Survival of an HLA-mismatched, bioengineered RPE implant in dry age-related macular degeneration

doi:10.1016/j.stemcr.2022.01.001

Clinical Trial

. 2022 Mar 8;17(3):448-458.

doi: 10.1016/j.stemcr.2022.01.001. Epub 2022 Feb 3.

Survival of an HLA-mismatched, bioengineered RPE implant in dry age-related macular degeneration

Amir H Kashani¹, Jane S Lebkowski², David R Hinton³, Danhong Zhu⁴, Mohamed A Faynus⁵, Sanford Chen⁶, Firas M Rahhal⁷, Robert L Avery⁸, Hani Salehi-Had⁹, Clement Chan¹⁰, Neal Palejwala¹¹, April Ingram², Wei Dang¹², Chih-Min Lin¹², Debbie Mitra¹³, Juan Carlos Martinez-Camarillo¹, Jeff Bailey⁵, Cassidy Arnold⁵, Britney O Pennington⁵, Narsing Rao¹³, Lincoln V Johnson², Dennis O Clegg¹⁴, Mark S Humayun¹⁵

Affiliations

¹ Wilmer Eye Institute, Johns Hopkins University, 600 N. Wolfe Street, Baltimore, MD 21087 USA.
² Regenerative Patch Technologies, 150 Gabarda Way, Portola Valley, CA 94028, USA.
³ Department of Pathology, Keck School of Medicine, University of Southern California, 1441 Eastlake Avenue, 7503, Los Angeles, CA 90033, USA; USC Roski Eye Institute, USC Ginsburg Institute for Biomedical Therapeutics and Department of Ophthalmology, Keck School of Medicine, University of Southern California, 1450 San Pablo, Los Angeles, CA 90033, USA.
⁴ Department of Pathology, Keck School of Medicine, University of Southern California, 1441 Eastlake Avenue, 7503, Los Angeles, CA 90033, USA.
⁵ Regenerative Patch Technologies, 150 Gabarda Way, Portola Valley, CA 94028, USA; Center for Stem Cell Biology and Engineering, Neuroscience Research Institute, Mail Code 5060, University of California, Santa Barbara, CA 93016, USA.
⁶ Orange County Retina Medical Group, 1200 N. Tustin Avenue, Suite 140, Santa Ana, CA 92705, USA.
⁷ Retina-Vitreous Associates Medical Group, 9001 Wilshire Boulevard, Suite 301, Beverly Hills, CA 90211, USA.
⁸ California Retina Consultants, 525 E. Micheltorena Street, Santa Barbara, CA 93103, USA.
⁹ Retina Associates of Southern California, 7777 Edinger Avenue, Suite 234, Huntington Beach, CA 92647, USA.
¹⁰ Southern California Desert Retina Consultants, University Park, 36-949 Cook Street, Suite 101, Palm Desert, CA 92211, USA.
¹¹ Retinal Consultants of Arizona, 15401 North 29th Avenue, Phoenix, AZ 85053, USA.
¹² Center for Biomedicine and Genetics, Beckman Research Institute of City of Hope, 1500 East Duarte Road, Duarte, CA 91010, USA.
¹³ USC Roski Eye Institute, USC Ginsburg Institute for Biomedical Therapeutics and Department of Ophthalmology, Keck School of Medicine, University of Southern California, 1450 San Pablo, Los Angeles, CA 90033, USA.
¹⁴ Center for Stem Cell Biology and Engineering, Neuroscience Research Institute, Mail Code 5060, University of California, Santa Barbara, CA 93016, USA.
¹⁵ USC Roski Eye Institute, USC Ginsburg Institute for Biomedical Therapeutics and Department of Ophthalmology, Keck School of Medicine, University of Southern California, 1450 San Pablo, Los Angeles, CA 90033, USA; Department of Biomedical Engineering, Denney Research Center (DRB) 140, University of Southern California, 1042 Downey Way, Los Angeles, CA 90089, USA. Electronic address: humayun@usc.edu.

PMID: 35120620
PMCID: PMC9039755
DOI: 10.1016/j.stemcr.2022.01.001

Clinical Trial

Survival of an HLA-mismatched, bioengineered RPE implant in dry age-related macular degeneration

Amir H Kashani et al. Stem Cell Reports. 2022.

. 2022 Mar 8;17(3):448-458.

doi: 10.1016/j.stemcr.2022.01.001. Epub 2022 Feb 3.

Authors

Affiliations

¹ Wilmer Eye Institute, Johns Hopkins University, 600 N. Wolfe Street, Baltimore, MD 21087 USA.
² Regenerative Patch Technologies, 150 Gabarda Way, Portola Valley, CA 94028, USA.
³ Department of Pathology, Keck School of Medicine, University of Southern California, 1441 Eastlake Avenue, 7503, Los Angeles, CA 90033, USA; USC Roski Eye Institute, USC Ginsburg Institute for Biomedical Therapeutics and Department of Ophthalmology, Keck School of Medicine, University of Southern California, 1450 San Pablo, Los Angeles, CA 90033, USA.
⁴ Department of Pathology, Keck School of Medicine, University of Southern California, 1441 Eastlake Avenue, 7503, Los Angeles, CA 90033, USA.
⁵ Regenerative Patch Technologies, 150 Gabarda Way, Portola Valley, CA 94028, USA; Center for Stem Cell Biology and Engineering, Neuroscience Research Institute, Mail Code 5060, University of California, Santa Barbara, CA 93016, USA.
⁶ Orange County Retina Medical Group, 1200 N. Tustin Avenue, Suite 140, Santa Ana, CA 92705, USA.
⁷ Retina-Vitreous Associates Medical Group, 9001 Wilshire Boulevard, Suite 301, Beverly Hills, CA 90211, USA.
⁸ California Retina Consultants, 525 E. Micheltorena Street, Santa Barbara, CA 93103, USA.
⁹ Retina Associates of Southern California, 7777 Edinger Avenue, Suite 234, Huntington Beach, CA 92647, USA.
¹⁰ Southern California Desert Retina Consultants, University Park, 36-949 Cook Street, Suite 101, Palm Desert, CA 92211, USA.
¹¹ Retinal Consultants of Arizona, 15401 North 29th Avenue, Phoenix, AZ 85053, USA.
¹² Center for Biomedicine and Genetics, Beckman Research Institute of City of Hope, 1500 East Duarte Road, Duarte, CA 91010, USA.
¹³ USC Roski Eye Institute, USC Ginsburg Institute for Biomedical Therapeutics and Department of Ophthalmology, Keck School of Medicine, University of Southern California, 1450 San Pablo, Los Angeles, CA 90033, USA.
¹⁴ Center for Stem Cell Biology and Engineering, Neuroscience Research Institute, Mail Code 5060, University of California, Santa Barbara, CA 93016, USA.
¹⁵ USC Roski Eye Institute, USC Ginsburg Institute for Biomedical Therapeutics and Department of Ophthalmology, Keck School of Medicine, University of Southern California, 1450 San Pablo, Los Angeles, CA 90033, USA; Department of Biomedical Engineering, Denney Research Center (DRB) 140, University of Southern California, 1042 Downey Way, Los Angeles, CA 90089, USA. Electronic address: humayun@usc.edu.

PMID: 35120620
PMCID: PMC9039755
DOI: 10.1016/j.stemcr.2022.01.001

Abstract

Cell-based therapies face challenges, including poor cell survival, immune rejection, and integration into pathologic tissue. We conducted an open-label phase 1/2a clinical trial to assess the safety and preliminary efficacy of a subretinal implant consisting of a polarized monolayer of allogeneic human embryonic stem cell-derived retinal pigmented epithelium (RPE) cells in subjects with geographic atrophy (GA) secondary to dry age-related macular degeneration. Postmortem histology from one subject with very advanced disease shows the presence of donor RPE cells 2 years after implantation by immunoreactivity for RPE65 and donor-specific human leukocyte antigen (HLA) class I molecules. Markers of RPE cell polarity and phagocytosis suggest donor RPE function. Further histologic examination demonstrated CD34⁺ structures beneath the implant and CD4⁺, CD68⁺, and FoxP3⁺ cells in the tissue. Despite significant donor-host HLA mismatch, no clinical signs of retinitis, vitreitis, vasculitis, choroiditis, or serologic immune response were detected in the deceased subject or any other subject in the study. Subretinally implanted, HLA-mismatched donor RPE cells survive, express functional markers, and do not elicit clinically detectable intraocular inflammation or serologic immune responses even without long-term immunosuppression.

Keywords: allogeneic; clinical trial; geographic atrophy; histology; implant; macular degeneration; parylene; polarized; retinal pigmented epithelium; stem cells.

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Figures

**Figure 1**
Color fundus photographs of subject 125 at baseline and 1 year after CPCB-RPE1 surgical implantation into the subretinal space (A) Preoperative photograph demonstrates variable areas of depigmentation in the central macula consistent with geographic atrophy (GA) in advanced dry age-related macular degeneration. (B) Postoperative fundus photographs of the same region at 1 year after CPCB-RPE1 implantation demonstrates the presence of the implant and its associated pigmented cells covering a large portion of the GA lesion. One edge of the pigmented implant is denoted by a white arrow for reference. The retinotomy site is denoted by a yellow arrow. (C) Fundus photograph of the nonimplanted eye at baseline. (D) Fundus photograph of the nonimplanted eye 1 year later.

**Figure 2**
Retinal histopathology in subject 125 at 2 years post-implantation shows RPE survival and phagocytotic activity (A) H&E staining of implanted retina. The parylene membrane (black arrow) that forms the basement membrane-like scaffold for the RPE cells appears as a translucent rectangular object with alternating thin (6 μm) and ultrathin (0.4 μm) regions on H&E images but is not directly visible in subsequent fluorescence images. H&E staining within the area of the implant demonstrated a monolayer of pigmented RPE cells associated with the parylene membrane; RPE cells also were occasionally observed to be associated with the underside of the membrane (green arrow) as a result of growth of RPE cells around the edge of the membrane onto the bottom surface during implant production. The retina overlying the implant exhibits severe disorganization of outer retinal layers consistent with geographic atrophy. (B) Immunofluorescence for RPE65 (green) is present on the implant RPE cells. (C) Immunofluorescence of a similar region demonstrates that donor RPE cells express Na⁺/K⁺-ATPase (red) in a largely apical distribution consistent with mature and functional RPE. (D–F) Immunohistochemical identification of donor RPE. (D) Human leukocyte antigen serotype A2 (HLA-A2) immunoreactivity (red) in donor RPE cells closely associated with the parylene membrane (arrowhead). The HLA-A2 serotype is specifically expressed by donor, but not recipient, cells. Fluorescence associated with the parylene membrane is a consequence of the Superboost staining procedure and non-specific binding of the Tyramide solution. (E) Immunostaining for bestrophin (BEST1, red) in an adjacent section to that shown in (D) confirms the identity of cells associated with the parylene membrane (arrowhead) as RPE cells. (F) Secondary antibody control shows only artifactual staining associated with the parylene membrane (arrowhead). (G) Yellow immunofluorescence represents red-stained phagosomes (rhodopsin) in green-stained cytoplasm (RPE65) within the donor RPE of the implant in small granules suggestive of the presence of phagosomes containing photoreceptor outer segments. Rhodopsin staining, normally associated with rod photoreceptors, is present in outer segment-like rosette structures in the overlying atrophic retina. (H) Phase-contrast image of implant area showing the pigmented RPE cells along the entire length of the CPCB-RPE1 implant. Blue fluorescence in (B)–(D) indicates DAPI staining of cell nuclei.

**Figure 3**
Retinal histopathology of cellular immune responses in subject 125 at 2 years post-implantation (A–C) The parylene membrane that forms the basement membrane-like scaffold for the RPE cells appears as a translucent rectangular object with alternating thin (6 μm) and ultrathin (0.4 μm) regions. All sections were stained with hematoxylin and counterstained as follows: (A) CD68 (red), a marker of macrophages, is present in the retina and choroid; (B) CD8 (red), a marker of cytotoxic T lymphocytes, also is observed occasionally in the choroid and in the retina near the implant; (C) CD4 (red), a marker of Th lymphocytes, is distributed throughout the retina and choroid and fluorescence imaging of double-labeled CD4⁺ and FOXP3⁺ cells (red-rimmed cells with yellow nuclei) found in the retina (inset). DAPI (blue) was used as a counterstain to label nuclei in the inset in (C).

**Figure 4**
Histopathology of intraretinal gliosis and sub-implant material in subject 125 at 2 years post-implantation The parylene membrane that forms the basement membrane-like scaffold for the RPE cells appears as a translucent rectangular object with alternating thin (6 μm) and ultrathin (0.4 μm) sections. (A) Staining for glial fibrillary acidic protein (GFAP, red) demonstrates diffuse gliosis in the retina, but no staining in the subimplant space. (B) Trichrome Masson staining demonstrates staining of collagen (blue) beneath the implant that is consistent with the staining of the native choroid below it. (C) CD34, an endothelial cell marker (red) associated with vascular-like structures, is present in the sub-implant tissue immediately adjacent to the implant. There is also staining of native choroidal vessels beneath Bruch's membrane. Bottom row: gliosis in the area of geographic atrophy in the non-implanted, contralateral control eye of subject 125 with severe, advanced dry age-related macular degeneration. (D) GFAP (red) counterstained with hematoxylin demonstrates diffuse gliosis of the retina. (E) Trichrome Masson staining in area of geographic atrophy. (F) CD34, a marker of endothelial cells (red), counterstained with hematoxylin demonstrates staining of intraretinal and choroidal vessels; no staining is observed in the subretinal space. (A, C, D, and F) Counterstained with hematoxylin (blue) to identify cell nuclei.

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References

1. Algvere P.V., Berglin L., Gouras P., Sheng Y., Kopp E.D. Transplantation of RPE in age-related macular degeneration: observations in disciform lesions and dry RPE atrophy. Graefes Arch. Clin. Exp. Ophthalmol. 1997;235:149–158. - PubMed
1. Benner J.D., Sunness J.S., Ziegler M.D., Soltanian J. Limited macular translocation for atrophic maculopathy. Arch. Ophthalmol. 2002;120:586–591. - PubMed
1. Binder S., Krebs I., Hilgers R.D., Abri A., Stolba U., Assadoulina A., Kellner L., Stanzel B.V., Jahn C., Feichtinger H. Outcome of transplantation of autologous retinal pigment epithelium in age-related macular degeneration: a prospective trial. Invest. Ophthalmol. Vis. Sci. 2004;45:4151–4160. - PubMed
1. Brant Fernandes R.A., Koss M.J., Falabella P., Stefanini F.R., Maia M., Diniz B., Ribeiro R., Hu Y., Hinton D., Clegg D.O., et al. An innovative surgical technique for subretinal transplantation of human embryonic stem cell-derived retinal pigmented epithelium in yucatan mini pigs: preliminary results. Ophthalmic Surg. Lasers Imaging Retina. 2016;47:342–351. - PubMed
1. Cahill M.T., Mruthyunjaya P., Bowes Rickman C., Toth C.A. Recurrence of retinal pigment epithelial changes after macular translocation with 360 degrees peripheral retinectomy for geographic atrophy. Arch. Ophthalmol. 2005;123:935–938. - PubMed

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[1] Algvere P.V., Berglin L., Gouras P., Sheng Y., Kopp E.D. Transplantation of RPE in age-related macular degeneration: observations in disciform lesions and dry RPE atrophy. Graefes Arch. Clin. Exp. Ophthalmol. 1997;235:149–158. - PubMed

[2] Algvere P.V., Berglin L., Gouras P., Sheng Y., Kopp E.D. Transplantation of RPE in age-related macular degeneration: observations in disciform lesions and dry RPE atrophy. Graefes Arch. Clin. Exp. Ophthalmol. 1997;235:149–158. - PubMed

[3] Benner J.D., Sunness J.S., Ziegler M.D., Soltanian J. Limited macular translocation for atrophic maculopathy. Arch. Ophthalmol. 2002;120:586–591. - PubMed

[4] Benner J.D., Sunness J.S., Ziegler M.D., Soltanian J. Limited macular translocation for atrophic maculopathy. Arch. Ophthalmol. 2002;120:586–591. - PubMed

[5] Binder S., Krebs I., Hilgers R.D., Abri A., Stolba U., Assadoulina A., Kellner L., Stanzel B.V., Jahn C., Feichtinger H. Outcome of transplantation of autologous retinal pigment epithelium in age-related macular degeneration: a prospective trial. Invest. Ophthalmol. Vis. Sci. 2004;45:4151–4160. - PubMed

[6] Binder S., Krebs I., Hilgers R.D., Abri A., Stolba U., Assadoulina A., Kellner L., Stanzel B.V., Jahn C., Feichtinger H. Outcome of transplantation of autologous retinal pigment epithelium in age-related macular degeneration: a prospective trial. Invest. Ophthalmol. Vis. Sci. 2004;45:4151–4160. - PubMed

[7] Brant Fernandes R.A., Koss M.J., Falabella P., Stefanini F.R., Maia M., Diniz B., Ribeiro R., Hu Y., Hinton D., Clegg D.O., et al. An innovative surgical technique for subretinal transplantation of human embryonic stem cell-derived retinal pigmented epithelium in yucatan mini pigs: preliminary results. Ophthalmic Surg. Lasers Imaging Retina. 2016;47:342–351. - PubMed

[8] Brant Fernandes R.A., Koss M.J., Falabella P., Stefanini F.R., Maia M., Diniz B., Ribeiro R., Hu Y., Hinton D., Clegg D.O., et al. An innovative surgical technique for subretinal transplantation of human embryonic stem cell-derived retinal pigmented epithelium in yucatan mini pigs: preliminary results. Ophthalmic Surg. Lasers Imaging Retina. 2016;47:342–351. - PubMed

[9] Cahill M.T., Mruthyunjaya P., Bowes Rickman C., Toth C.A. Recurrence of retinal pigment epithelial changes after macular translocation with 360 degrees peripheral retinectomy for geographic atrophy. Arch. Ophthalmol. 2005;123:935–938. - PubMed

[10] Cahill M.T., Mruthyunjaya P., Bowes Rickman C., Toth C.A. Recurrence of retinal pigment epithelial changes after macular translocation with 360 degrees peripheral retinectomy for geographic atrophy. Arch. Ophthalmol. 2005;123:935–938. - PubMed

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Survival of an HLA-mismatched, bioengineered RPE implant in dry age-related macular degeneration

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Survival of an HLA-mismatched, bioengineered RPE implant in dry age-related macular degeneration

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