Preexisting immunity against adeno-associated virus (AAV) is a major challenge facing AAV gene therapy, resulting in the exclusion of patients from clinical trials. Accordingly, proper assessment of anti-AAV immunity is necessary for understanding clinical data and for product development. Previous studies on anti-AAV prevalence lack method standardization, rendering the assessment of prevalence difficult. Addressing this need, we used clinical assays that were validated according to guidelines for a comprehensive characterization of anti-AAV1,-AAV2,-AAV5, and-AAV8 immunity in large international cohorts of healthy donors and patients with hemophilia B. Here, we report a higher than expected average prevalence for anti-AAV8 ($40%) and anti-AAV5 ($30%) neutralizing antibodies (NAbs), which is supported by strongly correlating anti-AAV IgG antibody titers. A similar anti-AAV8 NAb prevalence was observed in hemophilia B patients. In addition, a high co-prevalence of NAbs against other serotypes makes switching to gene therapy using another serotype difficult. As anti-AAV T cell responses are believed to influence transduction, we characterized anti-AAV T cell responses using interleukin-2 (IL-2) and interferon-g (IFN-g) ELISpot assays, revealing a similar prevalence of IFN-g responses ($20%) against different serotypes that did not correlate with NAbs. These data, along with the long-term stability of NAbs, emphasize the need to develop strategies to circumvent anti-AAV immunity.
Patients with preexisting anti-adeno-associated virus serotype 8 (AAV8) neutralizing antibodies (NAbs) are currently excluded from AAV8 gene therapy trials. Therefore, the assessment of biologically relevant AAV8-NAb titers is critical for product development in gene therapy. However, standardized assays have not been routinely used to determine anti-AAV8-NAb titers, contributing to a wide range of reported anti-AAV8 prevalence rates. Using a clinical in vitro NAb assay in a separate study, a higher than expected anti-AAV8-NAb prevalence of about 50% was found in international cohorts. This comparative study has a translational character, confirming the biological relevance of anti-AAV8-antibody titers measured by this assay. The significance of low-titer anti-AAV8 NAbs is shown, along with the relevance of the in vitro assay cutoff (1:5) compared with other assays. Importantly, internally standardized reagents and purified AAV8 constructs containing 90% full capsids were used to reduce the effect of empty capsids. It was found that even very low anti-AAV8-NAb titers (<1:5) could efficiently hinder transduction in vivo, demonstrating the importance of sensitive NAb assays for clinical applications. The in vitro NAb assay was found to be more sensitive than an in vivo NAb assay and thus more suitable for patient screening. Additionally, the study showed that anti-AAV8-NAb titers <1:5 were very rare, further supporting the in vitro assay. However, assays using a lower cutoff may still be useful to explain potential variances in transgene expression. These findings support the relevance of the higher than expected prevalence of anti-AAV8 NAbs, highlighting the need for strategies to circumvent preexisting anti-AAV8 NAbs.
BackgroundThe application of antisense molecules, such as morpholino oligonucleotides, is an efficient method of gene inactivation in vivo. We recently introduced phosphonic ester modified peptide nucleic acids (PNA) for in vivo loss-of-function experiments in medaka embryos. Here we tested novel modifications of the PNA backbone to knockdown the medaka tcf3 gene.ResultsA single tcf3 gene exists in the medaka genome and its inactivation strongly affected eye development of the embryos, leading to size reduction and anophthalmia in severe cases. The function of Tcf3 strongly depends on co-repressor interactions. We found interactions with Groucho/Tle proteins to be most important for eye development. Using a dominant negative approach for combined inactivation of all groucho/tle genes also resulted in eye phenotypes, as did interference with three individual tle genes.ConclusionsOur results show that side chain modified PNAs come close to the knockdown efficiency of morpholino oligonucleotides in vivo. A single medaka tcf3 gene combines the function of the two zebrafish paralogs hdl and tcf3b. In combination with Groucho/Tle corepressor proteins Tcf3 acts in anterior development and is critical for eye formation.Electronic supplementary materialThe online version of this article (10.1186/s12896-017-0411-0) contains supplementary material, which is available to authorized users.
BackgroundSynthetic antisense molecules have an enormous potential for therapeutic applications in humans. The major aim of such strategies is to specifically interfere with gene function, thus modulating cellular pathways according to the therapeutic demands. Among the molecules which can block mRNA function in a sequence specific manner are peptide nucleic acids (PNA). They are highly stable and efficiently and selectively interact with RNA. However, some properties of non-modified aminoethyl glycine PNAs (aegPNA) hamper their in vivo applications.ResultsWe generated new backbone modifications of PNAs, which exhibit more hydrophilic properties. When we examined the activity and specificity of these novel phosphonic ester PNAs (pePNA) molecules in medaka (Oryzias latipes) embryos, high solubility and selective binding to mRNA was observed. In particular, mixing of the novel components with aegPNA components resulted in mixed PNAs with superior properties. Injection of mixed PNAs directed against the medaka six3 gene, which is important for eye and brain development, resulted in specific six3 phenotypes.ConclusionsPNAs are well established as powerful antisense molecules. Modification of the backbone with phosphonic ester side chains further improves their properties and allows the efficient knock down of a single gene in fish embryos.
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known to activate the canonical NF-κB pathway similar to TNF. The exact mechanism of the entire signaling cascade is still under investigation. The involvement of linear ubiquitylation as upregulating component has already been shown recently in some cell lines, but not in human embryonic kidney 293 (HEK293) cells. The downregulating function of the ABIN-1 (A20 binding and inhibitor of NF-κB) as linear ubiquitylation antagonist has been shown in combination with some NF-κB-inducing pathways, but not with TRAIL. We performed luciferase and western blot assays using HEK293 cells stimulated with either TRAIL (or TNF as a control) to analyze the involvement of linear ubiquitin chain assembly complex (LUBAC) components and the impact of ABIN-1 and ABIN-1-MAD (truncated form without A20 binding site) on NF-κB signaling. For overexpression experiments, we added plasmids of ABIN-1 and ABIN-1-MAD or LUBAC components HOIP, HOIL-1, or SHARPIN (single and combinations). For downregulation experiments five pairs of either SHARPIN, HOIL-1, or HOIP targeting miRNAs or one miRNA for ABIN-1 were designed and added. ABIN-1 and its truncated form ABIN-1-MAD reduced the NF-κB induction significantly indicating its involvement as antagonist (independent of deubiquitinase A20) of linear ubiquitylation in TRAIL-induced NF-κB signaling. In opposition, knockdown of ABIN-1 using a specific ABIN-1 miRNA led a clear increase of NF-κB signaling. Addition of single LUBAC components or combinations (except for SHARPIN with HOIL-1) resulted in clearly stronger NF-κB inductions. MiRNAs targeting LUBAC components significantly reduced NF-κB activation. Thus, in HEK293 cells linear ubiquitylation by LUBAC critically upregulates and ABIN-1 downregulates TRAIL-induced NF-κB signaling and may be interesting targets for future pathological therapies.
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