A novel AAV9 random peptide library to select for endothelial cell – directed gene transfer vectors

Endothelial cells play a central role in vascular diseases and represent therefore a clinically relevant cell type for gene therapeutic approaches. However, the endothelium is difficult to transduce with wtAAV vectors at feasible levels. The potential of random peptide libraries displayed on AAV2 to select for AAV2 vectors with improved efficiency of endothelial-directed gene transfer has been demonstrated. AAV9, however, may have advantages over AAV2 because of a lower prevalence of neutralizing antibodies in humans and more efficient gene transfer in vivo. The present study provides evidence that random peptide libraries can be displayed on AAV9 and can be utilized to select for AAV9 capsids redirected to human endothelium, as previously shown for AAV2. An AAV9 peptide display library which ensures that the displayed peptides correspond to the packaged genomes was generated. Four consecutive selection rounds on human coronary artery endothelial cells (HCAEC) performed in vitro yielded AAV9 library capsids with distinct peptides that strongly outperformed transduction of wild type AAV9. A central point of this study is posed by the finding that incorporation of sequences selected from AAV2 libraries into AAV9 capsids could not increase transduction as efficient as peptides selected in the AAV9 library context, justifying the generation and selection of an AAV9 library. Furthermore, AAV9 vectors with targeting sequences selected from AAV9 libraries revealed an increased transduction efficiency in presence of neutralising human intravenous immunoglobulins suggesting a reduced immunogenicity and a better suitability of AAV9 as a vector backbone. However, enriched peptides did not restrict AAV9 specificity towards HCAEC. The attempt to restrict transgene expression to this cell type by transcriptional targeting using a murine endothelium-specific promoter may be promising as an additional targeting level. To determine the potential of selected AAV on endothelial cells in the intact natural vascular context, murine mesenteric arteries and human umbilical veins were incubated in vivo or in situ, respectively, using the most efficient AAV9 vector. Analysis revealed a highly efficient transduction of human umbilical vein endothelial cells (HUVEC) by the vector mutant. Similar to the negligible transduction by wtAAV9 vectors, no transduction of murine mesenteric artery endothelial cell (MMAEC) could be detected. A closer comparative analysis of two selected vectors displaying peptides with divergent sequences revealed a HSPG-independent transduction of and the partial use of a common transduction. The results obtained in the present study permit the conclusion that the novel AAV9 peptide library is functional and can be used to select for vectors for future preclinical and clinical gene transfer applications

Peptide Ligands Incorporated into the Threefold Spike Capsid Domain to Re-Direct Gene Transduction of AAV8 and AAV9 In Vivo

Efficiency and specificity of viral vectors are vital issues in gene therapy. Insertion of peptide ligands into the adeno-associated viral (AAV) capsid at receptor binding sites can re-target AAV2-derived vectors to alternative cell types. Also, the use of serotypes AAV8 and -9 is more efficient than AAV2 for gene transfer to certain tissues in vivo. Consequently, re-targeting of these serotypes by ligand insertion could be a promising approach but has not been explored so far. Here, we generated AAV8 and -9 vectors displaying peptides in the threefold spike capsid domain. These peptides had been selected from peptide libraries displayed on capsids of AAV serotype 2 to optimize systemic gene delivery to murine lung tissue and to breast cancer tissue in PymT transgenic mice (PymT). Such peptide insertions at position 590 of the AAV8 capsid and position 589 of the AAV9 capsid changed the transduction properties of both serotypes. However, both peptides inserted in AAV8 did not result in the same changes of tissue tropism as they did in AAV2. While the AAV2 peptides selected on murine lung tissue did not alter tropism of serotypes 8 and -9, insertion of the AAV2-derived peptide selected on breast cancer tissue augmented tumor gene delivery in both serotypes. Further, this peptide mediated a strong but unspecific in vivo gene transfer for AAV8 and abrogated transduction of various control tissues for AAV9. Our findings indicate that peptide insertion into defined sites of AAV8 and -9 capsids can change and improve their efficiency and specificity compared to their wild type variants and to AAV2, making these insertion sites attractive for the generation of novel targeted vectors in these serotypes

Design of novel peptide insertion sites in capsid regions adjacent to the threefold-spike in serotypes AAV8 and AAV9.

<p><b>A:</b> Sequence alignment of surface exposed capsid domains encoded by the <i>cap</i> gene of the AAV serotypes 2, 8, and 9. Parts of the heparin binding domain 484-RQQR-487 and the integrin binding motif 511-NGR-513 are strongly conserved among the three serotypes. While AAV8 and -9 both contain an R at position 532, marked differences in the sequences are apparent at positions 585–588. Domains highlighted in B are tagged by colored rectangles. Alignment of the serotype sequences was carried out using BioEdit Sequence Alignment Editor Software. <b>B:</b> Capsid domains of AAV2 known to be involved in receptor binding are highlighted in blue (R484; R487), yellow (511-NGR-513), green (K532), and red (R585; R588). Surface rendering and mapping of the threefold-spike region was performed using PyMOL with the crystal structure of AAV2 supplied as template (PDB ID: 1lp3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023101#pone.0023101-Xie1" target="_blank">[50]</a>). <b>C:</b> Design of AAV8 and -9 constructs for the incorporation of oligonucleotides encoding targeting peptides into the <i>cap</i> gene. The amino acid sequence of the <i>cap</i> gene for each corresponding serotype (single letter amino acid code) is depicted in black letters; differences compared to the respective wild type sequence are shown in blue letters. Red indicates seven additional amino acid residues from the insertion of oligonucleotides encoding a known peptide ligand for the re-direction of AAV serotypes. <b>D:</b> Model of the VP-3 capsid protein of AAV2 and localization of R588 and <b>E:</b> model of the VP-3 protein of AAV8 capsid and localization of R590. The potential peptide insertion site is depicted in yellow. Basic amino acids are depicted in red, acidic amino acids in blue. The VP-3 model of AAV2 and AAV8 was generated using Cn3D with coordinates PDB ID: 1lp3 for AAV2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023101#pone.0023101-Xie1" target="_blank">[50]</a> and PDB ID: 2QA0 for AAV8 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023101#pone.0023101-Nam1" target="_blank">[37]</a> serving as template.</p

Bioluminescence imaging of gene transduction by vectors derived from AAV2, -8 and -9 displaying peptide ligands.

<p>In vivo bioluminescence imaging of transgene expression in FVB mice injected intravenously with rAAV-luciferase vectors harboring wild type capsid or capsids displaying a targeting peptide selected in the structural context of AAV2. Images were taken 28 days after vector injection, when whole animal bioluminescence intensities (BLI) reached peak values after injection of luciferase substrate. BLI ranges from 10⁵–10⁸ relative light units per animal (photons/sec/cm²).</p

Titers of recombinant AAV vectors.

<p>*vector genomes of viral stocks were determined by quantitative real-time PCR.</p

Biodistribution analysis of tropism-modified vectors.

<p>Vector genome copy numbers in various tissues were quantified by quantitative real-time PCR using CMV-promoter-specific primers. Tissues were harvested 28 days after intravenous injection of capsid-modified or wild type vectors, respectively. Recovered vector genome numbers are shown for <b>A</b> liver, <b>B</b> cardiac, and <b>C</b> lung tissue. (n = 3 animals per group).</p

In vivo bioluminescence of gene expression in breast cancer tissue of PymT-transgenic mice.

<p>Luciferase vectors derived from AAV2, -8 and -9 displaying breast cancer-directed peptides (ESGLSQS) and respective wild type capsid vectors were injected intravenously into tumor-bearing mice (n = 3 per group). Images were taken 14 days after vector injection. BLI ranged from 10⁵–10⁸ relative light units per animal (photons/sec/cm²).</p

Breast cancer-directed gene delivery by AAV capsids displaying ESGLSQS depends on the underlying capsid serotype.

<p><b>A:</b> Luciferase activities in PymT-induced breast cancer tissue were determined as relative light units (RLU) per mg protein 14 days after injection of AAV2, -8 and -9 displaying breast cancer-directed peptides (ESGLSQS) or respective wild type capsid variants (n = 3). <b>B and C:</b> the amount of vector genomes recovered in tumor, liver and cardiac tissue was analyzed by quantitative real-time PCR using the tissue samples as described in A (n = 3 animals per group). <b>B:</b> ratio of vector genome copy numbers tumor/liver (n = 3 animals per group); <b>C:</b> ratio of vector genome copy numbers tumor/heart. <b>D and E:</b> ratios of gene transduction using AAV vectors of serotypes 2, 8 and 9 with insertion of the ESGLSQS peptide and respective wild type variants. Values were calculated as <b>D:</b> tumor/liver expression ratios or <b>E:</b> tumor/heart expression ratios, respectively. <b>F, G and H:</b> Luciferase activities in various tissues were detected as relative light units (RLU) per mg protein for AAV-2 (<b>F</b>) for AAV-8 (<b>G</b>) and AAV-9 (<b>H</b>) displaying breast cancer-directed peptides (ESGLSQS) or respective AAV wild type capsid variants (n = 3 animals per group).</p

Ex vivo determination of transgene expression after systemic administration of AAV2, AAV8 and AAV9 vectors displaying peptide ligands.

<p>Luciferase gene expression in various tissues was analyzed 28 days after intravenous injection of rAAV vectors into FVB mice. Capsid insertion of the random VRRPRFW peptide did not result in transgene expression in any organ, while the lung-directed peptide PRSTSDP and the breast tissue-directed peptide ESGLSQS resulted in specific changes of tissue tropism. <b>A</b> shows results for AAV2-, <b>B</b> for AAV8-, and <b>C</b> for AAV9-injected animals. Values below 10³ RLU/mg protein are not shown because this indicates the threshold beyond which luciferase expression data could be reproducibly delineated from background signal. * = p<0.05; ** = p<0.01; *** = p<0.001 capsid displaying peptide versus respective wild type control (n = 3 animals).</p