(T1284) ENGINEERING A NON-VIRAL SYSTEM FOR TRANSCRIPTION FACTOR BASED DIRECT DIFFERENTIATION OF CENTRAL NERVOUS SYSTEM CELL TYPES USING THE TRANSGENIC SYSTEM MADR
Postdoctoral Scientist Cedars-Sinai Health System West Hollywood, California, United States
Abstract: Transcription factor based direct differentiation of cells in the Central Nervous System (CNS) is a novel method to study mature populations of specific CNS cell types in vitro and could be used in cell therapy for neurological diseases in vivo. MADR is a non-viral transgenic technology which uses dual recombinase to stably incorporate a donor cassette between recombinase sites. This project combined the MADR transgenic system with a doxycycline (Dox) inducible “tet-on” element to create the inducible MADR system as an alternative to viral based transduction, in order to control for many of the off-target safety concerns associated with viruses. Testing constructs both in vitro and in vivo using staining and sequencing confirmed tight control over the inducible transcription factors. Using this methodology, we compared a number of TF cocktails. A combination of CTIP2, DLX2, and Bcl-XL along with microRNA 9 and 124 for the differentiation of induced Medium Spiny Neurons (iMSNs) resulted in proneural differentiating cells in vitro but not mature MSNs as predicted in the literature. Misexpression of SOX9 and NFIA, two canonical Astrocyte TFs, resulted in the increase of astrocytic genes in vitro but did not differentiate astrocytes in vivo. The misexpression OLIG2 and SOX10 drove the direct differentiation of Oligodendrocyte Precursor Cells (OPCs) in vivo in as little as 7 days. Taken together, this flexible and consistent approach allows for rigorous, parallel characterization of TF cocktails.
