Characterization of AAV vector particle stability at the single-capsid level

Abstract

Virus families have evolved different strategies for genome uncoating, which are also followed by recombinant vectors. Vectors derived from adeno-associated viruses (AAV) are considered as leading delivery tools for in vivo gene transfer, and in particular gene therapy. Using a combination of atomic force microscopy (AFM), biochemical experiments, and physical modeling, we investigated here the physical properties and stability of AAV vector particles. We first compared the morphological properties of AAV vectors derived from two different serotypes (AAV8 and AAV9). Furthermore, we triggered ssDNA uncoating by incubating vector particles to increasing controlled temperatures. Our analyses, performed at the single-particle level, indicate that genome release can occur in vitro via two alternative pathways: either the capsid remains intact and ejects linearly the ssDNA molecule, or the capsid is ruptured, leaving ssDNA in a compact entangled conformation. The analysis of the length distributions of ejected genomes further revealed a two-step ejection behavior. We propose a kinetic model aimed at quantitatively describing the evolution of capsids and genomes along the different pathways, as a function of time and temperature. This model allows quantifying the relative stability of AAV8 and AAV9 particles.

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

“…These results are consistent with the reported amount of linear ssDNA ejected from mostly intact capsids, as described by Bernaud et al. 21 On the other hand, the accessibility of vector DNA is not an issue since all vector genomes can be released by the treatment of a lysis buffer along with proteinase K digestion. This result also suggests that the majority of vector genomes are shielded by the capsid, even after heat denaturation as high as 95°C.…”

“…The thermal stability of AAV particles has been the subject of recently published studies, which is imperative to AAV genome stability both in vitro and in vivo . 19 , 20 , 21 , 22 Studies have shown that capsid stability is notably preserved in temperatures of 4°C–55°C and a pH ranging from 5.5 to 8.5. 23 This stability is maintained in the face of interactions with human or mice serum, as well as nonorganic materials, like glass, polystyrene, polyethylene, polypropylene, and stainless steel.…”

“… 24 Additionally, studies employing electron microscopy (EM), atomic force microscopy (AFM), and fluorometric assays have demonstrated that the biophysical and ultrastructural properties of the AAV capsid fluctuate with thermal changes in a given setting. 21 , 25 , 26 Other recent studies have indicated that AAV capsid stability may vary by serotype and the nature (single stranded or double stranded) and length of the vector DNA packaged inside. 19 , 21 , 25 However, it has also been suggested that AAV vector stability was governed by VP3 alone, primarily due to its ratio of basic/acidic amino acids, and was independent of VP1 and VP2 or the genome packaged.…”

“… 21 , 25 , 26 Other recent studies have indicated that AAV capsid stability may vary by serotype and the nature (single stranded or double stranded) and length of the vector DNA packaged inside. 19 , 21 , 25 However, it has also been suggested that AAV vector stability was governed by VP3 alone, primarily due to its ratio of basic/acidic amino acids, and was independent of VP1 and VP2 or the genome packaged. 20 This apparent discrepancy in the findings might originate from the variance in measurement methods utilized, and it remains unclear how these physical measures relate to the biological properties of the viral particles.…”

Transduction optimization of AAV vectors for human gene therapy of glaucoma and their reversed cell entry characteristics

“…Factors affecting transduction are continually being uncovered. A recent publication combining atomic force microscopy (AFM) with biochemical experiments showed that physical properties and stability of the ssAAV particles have an influence on genome ejection pathways which in turn, can also contribute to different serotypes' transduction efficiencies [64]. At the present time we do not know whether the recently discovered AAVR receptor [65] is present in the TM, or whether its presence would add to the efficiency of the current serotypes.…”