Design and initial characterization of a novel mini-promoter for gene therapies targeting the central nervous system.

Choosing an optimal combination of adeno-associated virus (AAV) capsid and promoter is key to achieving precise, efficient, and safe gene delivery. However, the limited packaging capacity of AAV can make incorporating large transgenes with strong promoters challenging. To address this, we screened several promoter designs derived from portions of the 5' untranslated regions (UTRs) of the human ERCC6 and ERCC8 DNA repair genes to generate a novel 126-base pair (bp) mini-promoter (CP040). We evaluated the expression capabilities of CP040 and its components in vitro and demonstrated that intravenous (i.v.) administration of AAVDJ vector harboring enhanced green fluorescent protein (eGFP) driven by the CP040 promoter (AAVDJ-CP040-eGFP) in neonatal mice resulted in strong central nervous system (CNS) expression 6 weeks later. CP040 expression was compared with the chicken beta actin (CBA), human elongation factor 1 alpha (EF1α), and human synapsin 1 (hSYN) promoters following intracerebroventricular (ICV) and intrathecal (IT) AAVDJ administrations. The CP040 mini-promoter yielded comparable expression levels to CBA and hSYN promoters and higher levels than EF1α, with lower off-target expression in liver and kidneys. These initial characterizations support CP040 as a promising candidate to enhance AAV-mediated gene therapy targeting the CNS when cargo sequences cannot fit within AAV size constraints.