Novel random peptide libraries displayed on AAV serotype 9 for selection of endothelial cell-directed gene transfer vectors

Abstract

We have demonstrated the potential of random peptide libraries displayed on adeno-associated virus (AAV)2 to select for AAV2 vectors with improved efficiency for cell type-directed gene transfer. AAV9, however, may have advantages over AAV2 because of a lower prevalence of neutralizing antibodies in humans and more efficient gene transfer in vivo. Here we provide evidence that random peptide libraries can be displayed on AAV9 and can be utilized to select for AAV9 capsids redirected to the cell type of interest. We generated an AAV9 peptide display library, which ensures that the displayed peptides correspond to the packaged genomes and performed four consecutive selection rounds on human coronary artery endothelial cells in vitro. This screening yielded AAV9 library capsids with distinct peptide motifs enabling up to 40-fold improved transduction efficiencies compared with wild-type (wt) AAV9 vectors. Incorporating sequences selected from AAV9 libraries into AAV2 capsids could not increase transduction as efficiently as in the AAV9 context. To analyze the potential on endothelial cells in the intact natural vascular context, human umbilical veins were incubated with the selected AAV in situ and endothelial cells were isolated. Fluorescence-activated cell sorting analysis revealed a 200-fold improved transduction efficiency compared with wt AAV9 vectors. Furthermore, AAV9 vectors with targeting sequences selected from AAV9 libraries revealed an increased transduction efficiency in the presence of human intravenous immunoglobulins, suggesting a reduced immunogenicity. We conclude that our novel AAV9 peptide library is functional and can be used to select for vectors for future preclinical and clinical gene transfer applications.

Directed evolution of novel adeno-associated viruses for therapeutic gene delivery

“…Furthermore, the anti-AAV9 antibody ADK9 did not neutralize the variants. 31 These results indicate that it may be possible to simultaneously select for two independent properties: antibody resistance and maintenance of existing or engineering of novel tropism.…”

“…In addition, a chimeric variant isolated by Yang et al 29 from an in vivo selection to identify a muscle-targeting variant (described in more detail below) exhibited a similar level of in vitro resistance to intravenous immunoglobulin as compared to AAV8, and a higher level of resistance compared to AAV2. Finally, Varadi et al 31 demonstrated that random peptide insertions can alter AAV immunoreactivity, as AAV9-SLRSPPS and AAV9-RDVRAVS vectors exhibited enhanced in vitro transduction in the presence of intravenous immunoglobulin compared with wild-type AAV2 and AAV9. Furthermore, the anti-AAV9 antibody ADK9 did not neutralize the variants.…”

“…38 Recent selection of an AAV9 random peptide insertion library for the capacity to transduce human coronary artery endothelial cells yielded a variant with a 200-fold improved infection efficiency compared with AAV9 on postnatal human umbilical vein endothelial cells. 31 In addition to AAV peptide display, other forms of mutagenesis can alter receptor binding. As a proof of concept, Maheshri et al 23 showed that directed evolution can be applied to modulate the affinity of AAV for its primary receptor.…”

“…30 This technique has recently been extended to AAV9. 31 Defined peptideencoding sequences can also be inserted into random locations of the AAV2 cap ORF via transposon mutagenesis. 32 This approach was used to incorporate hexahistadine tags randomly throughout the AAV2 capsid to explore AAV clones capable of immobilized metal affinity chromatography purification.…”

Vascular-targeted recombinant adeno-associated viral vectors for the treatment of rare diseases

“…by error-prone PCR, or they comprise pools of random peptides that are displayed on the AAV capsid in certain relevant positions. [15][16][17][18][19] In theory, such libraries allow to select for particles with the ability to target any unique structure on the cell surface. 15,[20][21][22] The screening of random AAV display peptide libraries is the only approach to select peptides directly within the structural constraints of the assembled AAV capsid.…”