A variety of gene therapies for retinal disease are in development. These include gene replacement for inherited retinal diseases (IRDs) like CNGA3 or CNGB3 for achromotopsia (AGTC and MeiraGTX) and Rep-1 for choroideremia (Biogen, 4D Molecular Therapeutics, Spark Therapeutics). There are also biofactory approaches for age-related macular degeneration (AMD), like RGX-314 (Regenxbio) ADVM-022 (Adverum Biotechnologies), and 4D-150 (4D Molecular Therapeutics) for wet AMD and GT005 (Gyroscope Therapeutics) or HMR59 (Hemera Biosciences) for dry AMD. However, developing gene therapies in ophthalmology has come with its fair share of challenges, among them inflammation in treated eyes.

Retinal Physician convened a roundtable including Glenn C. Yiu, MD, PhD, Kathryn L. Pepple, MD, PhD, and Arshad Khanani, MD, MA, to discuss the challenges facing these trials. Dr. Yiu is a professor at University of California, Davis, and is involved in translational gene therapy research. Dr. Pepple is an associate professor at the University of Washington in the department of ophthalmology, and she is a uveitis expert and clinician scientist with a research lab where she studies animal models to understand uveitis and inflammation. Dr. Khanani is the director of clinical research and an associate professor at University of Nevada, Reno, and a managing partner of Sierra Eye Associates. He is a world-class clinical trialist involved in several clinical trials in gene therapy.

Glenn C. Yiu, MD, PhD: Arshad, you have been involved in several of these clinical trials. What do you feel are the major challenges facing gene therapy trials today?

Arshad Khanani, MD, MA: Gene therapy is a paradigm shift in treatment of retinal diseases.¹ For IRDs, because there are no treatments, the bar is different than for wet AMD, diabetic retinopathy (DR), or even diabetic macular edema (DME). We have good treatments for AMD and DR with great efficacy and a very good safety track record, and gene therapy is showing to have very good efficacy for vascular retinal diseases, which is great. But the problem has been safety. We have seen pigmentary changes in subretinal gene therapy. We have seen intraocular inflammation with intravital gene therapy. We have seen some signs of inflammation even with suprachoroidal gene therapy. Gene therapy associated inflammation is something that we need to address as a field. The question is, is this happening because of inadequate prophylaxis, or is it because we are exposing the tissues of the eye that are proinflammatory, like the ciliary body or the iris, to viral vectors?

What we have learned is that dose ranging is very important for gene therapy. We should always start with a low dose, and the challenge is getting the right dose so that we maximize efficacy and minimize safety events. Gene therapy is irreversible, so we need to make sure that we don’t expose our patients to high doses where we may start having dose-limiting toxicities like were seen in Adverum’s INFINITY program. Another unanswered question is about prophylaxis: should it be intravitreal, topical, or by mouth, and how long do we need to treat? Subretinal gene therapy does seem not to cause gene therapy associated uveitis or inflammation acutely, but we do see pigmentary changes.

Glenn C. Yiu, MD, PhD: That is a good point about subretinal delivery reaching a space that is more immune privileged than intravitreal delivery. Some of our own work on suprachoroidal delivery suggests that the viral vector is outside the blood-retinal barrier and potentially also more immunogenic.^2,3 This segues well to Dr. Pepple: do you think that gene therapy associated uveitis is just another form of uveitis, or from your animal models do you think it needs to be managed differently?

Kathryn L. Pepple, MD, PhD: The vision science community is not alone in grappling with gene therapy associated inflammation. Groups targeting other organ systems are also working to identify the mechanisms responsible for undesired immune responses impacting successful gene therapy.⁴ I think we can learn from our colleagues in other fields, but it will be important to verify if mechanisms identified in other tissues are relevant to the fairly unique immune microenvironment of the eye.⁵

Whether gene therapy inflammation is like other forms of uveitis is a great question. Furthermore, should it be classified as an infectious or autoimmune form of uveitis? In some ways, it could be considered an iatrogenic form of infectious uveitis. With AAV gene therapy, we’re intentionally exposing the eye to a high dose of viral particles. Viral capsid proteins and genomic material can activate ocular innate immune sensors and trigger an acute inflammatory state with poorly understood sequela. My lab has been studying the chronic immune meditated uveitis that results from the intravitreal injection of heat killed mycobacteria (HKMtb) which is a robust innate immune stimulus. We consider this model a form of postinfectious uveitis, and we have shown that the chronic uveitis that develops depends on a T cell mediated adaptive immune response. Now AAV is not as potent an innate stimulus as HKMtb, but we, and others, have also identified that T cells accumulate in eyes after intravitreal and subretinal gene therapy injection, suggesting a similar postinfectious mechanism may play a role here.^6,7

Could there be a role for immune modulation strategies that are effective in uveitis as treatment of gene therapy uveitis? This is an important question, and preclinical animal model studies will be helpful in screening options we use routinely for our uveitis patients to determine their effectiveness in preventing gene therapy associated inflammation. However, there may be unique prevention strategies that aren’t available to other patients with uveitis. In the case of gene therapy associated inflammation, we know exactly when the innate stimulus and new antigens will be introduced into the eye, so we can potentially modulate the immune responses at the time of treatment and hopefully prevent long-term adaptive immune responses. Strategies such as vector modification to inhibit innate recognition⁸ or short-term T cell costimulation inhibition at the time of gene therapy administration⁹ to prevent adaptive immune responses. These and other options could be exciting to look forward to in the future.

Glenn C. Yiu, MD, PhD: That is a very good point that in other forms of uveitis we don’t know the mechanism but here we know the mechanism. We can create an animal model; we can put virus into a mouse and study it and figure out a good way to mitigate inflammation. Arshad, what are different companies and clinical trial organizers or designers doing to try to address this issue of inflammation?

Arshad Khanani, MD, MA: The issue of inflammation is very important for intravitreal and now suprachoroidal gene therapy. In suprachoroidal programs, we didn’t have prophylaxis and yet there was inflammation. If something like 20% of your patients have inflammation, even though it’s mild and it goes away with steroids, that’s not something we want to see in a commercially available product. The suprachoroidal program is now implementing topical or local steroid prophylaxis based on that learning. For intravitreal delivery, we saw in the OPTIC study with ADVM-022 and then also INFINITY in DME that the high dose (6x10¹¹ vg/eye) was too high, and those patients have required chronic long-term steroid. There were also cases of hypotony, choroidals, and vision loss in the high dose patient population in INFINITY.

Now, we know that dose matters. The less virus you put in, the less immune response there will be. Lowering the dose is the key. Most of these programs did start at low doses, sometimes even lowering the doses in the next cohort because even with low dose we’re seeing very good efficacy. So, the 2 learnings have been to lower the dose as much as possible and to introduce prophylaxis. As a field, however, we are still trying to determine how long to give prophylaxis. Some programs are using anti-inflammatory for as long as 5 months.

Glenn C. Yiu, MD, PhD: It seems like a lot of these gene therapy trials love to use steroids — topical, intravitreal, oral. Kathryn, do you think that there’s room for immunomodulatory therapies?

Kathryn L. Pepple, MD, PhD: This is a great question particularly when you look at certain patient populations like the pediatric population. They are at such high risk of adverse outcomes from high-dose oral corticosteroids, with potential changes in their growth, and then local corticosteroids, with really concerning response rates with ocular hypertension and cataract development. Finding alternatives for those patients would be impactful. It is not the standard of care in uveitis to continue with long-term corticosteroids, but it took time and study to determine the appropriate immunomodulatory agents to bring in from other fields for treatment of uveitis. We’ve started to realize as a field now that inflammation is probably going to be something we need to plan for in gene therapy to improve safety and efficacy in gene therapy studies. We need to determine the best immunomodulatory approaches that could provide some additional benefit to our patients.

There are some interesting animal model studies that could suggest certain immunomodulatory agents that we should consider from our experimental animal models in uveitis. Phoebe Lin published a paper on a study that compared methotrexate and mycophenolate for modulation of autoimmune uveitis.¹⁰ They found impacts on the gut microbiome as well as gut microbiome independent impacts on uveitis. There was maybe a suggestion that mycophenolate could stimulate some regulatory T cell (T reg) expansion. It would be very impactful if inflammatory mechanisms in gene therapy associated uveitis were linked to T cell populations, particularly against transgenes. There is some evidence that sirolimus could influence T reg expansion as well. It will come down to testing for efficacy and additional benefit with certain immunomodulatory agents. We will need to narrow down to a small number of agents that are reasonable to propose for use in clinical trials to get away from some of these chronic high-dose corticosteroids or intense local steroid approaches.

Glenn C. Yiu, MD, PhD: Great. I also wanted to touch on surgery with you, Arshad, because you’re a surgeon. Some of these gene therapies require subretinal injections. What are some of the strategies to potentially mitigate the potential for inflammation from the surgery standpoint? Do you know of any things that the companies are doing, any best practices for delivering gene therapy subretinally?

Arshad Khanani, MD, MA: There are 2 ways to deliver gene therapy surgically. Number one is to perform vitrectomy and then deliver the dose in a bleb directly. In those programs, there could be leakage of gene therapy material into the vitreous. You do an air-fluid exchange to clean up the vector from the eye. And we have not seen gene therapy associated inflammation with subretinal gene therapy, and the postoperative care is just like a regular postoperative steroid taper over 4 weeks. Other programs are doing a balanced saline solution bleb followed by a gene therapy infusion, so you don’t get a leak. Those programs are not doing air-fluid exchange and investigators are not seeing inflammation.

The subretinal space is an immune-privileged space, and we are not seeing inflammation. I think there are 2 phases of inflammation of gene therapy: the acute phase where you have the viral capsid and a second phase where there is pigment release from these pigmented cells, whether it be the retinal pigment epithelium, the ciliary body, or the iris. The question is, how do we prevent that? In a subretinal space it doesn’t go anywhere, so you see atrophy and pigmentary changes. But in intravitreal gene therapy, you see these pigmented cells and white blood cells just floating around. The goal is to have more cell-specific vectors. If we can avoid hitting the cells that have pigment, in my opinion at least, the iris and the ciliary body, we may be able to go forward with intravitreal gene therapy. Therefore, lower doses of intravitreal gene therapy are now being utilized in clinical trials.

Glenn C. Yiu, MD, PhD: I agree that we don’t want to convert all these patients to uveitis patients.

Kathryn L. Pepple, MD, PhD: Yes; there are definitely not enough uveitis specialists to go around to handle that kind of approach.

Glenn C. Yiu, MD, PhD: I wanted to ask one more question: what do we have in the future in terms of development? Kathryn, do we know of anything besides steroid and immunomodulatory therapies or there are new strategies for beyond what we have now to avoid inflammation?

Kathryn L. Pepple, MD, PhD: In systemic life-threatening diseases, there’s greater acceptance of fairly aggressive approaches to dealing with the immune response and how it’s limiting gene therapy applications. I’ve been impressed by the creativity in other fields. There’s a group developing CAR T cells that are T reg generating. They recognize adeno-associated virus capsid, and the CAR T cells are actually regulatory, so where they recognize capsid, they generate T reg cells and modulate the immune responses that they’re seeing in those tissues.¹¹ That’s very high-level sci-fi immunology. I can’t imagine applying a cell-based product in addition to gene therapy in the eye; that’s going pretty far. You’d also have to get rid of preexisting neutralizing antibodies. There is an IgG-cleaving endopeptidase that can be infused that can deplete the preexisting IgG. This would be more for systemic and diseases as well but might help if you were having trouble redosing potentially in the ocular space. Another avenue are things like using rituximab and IVIG to overwhelm those systems.

For ocular disease, what’s going to save the day is refining the technique, decreasing injury associated with delivery of gene therapy, decreasing other forms of alarmins that are happening at the time of these gene therapy approaches, targeted immune modulation, and possibly supplementing with things like costimulation inhibition at the time of administration. These things could narrow the window of immune suppression required, so that patients aren’t on immune suppression forever, because we don’t want that.

Glenn C. Yiu, MD, PhD: I agree. Especially in the ophthalmic field we might need to rely on simpler methods like cell-specific targeting. Connie Cepko published a paper a few years ago that showed that if you use a photoreceptor-specific promoter rather than a general promoter, you cause less damage and maybe less inflammation. There are also AAVs that are theoretically designed to be cloaked from the immune system so they can evade the immune system.^8,12 That’s an exciting new technology. So, any last comments from either of you about what you want the audience to take away from this discussion?

Arshad Khanani, MD, MA: Gene therapy is a paradigm shift, it’s very exciting as a field. I think it will be the future. We are not there yet, but we are getting close. The bottom line is that through collaboration, we are learning how to make gene therapy safer for our patients, and patients can benefit from gene therapy, whether it’s for IRDs or common retinal diseases. I think we’re going to get there. I’m very hopeful.

Kathryn L. Pepple, MD, PhD: I can only agree with that. This is an amazing technology for patients. With gene therapy, we can hope to restore sight and to prevent blindness in a way never possible before. Any approach that we can develop that improves therapeutic delivery and improves patient outcomes will be beneficial. Looking at inflammation not as a barrier to this great technology but as something that everybody is dealing with, and planning for it, is going to help make this an even better treatment in the long run. RP

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