Retinal microglia and their immunological effects in AAV-based ocular gene therapy
Introduction
Gene therapies using adeno-associated viruses (AAVs) are among the most promising strategies to treat inherited retinal diseases. Although AAV vectors are considered to be safe, they are recognized as foreign invaders and elicit host-cell responses even in immune privileged areas such as the eye. A deep understanding of these immune processes in the retina is important for eliminating immune-related side effects and toxicity that negatively impact gene therapy. Microglia, the resident immune cells of the retina, are not only bystanders but can also trigger retinal degeneration and potentially serve as targets for therapy. Our preliminary data together with recent findings from collaborators indicate that microglia are early responders to locally injected AAVs. Beside the impact of virus titer on the immunogenicity of AAVs, capsid protein variants, driving promoters and the transgene product also seem to be decisive factors. Our hypothesis is that microglia acquire a cytotoxic phenotype after AAV transduction that results in chronic inflammation and reduced transgene expression. In this project, we will perform an in-depth analysis of retinal microglia at different stages after AAV transduction in response to different titers, capsid variants, ubiquitous and endogenous promoters as well as vectors containing different transgenes. We also aim to perform an experimental therapy in retinoschisin (Rs1h)-deficient mice under conditions of microglia modulation using minocycline. This study will not only help to decipher toxic mechanism and pinpoint triggers for harmful retinal immune responses but also help to elucidate whether immunomodulation is a beneficial approach for improving gene therapy efficacy and durability.
Figure 1
Figure 1: Intravitreal injections of recombinant adeno-associated viruses (rAAVs) trigger microglia activation, resulting in migration and accumulation of these cells in the inner retina, particularly in the ganglion cell layer (GCL). Immunogenic components of rAAV that could influence the microglial immune response are (1) AAV capsid variants, (2) vector DNA including the promoter and/or the transgene sequence and (3) the transgene protein. Identifying the factors that determine the immunogenicity of the rAAVs is crucial to achieve long-term expression of the therapeutic gene. BM, Bruch’s membrane; OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer and NFL, nerve fiber layer.
Clinical Relevance
Severe ocular inflammation occurring in viral gene therapy of the retina, triggered by innate and adaptive immune responses to AAVs and cargo transgenes may build substantial hurdles to the clinical development in progress for many visually impaired patients. The identification of immune sensing triggers and potential molecular targets for eliminating immune-related side effects that impact gene therapy will help to understand the nature of inflammation and to improve interventions during gene therapy to control or prevent inflammation in patients with inherited retinal degenerations.
Approach
Aim 1: Analyze and understand retinal microglia responses after AAV2 transduction related to vector titers, capsid variants, promoters and transgenes.
Figure 2: C57BL/6J mice will be intravitreally (ivt.) injected with rAAV2 that differ in their capsid (7m8 vs GL), promoter (CAG vs Rs1) and transgene (EGFP vs. Rs1) at three different vector titers. Early and late phase microglia responses will be analyzed.
Methods:
Optomotor response to assess visual acuity
OCT and FFA to determine retinal thickness, structure and vascular integrity
IHC of retinal cryosections and flat mounts to assess:
structural changes of the retina (anti-Cone arrestin, Rho and ZO-1) and cell death (TUNEL)
microglia reactivity and morphology (anti-Iba1)
transduction efficacy of Rs1 and EGFP (+ WB)
qPCR and ELISA to assess inflammatory marker expression
RNA-Seq of isolated retinal microglia and pathway analysis
Aim 2: Analysis of microglia reactivity and validation of AAV retinal gene therapy vectors on a human-based retina-on-a-chip as a new translational model.
Figure 3: Human-based retina-on-a-chip. Schematics showing the hiPSC-derived photoreceptor cells and hARPE19 cells in the top channel and the hiPSC-derived endothelial cells and human microglia in the bottom channel. The two-channel structure allows the cells to subject to different media and fluid shear stress caused by media flow. The two channels are separated by a porous membrane.
Methods:
Live-cell imaging and immunostainings to assess:
rAAV transduction efficacy
Microglia reactivity and morphology
Blood retinal barrier penetration and cell migration
qPCR and ELISA to analyze inflammation and pathways based on findings from Aim1
Aim 3: To perform an experimental AAV2 vector-based gene augmentation therapy in Rs1h-deficient mice under conditions of potent immunomodulation using minocycline.
Rs1h-/y mice and WT littermates will be intravitreally (ivt.) injected with rAAV2 identified in Aim1/2. Additionally, mice receive intraperitoneal injections of minocycline constantly during the gene therapy phase.
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Prof. Dr. Thomas Langmann
Clinic of General Ophthalmology | Lab. for Experimental Immunology of the Eye
Office Anja Volkmann Lab head Dr. Anne Wolf PostDocs Dr. Sarva Keihani Dr. Mona Tabel PhD students Urbanus Kinuthia Mandy Hector Nils Laudenberg Julia Hofmann Justus Dick Master student Anna Gröger Medical students Leo Bahlmann Melis Cansu Cömert Technician Eva Scheiffert
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