Combining modeling approaches for rapid preclinical evaluation of retinal gene therapy

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The pathway to developing genetic treatments for rare, monogenic ocular disorders has historically been impeded by limited options in preclinical testing pipelines. In this issue of Molecular Therapy Advances, Dr. Aman George and colleagues navigate this challenge by presenting a proof-of-principle investigation of an adeno-associated virus (AAV)-based gene therapy strategy for oculocutaneous albinism type 1A (OCA1A).¹ Building on prior work² using a constitutive promoter, their approach delivers a human TYROSINASE (TYR) transgene under the stringent transcriptional control of a human BESTROPHIN (VMD2) promoter to restore melanin synthesis. Rather than relying on a single experimental framework, the authors combine several: in vitro studies in human induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) monolayers from OCA1A patients with in vivo validation in albino rat eyes. This rigorous approach exemplifies the effective integration of diverse preclinical models, using them in a manner tailored to their specific purposes to capitalize on their unique biological advantages while addressing their inherent limitations (Table 1).

Table 1.

Fit-for-purpose application of complementary preclinical models in evaluating retinal gene therapy candidates

Preclinical model	Primary modeling purpose	Key biology captured	Limitations
Human patient iPSC-RPE monolayers (human NAM)	Mechanism-driven proof of concept and human fidelity	Captures patient-specific genetic backgrounds; demonstrates human TYR expression and melanosome formation; may comply with the FDA Plausible Mechanism Framework.	Fails to fully replicate mature tissue barriers; cannot model integrated multicellular pathology or systemic immunogenicity; developmental maturity and aging
Albino rat eye	Tissue barrier transport, multicellular pathology and safety profile	Preserves intact anatomical tissue layers and fluid dynamics; mimics disease progression; effectively uncovers macro-level toxicities (e.g., RPE-photoreceptor interactions), and local inflammatory reactions.	Cross-species differences in promoter activity and ocular scale; lack of patient-specific human genetic context; high transduction heterogeneity.

iPSC induced pluripotent stem cell derived; NAM, new approach methodology; TYR, TYROSINASE gene; FDA, Food and Drug Administration; RPE, retinal pigment epithelial cells.

New-approach methodologies (NAMs), including organ chips, microphysiological systems, and organoids, are increasingly being employed in translational medicine to accelerate preclinical studies.^3,4 The Food and Drug Administration’s recent draft guidance on the Plausible Mechanism Framework⁵ encourages sponsors to leverage established, biologically plausible mechanisms to accelerate the preclinical development of gene therapies for rare diseases with a known genetic cause. For rare conditions like OCA1A, where the complete loss of metabolic function stems directly from autosomal recessive mutations in the TYR gene, human NAMs provide a powerful means to rapidly demonstrate the therapeutic mechanism of action of a gene therapy. While advanced NAMs offer transformative potential to eventually reduce or replace conventional animal testing, their routine integration into commercial pharmaceutical pipelines requires addressing persistent validation hurdles.⁴ A current focus for translational scientists is systematically benchmarking these in vitro human systems against established in vivo animal models. This comparison aims to ensure that their predictive value remains valid for translational science, which inherently involves measuring safety and efficacy in complex, multi-scale biological systems.

The primary justification for maintaining a multi-model hybrid pipeline lies in the inherent structural shortfalls of current human NAMs. Despite their high genetic fidelity to human disease for some conditions, standard in vitro NAMs have yet to accurately model the complex, mature tissue barriers that dictate drug transport, clearance, and physiological resistance in an intact organ. Within the retina, these dynamic physiological barriers, including the outer blood-retinal barrier and the structural interactions between the inner retina, the RPE, and the underlying vascularized choroid, cannot be fully mimicked in simplified monolayer formats. Consequently, numerous mechanisms governing efficient delivery of genomic medicine payloads continue to be most effectively studied in live animals.^3,6 The rat persists as a powerful small animal model owing to its well-characterized ocular anatomy, predictable surgical handling, and established vascular physiology.

Furthermore, human NAMs often fail to accurately represent the complex processes involved in retinal development and the multicellular mechanisms underlying diseases. When an intervention induces an immunogenic response or localized cytotoxicity, the physiological cascade involves interactive loops between structural, vascular, and infiltrating immune cells that isolated cellular systems cannot fully capture. For the in vivo studies by George et al., although the AAV successfully induced pigmentation in the rat RPE, histopathological profiling was not intended to comprehensively evaluate vector biodistribution or systemic inflammatory markers following suprachoroidal injection. Future investigations using these animal models may identify systemic tissue susceptibilities and delivery risks that are not detectable through current human in vitro models. Additionally, the dosing and timing of intervention aimed at achieving functional rescue in various disease models could be explored in small animals, as was done in recent work using a similar AAV strategy driven by the RPE65 promoter.⁷ Furthermore, subsequent studies could elucidate how the suprachoroidal route of administration compares with intravitreal and subretinal injections, the latter of which are used to deliver Luxturna™ and other gene therapies.

Looking forward, it is highly probable that it will take another 5 to 10 years to establish more mature, complex human NAMs, such as vascularized, multi-lineage retinal organoids-on-a-chip,⁸ that effectively recreate systemic tissue barriers and integrated immune microenvironments. Achieving this will require prolonged, iterative validation and extensive benchmarking against definitive animal studies. In this interim period, the field of genomic medicine will increasingly rely on hybrid modeling strategies. Rather than viewing animal systems and NAMs as mutually exclusive, preclinical testing will focus on deploying them side-by-side in a complementary, fit-for-purpose manner.

To safely guide this transition, advanced multi-scale evaluation toolkits must be routinely applied to track therapeutic effects from the genome to the whole organ. Recent advances in imaging and genomic analyses have elucidated complex spatiotemporal effects of somatic cell genome editing interventions.³ In particular, combining human-specific genomic analyses and microphysiological monitoring in patient-derived NAMs proved to be fruitful, and subsequent work could be benchmarked via bridging studies to small and large animal studies. As such studies prove successful in advancing drug candidates toward the clinic, regulatory authorities will likely view validated NAMs as truly enabling Investigational New Drug (IND) applications (i.e., IND-enabling). This elegant study by George et al. highlights the near-term future of gene therapy development: an era in which mixing and matching complementary platforms allows researchers to demonstrate proof-of-principle in human cells and animals in parallel, ultimately moving genomic medicines to patients faster.

Acknowledgments

This work was supported in part by the National Institutes of Health (NIH) through U19NS132296 and R35 GM119644, as well as by support from the Retina Research Foundation Kathryn and Latimer Murfee Chair Professorship.

Declaration of interests

K.S. is on the advisory boards of Andson Biotech and Bharat Biotech.