Development of Genetic Tools to Manipulate Glycolytic Flux for Potential Gene Therapy

Dennis Le

Authors:  Dennis Le, Zach Salyers, and Terence Ryan

Faculty Mentor:  Terence Ryan

College:  College of Health and Human Performance

Abstract

Peripheral arterial disease (PAD) – the narrowing of blood vessels in the limb from plaque buildup – is the third leading cause of atherosclerotic cardiovascular mortality. Critical limb ischemia (CLI) – the most severe form of PAD – requires urgent care. In absence of effective revascularization, the mortality rates of CLI patients exceed 50% by 5 years with CLI conditions. This project aimed to develop and validate genetic therapy tools for potential CLI treatment through one of the underlying biological changes experienced among PAD patients. CLI muscle biopsies from Ryan Lab revealed a decreased expression of PFKFB3 (6-phosphofructo-2-kinase/2,6-bisphosphatase 3). PFKFB3 – a bifunctional enzyme regulating an allosteric glycolytic activator – has an established upregulating role in the anaerobic energy production within our cells. PFKFB3 knockdown and overexpression vectors were constructed and evaluated to ensure tissue specificity and gene manipulation. In mouse tibialis anterior (TA) muscle, the downregulating vector revealed an overall 62.9% knockdown of PFKFB3 mRNA while the upregulating vector showed a 110-fold increase (P < 0.05). Verifying the tools’ functionality and specificity confirmed our goal of manipulating the targeted cells and their genome before conducting any biological study of hindlimb ischemic mice mimicking CLI conditions with implementation of the PFKFB3 vector in the TA.

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Tanja Taivassalo
Tanja Taivassalo (@guest_1404)
1 year ago

very interesting poster and great presentation Dennis!
I look forward to seeing your final results.
This work could be very relevant for myophosphorylase deficiency (McArdel’s disease – a glycogen storage disease) as well!

Dennis Le
Dennis Le (@guest_4478)
Reply to  Tanja Taivassalo
1 year ago

Thank you Dr. Taivassalo!

I haven’t thought about its potential clinical significance with McArdel’s disease! Upon finishing all the biological studies measuring muscle function and various AAVs under HLI mice, perhaps I can talk to Dr. Ryan about exploring that as well!

Elisabeth Barton
Elisabeth Barton (@guest_2874)
1 year ago

Really interesting approach Dennis! Glad that the validation of up and down regulation is achieved. 100 fold increase seems like very strong expression.

Dennis Le
Dennis Le (@guest_4642)
Reply to  Elisabeth Barton
1 year ago

Yes it is indeed! Replicating the cDNA version of the murine mRNA PFKFB3 and putting it in a plasmid for overexpression really seemed to do the trick!

Dennis Le
Dennis Le (@guest_3784)
1 year ago

Hello!

I would love to chat about topic and/or answer questions about the project and current projects going on now! Here is my link to a Zoom:

https://ufl.zoom.us/j/125523641

Thanks for checking this out!

Thank you,
Dennis Le

Christian McLaren
Christian McLaren (@guest_4836)
1 year ago

Hello Dennis, very interesting presentation! You did a great job at explaining technical concepts into a more digestible form to the audience! Great job!

Dennis Le
Dennis Le (@guest_5166)
Reply to  Christian McLaren
1 year ago

Thank you I appreciate it!! It’s all about the big picture before getting into the nitty gritty right?

Sierra Budd
Sierra Budd (@guest_7096)
1 year ago

Great poster and explanation! Why did you choose the tibialis anterior muscle for the injections?

Dennis Le
Dennis Le (@guest_7352)
Reply to  Sierra Budd
1 year ago

Hi Sierra!

We chose the tibialis anterior (TA) because of a few reasons:

-The hindlimb ischemic (HLI) mice mimicking the CLI conditions will have less blood flow and oxygen perfusion downstream of the limb, so their muscles distal to/farther from their bodies will have exacerbated problems with functionality, contractility, and overall use of the leg due to the hypoxic conditions (more oxygen demand than supply, so an energy deficient environment).
– Depending if its the up- or down-regulating AAV vector. The AAV injections were either muscle-specific (HSA) or vascular-specific (Cdh5) that will respectively bind to the actin of the muscles or the capillaries in the muscle to see if theres improved survivability (or exacerbated injury) in the muscles under femoral artery ligation (FAL) for HLI mice.
– The TA is one of the farthest muscle group from the FAL that can be collected easily and histologically analyzed and stored for further genomic analyses (i.e. RNA count, protein levels).

Sierra Shepherd
Sierra Shepherd (@guest_7154)
1 year ago

Great poster and presentation! This is some very important research being done, and I hope your results lay the foundation for future studies, really good job!

Dennis Le
Dennis Le (@guest_7378)
1 year ago

Once again thank you for checking out the research here! For any further questions or discussion, please feel free to message me at

ledennis@ufl.edu