Dual CRISPR/Cas9 exon deleting approach for the treatment of USH2A-associated disorders - PROJECT SUMMARY/ABSTRACT
Dual sensory loss is defined as a combination of age-related vision loss, e.g., age-related macular degenera-
tion (AMD) and age-related hearing loss (ARHL) that co-occurs in people age >65. AMD, dry and wet forms,
and ARHL are multifactorial diseases that share etiologies such as smoking and complement dysregulation.
Importantly, the amplification loop of the complement alternative pathway (AP) is crucial to control, as it is re-
sponsible for the majority of complement activation on cell surfaces and extracellular membranes. The AP is
inhibited by circulating complement protein factor H (fH). One of the mechanisms by which complement is acti-
vated is natural antibodies (nAbs) binding to neoepitopes, or damage-associated molecular patterns, on dam-
aged tissues in response to injury. The complement effector molecules and nAbs activate macrophages and
other immune cells, intensifying the inflammatory state that can lead to neurodegeneration. Two well-studied
fusion proteins, CR2-fH and B4-scFv-fH, will be used to target the inhibitory domain of fH to sites of tissue
damage. The complement receptor 2 (CR2) domain binds to complement fragment deposited sites, and the
single chain antibody B4 (B4-scFv) domain binds to modified annexin IV exposed on damaged tissues. Both
CR2-fH and B4-scFv-fH have been shown to be efficacious in mouse models of neurodegenerative diseases
when administered systemically, or for CR2-fH locally and via gene therapy. Of interest is that complement and
macrophage activation are elevated in the subretinal space of wet AMD mouse models and in cochlear tissue
of aged mice. In addition, smoke-induced proinflammatory, neurodegenerative AP activation, and abnormal
macrophage activity, have been seen the eye and cochlea. Based on these observations, we hypothesize that
vector driven CR2-fH and injected B4-scFv-fH localize fH to damaged nerve tissues and mitigate wet AMD and
ARHL pathology, in part by reducing macrophage activation, and thereby reducing the complement-
macrophage inflammatory feedback loop. I will test this hypothesis in two specific aims, the results of which will
transition to my postdoctoral research. Aim 1 will provide proof of concept that the C3 promoter is modulated in
a complement-dependent manner and that circulating B4-scFv-fH localizes to damaged cochlear tissue in
smoke-exposed ARHL mice. In Aim 2, I will test the hypothesis that local delivery of AAV5-pC3-CR2-fH and
cochlear targeting of B4-scFv-fH can mitigate AMD pathology and prevent cochlear damage, respectively, in
part by reducing macrophage activation. In Aim 3, as a postdoctoral trainee, I aspire to train in a lab with exper-
tise in the aged human visual and auditory systems to expand my translational research in sensory loss. Over-
all, my goal for this proposal is to demonstrate that AP inhibition in dual sensory loss can ameliorate pathology
in part by reducing macrophage activation and reducing the proinflammatory microenvironment. My long-term
goal is to expand my research and research training in translational sensory neuroscience.
