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BACKGROUND. Systemic administration of adeno-associated virus (AAV) can trigger life-threatening inflammatory responses, including thrombotic microangiopathy (TMA), acute kidney injury due to atypical hemolytic uremic syndrome- like complement activation, immune-mediated myocardial inflammation, and hepatic toxicity. METHODS. We describe the kinetics of immune activation following systemic AAV serotype 9 (AAV9) administration in 38 individuals following 2 distinct prophylactic immunomodulation regimens. Group 1 received corticosteroids and Group 2 received rituximab plus sirolimus in addition to steroids to prevent anti-AAV antibody formation. RESULTS. Group 1 participants had a rapid increase in immunoglobulin M (IgM) and IgG. Increase in D-dimer, decline in platelet count, and complement activation are indicative of TMA. All Group 1 participants demonstrated activation of both classical and alternative complement pathways, as indicated by depleted C4 and elevated soluble C5b-9, Ba, and Bb antigens. Group 2 patients did not have a significant change in IgM or IgG and had minimal complement activation. CONCLUSIONS. This study demonstrates that TMA in the setting of AAV gene therapy is antibody dependent (classical pathway) and amplified by the alternative complement pathway. Critical time points and interventions are identified to allow for management of immune-mediated events that impact the safety and efficacy of systemic gene therapy., Introduction Adeno-associated virus-mediated (AAV-mediated) gene therapy has emerged as a promising treatment approach for a spectrum of monogenic genetic disorders (1-4). With the approval of the first recombinant AAV vector-based [...]

While adeno-associated viral (AAV) vectors are successfully used in a variety of in vivo gene therapy applications, they continue to be hampered by the immune system. Here, we sought to identify innate and cytokine signaling pathways that promote CD8+ T-cell responses against the transgene product upon AAV1 vector administration to murine skeletal muscle. Eliminating just one of several pathways (including DNA sensing via TLR9, IL-1 receptor signaling, and possibly endosomal sensing of double-stranded RNA) substantially reduced the CD8+ T-cell response at lower vector doses but was surprisingly ineffective at higher doses. Using genetic, antibody-mediated, and vector engineering approaches, we show that blockade of at least two innate pathways is required to achieve an effect at higher vector doses. Concurrent blockade of IL-1R1 > MyD88 and TLR9 > MyD88 > type I IFN > IFNaR pathways was often but not always synergistic and had limited utility in preventing antibody formation against the transgene product. Further, even low-frequency CD8+ T-cell responses could eliminate transgene expression, even in MyD88- or IL-1R1-deficient animals that received a low vector dose. However, we provide evidence that CpG depletion of vector genomes and including TLR9 inhibitory sequences can synergize. When this construct was combined with the use of a muscle-specific promoter, transgene expression in muscle was sustained with minimal local or systemic CD8+ T-cell response. Thus, innate immune avoidance/blockade strategies by themselves, albeit helpful, may not be sufficient to prevent destructive cellular responses in muscle gene transfer because of the redundancy of immune-activating pathways. [ABSTRACT FROM AUTHOR]

Food allergy (FA) is estimated to impact up to 10% of the population and is a growing health concern. FA results from a failure in the mucosal immune system to establish or maintain immunological tolerance to innocuous dietary antigens, IgE production, and the release of histamine and other mediators upon exposure to a food allergen. Of the different FAs, peanut allergy has the highest incidence of severe allergic responses, including systemic anaphylaxis. Despite the recent FDA approval of peanut oral immunotherapy and other investigational immunotherapies, a loss of protection following cessation of therapy can occur, suggesting that these therapies do not address the underlying immune response driving FA. Our lab has shown that liver-directed gene therapy with an adeno-associated virus (AAV) vector induces transgene product-specific regulatory T cells (Tregs), eradicates pre-existing pathogenic antibodies, and protects against anaphylaxis in several models, including ovalbumin induced FA. In an epicutaneous peanut allergy mouse model, the hepatic AAV co-expression of four peanut antigens Ara h1, Ara h2, Ara h3, and Ara h6 together or the single expression of Ara h3 prevented the development of a peanut allergy. Since FA patients show a reduction in Treg numbers and/or function, we believe our approach may address this unmet need. [ABSTRACT FROM AUTHOR]

Recombinant adeno-associated viruses (AAVs) have emerged as a widely used gene delivery platform for both basic research and human gene therapy. To ensure and improve the safety profile of AAV vectors, substantial efforts have been dedicated to the vector production process development using suspension HEK293 cells. Here, we studied and compared two downstream purification methods, iodixanol gradient ultracentrifugation versus immuno-affinity chromatography (POROS™ CaptureSelect™ AAVX column). We tested multiple vector batches that were separately produced (including AAV5, AAV8, and AAV9 serotypes). To account for batch-to-batch variability, each batch was halved for subsequent purification by either iodixanol gradient centrifugation or affinity chromatography. In parallel, purified vectors were characterized, and transduction was compared both in vitro and in vivo in mice (using multiple transgenes: Gaussia luciferase, eGFP, and human factor IX). Each purification method was found to have its own advantages and disadvantages regarding purity, viral genome (vg) recovery, and relative empty particle content. Differences in transduction efficiency were found to reflect batch-to-batch variability rather than disparities between the two purification methods, which were similarly capable of yielding potent AAV vectors. [ABSTRACT FROM AUTHOR]

Oral administration of antigen induces regulatory T cells (Treg) that can not only control local immune responses in the small intestine, but also traffic to the central immune system to deliver systemic suppression. Employing murine models of the inherited bleeding disorder hemophilia, we find that oral antigen administration induces three CD4+ Treg subsets, namely FoxP3+LAP-, FoxP3+LAP+, and FoxP3- LAP+. These T cells act in concert to suppress systemic antibody production induced by therapeutic protein administration. Whilst both FoxP3+LAP+ and FoxP3-LAP+ CD4+ T cells express membrane-bound TGF-β (latency associated peptide, LAP), phenotypic, functional, and single cell transcriptomic analyses reveal distinct characteristics in the two subsets. As judged by an increase in IL-2Rα and TCR signaling, elevated expression of co-inhibitory receptor molecules and upregulation of the TGFβ and IL-10 signaling pathways, FoxP3+LAP+ cells are an activated form of FoxP3+LAP-Treg. Whereas FoxP3-LAP+ cells express low levels of genes involved in TCR signaling or co-stimulation, engagement of the AP-1 complexmembers Jun/Fos and Atf3 is most prominent, consistent with potent IL-10 production. Single cell transcriptomic analysis further reveals that engagement of the Jun/Fos transcription factors is requisite for mediating TGFb expression. This can occur via an Il2ra dependent or independent process in FoxP3+LAP+ or FoxP3-LAP+ cells respectively. Surprisingly, both FoxP3+LAP+ and FoxP3-LAP+ cells potently suppress and induce FoxP3 expression in CD4+ conventional T cells. In this process, FoxP3-LAP+ cells may themselves convert to FoxP3+ Treg. We conclude that orally induced suppression is dependent on multiple regulatory cell types with complementary and interconnected roles. [ABSTRACT FROM AUTHOR]

Background: Gene editing has emerged as an exciting therapeutic development platform for numerous genetic and nongenetic diseases. Targeting lipid-modulating genes such as angiopoietin-related protein 3 (ANGPTL3) with gene editing offers hope for a permanent solution to lower cardiovascular disease risks associated with hypercholesterolemia. Results: In this study, we developed a hepatocyte-specific base editing therapeutic approach delivered by dual adeno-associated virus (AAV) to enable hepatocyte-specific targeting of Angptl3 to lower blood lipid levels. Systemic AAV9-mediated delivery of AncBE4max, a cytosine base editor (CBE), targeting mouse Angptl3 resulted in the installation of a premature stop codon in Angptl3 with an average efficiency of 63.3 ± 2.3% in the bulk liver tissue. A near-complete knockout of the ANGPTL3 protein in the circulation were observed within 2–4 weeks following AAV administration. Furthermore, the serum levels of triglyceride (TG) and total cholesterol (TC) were decreased by approximately 58% and 61%, respectively, at 4 weeks after treatment. Conclusions: These results highlight the promise of liver-targeted Angptl3 base editing for blood lipid control. [ABSTRACT FROM AUTHOR]