Abstract Details

Read the abstract and find the presentation below

Full text and presentation details

H - Clinical Translation of Gene and Cell Therapies -> Cell Therapy Product Engineering, Development and Manufacturing

1314: Directed Differentiation of Gene Edited iPSCs by Small-Molecule Inhibition of a Transgene-Encoded Protein

Type: Poster Session

Poster Board Number: 1314
Presentation Details
Session Title: Friday Poster Session
Start Time: 5/19/2023 12:00
End Time: 5/19/2023 14:00

Indoleamine 2,3-dioxygenase 1 (IDO1) is an inducible, heme-containing enzyme that is critically involved in tryptophan catabolism and known to be a prominent immune regulator. Cell therapies with increased IDO1 expression are of high interest for a variety of indications, including autoimmune disorders, inflammatory diseases, transplant recovery, and wound healing. In particular, iPSC-derived mesenchymal stem cells (iMSCs) engineered to overexpress IDO1 may be ideal for suppressing dysregulated immune cells while simultaneously promoting expansion of regulatory T lymphocytes and the M1 (pro-inflammatory) to M2 (anti-inflammatory) polarization of macrophages. Here, we report the development of an iMSC cell line containing an IDO1 transgene under the control of a JeT promoter that was inserted into the AAVS1 safe-harbor locus in mRNA-reprogrammed iPSCs. A clonal population of edited cells (i.e. IDO1-iPSCs) was isolated using single-cell sorting. Bi-allelic insertion of the IDO1 transgene was confirmed by amplicon sequencing. The IDO1-iPSCs were then differentiated to IDO1-iMSCs. During differentiation, we found that the IDO1-iPSCs showed an unexpected, cuboidal morphology and noticeably decreased proliferation rates relative to control iPSCs, possibly indicating that IDO1 was interfering with the differentiation process. Two small-molecule IDO1 inhibitors, Epacadostat, which binds to holo (heme-bound) IDO1 and inhibits the enzymatic function of the protein and IDO1-IN-5, which binds to apo (heme-dissociated) IDO1 and inhibits IDO1-dependent cell signaling, were added to the culture for the final five days of differentiation. The addition of both IDO1 inhibitors (each at a 10µM concentration) increased the proliferation of the partly differentiated IDO1-iPSCs (6 h reduction in doubling time) with a population doubling time approaching that of control iMSCs (29 h vs. 27 h). While both inhibitors could enhance proliferation independently, Epacadostat alone had a larger effect on population doubling time than the IDO1-IN-5 alone (6 h vs. 2 h reduction in doubling time), suggesting that the anti-inflammatory enzymatic function of IDO1 may be most responsible for decreased proliferation during differentiation of IDO1-iPSCs. Notably, we also found that when administering both IDO1 inhibitors to non-engineered control iMSCs, the population doubling time increased relative to untreated test cultures (34 h versus 27 h), suggesting a possible minimum IDO1 protein level required for optimal iMSC culture. These results suggest that small-molecule inhibition of transgene-encoded proteins may form a key element of directed differentiation process development for knock-in iPS cell lines, and may be useful for the scale-up manufacturing of IDO1-iMSCs in particular.

Raven Dance Klee1, Taeyun Kim2, Kyle M. Garland1, Christopher B. Rohde2, Matthew Angel1,2

1Eterna Therapeutics, Inc., Cambridge, MA,2Factor Bioscience, Inc., Cambridge, MA
 R.D. Klee: None.

Find This Presentation

This site uses cookies to offer you a better user experience and to analyze site traffic. By continuing to use this website, you accept our use of cookies.