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

A brain microvasculature endothelial cell-specific viral vector with the potential to treat neurovascular and neurological diseases

Gene therapy critically relies on vectors that combine high transduction efficiency with a high degree of target specificity and that can be administered through a safe intravenous route. The lack of suitable vectors, especially for gene therapy of brain disorders, represents a major obstacle. Therefore, we applied an invivo screening system of random ligand libraries displayed on adeno-associated viral capsids to select brain-targeted vectors for the treatment of neurovascular diseases. We identified a capsid variant showing an unprecedented degree of specificity and long-lasting transduction efficiency for brain microvasculature endothelial cells as the primary target of selection. A therapeutic vector based on this selected viral capsid was used to markedly attenuate the severe cerebrovascular pathology of mice with incontinentia pigmenti after a single intravenous injection. Furthermore, the versatility of this selection system will make it possible to select ligands for additional invivo targets without requiring previous identification of potential target-specific receptors

Pulmonary targeting of Adeno-associated viral vectors by next-generation sequencing-guided screening of random capsid displayed peptide libraries

Vectors mediating strong, durable, and tissue-specific transgene expression are mandatory for safe and effective gene therapy. In settings requiring systemic vector administration, the availability of suited vectors is extremely limited. Here, we present a strategy to select vectors with true specificity for a target tissue from random peptide libraries displayed on adeno-associated virus (AAV) by screening the library under circulation conditions in a murine model. Guiding the in vivo screening by next-generation sequencing, we were able to monitor the selection kinetics and to determine the right time point to discontinue the screening process. The establishment of different rating scores enabled us to identify the most specifically enriched AAV capsid candidates. As proof of concept, a capsid variant was selected that specifically and very efficiently delivers genes to the endothelium of the pulmonary vasculature after intravenous administration. This technical approach of selecting target-specific vectors in vivo is applicable to any given tissue of interest and therefore has broad implications in translational research and medicine

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

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

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

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