Lysosomal storage disorder; specific pharmacological chaperone is 1-deoxygalactonojirimycin; T cell stimulatory agent; broadly applicable, fast and efficient method for screening patients for responsiveness to SPC therapy
Inventors
Kaneski, Christine • Shin, Sang-hoon • Murray, Gary • Ludwig, Karin • Lockhart, David • Castelli, Jeff
Assignees
Bpcr LP • Amicus Therapeutics Inc • National Institutes of Health NIH
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Publication Number
US-7851143-B2
Publication Date
2010-12-14
Expiration Date
2027-05-16
Abstract
Provided are in vitro and in vivo methods for determining whether a patient with Fabry disease will respond to treatment with a specific pharmacological chaperone.
Core Innovation
The invention provides methods to determine whether a patient with Fabry disease will benefit from treatment with a specific pharmacological chaperone (SPC) such as 1-deoxygalactonojirimycin (DGJ). It exemplifies two methods—an in vitro assay using T cells and an in vivo assay using white blood cells—to evaluate α-galactosidase A (α-GAL) responsiveness to SPC therapy. The invention also offers an improved method for diagnosing Fabry disease in patients suspected of having the disease by measuring α-GAL activity in T cells.
Fabry disease is a lysosomal storage disorder caused by an X-linked inherited deficiency of lysosomal α-galactosidase A enzyme, leading to accumulation of glycosphingolipids such as globotriaosylceramide (GL-3) in cells. This accumulation causes severe symptoms including kidney failure and increased risk of heart attack and stroke. Most mutations are missense mutations resulting in unstable α-GAL that is misfolded and retained/degraded in the endoplasmic reticulum (ER), preventing proper lysosomal trafficking and function.
The problem addressed is the difficulty in diagnosing Fabry disease accurately due to its heterogeneous clinical presentation, delayed diagnosis, and challenges in predicting patient responsiveness to SPC therapy. Since only some mutations produce α-GAL proteins amenable to stabilization by SPCs, there was no reliable, broadly applicable method to screen patients for SPC responsiveness prior to treatment, particularly using viable cell populations. Thus, there was a need for fast, efficient, and non-invasive assays to evaluate enzyme enhancement by SPCs in patient cells to facilitate treatment decisions and improve diagnosis.
Claims Coverage
The patent includes one independent claim that focuses on a method for determining the responsiveness of a patient to SPC therapy by measuring protein activity in patient cells with and without exposure to a pharmacological chaperone. The main inventive features are extracted as follows.
Method for screening responsiveness to a specific pharmacological chaperone
A method for determining whether a patient deficient in protein activity will respond to treatment with a specific pharmacological chaperone by contacting patient-derived cells with the SPC and comparing protein activity with and without the SPC.
Use of T lymphocytes for in vitro assay of SPC response
Obtaining T lymphocytes by separating and washing white blood cells from a blood sample, followed by establishing a T cell-enriched culture to assess α-GAL activity in presence or absence of SPC.
Culturing T cells with 1-deoxygalactonojirimycin for enhancement assay
Culturing T lymphocytes in the absence or presence of 1-deoxygalactonojirimycin (DGJ) for about 1–3 days, preferably about 3 days, to detect changes in α-GAL activity.
Fluorometric assay for α-GAL activity measurement
Determining α-GAL activity by a fluorometric assay that quantifies hydrolysis of a substrate in lysates from T lymphocytes, comparing activity with and without DGJ.
Criteria for assessing meaningful increase in α-GAL activity
Measuring the increase in α-GAL activity after exposure to DGJ according to specified thresholds based on baseline activity levels to decide if the patient will respond to SPC therapy.
Applicability to missense mutations causing deficiency
The determination method applies particularly when the deficiency in enzyme activity is caused by missense mutations in the encoding gene.
Specific application to Fabry disease and α-GAL enzyme
The method is specified for lysosomal storage disorder Fabry disease and the lysosomal enzyme α-galactosidase A.
The independent claim covers a method for assessing SPC responsiveness by comparing protein activity in patient cells exposed and unexposed to a SPC, specifically using T lymphocytes cultured with 1-deoxygalactonojirimycin and measuring α-GAL activity via a fluorometric assay with defined criteria for responsiveness, particularly applying to Fabry disease caused by missense mutations.
Stated Advantages
The in vitro T cell assay is fast and highly sensitive, enabling quicker and less invasive screening compared to fibroblast-based assays.
The method allows accurate determination of patient eligibility for SPC therapy, preventing unnecessary and costly treatments.
The assay improves diagnostic accuracy for Fabry disease, especially in females, reducing misdiagnoses.
The in vivo assay offers a reliable, non-invasive method to assess treatment response in actual physiological conditions.
Use of T cells enables testing of enhancement for multiple α-GAL mutations, facilitating personalized treatment decisions.
The assays provide a platform extendable to other genetic diseases and protein deficiencies beyond Fabry disease.
Documented Applications
Determining whether a patient with Fabry disease will respond to treatment with specific pharmacological chaperones (SPC therapy).
Diagnosing Fabry disease in patients suspected of having the disease by measuring α-GAL activity in T cells.
Screening patient T cells in vitro to evaluate eligibility for SPC therapy prior to treatment initiation.
In vivo assessment of α-GAL activity enhancement in white blood cells following SPC administration to guide dosing and treatment decisions.
Differentiating Fabry disease patients with rescuable mutations for personalized therapeutic approaches.
Potential application to other lysosomal storage disorders and genetically defined diseases involving protein misfolding or enzyme deficiency.
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