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Activating TFEB and ADAM17 using CRISPRa to treat neurodegenerative diseases

Siddiqui, Abdullah Najum (2021) MOBN02 20202
Degree Projects in Molecular Biology
Popular Abstract
Enhancing Therapeutic Genes to cure Neurodegenerative diseases

Alzheimer’s and Parkinson’s are the leading cause of neurodegenerative disease around the world. Research so far has only progressed towards reducing the symptoms linked with these diseases with no cure having been developed. Alzheimer’s and Parkinson’s share similar pathology in the way that they develop and form protein clumps which interfere with neuron to neuron signaling. The aim in this study is to enhance two genes, the transcription factor EB (TFEB) and A desintegrin and metalloprotease 17 (ADAM17), to remove the protein clumps in neurons and maintain cellular homeostasis. TFEB is a master gene for lysosomal biogenesis, which breaks down the protein aggregates... (More)
Enhancing Therapeutic Genes to cure Neurodegenerative diseases

Alzheimer’s and Parkinson’s are the leading cause of neurodegenerative disease around the world. Research so far has only progressed towards reducing the symptoms linked with these diseases with no cure having been developed. Alzheimer’s and Parkinson’s share similar pathology in the way that they develop and form protein clumps which interfere with neuron to neuron signaling. The aim in this study is to enhance two genes, the transcription factor EB (TFEB) and A desintegrin and metalloprotease 17 (ADAM17), to remove the protein clumps in neurons and maintain cellular homeostasis. TFEB is a master gene for lysosomal biogenesis, which breaks down the protein aggregates through digestive enzymes. ADAM17 hosts a-secretase activity, a proteolytic enzyme required to cleave proteins into soluble monomers. It has been demonstrated that overexpression of TFEB has neuroprotective roles in removing protein aggregates in rodent models, while overexpression of ADAM17 have negative impact on insoluble protein formation which polymerize and develop into clumps.

The CRISPRa tool was adopted to target and enhance the two genes. In this technique, a deactivated Cas9 (dCas9) enzyme uses an RNA based guide (gRNA) to locate the promoter sequence of the gene of interest. Powerful transcription factors linked with the dCas9 (dCas9VTR) allows for the enhanced transcription of the gene that follows. For certain genes, a combination of 2 sgRNA gives powerful gene enhancing capabilities. Screening assays were developed using HEK293T cell lines to establish top sgRNA combinations. These cell lines were transduced with plasmids that had the promoter sequence of the gene of interest attached to a reporter gene. With the intent of studying rodent models in the future, the promoter sequence targeted was of the rat TFEB, however, the ADAM17 promoter sequence is the same in rats and humans. Plasmids with the dCas9VTR gene, sgRNA sequence and BFP2 transfection control were also transduced into these cell lines. Images that show the sgRNA with the highest expression of the reporter gene as compared to its control can be seen in figure 1. No reporter gene is observed in rTFEB control but deductible amounts of reporter gene can be seen in ADAM17 control which could likely be due to HEK293T’s own transcription factors identifying the ADAM17 promoter sequence.

The dCas9VTR gene along with its corresponding sgRNA sequence will be encompassed into adeno-associated viral vectors (rAAV) for delivery into cells. Therefor cell lines exhibiting neurodegenerative pathology needed to be developed for functional validation. C6 cell lines transfected with a-synuclein (a-syn) expressing plasmids were readied. A protocol was developed to transfect these cell line with Bovine Serum Albumin (BSA), a-syn monomers and pre-formed a-syn fibrils (PFF). The idea behind the transfection of these protein into a-syn C6 cells is to analyze the formation of aggregates using X-34 dye (amyloid dye). Previous studies show PFF fragments triggering a-syn aggregation as they modify the normal endogenous a-syn by ubiquitination and hyperphosphorylation at ser 129. Future perspective is to transfect the C6 cell lines that express aggregates with the therapeutic genes via AAV vectors.

Master’s Degree Project in Molecular Biology with Specialization in Molecular Genetics and Biotechnology. 45 credits 2021
Department of Biology, Lund University
Supervisors: - Cecilia Lundberg: cecilia.lundberg@med.lu.se, professor at CNS Gene Therapy, BMC A1154
Luis Quintino: luis.quintino@med.lu.se, postdoctoral fellow at CNS gene therapy, BMC A1153 (Less)
Please use this url to cite or link to this publication:
author
Siddiqui, Abdullah Najum
supervisor
organization
course
MOBN02 20202
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
9060855
date added to LUP
2021-07-05 10:56:22
date last changed
2021-07-05 10:56:22
@misc{9060855,
  author       = {{Siddiqui, Abdullah Najum}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Activating TFEB and ADAM17 using CRISPRa to treat neurodegenerative diseases}},
  year         = {{2021}},
}