Transplanted organoids empower human preclinical assessment of drug candidate for the clinic

Abstract

Pharmacodynamic (PD) studies are an essential component of preclinical drug discovery. Current approaches for PD studies, including the analysis of novel kidney disease targeting therapeutic agents, are limited to animal models with unclear translatability to the human condition. To address this challenge, we developed a novel approach for PD studies using transplanted, perfused human kidney organoids. We performed pharmacokinetic (PK) studies with GFB-887, an investigational new drug now in phase 2 trials. Orally dosed GFB-887 to athymic rats that had undergone organoid transplantation resulted in measurable drug exposure in transplanted organoids. We established the efficacy of orally dosed GFB-887 in PD studies, where quantitative analysis showed significant protection of kidney filter cells in human organoids and endogenous rat host kidneys. This widely applicable approach demonstrates feasibility of using transplanted human organoids in preclinical PD studies with an investigational new drug, empowering organoids to revolutionize drug discovery.

Fig. 1.. Transplantation of human kidney organoids in rats promotes vascularization by the rat host systemic circulation.

(A) Schematic of organoid protocol with transplantation and scRNA-seq time points. (B) Stacked bar graph showing cell type proportions of in vivo maturation across six time points (three replicate organoids per time point, 143,475 cells). All replicates for each time point are biological replicates of a single differentiation. The replicates show an increase in podocytes, tubular cells, endothelial cells, and off-target cell types after a prolonged in vivo maturation period. (C) Averaged cell type proportions after 2 weeks (light gray) and 4 weeks (dark gray) of in vivo maturation. Differences between cell type proportions tested with Dirichlet regression with significantly different comparisons are marked with one asterisk (P < 0.05) or two asterisks (P < 0.001). Error bars represent +SEM. (D) Boxplots [representing interquartile range (IQR) and median and with whiskers extending to ±1.5× IQR] of pseudotime, a measure of maturation inferred by a trajectory analysis, for podocytes from human samples (gray, 563 cells) and transplanted organoids (red, 10,753 cells). Podocytes from organoids harvested 4 weeks posttransplant had significantly increased pseudotime compared to podocytes from 2 weeks posttransplant organoids for all time points (P = 1.5 × 10⁻³⁰). (E) High-power view showing RECA-1–expressing rat endothelium–derived vascularization of a human organoid glomerulus. Red, RECA-1; green, synaptopodin; blue, nephrin. (F) Transplanted organoids develop a vascular network containing CD31/PECAM1-expressing human endothelial cells (top). A higher-magnification image shows CD31/PECAM1-positive human endothelial cells invaginating into a human organoid glomerulus (bottom). Red, RECA-1; green, CD31/PECAM1.

Fig. 2.. In vitro drug evaluation in human kidney organoids.

(A) Current-voltage relationship, 500-ms voltage ramp from −80 mV to +80 mV before/after GFB-887 (0.1 μM) or ML204 (100 μM). (B) Concentration-dependent inhibition of human TRPC5 after GFB-887 at +80 mV. Means ± SEM (n = 3 to 4 measurements per concentration). IC₅₀, median inhibitory concentration. (C) Up-regulation of human TRPC5 mRNA expression during organoid differentiation in vitro. Error bars, SD. BLOQ, below limit of quantitation. (D) Double labeling of podocytes with synaptopodin (SYNPO, green) and TRPC5 (red). (E) CsA and GFB-887 protect podocytes from PS-induced injury, region of interest in blue. Inset: Scale of representative images for injury quantification (top left). Representative images of glomeruli for podocyte injury quantification in organoids treated with DMSO (vehicle), PS, PS + CsA, PS + GFB-887, or PS + CsA + GFB-887. Cyan, synaptopodin; red, phalloidin. (F) Quantification of PS-induced actin aggregation. GFB-887 and CsA are nonadditive. DMSO versus PS, P < 0.0001; PS + CsA versus PS, P = 0.0021; PS + GFB-887 versus PS, P = 0.0087; PS + CsA + GFB-887 versus PS, P = 0.0397. A.U., arbitrary units. Means ± SEM. (G) Mechanism of action of GFB-887. PS drives TRPC5 activity, which induces Ca²⁺-mediated activation of calcineurin, promoting synaptopodin degradation and subsequent Rac1 activation. Rac1 promotes TRPC5 activity, establishing a vicious cycle that is blocked by GFB-887 or CsA.

Fig. 3.. PD studies in rats with transplanted human kidney organoids bolster confidence in GFB-887, an investigational new drug.

(A) Organoids differentiated for 14 days in vitro were transplanted under the kidney capsule of athymic male rats and followed for 2 (left) or 4 (right) weeks before oral dosing with GFB-887 (10 mg/kg) for three consecutive days. Extracted organoid and kidney samples were normalized to sample weights, and the final drug values were calculated as nanograms per gram tissue; nanograms per milliliter refers to GFB-887 concentration per milliliter of plasma. Oral dosing of GFB-887 resulted in equivalent drug exposure in organoids and rat plasma, thereby showing that the organoids had functional connectivity to the host vasculature at 2 weeks after transplantation. Organoid GFB-887 levels did not further increase at 4 weeks after transplantation. Data show means ± SEM from at least four independent measurements. (B) Superresolution imaging reveals PS-induced loss of synaptopodin protein abundance in transplanted organoids, which was prevented by oral dosing of GFB-887; blue, nuclei; green, synaptopodin; red, RECA-1; HBSS, Hank’s balanced salt solution vehicle control. (C) Quantification of PS-induced podocyte injury and protection by GFB-887 in transplanted organoids. (D) Quantification of PS-induced podocyte injury and protection by GFB-887 in endogenous rat kidney adjacent to transplanted organoids. For both (C) and (D), synaptopodin mean intensity in podocytes was quantified for HBSS vehicle, PS, coperfusion PS + GFB-887, or PS perfusion after oral dosing of GFB-887. Data show means ± SEM; for all treatment conditions versus PS, P < 0.0001; ordinary one-way analysis of variance (ANOVA). ****P < 0.0001.