Huntington's disease (HD) is a genetic neurodegenerative disorder characterised by progressive loss of medium spiny neurons (MSNs) in the striatum. Off-the-shelf allogeneic human PSC-derived cell therapy products for HD are scalable but are not fully tolerated by the host immune system in the absence of immunosuppression, and prolonged use of immunosuppressive drugs carries significant risks. Immune-evasive strategies for allogeneic grafts (e.g HLA-cloaking) are under investigation. However, such genetic modifications may impair the capacity of the donor line to differentiate into therapeutically relevant grafts. Here, we address this problem in vitro.

Macaca fascicularis iPSCs (Mac-iPSC) were produced and B2M and CIITA were inactivated using CRISPR–Cas9. These MHC-cloaked iPSCs expressed canonical pluripotency markers. We then assessed their neural differentiation and maturation capacity. Striatal progenitors were derived and cryopreserved from both wild-type (WT) and MHC-cloaked Mac-iPSCs. We compared the ability of each donor line to produce MSN precursors in 2D cultures (MSNp), mature into MSNs or astrocytes in 3D spheroids. We confirmed absence of MHC class I expression in all derivatives from the MHC-cloaked line and lack of MHC class II in purified astrocytes derived from those cloaked cells. To mitigate the “missing-self” NK response against MHC-cloaked derivatives, we evaluated expression of membrane-bound NK-inhibitory ligands. Transgene expression was assessed after lentiviral transduction of MSNp matured into striatal spheroids.

Our next goal is to compare, in vivo in nude rodents, the survival and maturation of single-cell MSN progenitor suspensions versus MSN spheroid suspensions after grafting, and to determine which format yields therapeutically relevant, MSN-containing grafts. We are currently conducting pilot experiments to test the ability of spheroids of different sizes to tolerate injection through needles of different gauges.