an anti-progerin-specific detection antibody conjugated to a fluorescent tag, and was performed in a 96-well plate (Figure 1). The progerin-bound detection antibody was eluted and read as a single molecule using a confocal laser.

[0069] The SMC assay for measuring progerin was as previously described (Todd et al.), except that the magnetic microparticles (MPs) coated with capture antibody and detection reagent were specific for the progerin assay. Briefly, progerin capture antibody (mouse Mab anti-lamin A+C, clone: ​​131C3 Abcam, epitope between amino acid residues 319-566, common to lamin A, C and progerin) was coated onto MPs at 25 μg antibody / mg MP. Samples were exposed to the MP-capture antibody, which specifically bound to the analyte, and the plate was washed once to remove unbound material. Next, fluorescently labeled progerin-specific detection antibody (mouse Mab, clone: ​​13A4, Millipore, epitope between amino acids 604-611 of progerin spanning the deletion region of lamin A) was pre-diluted to 2,000 ng / mL in assay buffer, then filtered through a 0.2 μm filter (MilliporeSigma) and added to the wells. The plate was then washed four times to remove unbound detection antibody. All MPs were then transferred from the original 96-well assay plate to a new plate to prevent elution of non-specific detection antibody bound to the plate. Progerin-bound detection antibody was then eluted by pH shift with a proprietary glycine buffer, transferred to a new plate, and detected by SMC using Erenna Immunoassay System software (SgxLink™). Raw signal data and interpolated data obtained from SgxLink were transferred to Excel, where precision was calculated for replicate wells. Acceptable specifications for raw signal and interpolated values ​​were precision and recovery bias ≤ 20%.

[0070] A 12-point standard curve of recombinant progerin (Abcam 93918) diluted in EMD standard diluent (a synthetic serum-based solution containing a proprietary mixture of Tris buffer and carrier protein (emdmillipore.com)) was run in triplicate on each plate. The standard curve was used to measure progerin concentrations in unknown samples. Samples were diluted in standard diluent such that the expected progerin fell within the dynamic range of the standard curve. All reported progerin and SD are dilution corrected. All samples were tested in duplicate unless specified as triplicate.

[0071] Signals below background plus two standard deviations were reported as not detected (ND). The lower limit of quantification (LLoQ) was predefined as the lowest point on the standard curve, from top to bottom, that was recovered within 20% of the expected value and had a percent coefficient of variation (CV) of 20% or less.

[0072] To ensure similar progerin quantification between high, medium, and low assays, the same interassay quality control (QC) set of endogenous plasma was assayed in duplicate on each plate. Endogenous plasma progerin was obtained from a homozygous transgenic mouse model of HGPS expressing the human LMNA gene with the classical pG608G,c.1824 C>T mutation (Murtada et al.). This mouse plasma contained high levels of progerin, allowing QC generation without using precious human HGPS plasma reserves. Aliquots were diluted with normal human plasma and stored at -80°C. Acceptability parameters for the QC plate were fixed as the mean ± 20% of the first 24 QC runs during assay development. Mean progerin for high and medium QC was 188,206 ± 10,580 pg / mL (%CV = 5.6) and 19,513 ± 1,235 pg / mL (%CV = 6.3), respectively. Low QCs were always below the LLoQ. To mirror the 1:25 dilution protocol for clinical trial samples, QCs were filtered and diluted 25-fold on the same day the assay was performed. Mean assay progerin for high and medium QCs in the clinical trial sample plate (N=39) was 7,303 ± 609 pg / mL (%CV=3.8) and 755 ± 90 pg / mL (%CV=5.7), respectively. Thus, high and medium QCs were always within the analytical measurement range.

[0073] Valid runs required that the lower limit of quantitation calculated from the standard curve was 59 pg / mL or less and that controls were within 20% of the established high and medium QCs.

[0074] Once the final assay conditions were established, samples with interpolated concentrations above the upper limit of quantitation (ULoQ) or with %CVs above 20% were retested using appropriate dilutions to ensure that progerin amounts fell within the quantitation limits of the assay. Additionally, any plates that did not meet the LLoQ of the standard curve (599 pg / mL) or whose QCs (high and medium, with the low control below the LLoQ) fell outside 20% of the mean and SD were repeated.

[0075] Dilution Linearity The linearity of the dilutions was calculated by dividing the dilution-corrected progerin values ​​in the dilution series by the progerin value of the preceding dilution and expressed as a percentage: DL (dilution linearity) = [actual value] B / [Actual value] A * 100, where B is the dilution correction value of the higher dilution and A is the dilution correction value of the preceding dilution series.

[0076] Freeze-thaw sample stability Plasma samples frozen at -80°C were thawed at room temperature (RT) for 15 min, mixed thoroughly, and then aliquoted to create three additional aliquots for this study. All aliquots were then refrozen at -80°C for additional freeze-thaw testing. On the day of testing, the assigned aliquots were thawed to room temperature for 15 min and refrozen at -80°C for up to four freeze-thaw cycles. The final thawed samples were diluted and evaluated in triplicate. All samples were tested on the same day. Stability of freeze-thaw samples was passed if the percentage difference between freeze-thaw cycles 2 and 5 was ±20% (industry standard).

[0077] The percentage difference (%) between freeze-thaw cycles 3, 4, and 5 and cycle 2 for each sample was calculated using the following formula:

number

[0078] Detection of native and recombinant progerin Healthy non-HGPS plasma samples (commercially available) were diluted 50-fold in standard diluent and then spiked with either recombinant progerin, which produces 1,000 pg / mL of 10% by volume of the standard dilution, or HGPl 110, designed to deliver 1,000 pg / mL of progerin. The calculated actual progerin spike was 1,019 pg / mL, determined from the control value of the buffer-spiked sample HGPl 110.

[0079] Spike recovery (%) = (progerin in spiked sample (pg / mL) - endogenous progerin (pg / mL) / expected spiked progerin (pg / mL).

[0080] Lonafarnib interference assay Lonafarnib active pharmaceutical ingredient (API) (batch number MK-6336-000R025) was obtained from the PRF Cell and Tissue Bank. HGPS plasma was spiked with 2 μL of either dimethyl sulfoxide (DMSO) or lonafarnib dissolved in DMSO at 7.92 μg / mL. Percent interference (%) = [(mean concentration of spiked samples - mean concentration of controls) / mean concentration of controls] x 100.

[0081] Human clinical data and samples Human clinical data were obtained from The PRF International Progeria Registry, Diagnostics Program, and Medical and Research Database (available online at progeriaresearch.org). HGPS plasma and serum were obtained from The PRF Cell and Tissue Bank (available online at progeriaresearch.org). Clinical trial samples were provided to the bank by Boston Children's Hospital (BCH) with informed consent or approval from the BCH Clinical Trials Committee. Test samples were collected in the fasting state in the morning immediately prior to treatment administration as trough samples, except for pK samples, which were collected at the trough and at designated times thereafter after the trough. Non-test samples were not time-restricted. Several non-HGPS healthy human plasma samples were obtained from ProMedDx. HGPS and non-HGPS plasma sample identifiers were prefixed with HGPl and NPl, respectively.

[0082] Patients with HGPS had been genetically confirmed to have a progerin-producing mutation in the LMNA gene. Samples from patients with classic HGPS (c.1824C>T) contributed to the main analyses, and comparisons with non-classic HGPS were used for subgroup comparisons.

[0083] For plasma isolation, blood was collected into sodium heparin (either heparin or CPT™ Vacutainers) and centrifuged at 1500xg for 15 min at 4°C, or collected into K2EDTA and centrifuged at 1300g for 10 min at room temperature (RT) (all BD Biosciences). For serum isolation, blood was allowed to clot for 60 min (± 10 min) at room temperature and then centrifuged at 1100xg for 10 min at room temperature. All samples were aliquoted, stored at -80°C, and shipped on dry ice to EMD Millipore for analysis.

[0084] For analysis, samples were completely thawed in less than 5 min, mixed gently, diluted with standard diluent, and then transferred to filter plate wells (MilliporeSigma MSBVN1210). Patient plasma volumes required for analysis were 200 μl or 100 μl (non-HGPS 1:1 and 1:2) and 8 μl (HGPS 1:25) in duplicate. Filter plates containing samples were centrifuged for 10 min to force the samples through the filters. The filtrates were then evaluated as described.

[0085] Study drug dosage and administration The clinical trial study samples were divided into three categories, named after the initial letters ProLon1 (Gordon et al., 2014; Gordon et al., 2012), Triple Therapy (Gordon et al., 2016), and ProLon2 (Gordon et al., 2018), and were sequentially conducted as open-label, single-center trials at Boston Children's Hospital (see details of the previously published study). In all clinical trials, patients received lonafarnib (Schering-Plough Research Institute or Merck & Co, Inc. or Eiger Biopharmaceuticals) orally every 12 ± 2 hours in capsules or liquid suspension. The dose of lonafarnib was 150 mg / m 2 However, ProLon1 was administered at 115 mg / m 2 Except that it was.

[0086] In the triple therapy trial, oral pravastatin (Pravachol, Bristol-Meyers Squibb) and zoledronic acid (Zometa, Novartis, Inc.) were administered with lonafarnib.

[0087] Sample blinding and inclusion criteria Once assay development was established, all samples were evaluated in a blinded fashion by EMD Millipore technical staff. Replicate samples were assigned different random ID numbers. ID assignments without the HG or nonHG prefix were submitted for analysis and converted to HG and nonHG identifiers.

[0088] For inclusion in the longitudinal analysis, sample sets required baseline and end-of-study on-therapy plasma samples. To compare lonafarnib doses, ProLon1 inclusion included baseline pretreatment, 4 months (dose 115 mg / m 2 ), and the sample at the end of the test (dosage 150 mg / m 2 ) was required. Sample sets required at least one trough plasma sample and one post-trough plasma sample for inclusion in the pK analysis.

[0089] statistical analysis Data summaries were primarily descriptive, and comparisons between sample sets were performed using paired Student's T-tests, two-sample T-tests, and Pearson correlation. Descriptive statistics included sample sizes, means, and standard deviations. The statistical significance level was two-sided p<0.05. No adjustments for multiple comparisons were made.

[0090] To assess the relationship between plasma progerin concentrations and survival, we performed two analyses, adjusting for both baseline age and sex. The first implemented a joint model with a time-dependent slope parameterization, thereby assessing the effect of current progerin levels on survival while adjusting for changes in progerin. The second implemented a time-dependent Cox model, treating progerin as a time-dependent covariate. Because patients began treatments shortly after baseline that would be expected to affect progerin levels, we did not assess survival outcomes by baseline progerin alone, as this would be biased.

[0091] To quantify the gains in life expectancy associated with reductions in progerin and duration of lonafarnib therapy, we used the methodology of Yang et al. (2021), which proposes a dynamic model (Dafni, 2011) using a series of landmark time points based on conditional restricted mean survival times (cRMST). The analysis also adjusted for sex and age at baseline.

[0092] Descriptive statistics were performed using Excel. SAS version 9.4 was used for time-dependent Cox models, and the JM and dynpred packages in R version 4.0.2 were used for joint modeling and the cRMST dynamic model, respectively.

[0093] Example 2: HGPS-specific plasma progerin assay This example describes the development and validation of an immunoassay for the specific and sensitive detection of progerin in plasma samples.

[0094] Development and validation of a quantitative assay for the detection of plasma progerin Standard curve and reporting range: To generate an SMC progerin immunoassay standard curve and determine a reliable reporting range, recombinant progerin was spiked into buffer at 30 ng / mL and serially diluted in 2-fold steps to generate a standard curve from 30,000 to 29 pg / mL (Figure 2, panels A and B). Signals were interpolated with SGXLink software. The dynamic detection range was 59 pg / mL (LLoQ) to 30,000 pg / mL (ULoQ). The mean (measured concentration) / (expected concentration) was 100% (range 95% to 106), with a linear response (R) from 59 pg / mL to 30,000 pg / mL. 2 = 0.9987). As a result, all samples were diluted with standard diluents to fall within this range. Specifically, non-HGPS samples were evaluated at 1:1 or 1:2, and HGPS samples were below the ULoQ when diluted 1:25.

[0095] Dilution Linearity: To evaluate the ability of the assay to consistently measure plasma progerin through a range of sample dilutions in HGPS, human HGPS plasma samples (N=3) were quantified in triplicate at 5x, 25x, and 125x dilutions in assay buffer (Table 1). All patient samples were quantifiable at all dilutions tested, with final progerin ranging from 15,835 pg / mL to 50,610 pg / mL. The mean linearity of dilution was 98.2% (range 92-110). Non-HGPS plasma (N=4) was evaluated for linearity 1:2 versus neat to maintain LLoQ excess.

[0096] [Table 1]

[0097] Intra- and Inter-Assay Variability: The precision of the SMC progerin immunoassay was determined to reliably quantify plasma samples within runs (intra-assay) and between days (inter-assay) using multiple replicates on multiple days. Intra-assay variability (Table 2) showed an average CV of 7% (range 1-16), and inter-assay variability (Table 3) showed an average CV of 12% (range 7-18). Furthermore, high and medium QCs run in duplicate for each clinical trial sample plate (N=39) demonstrated excellent intra-assay precision. The dilution-corrected average high QC value was 182,593 ± 15,229 pg / mL progerin with a CV of 3.8%, and the medium QC value was 18,880 ± 2,260 pg / mL progerin with a CV of 5.8%.

[0098] [Table 2] [Table 3]

[0099] Evaluation of an HGPS-specific plasma progerin assay: Cross-reactivity with lamin A and completeness of sample freeze-thawing Progerin assay does not cross-react with lamin A: To assess potential cross-reactivity between the detection of progerin and lamin A in the assay, two 12-point sample curves, one containing human recombinant lamin A and one containing human recombinant progerin, were run in triplicate in the SMC progerin immunoassay starting at 10,000 pg / mL and serially diluted 2-fold to 9.8 pg / mL, with a buffer-only anchor point of 0 pg / mL for each curve. No cross-reactivity to lamin A was detected in the assay. The recovery range of progerin was 97-111% of expected values ​​at the various concentrations tested, with R 2 = 0.9991. Therefore, no signal was generated by the presence of lamin A competing or interfering with the quantification of progerin.

[0100] Spiked recovery and detection of native and recombinant progerin: To evaluate the ability of the assay to measure recombinant progerin compared to native progerin, either was spiked into three different healthy non-HGPS donor plasma samples that were diluted 50-fold in standard diluent prior to spiking. Both native and recombinant progerin showed similar progerin recoveries within the expected range, with average recoveries of 96±6% and 120±15%, respectively (Table 4).

[0101] [Table 4]

[0102] Freeze-thaw stability: Samples presented in this study were frozen prior to analysis. Only pre-frozen samples are expected to be routinely evaluated in clinical trials to perform off-site assays. Freeze-thaw analysis through four freeze-thaw cycles showed no differences between progerins (all p<0.05).

[0103] Classical and non-classical genotypes Both classical and nonclassical HGPS are progerin-producing; classical is due to internal splice site optimization, and nonclassical is due to canonical splice site deoptimization.Data from children with the classical genotype (G608G, c.1824C>T; N=74) were compared with nonclassical HGPS patient samples (LMNA c.1822G>A, p.G608Sx2, c.1968+1G>A, c.1968+2T>A, c.1968+5G>C) (N=5; mean±SD=28,402±11,601 pg / mL). When baseline off-therapy progerin was compared to classical patient samples (N=74, mean ± SD=33,249 ± 12,299 pg / mL), progerin levels in all nonclassical patients fell within the range of classical levels, with no overall differences detected (p=0.39). Similarly, the reduction in progerin from baseline to end of study with therapy (40.3-57.5%) was within the range of classical patients, with end-of-study values ​​not different from classical patients (p=0.85).

[0104] Similarity between sodium heparin-isolated plasma, K2EDTA-isolated plasma, and serum in the determination of progerin To evaluate whether the assay is suitable for progerin detection across different blood collection tube types, a comparative analysis of blood collected in triplicate from the same patient blood draw (N=3 patients) in tubes containing sodium heparin (plasma), K2EDTA (plasma), and no anticoagulant (serum) was performed: p=0.98 for sodium heparin plasma vs. EDTA plasma, p=1.00 for sodium heparin plasma vs. serum, and p=0.97 for EDTA vs. serum.

[0105] Plasma progerin detection in treatment-naive patients Non-HGPS plasma: Non-HGPS plasma (N=69; 39 M (male), 30 F (female)) was evaluated (Figure 3, panel A). The sample set consisted of 33 donors evaluated for HGPS or non-HGPS progeroid laminopathy (PL) using LMNA±ZMPSTE24 mutation analysis, 32 healthy relatives who were evaluated for mutations, and 4 plasma samples from commercially purchased healthy controls. None of the donors evaluated for HGPS or PL had progerin-producing mutations. Overall mean progerin was 351±251 pg / mL (range=ND-1534). The mean donor age was 23.3±18.7 years (range 0.2-71.3). There was no correlation between donor age and progerin (r=0.3, p=0.81).

[0106] Plasma progerin in drug-naive patients with HGPS: Plasma progerin profiles were assessed in children with HGPS at baseline before initiation of study drug treatment (Figure 3, Panel B). Mean progerin was 33,249 ± 12,299 pg / mL overall (N = 74), 33,237 ± 11,797 (N = 37) in women, and 33,285 ± 13,035 (N = 37) in men. No association was observed between progerin and gender. Mean progerin in HGPS was 95-fold increased compared to non-HGPS (p < 0.0001). In the individual studies, mean baseline treatment-naive progerin levels were 27,572 ± 7,542 pg / mL for ProLon1 (N = 26), 31,464 ± 16,958 pg / mL for triple therapy (N = 13), and 38,154 ± 11,546 pg / mL for ProLon2 (N = 35). There were no significant differences in progerin when comparing ProLon1 or ProLon2 with triple therapy (p = 0.44 and 0.12, respectively), but baseline levels were higher with ProLon2 than with ProLon1 (p < 0.0001). Subgroup comparisons of age- and sex-matched controls also demonstrated highly significant differences between HGPS (progerin = 34,512 ± 14,432 pg / mL, N = 25) and non-HGPS control levels (progerin = 377 ± 226 pg / mL, N = 13, p < 0.0001).

[0107] Plasma progerin and age: Most patients provided a single baseline sample immediately prior to administration of clinical trial drug at the BCH study site, but some patients also provided blood samples as part of a natural history study conducted at the National Institutes of Health (NIH) Clinical Center prior to BCH study provision (Merideth et al.); thus allowing for long-term evaluation of progerin independent of drug therapy. These samples were subsequently submitted to the PRF Cell and Tissue Bank. Blood samples from the NIH study were processed similarly to clinical trial samples. The mean blood sampling interval was 1.6 ± 0.5 years (range 0.9-2.3). Mean plasma progerin (N = 13) at initial and follow-up was 29,221 ± 7,772 pg / mL and 33,272 ± 11,959 pg / mL, respectively, with no significant difference in progerin over time (p = 0.14).

[0108] Example 3: Plasma Progerin Assay to Determine Efficacy of HGPS Treatment This example explores the feasibility of using the developed progerin immunoassay to determine the efficacy of treatments for HGPS, such as the FTI lonafarnib.

[0109] Exogenous lonafarnib does not interfere with progerin assay: To assess whether the changes in plasma progerin truly reflect the effects of lonafarnib therapy on plasma progerin levels, we assessed whether lonafarnib itself interferes with progerin assay measurements. Because the Cmax determined from a pharmacokinetic study in children with HGPS treated with lonafarnib during clinical trials was 2.64 μg / mL (Gordon, 2012), plasma samples were spiked with 7.92 μg / mL of lonafarnib, three times the Cmax. Mean progerin was unaffected by spiking with lonafarnib in DMSO compared to the control condition (p=0.198; Table 5).

[0110] [Table 5]

[0111] Progerin changes with lonafarnib therapy: ProLon1 was the first clinical trial to administer lonafarnib to children with HGPS and had several differences from triple therapy and ProLon2. Patients visited the study site every 4 months and had blood drawn for a total of 2.2 ± 0.1 years (7 visits), compared with every 6 months for a total of 3.5 ± 0.2 years (5 visits) for triple therapy and 2.4 ± 0.6 years for ProLon2 (baseline + end of study) (2 visits). Additionally, patients in ProLon1 received 115 mg / m 2 for 4 months, then 150 mg / m 2 Both the Triple Trial and ProLon2 increased patients to 150 mg / m 2 Patients were started at 18 weeks of age and 18 weeks of age at baseline. Sample numbers varied by individual study and visit; comparisons between study visits included only matched plasma sets. Changes in progerin were compared within and between separate but consecutive studies (Figure 4, Panels A-D). Overall, mean plasma progerin during therapy decreased 38% from untreated baseline, from 32,726 ± 12,659 to 20,211 ± 10,190 pg / mL (p<0.0001).

[0112] Changes in ProLon1 progerin levels with lonafarnib therapy: Mean progerin levels were 115 mg / m 2 At month 4, there was a 48% reduction from baseline during treatment with lonafarnib (N=25, p<0.0001, Figure 4, Panel A). 2 For the remaining 5 patients, the mean reduction from baseline at each visit ranged from 50% to 62% (N = 22-25, all p < 0.0001). 2 from 150 mg / m 2 At the eighth-month study visit, four months after switching to lonafarnib at 115 mg / m, progerin levels further decreased to near-significant levels (N=25, p=0.06), and at four subsequent study visits,2 Dosage exceeds 150 mg / m 2 Progerin levels decreased significantly during treatment (N = 22-25, all p < 0.05). 2 There was no significant difference between the mean progerin dose at the first visit and the mean progerin dose at subsequent visits at the same dose (N = 22-25, all p ≥ 0.05). This dose response resulted in a median progerin dose of 115 mg / m 2 More than 150mg / m 2 The ability of the assay to detect incremental but significant benefits of dosing is demonstrated.

[0113] Changes in progerin levels with triple therapy: At the first on-therapy study visit after 6 months, mean progerin levels were reduced by 41% from baseline (N=13, p=0.0018; Figure 4, Panel B). Thereafter, mean progerin levels remained significantly reduced by 35-47% from baseline (N=12-13 patients, 3-5 visits, p=0.0015-0.0058).

[0114] After the formal triple therapy period was completed, pravastatin and zoledronic acid were discontinued, and 10 patients completed an additional 4.1 ± 0.5 years of lonafarnib monotherapy extension (range 3.41 to 4.65; Figure 4, Panel B). In these 10 patients, mean progerin levels were not significantly different between consecutive triple therapy and on-treatment lonafarnib monotherapy extension study visits (p = 0.99).

[0115] Changes in ProLon2 progerin with lonafarnib therapy: Mean progerin decreased by 36.7% from baseline over the treatment period (N=26, p<0.0001; Figure 4, Panel C).

[0116] Effect of Long-Term Therapy with Lonafarnib on Progerin: A subgroup of 13 subjects continued treatment with lonafarnib without interruption as part of ProLon1, the triple therapy trial, and the lonafarnib monotherapy extension study of the triple trial (Figure 4, Panel D). This constituted a mean of 9.8 ± 0.5 years (range 9.0-10.3 years) of continuous lonafarnib treatment. The mean progerin dose was 115 mg / m 2 During treatment with 150 mg / m 2 The mean reduction from baseline during treatment ranged from 56 to 74% (p<0.0001). 2 Compared to the first time point (time point #3 at 0.66 years), time points 5, 8, and 9 were significantly lower (p<0.05), while all other time points (4, 6, 7, 10-13) were similar to time point #3 (p>0.05).

[0117] Circadian variation of progerin in a pharmacokinetic study: lonafarnib dose 115mg / m 2 and 150 mg / m 2 During ProLon1 (Gordon, 2012) and triple therapy (Gordon, 2016), plasma was collected at trough and 1, 2, 4, 6, and 8 hours post-dose for pK studies. Within each of these sample sets, progerin was administered at 150 mg / m 2 There was no significant change between the post-administration and trough levels in ProLon1 and triple therapy, indicating that Progerin does not exhibit circadian variation (all p ≥ 0.05). 2 With this lonafarnib dose, mean progerin trough levels were similar 1 hour after administration (p=0.2647) but significantly lower than 2, 4, and 8 hours after administration, with an average difference of approximately 3,000 pg / mL (all p<0.05).

[0118] Example 4: Progerin levels are strongly associated with survival In this example, we evaluate whether plasma progerin is suitable to serve as a predictive biomarker of survival. First, the change in risk of death relative to changes in progerin measurements was estimated directly from a joint model relating these two measurements. All patients with plasma samples were included, regardless of whether they underwent therapy, duration of therapy, or follow-up period (N=74, 26 deaths, 47 alive as of April 1, 2022). Progerin levels were significantly associated with risk of death (p<0.0001; Figure 5). For example, a 1,000 pg / mL decrease in plasma progerin was associated with a 6.3% decrease in risk of death (95% CI 4.7-7.8%), whereas a 10,000 pg / mL decrease in plasma progerin was associated with a 47.6% decrease in risk of death (95% CI: 38.4-55.4%). Progerin was modeled as a function of duration of therapy, duration of therapy squared, sex, and age at baseline. The joint models were adjusted for duration of therapy. Additional analyses using standard time-dependent Cox modeling that does not account for potential measurement error of the longitudinal covariate (progerin), as well as subgroup analyses using only patients with at least one on-therapy plasma sample, yielded similar results (Figure 6, Table 6).

[0119] [Table 6]

[0120] We next evaluated the relationship between the duration of lonafarnib treatment and the change in life expectancy by the degree of change in plasma progerin (Figure 7, Table 7). This analysis included samples from patients who had a baseline pre-therapy and at least one progerin measurement during treatment (N=65 patients). Life expectancy improved over time with both the degree of plasma progerin reduction and the time spent with low progerin levels. Figure 4 graphs three representative broad examples of progerin reduction during therapy (1,000, 10,000, and 15,000 pg / mL) over 10 years of treatment. Patients' lives were extended in all three cases.

[0121] [Table 7]

[0122] JPEG2025501303000010.jpg143153

[0123] In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are merely preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

1. 1. A method for prognosis of Hutchinson-Gilford progeria syndrome (HGPS) in a subject, comprising: providing a sample from a subject; Quantifying the concentration of progerin in the sample by a quantitative immunoassay, where the quantitative immunoassay detects progerin but does not detect wild-type Lamin A protein; and comparing the concentration of progerin in the sample with a HGPS positive control, wherein a concentration of progerin in the sample that is less than the concentration of progerin in the HGPS positive control indicates an increase in life expectancy of the subject; The method comprising:

2. The method of claim 1 , wherein the sample is a liquid sample selected from plasma, serum, urine, and saliva.

3. Quantitative immunoassays: contacting the sample with a capture antibody that binds both wild-type lamin A and progerin to obtain a mixture of captured lamin A and captured progerin; Separating the mixture of captured lamin A and captured progerin from the sample; contacting the mixture of captured lamin A and captured progerin with a progerin-specific detection antibody that comprises a detectable label, thereby binding the detection antibody to the captured progerin, but not to the captured lamin A; Separating the detectable label from the mixture with the captured Lamin A; and Detecting the detectable label and thereby quantifying the amount of progerin in the sample; The method of claim 1 or 2, comprising:

4. The method of any one of claims 1 to 3, wherein the control is obtained from the same subject at an earlier time point.

5. The method according to any one of claims 1 to 4, wherein the lower limit of quantification of the quantitative immunoassay is 59 pg / ml.

6. The method according to any one of claims 1 to 5, wherein the upper limit of quantification of the quantitative immunoassay is 30,000 pg / ml.

7. The method of any one of claims 1 to 6, further comprising repeating the method one or more times after the subject has been treated with HGPS, wherein a decrease in progerin levels after treatment indicates an increase in life expectancy.

8. The method of claim 7, wherein the reduction in progerin levels after treatment is 35-62%.

9. The method of claim 7, wherein a greater decrease in progerin concentration indicates a greater increase in life expectancy.

10. 1. A method for determining the efficacy of a treatment for Hutchinson-Gilford Progeria Syndrome (HGPS), comprising: providing a pre-treatment sample from the subject, the pre-treatment sample being taken prior to administration of a therapeutic agent for HGPS; determining the concentration of progerin in the pre-treatment sample by a quantitative immunoassay, where the quantitative immunoassay detects progerin but does not detect wild-type Lamin A protein; providing a post-treatment sample from the subject taken during or after administration of a therapeutic agent for HGPS; Determining the concentration of progerin in the post-treatment sample by quantitative immunoassay; and comparing the progerin concentration before treatment to the progerin concentration after treatment, wherein a significant decrease in the progerin concentration in the post-treatment sample indicates that the treatment is effective; The method comprising:

11. The method of claim 10, wherein the pre-treatment and post-treatment samples are liquid samples selected from plasma, serum, urine, and saliva.

12. The method of claim 10 or claim 11, further comprising providing one or more additional post-treatment samples taken from the subject at time points after the initial post-treatment sample, wherein a continued decrease or further decrease in progerin concentration indicates continued effectiveness of the treatment.

13. Quantitative immunoassays: contacting the sample with a capture antibody that binds both wild-type lamin A and progerin to obtain a mixture of captured lamin A and captured progerin; Separating the mixture of captured lamin A and captured progerin from the sample; contacting the mixture of captured lamin A and captured progerin with a progerin-specific detection antibody that comprises a detectable label, thereby binding the detection antibody to the captured progerin, but not to the captured lamin A; Separating the detectable label from the mixture with the captured Lamin A; Detecting the detectable label and thereby quantifying the amount of progerin in the sample; The method according to any one of claims 10 to 12, comprising:

14. The method according to any one of claims 10 to 13, wherein the lower limit of quantification of the quantitative immunoassay is 59 pg / ml.

15. The method according to any one of claims 10 to 14, wherein the upper limit of quantification of the quantitative immunoassay is 30,000 pg / ml.

16. The method of any one of claims 10 to 15, wherein the treatment comprises a farnesyltransferase inhibitor.

17. 17. The method of claim 16, wherein the farnesyltransferase inhibitor is lonafarnib.

18. 1. A method for treating Hutchinson-Gilford Progeria Syndrome (HGPS) in a subject, comprising: providing a sample from a subject; Quantifying the concentration of progerin in the sample by a quantitative immunoassay, where the quantitative immunoassay detects progerin but does not detect wild-type Lamin A protein; comparing the concentration of progerin in the sample with a HGPS positive control, where the concentration of progerin in the sample indicates that the subject has HGPS; and if the subject has HGPS, administering to the subject a therapeutic agent for HGPS that reduces progerin levels; The method comprising:

19. 19. The method of claim 18, wherein the lower limit of quantification of the quantitative immunoassay is 59 pg / ml.

20. 20. The method of claim 19, wherein the upper limit of quantification of the quantitative immunoassay is 30,000 pg / ml.

21. 21. The method of claim 20, wherein treating HGPS comprises administering to the subject a farnesyltransferase inhibitor.

22. 22. The method of claim 21, wherein the farnesyltransferase inhibitor is lonafarnib.

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