Alpha-Synuclein Mutants and Uses of thereof
We have developed a first-of-its-kind stand-alone, easy, accurate, and cost-effective blood test for detecting early onset Parkinson’s disease (PD). It is based on amplifying the circulating α-synuclein seed in the patient’s blood and detecting it using Congo red blot assay. This technology uses novel α-synuclein mutants, which we have engineered and patented. Our blood test, developed using novel engineered α-synuclein mutants, has demonstrated high accuracy and specificity in detecting Parkinson’s Disease.
Parkinson's disease (PD) is a chronic and incurable neurodegenerative disease characterized by selective loss of dopamine-producing neurons in the brain, eventually manifesting severe motor symptoms in patients. PD is caused by the aggregation and accumulation of α-synuclein (α-Syn) protein. The available neuroimaging techniques (PET, SPECT, and MRI) cannot detect early-stage disease manifestations and further cannot differentiate between various forms of Atypical Parkinsonism. Ultra-low (undetectable) levels of α-Syn amyloid seeds are present in the biological fluids of PD patients before the appearance of clinical symptoms, which encouraged us to develop a robust, sensitive, and less invasive biochemical test for the early detection of PD.
We have developed a method to amplify these low concentrations of circulating α-Syn aggregates in the patient's blood. The drawbacks of the currently available commercial biochemical tests are the invasiveness (CSF-based), longer detection times, and the high false positive results (due to the inherent aggregation tendency of wild-type α-Syn as a substrate). Alternatively, using structure-guided protein engineering, we have strategically generated two mutants of α-Syn to overcome the limitations. Further, we have developed a high-throughput Congo Red Dot (CRD) assay to quantify the amplified product.
Our innovation encompasses two key aspects:
- Engineering the mutants of α-Syn devoid of intrinsic self-aggregation properties. These mutants aggregate solely in the presence of α-Syn amyloid seeds in the patient's blood. Patents for these mutants and their application have been filed in India and the US.
- Devising a high-throughput Congo Red Blot (CRB) Assay to determine amplified misfolded α-Syn protein.
Amyloid aggregation of α-Synuclein (α-Syn) leads to various synucleinopathies, including PD. These amyloids circulate in ultra-low, undetectable concentrations in the blood by crossing the blood-brain barrier. Due to the intrinsic aggregation property of α-Syn, it cannot be used for regular amplification of the misfolded proteins, leading to false positive results. We developed novel α-Syn mutants that do not self-aggregate and form amyloids only in the presence of α-Syn seeds, thereby significantly reducing the probability of false positive results.
There are currently no single blood-based biomarkers available for PD. CSF-based assays for detecting PD are expensive, invasive, and prone to high false positives. Similarly, imaging techniques such as DaTscan and PET scan using Fluorodopa F18 tracer have difficulty differentiating between PD and other NDs and need to be validated by other tests for confirmation of the disease pathology.
Key strengths of our technology include its stand-alone capability, simplicity, accuracy, cost-effectiveness, and non-invasive nature—enabling reliable diagnosis of Parkinson’s Disease.
We verified this technology using 43 PD patients and 15 control samples. The test is ~93% sensitive and with ~88% specificity to detect PD.
In India, PD diagnosis primarily relies on clinical assessments conducted by specialized doctors in clinics, utilizing subjective evaluations. These assessments often include observing the patient's response to medication used for treating PD, whose response varies in manifestation and interpretation. The presentation and progression of symptoms (due to α-synuclein aggregation) varies in every individual; this poses challenges in accurately assessing disease severity, leading to inadequate patient management. Doctors often prescribe different image-based diagnostic tests, including MRI, PET, and DaTScan. Most of these imaging techniques cannot conclusively detect PD but rather exclude the possibility of other diseases. Moreover, these tests are expensive and often utilize the radioactive tracer, which might have serious side effects. According to the latest reports, there are only 233 SPECT scanners and 225 PET scanners available in our country, which are located largely in metropolitan cities, restricting access for the majority of citizens.
With our blood test, we will be able to predict the different stages and forms of PD, providing a strong alternative for diagnosing and building a foundation to improve the treatment and quality of patient’s life of afflicted with Parkinson’s disease.