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Background and Aims: Current liver-directed gene therapies look for adeno-associated virus (AAV) vectors with improved efficacy. With this background, capsid engineering is explored. Whereas shuffled capsid library screenings have resulted in potent liver targeting variants with one first vector in human clinical trials, modifying natural serotypes by peptide insertion has so far been less successful. Here, we now report on two capsid variants, MLIV.K and MLIV.A, both derived from a high-throughput in vivo AAV peptide display selection screen in mice., Approach and Results: The variants transduce primary murine and human hepatocytes at comparable efficiencies, a valuable feature in clinical development, and show significantly improved liver transduction efficacy, thereby allowing a dose reduction, and outperform parental AAV2 and AAV8 in targeting human hepatocytes in humanized mice. The natural heparan sulfate proteoglycan binding ability is markedly reduced, a feature that correlates with improved hepatocyte transduction. A further property that might contribute to the improved transduction efficacy is the lower capsid melting temperature. Peptide insertion also caused a moderate change in sensitivity to human sera containing anti-AAV2 neutralizing antibodies, revealing the impact of epitopes located at the basis of the AAV capsid protrusions., Conclusions: In conclusion, MLIV.K and MLIV.A are AAV peptide display variants selected in immunocompetent mice with improved hepatocyte tropism and transduction efficiency. Because these features are maintained across species, MLIV variants provide remarkable potential for translation of therapeutic approaches from mice to men., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of American Association for the Study of Liver Diseases.)

Endothelial cells (EC) are targets in gene therapy and regenerative medicine, but they are inefficiently transduced with adeno-associated virus (AAV) vectors of various serotypes. To identify barriers hampering efficient transduction and to develop an optimized AAV variant for EC transduction, we screened an AAV serotype 2-based peptide display library on primary human macrovascular EC. Using a new high-throughput selection and monitoring protocol, we identified a capsid variant, AAV-V EC , which outperformed the parental serotype as well as first-generation targeting vectors in EC transduction. AAV vector uptake was improved, resulting in significantly higher transgene expression levels from single-stranded vector genomes detectable within a few hours post-transduction. Notably, AAV-V EC transduced not only proliferating EC but also quiescent EC, although higher particle-per-cell ratios had to be applied. Also, induced pluripotent stem cell-derived endothelial progenitor cells, a novel tool in regenerative medicine and gene therapy, were highly susceptible toward AAV-V EC transduction. Thus, overcoming barriers by capsid engineering significantly expands the AAV tool kit for a wide range of applications targeting EC.

AAV vectors poorly transduce Dendritic cells (DC), a feature invoked to explain AAV's low immunogenicity. However, the reason for this non-permissiveness remained elusive. Here, we performed an in-depth analysis using human monocyte-derived immature DC (iDC) as model. iDC internalized AAV vectors of various serotypes, but even the most efficient serotype failed to transduce iDC above background. Since AAV vectors reached the cell nucleus, we hypothesized that AAV's intracellular processing occurs suboptimal. On this basis, we screened an AAV peptide display library for capsid variants more suitable for DC transduction and identified the I/VSS family which transduced DC with efficiencies of up to 38%. This property correlated with an improved vector uncoating. To determine the consequence of this novel feature for AAV's in vivo performance, we engineered one of the lead candidates to express a cytoplasmic form of ovalbumin, a highly immunogenic model antigen, and assayed transduction efficiency as well as immunogenicity. The capsid variant clearly outperformed the parental serotype in muscle transduction and in inducing antigen-specific humoral and T cell responses as well as anti-capsid CD8 + T cells. Hence, vector uncoating represents a major barrier hampering AAV vector-mediated transduction of DC and impacts on its use as vaccine platform.

Unlabelled: Adeno-associated virus (AAV) is a helper-dependent parvovirus that requires coinfection with adenovirus (AdV) or herpes simplex virus 1 (HSV-1) to replicate. In the absence of the helper virus, AAV can persist in an episomal or integrated form. Previous studies have analyzed the DNA damage response (DDR) induced upon AAV replication to understand how it controls AAV replication. In particular, it was shown that the Mre11-Rad50-Nbs1 (MRN) complex, a major player of the DDR induced by double-stranded DNA breaks and stalled replication forks, could negatively regulate AdV and AAV replication during coinfection. In contrast, MRN favors HSV-1 replication and is recruited to AAV replication compartments that are induced in the presence of HSV-1. In this study, we examined the role of MRN during AAV replication induced by HSV-1. Our results indicated that knockdown of MRN significantly reduced AAV DNA replication after coinfection with wild-type (wt) HSV-1 or HSV-1 with the polymerase deleted. This effect was specific to wt AAV, since it did not occur with recombinant AAV vectors. Positive regulation of AAV replication by MRN was dependent on its DNA tethering activity but did not require its nuclease activities. Importantly, knockdown of MRN also negatively regulated AAV integration within the human AAVS1 site, both in the presence and in the absence of HSV-1. Altogether, this work identifies a new function of MRN during integration of the AAV genome and demonstrates that this DNA repair complex positively regulates AAV replication in the presence of HSV-1., Importance: Viral DNA genomes trigger a DNA damage response (DDR), which can be either detrimental or beneficial for virus replication. Adeno-associated virus (AAV) is a defective parvovirus that requires the help of an unrelated virus such as adenovirus (AdV) or herpes simplex virus 1 (HSV-1) for productive replication. Previous studies have demonstrated that the cellular Mre11-Rad50-Nbs1 (MRN) complex, a sensor and regulator of the DDR, negatively regulates AAV replication during coinfection with AdV, which counteracts this effect by inactivating the complex. Here, we demonstrate that MRN positively regulates AAV replication during coinfection with HSV-1. Importantly, our study also indicates that MRN also favors integration of AAV genomes within the human AAVS1 site. Altogether, this work indicates that MRN differentially regulates AAV replication depending on the helper virus which is present and identifies a new function of this DNA repair complex during AAV integration., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Adeno-associated viral vectors (rAAV) are frequently used in gene therapy trials. Although rAAV vectors are of low immunogenicity, humoral as well as T cell responses may be induced. While the former limits vector reapplication, the expansion of cytotoxic T cells correlates with liver inflammation and loss of transduced hepatocytes. Because adaptive immune responses are a consequence of recognition by the innate immune system, we aimed to characterize cell autonomous immune responses elicited by rAAV in primary human hepatocytes and nonparenchymal liver cells. Surprisingly, Kupffer cells, but also liver sinusoidal endothelial cells, mounted responses to rAAV, whereas neither rAAV2 nor rAAV8 were recognized by hepatocytes. Viral capsids were sensed at the cell surface as pathogen-associated molecular patterns by Toll-like receptor 2. In contrast to the Toll-like receptor 9-mediated recognition observed in plasmacytoid dendritic cells, immune recognition of rAAV in primary human liver cells did not induce a type I interferon response, but up-regulated inflammatory cytokines through activation of nuclear factor κB., Conclusion: Using primary human liver cells, we identified a novel mechanism of rAAV recognition in the liver, demonstrating that alternative means of sensing rAAV particles have evolved. Minimizing this recognition will be key to improving rAAV-mediated gene transfer and reducing side effects in clinical trials due to immune responses against rAAV., (Copyright © 2011 American Association for the Study of Liver Diseases.)

In chronic renal disease, tubulointerstitial fibrosis is a leading cause of renal failure. Here, we made use of one of the most promising gene therapy vector platforms, the adeno-associated viral (AAV) vector system, and the COL4A3-deficient mice, a genetic mouse model of renal tubulointerstitial fibrosis, to develop a novel bidirectional treatment strategy to prevent renal fibrosis. By comparing different AAV serotypes in reporter studies, we identified AAV9 as the most suitable delivery vector to simultaneously target liver parenchyma for endocrine and renal tubular epithelium for paracrine therapeutic expression of the antifibrogenic cytokine human hepatocyte growth factor (hHGF). We used transcriptional targeting to drive hHGF expression from the newly developed CMV-enhancer-Ksp-cadherin-promoter (CMV-Ksp) in renal and hepatic tissue following tail vein injection of rAAV9-CMV-Ksp-hHGF into COL4A3-deficient mice. The therapeutic efficiency of our approach was demonstrated by a remarkable attenuation of tubulointerstitial fibrosis and repression of fibrotic markers such as collagen1alpha1 (Col1A1), platelet-derived growth factor receptor-beta (PDGFR-beta), and alpha-smooth muscle actin (SMA). Taken together, our results show the great potential of rAAV9 as an intravenously applicable vector for the combined paracrine and endocrine expression of antifibrogenic factors in the treatment of renal failure caused by tubulointerstitial fibrosis.

Background: Genetic modification of capsid proteins by peptide insertion has created the possibility of using adeno-associated viral (AAV) vectors for receptor specific gene transfer (AAV targeting). The most common site used for insertion in AAV serotype 2 capsids are amino acid positions 587 and 588 located at the second highest capsid protrusion. Reasoning that peptide insertions at the most exposed position augments target receptor interaction, we explored position 453 as a new insertion site., Methods: Position 453 was identified in silico. Capsid mutants carrying the model ligand RGD-4C in position 453 with and without R585A/R588A substitutions were compared with respective mutants carrying the ligand in position 587. The accessibility of the inserted ligand was determined by an enzyme-linked immunosorbent assay, whereas the transduction efficiency and specificity of receptor binding were assayed by gene transfer and competition experiments, respectively. Vector biodistribution was determined in mice by quantitative polymerase chain reaction analysis., Results: Initially, RGD-4C, inserted at position 453, failed to efficiently bind its target receptor. R585 and R588, located at the neighboring peak and known to mediate primary receptor binding, were identified as interfering residues. R585A and R588A substitutions rendered position 453 mutants superior to those with the ligand in position 587 in target receptor binding and cell transduction efficiency. The in vivo biodistribution was independent of the insertion site, but directed by the inserted ligand when primary receptor binding was avoided., Conclusions: Position 453 emerged as a prominent site for the development of targeting mutants. Furthermore, we show for the first time that linearly distant residues can be critical for the efficiency of inserted peptide ligands., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

Targeted delivery of biological agents to atherosclerotic plaques may provide a novel treatment and/or useful tool for imaging of atherosclerosis in vivo. However, there are no known viral vectors that possess the desired tropism. Two plaque-targeting peptides, CAPGPSKSC (CAP) and CNHRYMQMC (CNH) were inserted into the capsid of adeno-associated virus 2 (AAV2) to assess vector retargeting. AAV2-CNH produced significantly higher levels of transduction than unmodified AAV2 in human, murine and rat endothelial cells, whereas transduction of nontarget HeLa cells was unaltered. Transduction studies and surface plasmon resonance suggest that AAV2-CNH uses membrane type 1 matrix metalloproteinase as a surface receptor. AAV2-CAP only produced higher levels of transduction in rat endothelial cells, possibly because the virus was found to be affected by proteasomal degradation. In vivo substantially higher levels of both peptide-modified AAV2 vectors was detected in the brachiocephalic artery (site of advanced atherosclerotic plaques) and aorta, whereas reduced levels were detected in all other organs examined. These results suggest that in the AAV2 platform the peptides are exposed on the capsid surface in a way that enables efficient receptor binding and so creates effective atherosclerotic plaque targeted vectors.

Virus-mediated gene delivery is restricted by the infectivity profile of the chosen vector. Targeting the vascular endothelium via systemic delivery has been attempted using peptides isolated in vitro (using either phage or vector display) and implicit reliance on target receptor expression in vivo. This has limited application since endothelial cells in vitro and in vivo differ vastly in receptor profiles and because of the existence of complex endothelial "zip codes" in vivo. We therefore tested whether in vivo phage display combined with adeno-associated virus (AAV) capsid modifications would allow in vivo homing to the endothelium residing in defined organs. Extensive in vivo biopanning in rats identified four consensus peptides homing to the lung or brain. Each was incorporated into the VP3 region of the AAV-2 capsid to display the peptide at the virion surface. Peptides that conferred heparan independence were shown to retarget virus to the expected vascular bed in vivo in a preferential manner, determined 28 days post-systemic injection by both virion DNA and transgene expression profiling. Our findings significantly impact the design of viral vectors for targeting individual vascular beds in vivo.