Does systemic inflammation prompt gene therapy uveitis?
Authors: Ying Kai Chan, Kathryn L. Pepple
Journal: Molecular Therapy (2021)

Ocular gene therapy is an exciting area of intense research and development, as highlighted by the 2017 approval of Luxturna. However, immune responses elicited by viral vectors remain a key concern. As reported in this issue of Molecular Therapy, Mishra et al.1Mishra A. Vijayasarathy C. Cukras C.A. Wiley H.E. Sen H.N. Zeng Y. Wei L.L. Sieving P.A. Immune function in X-linked retinoschisis subjects in an AAV8-RS1 phase I/IIa gene therapy trial.Mol. Ther. 2021; 29 (this issue): 2030-2040Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar evaluated patients with X-linked retinoschisis (XLRS) who received intravitreal gene therapy with AAV8-RS1 (ClinicalTrials.gov: NCT02317887) for signs of systemic inflammation before and after treatment.2Cukras C. Wiley H.E. Jeffrey B.G. Sen H.N. Turriff A. Zeng Y. Vijayasarathy C. Marangoni D. Ziccardi L. Kjellstrom S. et al.Retinal AAV8-RS1 Gene Therapy for X-Linked Retinoschisis: Initial Findings from a Phase I/IIa Trial by Intravitreal Delivery.Mol. Ther. 2018; 26: 2282-2294Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar Each participant’s immune activation state was determined by analysis of peripheral blood mononuclear cell (PBMC) composition and measurement of serum pro-inflammatory cytokine concentration. The authors concluded that the XLRS patients demonstrated a baseline proinflammatory immune phenotype and that intravitreal injection of AAV8-RS1 increased signs of systemic inflammation. This study is important because it proposes a new hypothesis to explain the incidence of gene therapy-associated uveitis (GTAU) in patients with XLRS, which has been reported in two separate adeno-associated virus (AAV) clinical-stage programs.2Cukras C. Wiley H.E. Jeffrey B.G. Sen H.N. Turriff A. Zeng Y. Vijayasarathy C. Marangoni D. Ziccardi L. Kjellstrom S. et al.Retinal AAV8-RS1 Gene Therapy for X-Linked Retinoschisis: Initial Findings from a Phase I/IIa Trial by Intravitreal Delivery.Mol. Ther. 2018; 26: 2282-2294Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar,3AGTC AGTC Announces Topline Safety Data for X-Linked Retinoschisis Phase 1/2 Study.2017http://ir.agtc.com/news-releases/news-release-details/agtc-announces-topline-safety-data-x-linked-retinoschisis-phaseGoogle Scholar Additionally, the authors studied a broader range of markers than most gene therapy trials, suggesting administration of intravitreal gene therapy products activates the systemic immune system. However, there are some important caveats in the study design that impact the strength of these conclusions. The first consideration is the comparability of the age-matched controls to the XLRS subjects. The control samples were not collected prospectively as part of the XLRS study, and this led to small but potentially important differences in sample collection, handling, and processing.4Gottfried-Blackmore A. Rubin S.J.S. Bai L. Aluko S. Yang Y. Park W. Habtezion A. Effects of processing conditions on stability of immune analytes in human blood.Sci. Rep. 2020; 10: 17328Crossref PubMed Scopus (5) Google Scholar Additionally, data on many important baseline factors (recent illness, injury, or vaccination) that could have contributed to the differences between the groups were not collected. The second consideration is the very broad characterization of immune function that was performed. Coupled with the small number of samples and the lack of correction for multiple comparisons, these results are exploratory. While not definitive, the study generates hypotheses for further studies on a critical, yet often overlooked, aspect of gene therapy: immunogenicity. Ocular gene therapy trials to date have typically focused on measuring antibody and cellular responses directed against AAV capsid for immunology testing. This study by Mishra et al.1Mishra A. Vijayasarathy C. Cukras C.A. Wiley H.E. Sen H.N. Zeng Y. Wei L.L. Sieving P.A. Immune function in X-linked retinoschisis subjects in an AAV8-RS1 phase I/IIa gene therapy trial.Mol. Ther. 2021; 29 (this issue): 2030-2040Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar cast a wider net and provided a broad characterization of immune status after receiving ocular gene therapy. The authors must be congratulated for this effort and for sharing this wealth of information with the ocular gene therapy community at large. Ideally, this line of investigation would provide clear evidence for a specific immune phenotype that could reliably predict the risk of developing GTAU. Unfortunately, a clear and well-defined high-risk phenotype was not identified among the six subjects that developed uveitis after treatment. The ability to draw definitive conclusions may have been limited by the diverse circumstances and conditions each patient experienced (e.g., different dose, different immunosuppression protocols). Importantly, the last three subjects enrolled in the trial were already immunosuppressed before the sample collection. A number of previous studies have identified multiple factors associated with the induction of GTAU or other signs of treatment related toxicities, the most important ones being high vector titer and intravitreal, rather than subretinal, delivery.5Chan Y.K. Wang S.K. Chu C.J. Copland D.A. Letizia A.J. Costa Verdera H. Chiang J.J. Sethi M. Wang M.K. Neidermyer Jr., W.J. et al.Engineering adeno-associated viral vectors to evade innate immune and inflammatory responses.Sci. Transl. Med. 2021; 13: eabd3438Crossref PubMed Scopus (42) Google Scholar,6Chan Y.K. Dick A.D. Hall S.M. Langmann T. Scribner C.L. Mansfield B.C. and for the Ocular Gene Therapy Inflammation Working GroupInflammation in Viral Vector-Mediated Ocular Gene Therapy: A Review and Report From a Workshop Hosted by the Foundation Fighting Blindness, 9/2020.Transl. Vis. Sci. Technol. 2021; 10: 3Crossref PubMed Scopus (5) Google Scholar Other factors that may contribute include the presence of empty viral capsids, self-complementary vectors, and vectors with high CpG content, which have all been associated with reproducible inflammation in animal model systems.7Bucher K. Rodríguez-Bocanegra E. Dauletbekov D. Fischer M.D. Immune responses to retinal gene therapy using adeno-associated viral vectors - Implications for treatment success and safety.Prog. Retin. Eye Res. 2020; (Published online October 15, 2020)https://doi.org/10.1016/j.preteyeres.2020.100915Crossref PubMed Scopus (42) Google Scholar However, even when the gene therapy dose, route, and content are held constant, not all eyes will experience an inflammatory response. This suggests that there are likely additional host factors contributing to risk. The current study provides tantalizing evidence that it could be baseline systemic inflammation. However, the impact of retinal degeneration on the local ocular immune microenvironment may also be an important consideration.8Yu C. Roubeix C. Sennlaub F. Saban D.R. Microglia versus Monocytes: Distinct Roles in Degenerative Diseases of the Retina.Trends Neurosci. 2020; 43: 433-449Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar Looking at the bigger picture, several gene therapies are being developed for ophthalmic disease indications that have a degenerative or inflammatory component, such as retinitis pigmentosa and age-related macular degeneration. As the authors suggest, can any retinal degeneration activate the systemic immune system or could this indicate an as-yet-unknown role for Rs1, a predominantly retinal protein, in the immune system? The possibility of a baseline proinflammatory immune phenotype, whether systemic or ocular, could impede successful viral gene therapy. Readouts from gene therapy trials will clarify the breadth and magnitude of this issue. Furthermore, safety studies during preclinical development of ocular gene therapy often utilize healthy large animals, which would miss this phenomenon. On the flip side, animal models may facilitate testing and further characterization of key hypotheses raised by this study, e.g., whether intravitreal AAV treatment of non-human primates (NHPs) also trigger similar systemic immune signatures and whether the XLRS mouse model also exhibit proinflammatory systemic immunity. Regardless of the underlying mechanisms, a pressing question for therapies in development remains: what approaches should the field undertake to mitigate inflammation? On the vector side, it would be critical to design and manufacture the vector in a way that minimizes immunogenicity (e.g., increasing potency and hence reducing the dose, reducing CpG content or inhibiting TLR9, depleting empty capsids). Of note, Mishra et al.1Mishra A. Vijayasarathy C. Cukras C.A. Wiley H.E. Sen H.N. Zeng Y. Wei L.L. Sieving P.A. Immune function in X-linked retinoschisis subjects in an AAV8-RS1 phase I/IIa gene therapy trial.Mol. Ther. 2021; 29 (this issue): 2030-2040Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar used a self-complementary vector, which has been implicated in heightened innate immune and T cell responses in mice when delivered systemically,9Martino A.T. Suzuki M. Markusic D.M. Zolotukhin I. Ryals R.C. Moghimi B. Ertl H.C.J. Muruve D.A. Lee B. Herzog R.W. The genome of self-complementary adeno-associated viral vectors increases Toll-like receptor 9-dependent innate immune responses in the liver.Blood. 2011; 117: 6459-6468Crossref PubMed Scopus (140) Google Scholar potentially contributing to the observed systemic immune activation. On the clinical side, it would be desirable to use an evidence-based and optimal immunosuppression protocol that balances the chance of the gene therapy working while being safe and tolerable to patients. Interestingly, the addition of potent immunosuppressants cyclosporine (typically reserved for use in organ transplant patients) and mycophenolate mofetil gave mixed results between subjects S10 and S11, illustrating the complexity of managing the immune responses. The characterization of altered markers of systemic immunity, whether before or after ocular gene therapy, may pave the way for more targeted immunosuppression. Very recently, a trial reported loss of vision and panuveitis in the treated eye of a patient, 30 weeks following intravitreal injection of an AAV vector expressing anti-VEGF.10Adams B. Adverum shares halved after trial patient goes blind in one eye after experimental gene therapy.Fierce Biotech. 2021; (April 29, 2021)https://www.fiercebiotech.com/biotech/adverum-shares-halved-after-trial-patient-goes-blind-one-eye-after-experimental-geneGoogle Scholar Clearly, we need to advance our understanding of inflammation during ocular gene therapy and rationally design mitigation strategies. Mishra et al.1Mishra A. Vijayasarathy C. Cukras C.A. Wiley H.E. Sen H.N. Zeng Y. Wei L.L. Sieving P.A. Immune function in X-linked retinoschisis subjects in an AAV8-RS1 phase I/IIa gene therapy trial.Mol. Ther. 2021; 29 (this issue): 2030-2040Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar have provided some important clues for solving this puzzle. Immune function in X-linked retinoschisis subjects in an AAV8-RS1 phase I/IIa gene therapy trialMishra et al.Molecular TherapyFebruary 15, 2021In BriefSystemic immune status was evaluated in X-linked retinoschisis subjects in an ocular AAV8-RS1 gene therapy trial. Baseline changes before dosing showed altered CD4/CD8 T cell ratios, dendritic cell subsets, and pro-inflammatory cytokine levels. Vector application caused systemic immune activation with an increase of activated cytotoxic lymphocytes, macrophages, and pro-inflammatory cytokines. Full-Text PDF Open Archive