Non-invasive Modulation of Microglia Gene Expression Using Peripherally Administered Antibody Conjugates

The ability to selectively modulate microglia activity by increasing or decreasing the expression of one or more target genes is critical for defining and targeting the molecular pathways that govern normal physiology and diverse disease phenotypes. Unfortunately, adeno-associated viruses (AAVs) and other viral vectors – the workhorses of many in vivo studies aimed at modulating gene expression – are limited by immunogenicity and toxicity, as well as their relatively low activity in microglia compared to other CNS cell types. In contrast, antisense oligonucleotides (ASOs) are attractive agents for this purpose, especially given their clear advantages relative to AAVs – namely reversibility, lack of toxicity and immunogenicity, and insensitivity to gene size. However, progress has been limited by the need for invasive routes of ASO administration, limited cellular uptake, and lack of cell-type specificity. A technology capable of efficiently delivering ASOs across the intact blood-brain barrier (BBB) and specifically into microglia following intravenous (IV) administration – resulting in selective modulation of microglia gene expression without associated toxicity – would be a significant breakthrough with wide-reaching implications. Therefore, we have developed bispecific antibodies (bAbs) that target either transferrin receptor 1 (TfR-1) or CD98hc – the heavy chain of the large neutral amino acid transporter (LAT1) – and mediate shuttling of IgGs across the BBB and into the brain parenchyma. We also find that pairing a CD11b- specific IgG with both shuttles results in them crossing the BBB and localizing to microglia in the mouse brain after IV administration. Moreover, we have demonstrated selective in vivo gene silencing in microglia using peripherally administered CD11b bAb-ASOs. Therefore, the first objective of this proposal is a proof-of-concept study aimed at testing the therapeutic efficacy of our first-generation, microglia-targeted bAbs conjugated to ASOs that decrease expression of the transcription factor PU.1. This gene (Spi1) is a master regulator of myeloid cells, and reduced expression is linked to delayed age onset of Alzheimer’s disease (AD) and microglial- mediated pro-inflammatory responses in rodents and humans. We will test the impact of lowering PU.1 on microglia activation and associated pathology using mouse models of AD (APP/PS1 and 5xFAD). The second objective is to develop second-generation bAbs targeting additional cell-surface proteins enriched on microglia. Therefore, in Aim 1, we will evaluate the efficacy of first-generation bAb-ASOs for reducing PU.1 expression and pro-inflammatory microglia phenotypes, and modulating associated pathology in two AD (APP/PS1 and 5xFAD) mouse models. Next, in Aim 2, we will evaluate the extent and cell-type specificity of silencing a ubiquitously expressed gene (Malat1) for second-generation bAb-ASOs that target different cell-surface proteins (e.g., TMEM119, P2RY12, and CX3CR1) enriched on microglia. A key expected outcome is the identification and characterization of bAb-ASOs that selectively modulate gene expression in microglia after peripheral administration, which has a staggering number of fundamental and therapeutic applications. Project Number: 1R01AG095417-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute on Aging (NIA) | Principal Investigator: Peter Tessier (+1 co-PI) | Institution: UNIVERSITY OF MICHIGAN AT ANN ARBOR, ANN ARBOR, MI | Award Amount: $655,198 | Activity Code: R01 | Study Section: Special Emphasis Panel[ZRG1 BBBT-X (81)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11204839