Improving adeno-associated viral (AAV) vector-mediated transgene expression in retinal ganglion cells: comparison of five promoters

Abstract

Recombinant adeno-associated viral vectors (AAVs) are an effective system for gene transfer. AAV serotype 2 (AAV2) is commonly used to deliver transgenes to retinal ganglion cells (RGCs) via intravitreal injection. The AAV serotype however is not the only factor contributing to the effectiveness of gene therapies. Promoters influence the strength and cell-selectivity of transgene expression. This study compares five promoters designed to maximise AAV2 cargo space for gene delivery: chicken β-actin (CBA), cytomegalovirus (CMV), short CMV early enhancer/chicken β-actin/short β-globulin intron (sCAG), mouse phosphoglycerate kinase (PGK), and human synapsin (SYN). The promoters driving enhanced green fluorescent protein (eGFP) were examined in adult C57BL/6J mice eyes and tissues of the visual system. eGFP expression was strongest in the retina, optic nerves and brain when driven by the sCAG and SYN promoters. CBA, CMV, and PGK had moderate expression by comparison. The SYN promoter had almost exclusive transgene expression in RGCs. The PGK promoter had predominant expression in both RGCs and AII amacrine cells. The ubiquitous CBA, CMV, and sCAG promoters expressed eGFP in a variety of cell types across multiple retinal layers including Müller glia and astrocytes. We also found that these promoters could transduce human retina ex vivo, although expression was predominantly in glial cells due to low RGC viability. Taken together, this promoter comparison study contributes to optimising AAV-mediated transduction in the retina, and could be valuable for research in ocular disorders, particularly those with large or complex genetic cargos.

Novel chemical tyrosine functionalization of adeno-associated virus improves gene transfer efficiency in liver and retina

“…This suggests either greater AAV2-Man(Y) uptake specifically in ganglion cells or better penetration of the vector through the tissue layers as a consequence of the mannose modification. This increased transduction efficiency in the GCL suggests that these mannosemodified vectors could be coupled with ganglion cell-specific promoters for gene therapy strategies targeting glaucoma or optic neuropathies [33,34] Taken together, these data show that the AAV2-Man(Y) capsid results in stronger and more widespread retinal transduction than the unmodified AAV2 capsid.…”

Reduction of retinal ganglion cell death in mouse models of familial dysautonomia using AAV-mediated gene therapy and splicing modulators

“…Using two complementary FD mouse models, mice were treated either with (1) an intravitreal injection of an AAV2 (highly tropic for RGCs) engineered to express a wild type copy of ELP1 [18][19][20] (Figure 1A) or (2) a novel small molecule splicing modulator to restore correct ELP1 splicing [16] (Figure 1B).…”

Intravitreal Administration of AAV2-SIRT1 Reverses Diabetic Retinopathy in a Mouse Model of Type 2 Diabetes

“…The AAV2-CBA-hGFP (UF11) is a standard control. This application has been previously used in Yin et al 18 and Nieuwenhuis et al 19 After 3 months, mice were euthanized, and eyes enucleated for analysis.…”