INTRACELLULAR DELIVERY OF CRISPR-CAS9 VIA A SYNTHESIZED LIPID-LIKE NANOPARTICLE LIBRARY FOR GENE EDITING
Bolinger, Justin.
2018
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Abstract: The field
of protein therapeutics is an emerging one. One of the evident challenges of protein
therapeutics is the method of delivering these proteins, which are often fragile or
prone to break down. Lipid nanoparticles (LNPs) represent a class of drug delivery
system that has shown promise for providing an efficient vehicle for protein
therapeutics. The hypothesis of this study was ... read morethat fabricated LNPs could carry out
CRISPR deliveries of model proteins at transfection efficiencies comparable to a
commercially available lipid. 20 LNPs were synthesized using a self-assembly fabrication
method to create a congener library. The LNPs comprising this library were confirmed
structurally and their physical stability profiles were determined. GFP-Cre was
delivered to HeLa-DsRed cells via these LNPs to determine their ability to enable
protein uptake by assessing induced GFP fluorescence. Similarly, Cas9:sgRNA complexes
targeting GFP were delivered to GFP-HEK cells to assess their GFP knockout efficiencies
by assessing GFP fluorescence. 20 formulated and 20 non-formulated LNPs were produced.
Transmission electron microscopy and dynamic light scattering analysis were used to
confirm that the fabricated LNPs had uniform size and shape as well as uniform stability
profiles and relatively homogenous particle size. For the delivery of GFP-Cre with the
20 formulated LNPs, 12 were able to induce GFP fluorescence at a rate of greater than
30%, with 10 performing similar to or better than the positive control, Lipofectamine
2000 (~45% GFP-positive). For the delivery of Cas9:sgRNA with the 20 formulated LNPs, 13
were able to induce GFP knockout at a rate of greater than 30%, with 6 performing
similar to or better than the positive control (~65%). MTT assay data showed 6
formulated LNPs which yielded high values for cell viability (greater than 90%). A
combination of these data gave us 5 formulated LNPs with high transfection efficiencies
and low cytotoxicities, making these LNPs attractive for further
research.
Thesis (M.S.)--Tufts University, 2018.
Submitted to the Dept. of Biomedical Engineering.
Advisor: Qiaobing Xu.
Committee: Sergio Fantini, and James Van Deventer.
Keyword: Biomedical engineering.read less - ID:
- w9505b883
- Component ID:
- tufts:25402
- To Cite:
- TARC Citation Guide EndNote
