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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session B

Poster H19 - Engineered Extracellular Vesicles loaded with Transcription Factors induce Motor-like Neuron Reprogramming

Thursday, October 24, 2024
2:45 PM – 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Peripheral nerve damage is a neurologic condition impacting the lives of over 20 million people in the United States, producing muscle weakness, pain, and muscular atrophy within periods of neuronal regeneration.

Previous studies found that fibroblast transfection with ASCL1, BRN2, and MYT1L (ABM), along with NGN2, ISL1, and LHX3 (NIL), facilitates their conversion into motor-like neurons. Transfection occurs via Tissue Nanotransfection (TNT), a method our lab developed to transfect cells in vivo. However, we hypothesized that TNT can be improved by delivering extracellular vesicles (EVs) loaded with ABM and NIL factors to amplify the reprogramming signal once injected into tissues after the initial transfection.

Materials and

Methods:  Initial experiments tested different ratios of each factor to maximize transfection and myoblast reprogramming. C2C12 cells were transfected with ABM and NIL at different concentrations by bulk electroporation, incubated for 24 hours, and analyzed via qRT-PCR to detect expression of the transfected factors. Reprogramming was then verified in cells transfected and incubated for 14 days by measuring expression of neuronal markers (MAP2, SNP1) and motor neuron markers (NKX6.2, VaCht).

EVs collected from cells transfected with the ABM and NIL cocktail were isolated from culture 24 hours, 7 days, and 14 days after initial transfection, RNA was extracted, and qRT-PCR detected the presence of the ABM and NIL cargo. C2C12 cells were then treated with the loaded EVs, and qRT-PCR was conducted after 24 hours to detect if the ABM and NIL factors in the EVs were uptaken by the cells.

Results, Conclusions, and Discussions: Upon these experiments' completion, ABM and NIL most effectively induce reprogramming when added in ratios of 1:2:2 and 1:2:2 respectively. This was noted by not only high levels of these factors’ expression after 7, 14, and 21 days, but also the upregulation of the motor neuronal markers indicating how reprogramming has occurred. This finding set the groundwork for further experimentation as induced cellular reprogramming can be optimized with a cocktail best suited for effective transfection and cellular conversion to motor-like neurons.

Additionally, analysis of the EVs collected after cellular transfection showed significant expression of the ABM and NIL factors after being isolated from transfected cells. Detecting this level of the transfected factors in the nanoparticles suggested that uptake of the genes into these carriers is feasible, and further suggested their ability to be used for further therapeutic use.

Uptake of the EV cargo was confirmed to be possible after treating the cells with the loaded EVs. Significant expression of ABM and NIL were detected in cells treated with the EVs as compared to the control, suggesting that myoblast cells are capable of uptaking these EVs and the cargo they carry.

Future studies will focus on injecting these loaded EVs in vivo after TNT to improve myoblast conversion to motor-like neurons. Using a mouse model, TNT will transfect muscle tissue with the ABM+NIL factors, and weekly injections of the engineered EVs will be given intramuscularly to study the development of motor-like neurons in living tissues.

Acknowledgements (Optional):