Throughout the years, several hundred million people with rare genetic disorders have been receiving only symptom management therapy. However, research and development efforts worldwide have led to the development of long-lasting, highly efficient, and safe gene therapy for a wide range of hereditary diseases. Improved viral vectors are now able to evade the preexisting immunity and more efficiently target and transduce therapeutically relevant cells, ensuring genome maintenance and expression of transgenes at the relevant levels. Hematological, ophthalmological, neurodegenerative, and metabolic therapeutic areas have witnessed successful treatment of hemophilia and muscular dystrophy, restoration of immune system in children with immunodeficiencies, and restoration of vision. This review focuses on three leading vector platforms of the past two decades: adeno-associated viruses (AAVs), adenoviruses (AdVs), and lentiviruses (LVs). Special attention is given to successful preclinical and clinical studies that have led to the approval of gene therapies: six AAV-based (Glybera® for lipoprotein lipase deficiency, Luxturna® for retinal dystrophy, Zolgensma® for spinal muscular atrophy, Upstaza® for AADC, Roctavian® for hemophilia A, and Hemgenix® for hemophilia B) and three LV-based (Libmeldy® for infantile metachromatic leukodystrophy, Zynteglo® for β-thalassemia, and Skysona® for ALD). The review also discusses the problems that arise in the development of gene therapy treatments, which, nevertheless, do not overshadow the successes of already developed gene therapies and the hope these treatments give to long-suffering patients and their families.

INTRODUCTION. The development of adeno-associated virus (AAV)-based gene therapy products in Russia requires establishing reference standards, which are used throughout the pharmaceutical development cycle, and monitoring their stability during the storage period. A preparation of empty capsids of AAV serotype 9 (AAV9) is an appropriate material for a reference standard for empty AAV9 capsids (AAV9 RS).AIM. This study aimed to develop analytical procedures to evaluate the AAV9 RS physicochemical quality parameters for its characterisation and to study its storage stability.MATERIALS AND METHODS. Empty AAV9 capsids were produced in HEK293 suspension culture using serum-free medium and optimised transfection parameters. The next steps involved AAV9 clarification, concentration, and purification by affinity chromatography with AAVx resin and diafiltration. The analysis of AAV9 samples used electrophoresis, transmission electron microscopy, enzyme-linked immunosorbent assay (ELISA), size-exclusion chromatography, dynamic light scattering, spectrophotometry, and bio-layer interferometry. The concentration of capsids was measured by ELISA. Analytical procedures for physical titre determination were developed using an AAV9 standard with a known physical titre. The stability study of the AAV9 RS involved storage at –80 °C for 9 months.RESULTS. Size-exclusion chromatography demonstrated the high purity of the established AAV9 RS, with at least 98% content of the viral capsid monomer. Dynamic light scattering, size-exclusion chromatography, and electron microscopy confirmed that the AAV9 RS was free of aggregates. The stability study showed that the AAV9 RS remained stable for 9 months. Dynamic light scattering and spectrophotometry were deemed optimal methods for routine quality analysis measuring the AAV9 RS physical titre, and bio-layer interferometry was recommended for regular analysis. The viral particle titres determined by these methods ranged from 1.48×10¹³ to 5.6×10¹³.CONCLUSIONS. The AAV9 RS established in this study is suitable for quality control of AAV9-based gene therapy products.

Inherited retinal disorders (IRD) have become a primary focus of gene therapy research since the success of adeno-associated virus-based therapeutics (voretigene neparvovec-rzyl) for Leber congenital amaurosis type 2 (LCA2). Dozens of monogenic IRDs could be potentially treated with a similar approach using an adeno-associated virus (AAV) to transfer a functional gene into the retina. Here, we present the results of the design, production, and in vitro testing of the AAV serotype 9 (AAV9) vector carrying the codon-optimized (co) copy of aryl hydrocarbon receptor-interacting protein like-1 (AIPL1) as a possible treatment for LCA4. The pAAV-AIPL1co was able to successfully transduce retinal pigment epithelium cells (ARPE-19) and initiate the expression of human AIPL1. Intriguingly, cells transduced with AAV9-AIPL1co showed much less antiviral response than AAV9-AIPL1wt (wild-type AIPL1) transduced. RNA-sequencing (RNA-seq) analysis of trans-differentiated ARPE-19 cells transduced with AAV9-AIPL1co demonstrated significant differences in the expression of genes involved in the innate immune response. In contrast, AAV9-AIPL1wt induced the prominent activation of multiple interferon-stimulated genes. The key part of the possible regulatory molecular mechanism is the activation of dsRNA-responsive antiviral oligoadenylate synthetases, and a significant increase in the level of histone coding genes’ transcripts overrepresented in RNA-seq data (i.e., H1, H2A, H2B, H3, and H4). The RNA-seq data suggests that AAV9-AIPL1co exhibiting less immunogenicity than AAV9-AIPL1wt can be used for potency testing, using relevant animal models to develop future therapeutics for LCA4.

Leber congenital amaurosis (LCA) is a severe inherited retinal disorder manifesting at birth or in early infancy, with a subset of cases linked to mutations in the aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) gene. Initially identified as the disease locus for LCA4, AIPL1 exhibits a retina-specific expression pattern. Its protein product is a unique member of the FKBP family, distinguished by its specific structural features and specialized functions. A wide spectrum of mutations in AIPL1 is associated with varying severities of retinal degeneration, implicating diverse pathogenic mechanisms. While the early onset and rapid progression of AIPL1-related disorders pose a therapeutic challenge, significant progress in gene therapy has unlocked promising avenues for effective treatment. This comprehensive review summarizes current findings to spark interest and pave the way for further studies in the therapy of AIPL1-caused retinal diseases.

Gene replacement therapy can be used for the treatment of hereditary retinopathies, such as retinol dehydrogenase 12 (RDH12)-associated Leber congenital amaurosis 13 (LCA13); however, the lack of animal models accurately mimicking the human disease phenotype requires the initial in vitro confirmation of therapy efficacy. Two synthetic serotypes (2.7m8 and PHP.S) of adeno-associated virus (AAV) were tested against the natural serotypes (5 and 9) with the aim of increasing the transduction efficiency and delivery of the green fluorescent protein (GFP) in HEK293 and ARPE-19 cells. The three most efficient serotypes were then used for the delivery of RDH12, followed by the assessment of its functional activity in the transduced cells. In the in vitro test system, a cassette encoding GFP and the wild-type (wt) RDH12 was delivered into ARPE-19 and HEK293 cells by rAAV 5, PHP.S, and 7m8 at 30K and 60K VG/cell. RDH12 mutants pThr155Ile (RDH12mut) and Met1* (RDH12sc) were used to mimic the RDH12-associated pathology. Transduction efficiency and protein expression were assessed by flow cytometry, fluorescence microscopy, and Western blotting. Percentages of AAV7m8-transduced GFP+ cells 1.5- and 6.4-times higher were observed as compared to AAV5 and AAV.PHP.S, respectively. 4-hydroxynonenal (4-HNE), more toxic to the cells with dysfunctional RDH12, was used on cells expressing the three RDH12wt versions. Following treatment with 100 μM 4-HNE, 2.6 (AAV5) and 8.8 (AAV7m8) times more cells co-expressing RDH12wt and GFP were alive as compared to the cells expressing only GFP. The number of live RDH12wt-expressing cells was also 32 and 9.6 times higher than that of RDH12sc-expressing cells and the negative control (NC), respectively. The developed approach enables the functional assessment of RDH12 replacement therapy only in rAAV-transduced cells and demonstrates that rAAV7m8 is the most efficient serotype for this purpose.

Chronic inflammation and subfertility converge on a number of ligand-dependent transcription factors induced by chemically heterogeneous environmental exposures. Foremost among them is the aryl hydrocarbon receptor (AhR), of the basic helix-loop-helix (bHLH)–PER-ARNT-SIM (PAS) superfamily. Identified first as the receptor for dioxins and long regarded as a mediator of xenobiotic toxicity, AhR is now established as a regulator of physiological homeostasis, responding to microbial, dietary, phytochemical substances and endogenously generated metabolites. In this review, we examine AhR ligands and the transcriptional programmes they elicit, organising the evidence around one distinction: whether a ligand is a substrate for the cytochrome P450 enzymes it induces, and therefore self-limiting, or whether it persists and converts a single exposure into a chronic stimulus. We follow the receptor through the gastrointestinal tract, skin and adipose tissue; through the eye, where biallelic loss-of-function genetic variants cause foveal hypoplasia and retinitis pigmentosa and through the reproductive axis, where AhR sets ovarian aromatase expression, follicular recruitment, spermatogenesis and trophoblast invasion. Spanning the good, the bad, the ugly and the blind, we argue that therapeutic progress depends not on deciding whether to activate or inhibit AhR, but on confining modulation to a defined tissue, ligand residence time and transcriptional output.