Engineering Novel Single-chain Variable Fragments for Therapeutic and Diagnostic Approaches in Parkinson's Disease

الموضوعات

طب

الملخص

The pathological accumulation of α-synuclein is a hallmark of neurodegenerative diseases such as Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), thus classified as synucleinopathies. Amongst the various approaches attempting to tackle the pathological features of synucleinopathies, antibody-based immunotherapy holds much promise. However, the large size of antibodies, requirement of high-doses and difficulty in crossing the blood-brain barrier are major hurdles. In this project, single-chain variable fragments (scFvs) were engineered against fibrillar α-synuclein. Six different scFv cDNA constructs, containing either cell-penetrating peptide (CPP), 12-Arginine, or no peptide, were cloned in E. coli expression constructs with or without GST tag. A series of optimization experiments were conducted to obtain desirable expression of the constructs in an E. coli expression system. Eventually, only two constructs, scFv-pF (no peptide) and scFv-pC (with CPP peptide) were used for further characterization. The two purified scFvs showed specific activity towards α-synuclein fibrils in comparison to monomers and other amyloidogenic proteins as assessed by dot blot and ELISA. An in vitro α-synuclein aggregation assay revealed that the scFvs inhibit the seeding of α-synuclein aggregation in a time-dependent manner, monitored by Th-S assay. Furthermore, the scFvs decreased α-synuclein seed-induced toxicity in a cell model of PD. In another cell model, the scFvs inhibited seeded aggregation of α-synuclein as inferred by decreased insoluble Serine 129 phosphorylated (pS129) α-synuclein. These results suggest that the α-synuclein fibril-specific scFvs can inhibit the aggregation of α-synuclein both in vitro and in cell models and prevent seeding-dependent toxicity. Thus, the scFvs described here have a high potential to be utilized towards immunotherapy as well as diagnosis of synucleinopathies.

روابط وملفات

التعريف والنوع

رقم الوثيقة

0fd78153-5a04-497b-8740-9f6c54529702

رقم العقد

0

نوع الوسائط

Crawler

نوع المحتوى

الرسائل العلمية

صيغة المصدر

رسائل ماجيستير

نوع الملف

pdf text

أسماء الملفات

0fd78153-5a04-497b-8740-9f6c54529702_1.pdf

بيانات النشر

ألقاب المؤلفين

[{"name_ar":"Omar El-Agnaf","title_ar":"اشراف","title_en":"Supervision"},{"name_ar":"Safa Salim","title_ar":"اعداد","title_en":"Preparation"}]

اللغة

English

المصدر والدورية

اسم المصدر

Engineering Novel Single-chain Variable Fragments for Therapeutic and Diagnostic Approaches in Parkinson's Disease

المحتوى والصفحات

عدد الصفحات

0

إشراف وإعداد

الإشراف

Omar El-Agnaf

الإعداد

Safa Salim

الاقتباسات الببليوغرافية