Development of a responsive gene therapy to promote clearance of polyglutamine aggregates Open Access

Abstract

Neurodegenerative diseases are devastating conditions characterized by protein toxicity and neuronal cell death, which is physically manifested as progressive cognitive and physical decline. Without a cure, current treatments focus on palliative care of symptoms. A responsive gene therapy targeted to neurodegenerative diseases, specifically Huntington’s disease, is a promising tool to address the disease pathology at a cellular level with high specificity and temporal control. This responsive gene therapy construct was engineered to have a sensingmodality in which activation is contingent on cellular status, neuronal stress induced by unfolded or misfolded proteins. When activated, the therapy construct is, subsequently, expressed. To target the mechanisms implicated in Huntington’s disease (HD), the Unfolded Protein Response (UPR) was selected and its ability to detect misfolded proteins harnessed, using the stress-sensitive 5’ untranslated region (UTR) of the activating transcription factor 4 (ATF4) gene. Upregulation of autophagy, the cell’s natural mechanism for protein clearance, by increased expression of Beclin 1 (BECN1), was chosen as the therapy. This project entailed evaluating the components of our gene therapy in the context of endoplasmic reticular stress, assessing these constructs in a cellular model of HD and, ultimately, determining the efficacy of the responsive gene therapy’s ability to clear polyglutamine aggregates.

Table of Contents

Abbreviations.......................................................................................................................1

Section 1: Background.........................................................................................................2

Introduction..................................................................................................................................2

Huntington’s Disease....................................................................................................................6

Sensing, ATF4 dependent sensing modality................................................................................10

Figure 1: UPR activation via the PERK pathway..........................................................................11

Figure 2: Regulation of ATF4 translation....................................................................................12

Molecular therapy, Beclin-1 Expression......................................................................................14

Figure 3: Macroautophagy mechanism........................................................................................14

Viral vectors in gene therapy.......................................................................................................17

Responsive gene therapy.............................................................................................................18

Figure 4: Gene therapy for unfolded protein regulation summary schematic.............................18

Section 2: Materials and Methods......................................................................................20

Cell Culture..................................................................................................................................20

Plasmids.......................................................................................................................................20

Table 1: Plasmid maps..................................................................................................................20

Transfection..................................................................................................................................22

UPR-Inducing Reagents................................................................................................................22

Imaging.........................................................................................................................................22

Image analysis...............................................................................................................................23

Statistics........................................................................................................................................23

Immunocytochemistry...................................................................................................................23

Adeno-associated virus production...............................................................................................24

Section 3: Results.................................................................................................................25

HD Model.......................................................................................................................................25

Figure 5: Huntingtin plasmid expression in HEK293 cells 32hr post-transfection........................25

Figure 6: Time-lapse of HTT expression........................................................................................26

Testing the ATF4 Sensor................................................................................................................26

Figure 7: Sensingconstruct.............................................................................................................27

Figure 8: Differential expression of ATF4-GFP versus cGFP..........................................................27

Figure 9: TM-induced increase in ATF4 expression in HEK293.....................................................28

Figure 10: TM-induced increase in ATF4 expression blocked by PERKi in HEK293.......................29

ATF4 SENSOR IN HD Model...........................................................................................................29

Figure 11: ATF4-GFP induction in response to HTT in HEK293 cells.............................................30

Molecular therapy, Beclin-1 Expression.........................................................................................30

Figure 12: Beclin 1 Immunocytochemistry.....................................................................................31

Responsive Gene Therapy...............................................................................................................31

Figure 13: ATF4-BECN1-GFP induction in response to HTT in HEK293 cells.................................32

Affect of BECN1 on HTT Expression...............................................................................................32

Figure 14: Effect of BECN1 treatment on HTT-Q20 expression in HEK293 cells.............................33

Figure 15: Effect of BECN1 treatment on HTT-Q150 expression in HEK293 cells...........................34

Figure 16: 1-way ANOVA.................................................................................................................35

Effective transduction of neuronal cultures with ATF4 sensor construct........................................35

Figure 17: Tunicamycin induction of AAV2-ATF4-GFP construct in neuronal culture....................36

Figure 18: Adeno-associated virus...................................................................................................36

Section 4: Discussion..............................................................................................................37

Section 5: Conclusion.............................................................................................................40

Section 6: Works Cited............................................................................................................41

Appendix A: Cell profiler image analysis...............................................................................44

Appendix B: Statistical Analysis.............................................................................................46

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Neuroscience
Keyword	Huntington's disease Beclin 1 Gene therapy ATF4
Committee Chair / Thesis Advisor	Dr. Claire-Anne Gutekunst, Emory University
Committee Members	Dr. David Lynn, Emory University Dr. Nicole Gerardo, Emory University

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