Preclinical Efficacy and Safety of a Human Embryonic Stem Cell-Derived Midbrain Dopamine Progenitor Product, MSK-DA01

Abstract

Parkinson's disease is characterized by the loss of dopaminergic neurons in the substantia nigra leading to disabling deficits. Dopamine neuron grafts may provide a significant therapeutic advance over current therapies. We have generated midbrain dopamine neurons from human embryonic stem cells and manufactured large-scale cryopreserved dopamine progenitors for clinical use. After optimizing cell survival and phenotypes in short-term studies, the cell product, MSK-DA01, was subjected to an extensive set of biodistribution, toxicity, and tumorigenicity assessments in mice under GLP conditions. A large-scale efficacy study was also performed in rats with the same lot of cells intended for potential human use and demonstrated survival of the grafted cells and behavioral amelioration in 6-hydroxydopamine lesioned rats. There were no adverse effects attributable to the grafted cells, no obvious distribution outside the brain, and no cell overgrowth or tumor formation, thus paving the way for a future clinical trial.

Keywords: GMP; Parkinson’s disease; cell therapy; dopamine neurons; human embryonic stem cells; human pluripotent stem cells; preclinical study; safety studies; transplantation.

Figure 1.. Testing of MSK-DA01 prior to preclinical efficacy and safety studies.

(A) Schematic diagram showing testing undertaken at different steps of manufacture and application of MSK-DA01. (B) Cycle quantification (Cq) values of POU5F1, LMX1A, PITX3 and TH in four lots (01–04) of cryopreserved MSK-DA01 assessed by qRT-PCR. PITX3 and TH were assessed at five days post thaw. WA09 (ES), reference (REF, a previous successful batch) and negative control (NTC, no template control) were included in the analysis. Data are represented as mean±SD (n=3/group). (C) Representative section through a graft 3 weeks post transplantation, immunostained for FOXA2, TH and human nuclear antigen (hNA). (D) Graft immunohistochemistry for NURR1, TH and human nuclear antigen (hNA) showing co-expression in most human cells. Rectangles indicate areas of magnification shown in the right panels. Scale bars=100 µm in left panel and 50 µm in right panels.

Figure 2.. MSK-DA01 rescued the motor asymmetry in the preclinical efficacy study.

(A) Number of amphetamine-induced rotations per minute at different timepoints post-grafting in the entire animal group. Male and female groups are also plotted separately. (B) Immunohistochemistry (IHC) for human nuclear antigen (hNA) and TH in representative brain sections from vehicle or grafted animals. Note the almost complete absence of endogenous TH⁺ processes in the unilateral 6-OHDA lesioned striatum that received vehicle. The grafted brain shows evidence of TH reinnervation in the putamen beyond the graft itself. (C) Stereological estimation of hNA⁺ cell number in the MSK-DA01 graft. (D) Stereological estimation of graft volume. (E) Correlation between human cell number and graft volume in all animals. (F) mature human dopamine neurons (hNA⁺TH⁺) in the graft. (G) IHC for TH shows graft-derived processes in the host striatum. (H) Stereological estimation of TH⁺ cell number in MSK-DA01 graft. (I) IHC for Ki67, FOXA2 and hNA in the graft. (J) Percentage of Ki67⁺ expressing human cells in male or female rats. (K) IHC for 5-hydroxy-tryptamine (5-HT) shows absence of serotonergic neurons in the graft. (L) IHC for GAD67 indicates the absence of GABA-ergic neurons in the graft. Scale bars=500 µm in B; 50 µm in F,G; 85 µm in I, 100 µm in K,L. Data are represented as median with interquartile range in A, C–D and mean±SD in H, J. Graft group, n=27 (M, n=13; F, n=14); Vehicle control group, n=14 (n=7 for each gender). *P<0.05, ** P<0.01, *** P<0.001, ****, P<0.0001 compared with the vehicle group.

Figure 3.. Biodistribution and graft analysis.

(A) Human genome copy number in the brain and other organs was estimated by DNA qPCR on days 30 and 180 post grafting, in the biodistribution study. All vehicle tissues are at the limit of quantification for the human DNA genome copy number for each tissue. Data represented as median with interquartile range. M= male; F= female. n=4–5 per group. *P<0.05, ** P<0.01 compared with vehicle group. (B) H&E staining of a representative section of a graft on day 180. Neuronal cell bodies and neuropil are visible in the magnified image on the right. (C) Immunohistochemistry for hNA in an adjacent section highlights the human cells, with a magnified image on the right. Hematoxylin nuclearstain. Scale bar values are indicated on the images.

Figure 4.. No tumor formation was observed in the MSK-DA01 tumorigenicity study.

(A) Teratoma formed in the brain by intra-striatal injection of 100% cells: H&E stain in the upper panels highlights the various tissue components, and IHC for hNA in the lower panels. Teratoma is composed of multiple epithelial-lined cysts (ELC), smooth muscle, fibrous connective tissue (FCT), cartilage, bone and neural tissues. The rectangles outline the regions shown on the right. (B) Representative sections of brain grafts in the 100% MSK-DA01 by H&E and IHC staining. (C) Representative sections of brain grafts in the MSK-DA01 spiked with 0.01% ES, and (D) 0.1% ES. The rectangle outlines the regions shown on the right. (E, F) Survival curves of male (M) and female mice (F) in the vehicle, 100% MSK-DA01, and spiking groups. n=12 M or F in the vehicle group; n= 18 M and 20 F in MSK-DA01; n=21 M or F in the 0.01% ES; n=17 M and 20 F in the 0.1% ES; n=11 M or F in the 100% ES groups. ***P<0.001; ****P<0.0001; n.s.: not significant. Scale bars =500 µm in the left panels and 50 µm the in the right panels in each set.

Figure 5.. Graft evolution over time.

(A) Graft volumes for MSK-DA01 on day 30 (n=18), 180 (n=18), 266 (n=30) and 100% ES on day 266 (n=4). (B) Percentage Ki67 in the MSK-DA01 grafts on different days and in the 100% ES group on day 266. (C) There is no clear correlation between the graft volume and the percentage of Ki67 in MSK-DA01 grafts at day 266 (r =−0.09608, P=0.6071, n=30). (D) Representative immunohistochemical images of MSK-DA01 grafts at different timepoints, immunostained for hNA. Hematoxylin nuclear stain. Graph showing the decreasing density of human cells per mm² over time. n=10 mice/group. (E) The presence of pHH3⁺Ki67⁺ cell (arrow), pHH3⁻Ki67⁺ cell (arrowhead) in the MSK-DA01 graft (left panel). The percentage of pHH3⁺Ki67⁺ in MSK-DA01 grafted cells on day 30 (n=15), 180 (n=11), 266 (n=30) post grafting (right panel). Data represented as median with interquartile range. Each circle represents one animal. (F) Some hNESTIN⁺ cells expressed Ki67 (arrow). NESTIN⁺ processes were observed in the graft (chevron). The data are represented as median with interquartile range. *P<0.05, ***P<0.001, ****P<0.0001. Scale bars=50 µm.