Phase 1 clinical study of an embryonic stem cell–derived retinal pigment epithelium patch in age-related macular degeneration

Abstract

Age-related macular degeneration (AMD) remains a major cause of blindness, with dysfunction and loss of retinal pigment epithelium (RPE) central to disease progression. We engineered an RPE patch comprising a fully differentiated, human embryonic stem cell (hESC)–derived RPE monolayer on a coated, synthetic basement membrane. We delivered the patch, using a purpose-designed microsurgical tool, into the subretinal space of one eye in each of two patients with severe exudative AMD. Primary endpoints were incidence and severity of adverse events and proportion of subjects with improved best-corrected visual acuity of 15 letters or more. We report successful delivery and survival of the RPE patch by biomicroscopy and optical coherence tomography, and a visual acuity gain of 29 and 21 letters in the two patients, respectively, over 12 months. Only local immunosuppression was used long-term. We also present the preclinical surgical, cell safety and tumorigenicity studies leading to trial approval. This work supports the feasibility and safety of hESC-RPE patch transplantation as a regenerative strategy for AMD.

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Figure 1: Generation of hESC-derived RPE for the manufacture of an advanced therapeutic medicinal product (ATMP) to treat AMD.
Figure 2: Characterization of hESC-derived RPE.
Figure 3: Preclinical mouse teratoma and pig transplantation studies.
Figure 4: Case 1.
Figure 5: Case 2.
Figure 6: Sequences of color photographs of the transplanted patch in cases 1 and 2.
Figure 7: Best-corrected visual acuity and reading speed in cases 1 and 2 (a) BCVA over 12 months for patient 1.

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Acknowledgements

We acknowledge H. Moore, Stem Cell Derivation Facility, Centre for Stem Cell Biology (CSCB), University of Sheffield for derivation of the original SHEF-1 hESC line and P. Keane and M. Cheetham for comments on the paper. We thank R. McKernan for support and input throughout the project. L.d.C. and P.J.C. received the following grants and donations and would like to acknowledge that they were used to fund the studies reported in this article: Anonymous Donor, USA, Establishment of The London Project to Cure Blindness - Donation. Lincy Foundation, USA, The London Project To Cure Blindness: Funding Towards The Production Of A Cell Based Therapy For Late Stage Age-Related Macular Degeneration - P12761. Macular Disease Society Studentship – Donation. MRC, Stem Cell Based Treatment Strategy For Age-Related Macular Degeneration (AMD) - G1000730. CIRM (California Institute of Regenerative Medicine) LA1_C2-02086. Pfizer Inc, The Development Plan For A Phase I/IIa Clinical Trial Implanting HESC Derived RPE for AMD - PF-05406388. Moorfields Biomedical Research Centre, National Institute for Health Research (NIHR) - BRC2_011. The Michael Uren Foundation R170010A.

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Contributions

L.d.C., P.T.L., P.W., and P.J.C. designed all of the animal studies and the clinical study, developed the methodology for these studies, collected the data, performed the analysis, and wrote the manuscript. L.d.C. performed the pig and human surgery. K.F., J.K., A.A., A.Ve., J.T.D., B.N., S.M.H., S.B.G., A.-J.F.C., A.Vu., C.M.R., M.B., M.F., J.S., T.H., and A.W. developed, isolated, and prepared the hESC-RPE and performed the engineering of the hESC-RPE patch; and assisted in designing and conducting the mouse and pig studies, collecting the data, performing the analysis, and writing the manuscript. A.A. and A.Vu. performed the mouse surgery. O.G., Y.H.L., A.A., A.T., G.F., M.W., A.G.R., G.E.H. and M.S.S. assisted in designing the clinical study, developing the methodology, collecting the data, performing the analysis, and writing the manuscript.

Corresponding author

Correspondence to Lyndon da Cruz.

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Competing interests

J.K., M.B., M.F., J.S., T.H., G.F., M.W., P.T.L., and P.W. were all employees of Pfizer during the period of this clinical trial. This study was sponsored by Pfizer Inc. L.d.C. and P.J.C. are named on two patents lodged by University College London Business. They are Patent Application No. PCT/GB2009/000917 (for the patch) and International Patent Application No. PCT/GB2011/051262 (for the surgical tool).

Integrated supplementary information

Supplementary Figure 1 The purpose-built introducer tool

The device for introducing the therapeutic patch into the sub-retinal space. The device consists of a Handle containing a mechanism driven by the Wheel which advances a flexible Rod through the Shaft that in turn pushes the therapeutic patch out of the Tip of the device. The surgeon rolls the Wheel forward to advance the Rod.

Supplementary Figure 2 TRA-1-60 flow cytometry on SHEF1.3 hESC, P0 and P1 RPE (Fluorophore - TO-PRO®-3)

A: Forward scatter (FSC) / side scatter (SSC) plots of hESC, P0 and P1 RPE. hESC and RPE lie in slightly different positions on this plot, with RPE being smaller (FSC) and more granular (SSC) than hESC.B: Overlays of IgM (black) and TRA-1-60 (red) staining for hESC, P0 RPE and P1 RPE. Positive staining is clearly visible for hESC, whereas the isotype control (IgM, negative) and TRA-1-60 samples clearly overlay for both P0 and P1 RPE. Percentages indicate the percentage of positive events occurring within the gate shown (M12, M18 or M16 in hESC, P0 and P1 RPE plots respectively).C: Percentage positive events recorded for IgM isotype control and TRA-1-60 in hESC, P0 RPE and P1 RPE. Average values are hESC 0.08% IgM, 93.5% TRA-1-60; P0 RPE 0.10% IgM, 0.30% TRA-1-60 and P1 RPE 0.09% IgM and 0.32% TRA-1-60. All values except for the TRA-1-60 stained hESC are below the lower limit of detection (1.03%).D: An example of the 0.3% of TRA-1-60 events that show slightly higher staining than the isotype control in P1 RPE (even though these are within the background limits of the assay) displayed on a FSC/SSC plot. (For example taking all TRA-1-60 events in gate M16 in figure 2B and displaying them as FSC/SSC). This is compared to a representative hESC FSC/SSC plot to show that the majority of these events are separated from hESC based on SSC ie they are more granular and very unlikely to be hESC cells.

Supplementary Figure 3 Propidium iodide (PI) staining of SHEF1.3 hESC dissociated and seeded into RPE culture conditions (Fluorophore - TO-PRO®-3)

A & B: On day 0 over 95% of hESC cells seeded into DMEM/CellStart (RPE conditions) or mTeSR1/Matrigel were viable as they excluded PI (A). By 2 days post seeding on average 96%±1% (n=5) of cells in DMEM conditions, including both adherent cells and those in the media (as very few actually adhered) were dead or dying, as judged by flow cytometry for PI. The same was observed on day 4 (97%±0.6% dead, n=3). By contrast, around 40-50% of cells in mTeSR conditions were dead at 2 days post seeding, demonstrating that under these conditions around half of SHEF1.3 hESC survive.C: Virtually all SHEF1.3 hESC seeded into DMEM/CellStart (RPE culture conditions) appear rounded and dead as well as staining positively for PI (red). By contrast, though dead cells are apparent, many of the cells seeded into mTeSR/Matrigel (MGL) conditions appear morphologically viable and do not stain with PI.D and E: Blue = HOECHST (nuclei) and green = TRA-1-60 (D) or Ki67 (E). By 6 weeks post seeding, a small number of live cells were occasionally visible in DMEM/CellStart conditions (4 out of 9 experiments). These did not appear morphologically like SHEF1.3 hESC cells (compare the large, elongated cells in D and E with single cell hESC on Matrigel (MGL) in C. These did not stain positively for TRA-1-60 (compare D with hESC on MGL in figure 4C) or KI67 (compare CellStart and MGL seeded cells in E).

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da Cruz, L., Fynes, K., Georgiadis, O. et al. Phase 1 clinical study of an embryonic stem cell–derived retinal pigment epithelium patch in age-related macular degeneration. Nat Biotechnol 36, 328–337 (2018). https://doi.org/10.1038/nbt.4114

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