Langmann, Thomas | Wolf, Anne - B 07

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.

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.

Aim 1: Analyze and understand retinal microglia responses after AAV2 transduction related to vector titers, capsid variants, promoters and transgenes.

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.

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.

Methods:

• same as for Aim1