The therapeutic potential of cells derived from embryonic stem cells (ESC) in the treatment of diseases characterized by tissue dysfunction or loss, such as myocardial infarction, diabetes, ischemic stroke, spinal cord injury, and even Parkinson and Alzheimer disease, has long been recognized.¹ At the same time, safety caveats with ESC-derived approaches have been numerous, arising both out of their complex biology and regulatory/ethical concerns. ESC’s capacity for unlimited and various renewal, for example, leads to concerns about potential development of teratomas and other unintended differentiation, as well as concomitant immune reactions.

Potential sources for cell-based therapeutics include not just human ESCs, but also adult stem cells and induced pluripotent stem cells (iPSCs).² While adult stem cells can differentiate into some specialized cell types, obtaining the large quantities needed for transplantation is problematic because they are difficult to harvest, and they have limited ability to divide and differentiate.³ Human ESCs (hESCs) offer several advantages. Lines of ESCs can be derived from single cells removed harmlessly from embryos generated during in vitro fertilization procedures, thereby avoiding ethical concerns pertaining to abortion-derived ESCs. ESCs divide readily, and because they are pluripotent, they can differentiate into a wide variety of cell types and have demonstrated less immunogenicity than adult tissues in animal models.⁴ As Schwartz et al¹ point out, however, safety and efficacy challenges remain for transplantation of ESCs into diseased environments with respect to engraftment, survival, and polarization. Induced pluripotent stem cells offer theoretically lower immunogenicity risk because they can be dedifferentiated from a patient’s own cells. That advantage comes with the potential detriment that source-cell genetic defects will be conveyed to differentiated iPSC offspring, although gene-correcting or gene-editing techniques may remediate such predispositions.

Until reprogramming technologies develop further, several factors favor diseases of the eye or other immunoprivileged sites as first-in-human targets for cell-based therapies. Among them are protection of the subretinal space by the blood-ocular barrier and by antigen-specific inhibition of cellular and humoral responses. Accessibility for local delivery allows therapeutic doses to be kept low, with minimal extraocular distribution risk. Furthermore, direct visualization of retinal function and structure is achievable through a variety of imaging techniques, accommodating noninvasive diagnosis, real-time therapy guidance, and quantification of therapeutic effects.

ORAL SUPPLEMENTATION AND AMD

Sponsored by the NIH, the Age-Related Eye Disease Study (AREDS) lent support in 2001 to the role of chronic oxidative stress and inflammation in the pathogenesis of AMD, showing an approximately 25% reduction in the risk of developing advanced AMD after 5 years of daily oral supplementation with antioxidant vitamins and minerals.⁵ AREDS included 3,640 participants (55 to 80 years of age) in 11 US centers receiving 1 of 4 treatments: (1) zinc alone (zinc 80 mg, as zinc oxide, and copper 2 mg, as cupric oxide); (2) antioxidants alone (vitamin C 500 mg, vitamin E 400 IU, and beta carotene 15 mg); (3) a combination of antioxidants and zinc; or (4) placebo. After an average follow-up of 6.3 years, there was a statistically significant odds reduction for the development of advanced AMD with antioxidants plus zinc (odds ratio [OR], 0.72; 99% confidence interval [CI], 0.52-0.98). When patients with small- or intermediate-size drusen were excluded, the OR was 0.66 for antioxidants plus zinc (99% CI, 0.47-0.91). This group receiving zinc and antioxidants was the only one to have reduced visual acuity loss.

AREDS2 was launched based on observational data suggesting that increased dietary intake of lutein + zeaxanthin (carotenoids), omega-3 long-chain polyunsaturated fatty acids (docosahexaenoic acid [DHA] + eicosapentaenoic acid [EPA]), or both might further reduce risk of progression to advanced AMD. The main components of the macular pigment are lutein and zeaxanthin, and DHA is a major structural component of the retina. Also, EPA may influence retinal function.⁶ The trial also evaluated 4 variations of the AREDS formulation, including elimination of beta carotene, lowering of zinc dose, or both. After a median follow-up of 5 years, compared with placebo there were no reductions in progression to advanced AMD in 1,608 participants and no apparent effect of beta carotene elimination or lower-dose zinc on progression to advanced AMD. Lung cancers, mostly in former smokers, were more common in those receiving beta carotene vs no beta carotene (2.0% vs 0.9%, P=.04).

AMD is known as a disease with a multifactorial etiology including many genetic variants influenced by nongenetic or environmental factors, such as diet and smoking. Also, genetic research has linked dysfunction of the alternative complement pathway (ACP) to the pathogenesis of AMD.⁷ Roche’s lampalizumab, an antigen-binding fragment of a humanized, monoclonal antibody directed against complement factor D, an enzyme with a role in activating/amplifying the ACP, is being tested in global (20+ countries) phase 3 SPECTRI and CHROMA trials (Clinicaltrials.gov identifiers NCT02247531 and NCT02247479, respectively).⁸ The trials, both with identical double-blind randomized design, investigate the efficacy and safety of 10-mg intravitreal injections of lampalizumab given every 4 weeks or 6 weeks vs sham injections in 1,800 patients with AMD-caused geographic atrophy (GA). The primary endpoint is the difference in mean change in GA lesion area at week 48, as measured by fundus autofluorescence. Two-year visual function is a secondary objective.

In September 2017, Roche announced that analysis at 48 weeks did not show a reduction in mean change in GA area for lampalizumab as compared with a placebo injection. Further dosing of patients was put on hold until results from CHROMA, the second phase 3 trial, are evaluated. The safety profile, it was also announced, was consistent with prior lampalizumab and other intravitreal therapy trials. At the American Academy of Ophthalmology (AAO) Annual Meeting in November 2017, Roche announced that CHROMA results were negative as well.

The first human trials of Ocata Therapeutics’ MA09-hRPE (later acquired by Astellas Pharma) and BioTime’s OpRegen cell-based therapies for advanced AMD are based on the premise that replacing the retinal pigment epithelium (RPE) cells essential to maintaining the health of the macula’s rods and cones will end disease progression and allow damaged or dormant light-sensitive cells to return to function. The immunoprivileged environment of the subretinal space, with its limited blood circulation, obviates the need for matched donors and allows establishment of a universal RPE cell bank. Ocata’s phase 1/2 safety studies of hESC MA09-hRPE among an initial cohort of 18 patients (9 with Stargardt disease [NCT01345006] and 9 with AMD [NCT01344993]) treated at 4 centers showed encouraging initial results.² Adverse events were not related to the cell therapy, but rather they were related to the surgical procedure and other factors. After transplantation, 72% of patients had increased subretinal pigment at the atrophic area border, and at 1 year, visual function was improved in 9 eyes and stable in 7. Optical coherence tomography (OCT) showed reconstitution or thickening of the RPE layer in some subjects. In a presentation of 2-year results at the 2017 AAO meeting, lead researcher Ninel Z. Gregori, MD, noted that in 24 of 26 subjects (92%), areas of subretinal pigmentation had developed, but that did not correlate with dose or visual acuity outcomes.

The related, phase 2 PORTRAY trial (NCT02563782) of hESC-derived RPE cells in AMD was aimed at assessing graft rejection strategies and secondarily at change in area of GA area and change in visual acuity. The trial was withdrawn prior to enrollment without explanation by the sponsor, Astellas Institute for Regenerative Medicine.

Degeneration of the RPE monolayer plays a role in both wet and dry AMD. An article in the New England Journal of Medicine included among its authors Shinya Yamanaka, MD, PhD,⁹ who received the 2012 Nobel Prize in Physiology or Medicine for showing that mature cells can be reprogrammed to become pluripotent. The report reviews a case study assessing the feasibility of treating wet AMD by transplanting a sheet of autologous RPE cells differentiated from induced pluripotent stem cells. A year after surgery to remove a neovascular membrane and implant the RPE sheet under the retina, the sheet remained intact and visual acuity was stable, although cystoid macular edema persisted. The patient’s score on the National Eye Institute Visual Functioning Questionnaire (VFQ)-25, on which scores range from 0 to 100 with higher scores indicating better visual function and general health, increased from 48.8 before surgery to 58.3 at 1 year post surgery, and the patient reported satisfaction with “brighter” vision. The authors noted, however, that it is possible that the improved VFQ score could have occurred without the surgery and that the brightening was likely attributable to the removal of the neovascular membrane.

An observed expansion of the graft area, the authors commented further, may indicate that the grafted cells survived. Also, good retinal integrity over the graft with a high-density area in optical coherence tomography images up to 1 year after surgery may indicate recovering inner and outer segments of photoreceptor cells.

BioTime’s OpRegen was granted fast-track designation by the FDA for advanced dry AMD with GA in 2015 based on animal AMD studies showing preserved visual function and retinal structure.¹⁰ A BioTime proprietary process produces higher yields of clinical grade hESC-derived RPE cells than are produced by spontaneous generation. It employs a strictly xeno-free (animal product-free) derived reagents protocol in adherence to regulations for embryo procurement, good tissue, manufacturing (GMP) and laboratory practices that minimize contamination concerns (Figure 1).¹¹

OpRegen was tested in Royal College of Surgeons rats (n=242) receiving 1 of 3 separate level single subretinal injections of RPE cells. The therapy demonstrated rescued visual function and preserved rod and cone photoreceptors long term compared with controls in a dose-dependent manner. Transplanted RPE cells were identified in the subretinal space and in the host RPE monolayer, often with internalized photoreceptor outer segments.

The OpRegen phase 1/2a trial (NCT02286089) tests 3 dosing levels (500 cells/µL, 100 µL; 2,000 cells/µL, 100 µL; 2,000 cells/µL, 50 µL among 3 cohorts of 3 patients, each with baseline best corrected visual acuity of 20/200 or less). In a second study stage, 6 patients with BCVA of 20/100 or less will receive dosing informed by the first 3 cohorts.

Included patients are 50 years of age or older, with non-neovascular AMD in both eyes, who have fundoscopic findings of GA in the macula, but without additional concomitant ocular disorders. The study is being conducted in Israel and the United States. The main objective is evaluation of the safety and tolerability of subretinal transplant of OpRegen hESC-derived RPE cells at 12 months. The study also explores the ability of OpRegen cells to engraft, survive, and moderate disease progression. Change in visual acuity, reading speed test, low luminance test, microperimetry, quality of life (NEI VFQ-25 Quality of Life) and centrally read GA lesion area will also be assessed at 12 months.

To date, imaging suggests cell engraftment in all patients, with signs of structural improvements at 9 months post procedure as evidenced by an observed thickening of the retina. In a recent presentation of preliminary results at the 2017 AAO conference, researcher Eyal Banin, MD, showed imaging suggesting healing in the injection site within a few weeks. BCVA remains stable, and subretinal pigmentation that correlates with irregular subretinal hyper-reflectance on optical coherence tomography (OCT) is evident in most patients (5 of 6), suggesting the presence of implanted cells in the subretinal space (Figure 2).

Whether this indicates engraftment and survival of the transplanted cells and improved photoreceptor health remains to be seen, Dr. Banin said. Subretinal transplantation of hESC-derived RPE appears to be well tolerated to date. If these trial results provide sufficient evidence for the safety of injecting OpRegen cells, subsequent research among AMD patients with better preserved vision at baseline will better test the ability of OpRegen to prevent disease progression and restore damaged or dormant photoreceptors. Approval to initiate treatment of cohort 4 is expected in the first 3 months of 2018.

The ongoing multicenter phase 2b PRELUDE study (NCT02659098) is assessing subretinal administration of CNTO 2476 in an estimated 285 patients with visual acuity impairment associated with GA secondary to AMD. Included study eyes have a BCVA of 20/80 to 20/800. Patients are randomized to 1 of 2 treatment arms or a sham surgical procedure. Patients in the intervention arms of PRELUDE, after an unmasked safety phase, are randomized double-blind to receive a single subretinal dose of human umbilical tissue-derived cells (CNTO 2476) at either 6 x 10⁴ cells or 3 x 10⁵ cells via a modified surgical procedure and a custom-designed delivery system. In an earlier phase study of CNTO 2476, rates of retinal perforation and retinal detachment were high.¹² Tolerability was good, however, and some subjects with vision loss secondary to GA had improved visual acuity. In PRELUDE, blinded outcomes assessors will evaluate safety and performance profile as well as BCVA. Study duration is about 5 years with evaluations at 6 months and 12 months, and annually thereafter.

PRELUDE’s primary efficacy outcome measures include the percentage of cases of successful delivery of CNTO 2476 cells to the subretinal space and the percentage of participants showing improvement from baseline of greater than or equal to 15 letters in BCVA at month 6. The primary safety outcome is the number of eyes with treatment-related ocular adverse events. The first primary analysis is estimated to occur in late December 2018.

CONCLUSION

Clinical research affirms the feasibility and safety of transplanting hESC-derived RPE cells in advanced dry AMD and in Stargardt disease. Signals of possible biologic activity suggest that stability and improvement of visual acuity may become evident as ongoing and future clinical trials progress. RP

REFERENCES

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