Novel 199 base pair NEFH promoter drives expression in retinal ganglion cells

Abstract

Retinal ganglion cells (RGCs) are known to be involved in several ocular disorders, including glaucoma and Leber hereditary optic neuropathy (LHON), and hence represent target cells for gene therapies directed towards these diseases. Restricting gene therapeutics to the target cell type in many situations may be preferable compared to ubiquitous transgene expression, stimulating researchers to identify RGC-specific promoters, particularly promoter sequences that may also be appropriate in size to fit readily into recombinant adeno associated viral (AAV) vectors, the vector of choice for many ocular gene therapies. In the current study we analysed EGFP expression driven by various sequences of the putative human NEFH promoter in order to define sequences required for preferential expression in RGCs. EGFP expression profiles from four different potential NEFH promoter constructs were compared in vivo in mice using retinal histology and mRNA expression analysis. Notably, two efficient promoter sequences, one comprising just 199 bp, are presented in the study.

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

“…We identified that AAV2 carrying CBA-and CMV promoters had ubiquitous expression in retinal cell types, moderate transgene expression and a relatively low expression efficacy compared to the other investigated promoters. The broad expression pattern by CMV in the retina has been observed before [13,26,27,32,88,89]. Furthermore, our finding that RGCs only make up 20-25% of the transduced cells is also consistent with a previous study that quantified the cellular tropism using this AAV serotype and promoter [32].…”

“…Neurofilament light polypeptide 2693 bp [25] Neurofilament heavy polypeptide 2251 and 2501 bp a [26,27] Doublecortin 2359 bp [29] Phosphodiesterase 6H 2005 bp [30] Purkinje cell protein 2 1652 bp [31] Gamma-synuclein promoter 1450 and 953 bp a [32,33] Interphotoreceptor-binding protein 1300 bp [34] Short promoter variant of glial fibrillary acidic protein 694 bp [136] Monocyte chemo attractant protein-1 560 bp [36] Short promoter variant of mouse cone arrestin 521 bp [37] Short promoter variant of human neurofilament heavy polypeptide 199 bp [27] bp base pair.…”

“…The promoter regulates the level of transgene expression and determines cellspecificity, as the activation state of the promoter is dependent on the transcription factor machinery present in the transduced cells. There has been great interest in identifying promoters for optimal transduction of different cell types within the retina [14,[24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43] and an ideal promoter would preferably be small, to make the incorporation of large and complex cargos possible.…”

AAV-PHP.eB transduces both the inner and outer retina with high efficacy in mice

“…Overall, the cell-specific tropisms of TV and IVT administration of AAV-PHP.eB-CMV-EGFP were similar ( Figures 1 and 2 ) to each other and to that of IVT AAV2/2-CMV-EGFP, a serotype commonly used for IVT delivery to RGCs. 18 , 37 , 38 , 39 , 58 , 59 …”

“…The evolution of AAV technology continues and is hallmarked by the frequent emergence of new and promising AAV serotypes. For example, systemic delivery of a gene-replacement therapy with AAV-PHP.B (the parental serotype of AAV-PHP.eB) provided significant benefit in an Ndufs4 knockout mouse model of Leigh syndrome, 59 where previous attempts using AAV1 63 and AAV2/9 64 serotypes resulted in less improvement. As such, the utility of AAV-PHP.B may represent a significant step toward the clinic for Leigh syndrome.…”