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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session D

Poster H18 - Optimizing the Physiology and Transduction Efficiency of Liver Organ-Tissue Equivalents to Study AAV-6 Gene Therapy Related Toxicity

Friday, October 25, 2024
3:30 PM - 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Adeno-associated virus (AAV) vectors are the primary platform used for delivering therapeutic genes for the treatment of a wide range of human diseases. Unfortunately, clinical trials using AAV vectors have been hindered by unanticipated immunological/inflammatory responses. This has led to lower levels or even loss of transgene expression, questioning the validity of animal models to accurately predict treatment safety and success in humans, especially in children. We have previously shown that human liver tissue equivalents (LTEs) manufactured using age-relevant liver cells at physiologically relevant frequencies, recapitulate the biology and function of native human liver, and that these LTEs can provide crucial information regarding transduction efficacy and toxicity of different AAV serotypes and transgenes. Nevertheless, we also demonstrated that transduction efficacy of liver cells prior to organoid formation results in higher LTE transduction levels when compared to transduction of pre-formed LTEs.

Materials and

Methods:  Here we hypothesize that providing fluidic flow mimicking normal tissue interstitial perfusion will enhance LTE oxygenation, increase longevity, stabilize metabolism, and increase AAV transduction rates in fully formed LTEs. To test this hypothesis, we used a perfusion plate platform specifically designed to deliver physiologically relevant interstitial perfusion, while providing finer oxygenation/gas exchange to LTEs in culture. LTEs transduced with AAV6-GFP prior to (Day 1) or after organoid formation (Day 5) were placed in static conditions or were subjected to interstitial perfusion and were subsequently analyzed. Non-transduced LTEs under the same conditions served as controls. Viability assays such as Live/Dead, ATP, and Toxilight were performed on Days 5 and 10. Media was collected from Days 5-10 to assess progression of LTE viability under perfused and static conditions. Staining, fluorescence imaging and GFP quantification were also performed.

Results, Conclusions, and Discussions: At Day 5, prior to initiation of perfusion, all LTEs exhibited high viability; however, by Day 10 all LTE groups showed poor viability. Quantification of the live vs. dead cell area in the LTEs demonstrated < 50% cell viability amongst all conditions - results that were confirmed by ATP and Toxilight analysis. While cell viability is a vital metric of analysis for this experiment, we were also interested in quantifying transduction of AAV6-GFP. Staining for GFP displayed enhanced GFP signal throughout the LTE in the perfused, Day 5 transduction condition. Live-Cell fluorescence imaging and analysis revealed the perfused, Day 1 transduction to have the greatest GFP intensity; however, statistics showed differences between conditions were non-significant. Since it was challenging to adapt the current static LTE culture system to the novel perfusion technology, it is possible that the results obtained do not fully reflect the power of interstitial perfusion to increase AAV transduction. We have identified several potential pitfalls with the automated system and will implement those in the next set of experiments. Only then can we reach appropriate conclusions about the role of perfusion in the health and transduction efficiency of human LTEs.

Acknowledgements (Optional):  National Science Foundation REU Site Award #2243993 (Atala, Schanck), NHLBI5R01HL166462-02, and Ann Austin Flynt Memorial