Design, Development, and Delivery of Novel Recombinant Proteins for Neuron-Targeted Therapy and Neuronal Differentiation

glucocerebrosidase;Gaucher"s disease;protein delivery;Chemical & Biomolecular Engineering

Gramlich, Paul A.Yarema, Kevin J. ;

Johns Hopkins University

关键词: glucocerebrosidase;    Gaucher";    s disease;    protein delivery;    Chemical & Biomolecular Engineering;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/39394/GRAMLICH-DISSERTATION-2015.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

This thesis dissertation research has aimed to develop novel protein therapeutics with potential to treat neurological diseases for which there are currently limited available treatments and no cures including Parkinson’s disease and neuronopathic Gaucher’s disease.Although the individual chapters of this thesis had varied goals, all involved designing and expressing recombinant proteins, purifying the proteins, facilitating targeted and efficient protein delivery, and demonstrating in vitro activity. The effective targeting of neuronal cell types requires effective delivery strategies.As a result, initial thesis work focused on the evaluation of cell-penetrating peptides (CPPs) and receptor-dependent neuronal membrane binding proteins to improve protein delivery.This work led to the discovery of a novel protein delivery vector, Tat-Tetanus Toxin Fragment C (Tat-TTC), a highly neuron-specific domain capable of dramatically enhancing the binding and internalization of recombinant proteins.Both fluorescent microscopy and quantitative fluorimetry were utilized to demonstrate that, with Tat-TTC, model protein GFP was bound and internalized by neuronal cell types at least an order of magnitude better than Tat-GFP or TTC-GFP alone.Since the Tat-TTC protein delivery vector was endocytosed, this research project addressed the issue of endosomal entrapment of cargo protein.Photochemical internalization (PCI) was used to facilitate the endosomal release of Tat-TTC-linked cargo presenting the option of using the delivery vector to target endosomes/lysosomes or cytosolic or nuclear intracellular targets. Cell-penetrating peptide Tat and PCI were next utilized in the design, production, and delivery of recombinant transcription factors Mash1, Lmx1a, and Nurr1 intended for neuronal differentiation.These transcription factors had been shown to transdifferentiate human fibroblasts to dopaminergic neurons (Caiazzo et al., 2011), the cell type lost in Parkinson’s disease (PD) (Thomas, 2010).We hypothesized that direct protein delivery of Tat-linked transcription factors had the potential to generate dopaminergic neurons that would be safe for clinical cell-replacement therapy.Four transcription factors, Tat-Mash1, Tat-Mash1-GFP, Tat-Lmx1a, and Tat-Lmx1a-GFP were successfully expressed and purified from insoluble HEK293F cell lysate.The transcription factors were delivered to cells and endosomal escape was facilitated with PCI.Induction of promoter-reporter constructs with purified protein was limited, a finding which led us to question the bioactivity of the resolubilized factors.To address this issue, a cell-free mammalian expression system capable of producing high concentrations of soluble transcription factor was identified.In the future, this strategy could prove invaluable for transcription factor production.Research into novel PD therapies led to my strong interest in Gaucher’s disease (GD).Mutations in the GBA1 gene encoding lysosomal enzyme glucocerebrosidase (GCase), leading to GD, has recently been shown to represent the greatest genetic risk factor for PD (Siebert et al., 2014).There is currently no available treatment for the neuronopathic type 2 and type 3 forms of GD.I aimed to develop a neuron-targeted recombinant GCase with the potential to cross the blood-brain barrier (BBB).Since the target for delivered protein was the lysosome, protein delivery vectors taken up by endocytosis were predicted to work well for GCase delivery, without any need for PCI.Seventeen GCase variants were designed and expressed in combination with a variety of protein delivery vectors.Cell-based assays using a GCase-knockout neuronal cell line facilitated the identification of a rabies-derived peptide (RDP) linked GCase utilizing an IgA hinge linker region to remove steric hindrance by GCase on RDP.RDP-IgAh-GCase was shown to target GCase-knockout neurons ~2.5X better than Tat-GCase.Both RDP-IgAh-GCase and Tat-GCase reduced substrate glucosylsphingosine accumulation to wild-type levels with 72 hours of protein treatment, indicating the enzymes were appropriately trafficked to lysosomes and functional.