Engineered NDI1 Gene Construct for AAV

Codon optimised NDI1 sequences, designed to encode specific amino acid changes to reduce immunogenicity profiles and a minimal polyadenylation signal, were synthesised by GeneArt, Inc. (Invitrogen, Paisley, UK) and cloned into pAAV-MCS (Agilent Technologies, CA, USA) downstream of a CMV promoter. The lead construct, pAAV-ophNdi1, contained 329 synonymous codon modifications and an I82V amino acid substitution (patent no. 10220102). An additional construct, pAAV-ophNdi1-HA, was created by cloning ophNdi1 with a C-terminal HA tag (synthesized by GeneArt®, Thermo Fischer Scientific, MA, USA) into pAAV-MCS. To enhance AAV packaging efficiency, 4.4 kb of bacteriophage lambda DNA [46 (link)] was inserted into the plasmid backbone of pAAV-ophNdi1 and the antibiotic resistance gene substituted for kanamycin. Cloning was verified by DNA sequencing. pAAV-Ndi1 and pAAV-CAG-EGFP vectors were cloned as previously described in [39 (link),47 (link)], respectively.

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Chadderton N., Palfi A., Maloney D.M., Carrigan M., Finnegan L.K., Hanlon K.S., Shortall C., O’Reilly M., Humphries P., Cassidy L., Kenna P.F., Millington-Ward S, & Farrar G.J. (2023). Optimisation of AAV-NDI1 Significantly Enhances Its Therapeutic Value for Correcting Retinal Mitochondrial Dysfunction. Pharmaceutics, 15(2), 322.

Publication 2023

pAAV-MCS GeneArt

Amino acid Amino acid substitution Antibiotic resistance Backbone Bacteriophage lambda Codon Gene Immunogenicity Kanamycin Ndi1 Plasmid Polyadenylation Vectors

Corresponding Organization : Trinity College Dublin

Other organizations : Royal Victoria Eye and Ear Hospital

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Variable analysis

independent variables

Codon optimized NDI1 sequences
Amino acid changes to reduce immunogenicity profiles
Minimal polyadenylation signal

dependent variables

Not explicitly mentioned

control variables

CMV promoter
PAAV-MCS plasmid backbone
Kanamycin resistance gene

positive controls

PAAV-Ndi1 vector
PAAV-CAG-EGFP vector

negative controls

Not explicitly mentioned

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