Targeting undruggable lung vascular enhancers with compact AAV-CAS12F crispr activation

The National Institute of Child Health and Human Development awarded Cincinnati Children's Hospital Medical Center $445,500 on September 1, 2026, under the Child Health and Human Development Extramural Research Projects program (CFDA 93.HDR) to develop and optimize a compact CRISPR activation system using the miniature Cas12f nuclease within an adeno-associated virus (AAV) vector for modulating lung vascular enhancers.

The project targets regulatory DNA elements that control cell- and tissue-specific gene expression but have been traditionally considered undruggable. The system packages both guide RNAs and nuclease within the approximately 4.7 kilobyte packaging capacity of AAV, avoiding the size constraints of commonly used Cas9 and Cas12a alternatives. The recipient will design and optimize the AAV-DenascaS12f-VPR system to modulate FOXF1 enhancer activity in vivo using FELS-GFP and FOXF1-2A-GFP reporter mouse lines to systematically evaluate how guide RNA placement and enhancer architecture influence activation efficiency and specificity. The recipient will then investigate therapeutic potential in the FOXF1-S52F alveolar capillary dysplasia heterozygous mouse model to determine whether concurrent activation of the FEL1 and FEL3 endothelial enhancers can restore functional FOXF1 transcription, enhance alveolar capillary density, and improve survival. The project establishes a compact, all-in-one CRISPR activation platform based on the miniature DenascaS12f nuclease as a broadly applicable tool for modulating regulatory DNA elements.

Work is performed in Cincinnati, Ohio. The period of performance runs from September 1, 2026, through August 31, 2028.

Abstract

Regulatory DNA elements control cell- and tissue- specific gene expression but have traditionally been considered undruggable due to absence of precise tools to modulate their activity. To address this challenge, we propose to develop a compact crispr-based modulation system using the miniature acidibacillus sulfuroxidans CAS12F (ASCAS12F) nuclease within an adeno-associated virus (AAV) vector. Unlike commonly used SPCAS9 and ASCAS12A, the small size of CAS12F enables both guide rnas and nuclease to fit within ~4.7 KB packaging capacity of AAV. To evaluate the therapeutic potential of the self- contained AAV modulation platform, we will investigate the enhancer-guided activation of FOXF1 in the established FOXF1 S52F mouse model, which recapitulates the key pathological manifestations of alveolar capillary dysplasia (ACD) and pulmonary arterial hypertension (PAH). In aim 1, we will design and optimize the AAV-DENASCAS12F-VPR system to modulate FOXF1 enhancer activity in vivo. Using the fels-GFP and FOXF1-2A-GFP reporter mouse lines, we will systematically evaluate how guide RNA placement and enhancer architecture influence activation efficiency and specificity. These studies will define generalizable rules for enhancer activation and validate a self-contained AAV CRISPRA platform. In aim 2, we will continue to investigate the therapeutic potential of this system in the FOXF1-S52F ACD heterozygous mouse model and determine whether concurrent activation of the two key endothelial enhancers, FEL1 and FEL3, can restore transcription from the functional FOXF1 allele, enhance alveolar capillary density, and improve survival. Overall, this project will establish a compact, all-in-one crispr activation (CRISPRA) system based on the miniature DENASCAS12F nuclease to selectively activate FOXF1 enhancers and position the novel system as a broadly applicable tool for modulating regulatory DNA elements.