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Selection of AAV capsid variants on iDC. (A) Quantification of NGS results. Variants that accumulated after two rounds of AAV2-based peptide library selection on iDC from different donors (Supplementary Fig. S3) were subjected to NGS using cap-specific primers to identify peptide sequences that mediated cell infection. Shown are sequences of peptide inserts present with amounts >1.5%. PTRLLP is excluded owning to its smaller size. (B) Phylogenic analysis of top AAV variants. Amino acid sequences of peptide inserts were aligned. A phylogenic tree was constructed using the maximum likelihood method. Selected variants were thereby classed into two main families. Numbers indicate bootstrap values.
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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 se...
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... entry was most efficient for AAV2 followed by AAV1 and AAV9. For the most efficient serotype, AAV2, we then determined the presence and quantity of AAV vectors in the cytosolic, membrane and nuclear fractions of iDC at 24 and 48 hrs p.i. (Supplementary Fig. S2). For both time points, highest vector genome numbers were measured in the mem- brane fraction, which also contains endosomes. ...
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... our two selection rounds: (i) usage of iDC from different donors, (ii) short incubation time, and (iii) recovery of viral genomes from the nuclear compartment (Supplementary Figs S3A,B). DNA isolated follow- ing the nuclear fraction was analyzed by next generation sequencing (NGS) to identify and quantify the variants that accumulated in iDC ( Fig. ...
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... analysis of the top variants, i.e. those that most strongly accumulated in the nuclear fraction of iDC, indicated that they could be classified into two families, "NNP" and "I/VSS", with NNPLPQR as the strong- est representative of the first, and VSSTSPR and ISSSTAR as lead candidates for the second (Fig. 2B). Of inter- est, although our library was pre-selected against HSPG-binding variants, all of the most frequently identified Figure 1. Early steps in iDC transduction by AAV vectors. (A) Entry efficiency of AAV vectors derived from different serotypes. iDC were incubated with AAV vectors encoding for enhanced green fluorescent protein ...
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... fractionation was performed using Subcellular Protein Fractionation Kit for Tissue (ThermoFisher Scientific). Purity of fractions on the protein level was confirmed by Western blot (Supplementary Fig. S2) using anti-Rab 5 (Santa Cruz sc 46692; 1:100), anti-Tubulin (SIGMA T5198; 1:5000), anti-Lamin B1 (Abcam antibody 16048; 1:5000), and anti-Calreticulin (Affinity BioReagents PA3-900, 1:100) antibodies, respectively. Factions were spiked with 1 ng of murine TOPO-GAPDH plasmid followed by DNA extraction (Blood & Tissue kit, Qiagen). ...
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Citations
... Several factors contribute to the limitations of AAV transduction efficiency. These include the natural tropism of AAV serotypes [54,55], which may not match the target cell type, as well as the presence of pre-existing neutralizing antibodies in the host that can inhibit vector entry [56][57][58]. Additionally, the relatively small packaging capacity of AAV vectors restricts the size of the transgene that can be delivered, limiting their utility for certain applications requiring larger genetic payloads. ...
Adeno-associated virus (AAV) has emerged as a pivotal tool in neuroscience research, owing to its remarkable versatility and efficiency in delivering genetic material to diverse cell types within the nervous system. This mini review aims to underscore the advanced applications of AAV vectors in neuroscience and their profound potential to revolutionize our understanding of brain function and therapeutic interventions for neurological disorders. By providing a concise overview of the latest developments and strategies employing AAV vectors, this review illuminates the transformative role of AAV technology in unraveling the complexities of neural circuits and paving the way for innovative treatments. Through elucidating the multifaceted capabilities of AAV-mediated gene delivery, this review underscores its pivotal role as a cornerstone in contemporary neuroscience research, promising remarkable insights into the intricacies of brain biology and offering new avenues for therapeutic intervention.
... These capsid proteins, required for rAAV internalization and intracellular processing, are essential for subsequent transgene expression. 64,65 These variable regions are the targets of rAAV capsid modifications in which directed evolution and rational design are the primary technological strategies used. 66,67 Briefly, directed evolution allows the creation of new proteins with improved functions through repeated cycles of mutation-selection from a functional capsid protein. ...
... They are derived from human blood monocytes cultured in the presence of granulocyte-macrophage colonystimulating factor (GM-CSF) and IL4, and are frequently used to study DC functions. 28,29 Incubation of moDCs with AAV2.MB453 or AAV2 vectors at a particle-per-cell ratio (GOI) of 2.5 Â 10 4 resulted in transgene expression from sc vector genomes; however, AAV2.MB453 outperformed AAV2 with regard to efficiency ( Figure 2A). Specifically, AAV2 led to an average of 18% transgene expressing cells (n = 5 donors), whereas AAV2.MB453 averaged 40% transgene positive cells. ...
... 30,31 To rule out that possible effects on gene expression are solely caused by the use of an AAV2 capsid modified by peptide insertion, we included the capsid variant AAV2.VSSTSPR as a further control. 28 This peptide insertion variant is derived from an AAV peptide display selection in moDCs and demonstrated significantly improved transduction efficiencies in moDCs as compared with AAV2, particularly in the presence of lipopolysaccharide, a strong inducer of DC maturation. 28 An up-or downregulation in gene expression by 2-fold was considered significant. ...
... 28 This peptide insertion variant is derived from an AAV peptide display selection in moDCs and demonstrated significantly improved transduction efficiencies in moDCs as compared with AAV2, particularly in the presence of lipopolysaccharide, a strong inducer of DC maturation. 28 An up-or downregulation in gene expression by 2-fold was considered significant. Compared with mock-treated moDCs, treatment with TLR2 agonists HKLM induced a strong upregulation of pro-inflammatory genes including IL1A, IL1B, IL6, IL8, and tumor necrosis factor (TNF) ( Figure S3B). ...
De novo immune responses are considered major challenges in gene therapy. With the aim to lower innate immune responses directly in cells targeted by adeno-associated virus (AAV) vectors, we equipped the vector capsid with a peptide known to interfere with Toll-like receptor signaling. Specifically, we genetically inserted in each of the 60 AAV2 capsid subunits the myeloid differentiation primary response 88 (MyD88)-derived peptide RDVLPGT, known to block MyD88 dimerization. Inserting the peptide neither interfered with capsid assembly nor with vector production yield. The novel capsid variant, AAV2.MB453, showed superior transduction efficiency compared to AAV2 in human monocyte-derived dendritic cells and in primary human hepatocyte cultures. In line with our hypothesis, AAV2.MB453 and AAV2 differed regarding innate immune response activation in primary human cells, particularly for type I interferons. Furthermore, mice treated with AAV2.MB453 showed significantly reduced CD8+ T cell responses against the transgene product for different administration routes and against the capsid following intramuscular administration. Moreover, humoral responses against the capsid were mitigated as indicated by delayed IgG2a antibody formation and an increased NAb50. To conclude, insertion of the MyD88-derived peptide into the AAV2 capsid improved early steps of host-vector interaction and reduced innate and adaptive immune responses.
... This remains controversial. Indeed, two previous studies showed an in vitro transduction of mouse DCs or human moDCs with poor efficiency compared to rAAV1 or rAAV2 [37,38]. Other studies have reported the ability of rAAV8 to transduce DCs in vitro [28] and in vivo, particularly spleen follicular DCs in mice and nonhuman primates [39,40]. ...
... Other studies have reported the ability of rAAV8 to transduce DCs in vitro [28] and in vivo, particularly spleen follicular DCs in mice and nonhuman primates [39,40]. In our study, the low detection of transgene-derived proteins and/or transcripts despite the high internalization rates of rAAV8-intact particles in moDCs suggests a postinternalization restriction mechanism as already described by Rossi et al. for the AAV2 serotype [38]. Aside from the hypothesis of an uncoating defect as discussed above, this restriction can be related to a failure in any other step of rAAV8 intracellular processing, such as trafficking impairment, defect in endosomal escape, partial nuclear import, accumulation of the vector in the nucleolus or a defect of the second DNA strand synthesis (reviewed by Berry and Asokan [41]). ...
Recombinant Adeno-Associated Virus (rAAV) is considered as one of the most successful and widely used viral vectors for in vivo gene therapy. However, host immune responses to the vector and/or the transgene product remain a major hurdle to successful AAV gene transfer. In contrast to antivector adaptive immunity, the initiation of the innate immunity towards rAAV is still poorly understood but is directly dependent on the interaction between the viral vector and innate immune cells. Here, we used a quantitative transcriptomic-based approach to determine the activation of inflammatory and anti-viral pathways after rAAV8-based infection of monocyte-derived dendritic cells (moDCs) obtained from 12 healthy human donors. We have shown that rAAV8 particles are efficiently internalized, but that this uptake does not induce any detectable transcriptomic change in moDCs in contrast to an adenoviral infection, which upregulates anti-viral pathways. These findings suggest an immunologically favorable profile for rAAV8 serotype with regard to in vitro activation of moDC model. Transcriptomic analysis of rAAV-infected innate immune cells is a powerful method to determine the ability of the viral vector to be seen by these sensor cells, which remains of great importance to better understand the immunogenicity of rAAV vectors and to design immune-stealth products.
... Effects on localization and transduction were followed by microscopy, and one mutant localized to the nucleus but did not induce the expression of their delivered gene of interest [84]. The transduction of monocyte-derived immature dendritic cells by AAV2 was observed with mAb20, Lamin B1 and DAPI staining [85]. ...
Research on adeno-associated virus (AAV) and its recombinant vectors as well as on fluorescence microscopy imaging is rapidly progressing driven by clinical applications and new technologies, respectively. The topics converge, since high and super-resolution microscopes facilitate the study of spatial and temporal aspects of cellular virus biology. Labeling methods also evolve and diversify. We review these interdisciplinary developments and provide information on the technologies used and the biological knowledge gained. The emphasis lies on the visualization of AAV proteins by chemical fluorophores, protein fusions and antibodies as well as on methods for the detection of adeno-associated viral DNA. We add a short overview of fluorescent microscope techniques and their advantages and challenges in detecting AAV.
... AAV vectors were produced and purified as described. 55,56 For AAV2::sOva and AAV2::eGFP (scGFP or ssGFP), the helper plasmid pRC alone was used 57 and combined with pCMV-sOva, pscGFP, 58 or pCMV-GFP, respectively, and pXX6. 55 AAV-Vac_Ova4 587 or AAV-Vac_Ova8 587 encoding for sc eGFP ( Figure S1; Table S1) were produced with unique capsids by using pRC-Ova_CD4 or pRC-Ova_CD8, respectively, combined with pscGFP and pXX6. ...
Immunotherapy has significantly improved treatment outcomes in various cancer entities. To enhance immunogenicity and efficacy, and to further broaden its applicability, co-administration of anti-tumor vaccines is considered as a promising strategy. Here, we introduce adeno-associated virus (AAV) vectors, widely used for in vivo gene therapy, as a potent cancer vaccine platform. Our AAV vector-based vaccine combines antigen display on the capsid surface with a vector-mediated antigen overexpression targeting different components of the immune system in a unique chronological order by a single intramuscular application. Thereby, both profound and long-lasting antigen-specific T and B cell immune responses were induced. Moreover, mice receiving the vaccine were protected against tumor growth, demonstrating its efficacy in two tumor models, including the low immunogenic and aggressive B16/F10-Ova melanoma model. Remarkably, this approach was even effective in conditions of a late tumor challenge, i.e., 80 days post-vaccination, between 88% (B16/F10-Ova melanoma) and 100% (EG7 thymoma) of mice remained tumor free. Thus, decorating AAV vector particles with antigens by capsid engineering represents a potent vaccine concept for applications in cancer immunotherapy. Its modular and versatile "plug-and-play" framework enables the use of tumor antigens of choice and the easy implementation of additional modifications to enhance immunogenicity further.
... 2,21 Additionally, the poor transduction of DCs by some AAVs might be partially due to inefficient vector uncoating, which limits their transduction capabilities. 36 In contrast, our vaccines, which were established based on an optimized AAV6 vector, provided CTL-based protection from tumor development in prevaccinated mice, both with model Ag OVA 19 and with TAA. Moreover, we observed significant expansion in the number of IFNg-producing CTLs after the restimulation of splenocytes with Ag for an additional 7 days before analysis (Figure 2). ...
We previously described therapeutic opportunities provided by capsid- and expression cassette-optimized adeno-associated virus serotype 6 (AAV6) vectors to suppress tumor growth in both solid and metastatic mouse models by using artificial ovalbumin (OVA) immunogen. In the current study, we further elucidated the mechanism of function of a novel AAV-based vaccine loaded with the melanoma tumor-associated antigens premelanosome protein gp100, tyrosinase (Tyr), tyrosinase-related protein 1 (TRP1), and dopachrome tautomerase (TRP2). We showed that the AAV6-based vaccine creates cellular and humoral antigen-specific responses, while antigen expression at the site of vaccine injection was temporal, and the clearance of antigen coincided with T cell infiltration. Our data revealed the superior protective immune response of optimized AAV6-TRP1 compared with other self-antigens in a disease-free mouse model. We further assessed the ability of AAV6-TRP1 to protect animals from metastatic spread in the lungs and to extend animal survival by inhibiting solid tumor growth. Flow cytometry-based analysis indicated significant infiltration of CD8+ T cells and natural killer (NK) cells in the tumor site, as well as changes in the polarization of intratumoral macrophages. Altogether, our data strongly support the use of optimized AAV vectors for cancer vaccine development.
... Once within the nucleus, AAV2 was shown to accumulate in the nucleoli in an infectious form [33]. The process of AAV uncoating is poorly understood and appears to be a limiting step in AAV transduction [34,35]. AAV may integrate into the host genome in the presence of Rep78, primarily at a locus on chromosome 19 designated AAVS1 [36,37]. ...
Discovered as a contaminant of adenovirus stocks in the 1960s, adeno-associated virus (AAV) is a mono-stranded DNA virus that depends on helper factors to replicate. Even though AAV is endemic in the human population (35–80%), it is remarkable that many issues concerning the natural infection by this virus remain unanswered. In this study, we reflect on the main basic aspects of AAV biology and provide an overview of the studies exploring the impact of AAV infection on human health, focusing on three major research areas including, (i) cervical and (ii) liver cancer, and (iii) reproductive system disorders. Conflicting results have been obtained into the association of AAV infection with the occurrence of adverse reproductive outcomes, such as placental complications, spontaneous abortion, and fertility disorders, or with a protective role in HPV-related cervical carcinogenesis. Noteworthy, recent reports have identified AAV insertional mutagenesis as a novel risk factor for the development of hepatocellular carcinoma. This latest finding raises concern regarding the widespread usage of AAV vectors in liver-targeted gene therapy.
... Moreover, also differential thermostability of natural AAV serotypes is well documented, 33,34 with two recent studies further indicating lower melting points for AAV2.GL, AAV2.NN, and other AAV2-based peptide insertion variants. 35,36 However, while peptide insertions per se as well as lower capsid thermostability can impact several aspects of AAV biology, including genome packaging, cellular binding/uptake, intracellular transport, and genome release, 35,37,38 it has (to our knowledge) not been investigated whether altered thermostability (with melting points >50°C) is predictive for a capsid's susceptibility to stressors and fragmentation at temperatures faced during vector production (£37°C). Additional experiments are therefore required to explain the occurrence and identity of the protein bands observed besides VP1-3 in Fig. 1b. ...
Adeno-associated viruses (AAV) represent highly attractive gene therapy vectors and potent research tools for the modulation of gene expression in animal models or difficult-to-transfect cell cultures. Engineered variants, comprising chimeric, mutated or peptide-inserted capsids, have strongly broadened the utility of AAVs by altering cellular tropism, enabling immune evasion, or increasing transduction efficiency. In this work, the performance of 50 of the most used, predominantly published, AAVs was compared on several primary cells, cell lines and iPSC-derived models from different organs, including adipose tissue, liver, lung, brain, and eyes. To identify the most efficient capsids for each cell type, self-complementary AAVs were standardized by digital PCR, arrayed on 96-well plates, and screened using high-content imaging. To enable best use of the data, all results are also provided in a web app. The utility of one selected AAV variant is further exemplified in a liver fibrosis assay based on primary hepatic stellate cells, where it successfully reversed an siRNA-induced phenotype. Most importantly, our comparative analysis revealed that a sub-selection of only five AAV variants (AAV2.NN, AAV9-SLRSPPS, AAV6.2, AAV6TM and AAV1P5) enabled efficient transduction of all tested cell types and markedly outperformed other well-established capsids, such as AAV2-7m8. These findings suggest that a core panel comprising these five capsid variants is a universally applicable and sufficient tool to identify potent AAVs for gene expression modulation in cellular systems.
... Components of the extracellular matrix, such as heparan sulfate proteoglycan (HSPG), mediate the initial cell contact, thereby supporting AAV binding to coreceptors [6] that induce clathrin-dependent endocytosis of the particles. Furthermore, AAV receptor (AAVR) and G protein-coupled receptor (GPR) 108 escort AAV's intracellular processing, [7,8] eventually leading to uncoating, i.e., the vector genome release from the viral capsid, [9,10] and formation of stable episomes. [11] The capsid has become the target of engineering, [4] aiming for improved transduction efficiencies and avoidance of vector loss in off-target tissues. ...
... We previously reported that capsid stability and uncoating efficiency correlates, [10] with the latter being a key determinant for transduction efficiency. We therefore performed a thermostability assay ( Figure S4). ...
... [14] Instead of combining natural AAV capsid sequence motifs or domains, AAV peptide display libraries explore the inherent features of a single AAV capsid backbone and genetically inserted random peptide sequences that affect capsid properties. These are exemplified by changes in thermal stability ( Figure S4), which correlate with uncoating efficiency, [10] a rate-limiting step in transduction, [9,10] and by changes in the host interaction both at the pre-and post-entry level (Figures S3 and 3). ...
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.
