rAAV Engineering for Capsid-Protein Enzyme Insertions and Mosaicism Reveals Resilience to Mutational, Structural and Thermal Perturbations

Abstract

Recombinant adeno-associated viruses (rAAV) provide outstanding options for customization and superior capabilities for gene therapy. To access their full potential, facile genetic manipulation is pivotal, including capsid loop modifications. Therefore, we assessed capsid tolerance to modifications of the structural VP proteins in terms of stability and plasticity. Flexible glycine-serine linkers of increasing sizes were, at the genetic level, introduced into the 587 loop region of the VP proteins of serotype 2, the best studied AAV representative. Analyses of biological function and thermal stability with respect to genome release of viral particles revealed structural plasticity. In addition, insertion of the 29 kDa enzyme β-lactamase into the loop region was tested with a complete or a mosaic modification setting. For the mosaic approach, investigation of VP2 trans expression revealed that a Kozak sequence was required to prevent leaky scanning. Surprisingly, even the full capsid modification with β-lactamase allowed for the assembly of capsids with a concomitant increase in size. Enzyme activity assays revealed lactamase functionality for both rAAV variants, which demonstrates the structural robustness of this platform technology.

Adeno-associated virus capsid protein expression in Escherichia coli and chemically defined capsid assembly

“…Our loop and C-terminal insertions did not interfere with in vitro assembly as detected by DLS and AFM and gave VLP yields comparable to that of wild type VP3. As the capsid is known to tolerate these modifications in vivo 39,40,53 , our result hints that the in-vitro assembly process in general reflects the cellular process. Since the loop-region is exposed on the capsid surface, this opens the possibility to produce VLPs with modified tropism.…”

Nanopore sequencing of native adeno-associated virus (AAV) single-stranded DNA using a transposase-based rapid protocol

“…Next, we were interested in the junction ITRs between two fused genomes and genome-backbone fusions, to eventually find hints on their origins. We previously described that in our pITR plasmid, just as in many other available ITR plasmids, the ITR adjacent to the transgene's polyadenylation signal harbours an 11 bp deletion within the B-C hairpins ( 6 ). In addition, the ITR complementary A-region is shorter in both ITRs compared to the ITR reference sequence.…”

“…Cells were co-transfected by plasmids pRepCap (lab code pZMB0504, encoding the replicases of AAV serotype 2 and the capsid of AAV serotype 9, Supplementary Figure S12 ), pHelper (Agilent Technologies, lab code pZMB0088, encoding AAV adenoviral helper functions, Supplementary Figure S12 ) and pITR (lab code pZMB0347, encoding fluorescence reporter mKate2 under control of a CMV promoter and human growth hormone polyadenylation signal, Supplementary Figure S14 ) with in total 37.5 μg DNA per dish in a molar plasmid ratio of 1:1:1. The pITR, the design of which has previously been published ( 6 ), provides the GOI to be packaged into viral capsids. Three days after transfection, cells were harvested by scraping, pelleted (5 min, 3000 rcf) and lysed by three freeze-thaw cycles (in 2.5 ml buffer per pellet from two dishes, 20 mM Tris, 150 mM NaCl, 10 mM MgCl 2, pH 7.5, range −80°C to 37°C).…”

“…Based on AAV biology, AAV vectors harbour an ssDNA, but this DNA can be designed to be self-complementary ( 4 ). For AAV applications with modified capsids such as tumor therapy ( 5 ), four-plasmid systems have been designed ( 6 ).…”

Effects of Thermally Induced Configuration Changes on rAAV Genome’s Enzymatic Accessibility

“…Inaccessibility of heat-treated virion undoubtedly is the main source of systemic error, yet previous studies did not consider this issue. For example, in a previous reported study, 44 , 45 heat-treated vectors are assumedly deemed 100% accessible by quantitative real-time PCR and used as the basis for the subsequent data analysis. Although this would not change the overall conclusion for these particular studies, it does indicate that the total amount of vector was underestimated.…”