Abstract
Purpose :
rAAV vectors are an important tool for mediating gene transfer to ocular tissues in therapeutic pre-clinical animal studies; however, the choice of an appropriate control for rAAV injection into the contralateral eye remains contentious with multiple possibilities existing including buffer only, reporter transgenes, scrambled expression cassettes, or empty capsids. The latter is conceptually attractive, where empty capsids should only effectively control for immunogenicity related to the injected materials but are challenging to isolate and purify reproducibly. Here we aim to generate a ‘minimal’ rAAV vector that packages the smallest possible non-coding sequence within a mature virion by progressively reducing the genome size between inverted terminal sequences (ITRs) and observing packaging efficiency and structural integrity, for use as a universal injection control.
Methods :
Plasmid constructs containing ITRs flanking a non-coding genome sequence ranging from 104-1070bp were cloned and packaged into unmodified rAAV2 serotype using a six-well, micro-scale virus production protocol (N=10 genome lengths; N=6 preps per length). Mean titer and SD were calculated for each construct length via picogreen assay. The presence and ratios of viral proteins 1-3 were confirmed via silver staining using denaturing SD-PAGE. Capsid maturity was evaluated using transmission electron microscopy.
Results :
All constructs were observed to package as assessed by picogreen assay with titers ranging from 7.14E122.58E12 vg/ml for the longest 1070bp construct, 2.91E12 9.84E11 vg/ml at the medium 442bp construct, and 6.0E115.12E11 vg/ml at 104bp, indicating reduced packaging efficacy nearing a minimal construct containing ITRs only. Assessment of virion structural integrity is ongoing.
Conclusions :
That our smallest-sized construct packaged less efficiently than our largest construct by approximately 2-log unit difference indicates that there may be a minimal size for genome incorporation and capsid maturation, which may impact the ability to generate a ‘minimal’ universal rAAV control. Upon delineation of the smallest possible ‘minimal’ virion, future steps will be to compare immunogenicity following intravitreal and subretinal injection versus established control approaches, such as scrambled transgene cassettes.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.