Vridhachalam, N. | bioRxiv Science

bioRxiv2026

Adeno-Associated Virus (AAV) Synthetic Inverted Terminal Repeats Enhance Tissue-Specific Transduction and Alter the Vector Induced Stress Response

While adeno-associated virus (AAV) vectors have shown therapeutic benefit in clinical applications, noted challenges include low transduction efficiencies, poor cellular targeting, and vector related adverse events. Recently, it was demonstrated that a rationally designed synthetic inverted terminal repeat (SynITR) altered the AAV vector-induced DNA damage response and abrogated apoptosis in human embryonic stem cells. To explore the utility of AAV-SynITR for diverse gene therapy applications, vector production, transduction, and the cellular response were evaluated in various contexts. Regarding production, SynITR preparations exhibited comparable titers to wtITR in a serotype/transgene-independent manner. Despite slightly decreased transduction efficiency in various cell lines, intravenous administration of AAV8 vectors showed SynITR enhanced transduction in a tissue-specific manner in liver (>7-fold) and kidney and pancreas (>2-fold) at equivalent vector copy numbers; however, no differences were observed in muscle/heart/spleen tissues. Interestingly, persistent {gamma}H2AX, a marker of aging/chronic inflammation, was abundant in the liver and spleen following wtITR (but not SynITR) transduction. In human corneas, SynITR enhanced transduction up to 16-fold over wtITRs. These data demonstrate that SynITRs elicit tissue-specific transduction enhancement and alter the cellular stress response. Importantly, the SynITRs offer an alternative context to elucidate wtITR biology for targeted, enhanced, and potentially safer human gene therapy.

molecular biology↗

bioRxiv2024

Microglial depletion decreases Müller cell maturation and inner retinal vascular density

The neuroretinal vascular system is comprised of three interconnected layers. The initial superficial vascular plexus formation is guided by astrocytes around birth in mice. The formation of the deep and intermediate vascular plexuses occurs in the second postnatal week and is driven by Muller-cell-derived angiogenic signaling. Previously, we reported that microglia play an important role in regulating astrocyte density during superficial vascular plexus formation. Here, we investigated the role for microglia in regulating Muller-cell-dependent inner retinal vascular development. Results from this study show that microglia closely interact with Muller cells and the growing inner retinal vasculature. Depletion of microglia resulted in reduced inner retinal vascular layers densities and decreased Vegfa isoforms transcript levels. RNA-seq analysis further revealed that microglial depletion significantly reduced specific Muller cell maturation markers including glutamine synthetase, responsible for glutamine biosynthesis, which is needed for angiogenesis. Thus, our study reveals an important role for microglia in Muller cell maturation and inner retinal angiogenesis.

cell biology↗