Lipid nanoparticles (LNPs) have demonstrated significant therapeutic value for non-viral delivery of mRNA and siRNA. While there is considerable interest in utilizing LNPs for delivering DNA (DNA-LNPs) to address a broad range of genetic disorders, acute inflammatory responses pose significant safety concerns and limit transgene expression below therapeutically relevant levels. However, the mechanisms and immune signaling pathways underlying DNA-LNP-triggered inflammatory responses are not well characterized. Through the use of gene-targeted mouse models, we have identified cGAS-STING and interferon-α/β receptor (IFNAR) pathways as major mediators of acute inflammation triggered by systemic delivery of DNA-LNPs. cGAS-STING activation induces expression of numerous JAK-STAT-activating cytokines, and we show that treatment of mice with the JAK inhibitors ruxolitinib or baricitinib significantly improves tolerability to systemically delivered DNA-LNPs. Furthermore, specific inhibition of IFNAR signaling enhances both DNA-LNP tolerability and transgene expression. Utilization of JAK inhibitors or IFNAR blockade represent promising strategies for enhancing the safety and efficacy of non-viral DNA delivery for gene therapy.

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.

Adeno-associated virus (AAV) vectors represent a novel tool for the delivery of genetic therapeutics and enable the treatment of a wide range of diseases. Success of this new modality is challenged, however, by cases of immune-related toxicities that complicate the clinical management of patients and potentially limit the therapeutic efficacy of AAV gene therapy. While significant progress has been made to manage immune-related liver enzyme elevations following systemic AAV delivery in humans, recent clinical trials utilizing high vector doses have highlighted a new challenge to AAV gene transfer-activation of the complement system. While current in vitro models implicate AAV-specific antibodies in the initiation of the classical complement pathway, evidence from in vivo pre-clinical and clinical studies suggests that the alternative pathway also contributes to complement activation. A convergence of AAV-specific, environmental, and patient-specific factors shaping complement responses likely contributes to differential outcomes seen in clinical trials, from priming of the adaptive immune system to serious adverse events such as hepatotoxicity and thrombotic microangiopathy. Research focused on the interplay of patient-specific and AAV-related factors driving complement activation is needed to understand and identify critical components in the complement cascade to target and devise strategies to mitigate vector-related immune responses.

Adeno-associated virus (AAV) vectors are used for correcting multiple genetic disorders. Although the goal is to achieve lifelong correction with a single vector administration, the ability to redose would enable the extension of therapy in cases in which initial gene transfer is insufficient to achieve a lasting cure, episomal vector forms are lost in growing organs of pediatric patients, or transgene expression is diminished over time. However, AAV typically induces potent and long-lasting neutralizing antibodies (NAbs) against capsid that prevents re-administration. To prevent NAb formation in hepatic AAV8 gene transfer, we developed a transient B cell-targeting protocol using a combination of monoclonal Ab therapy against CD20 (for B cell depletion) and BAFF (to slow B cell repopulation). Initiation of immunosuppression before (rather than at the time of) vector administration and prolonged anti-BAFF treatment prevented immune responses against the transgene product and abrogated prolonged IgM formation. As a result, vector re-administration after immune reconstitution was highly effective. Interestingly, re-administration before the immune system had fully recovered achieved further elevated levels of transgene expression. Finally, this immunosuppression protocol reduced Ig-mediated AAV uptake by immune cell types with implications to reduce the risk of immunotoxicities in human gene therapy with AAV.

COPYRIGHT © 2023 Rosenberg, Tourdot and Markusic. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. TYPE Editorial PUBLISHED 22 August 2023 DOI 10.3389/fimmu.2023.1219854

EDITORIAL article Front. Immunol., 22 June 2023Sec. Vaccines and Molecular Therapeutics Volume 14 - 2023 | https://doi.org/10.3389/fimmu.2023.1227231

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.

The liver is an important immune sentinel organ responsible for modulating adaptive immune responses to foreign dietary antigens derived from the gut. Mechanistically, this is achieved through maintenance of an anti-inflammatory microenvironment within the liver, which leads to antigen presentation by immature professional and non-professional antigen-presenting cells (APCs) and induction of antigen-specific immune tolerance. Within the liver, major histocompatibility complex (MHC) class I-restricted antigen presentation by hepatocytes and non-professional APCs results in suboptimal activation of antigen-specific CD8+ T cells, which can result in exhaustion or even clonal deletion, 1 Thomson A.W. Knolle P.A. Antigen-presenting cell function in the tolerogenic liver environment. Nat. Rev. Immunol. 2010; 10 (PubMed PMID: 20972472): 753-766https://doi.org/10.1038/nri2858 Crossref PubMed Scopus (597) Google Scholar ,2 Horst A.K. Neumann K. Diehl L. Tiegs G. Modulation of liver tolerance by conventional and nonconventional antigen-presenting cells and regulatory immune cells. Cell. Mol. Immunol. 2016; 13: 277-292https://doi.org/10.1038/cmi.2015.112 Crossref PubMed Scopus (173) Google Scholar whereas local MHC II-restricted antigen expression in professional and non-professional APCs within the liver induces both CD4+ T cell anergy and deletion and drives conversion of T effector cells into regulatory T cells (Tregs).

Liver directed gene delivery with AAV has made tremendous strides in the last decade and is poised to cure disorders such as hemophilia A and B. Although AAV is typically considered to be relatively innocuous, immunogenicity to the vector, its genome, and encoded transgene has resulted in innate and adaptive immunotoxicities as well as loss of the transgene product, which not only limit efficacy, but also raise important safety concerns. A major impediment to patient re-dosing with AAV is the formation of high-titer neutralizing antibodies (NAb) to the viral capsid following first administration. The right combination of immunosuppressive (IS) drugs have the capacity to control NAb formation, as well as mitigate innate and adaptive immune responses to both the vector and the transgene product. We tested hepatic gene delivery with AAV8 in C57BL/6 mice (n=8-10), using a dose (1X1011 vg/kg) that was previously shown to induce CD8+ T cells to the model transgene, ovalbumin (OVA). We compared (i) B cell depletion with α-CD20, 2 X 250μg doses spaced 3 weeks apart), (ii) α-CD20 combined with the mTOR inhibitor rapamycin (α-CD20+rapa, oral gavage with 4mg/kg rapa, 3X/week/5 weeks), (iii) α-CD20 combined with an antibody to B cell activating factor (α-CD20+α-BAFF, 2 X 60μg doses spaced 10 days apart), a cytokine that we confirmed to be crucial for B cell survival [(J Clin Invest 2021, 15;131(8)]. IS therapies administered concurrent with AAV8 gene therapy caused a high frequency of animals (40 - 75%) to develop transgene immunity (H-2 Kb SIINFEKL tetramer+), corresponding with loss of OVA expression in the liver and in circulation. OVA-CD8+ T cells had a short-lived effector memory phenotype in blood and spleen, characterized as CD44+CD62L-CCR7-KLRG1+CX3CR1+. Interestingly, there were much higher frequencies of OVA-CD8+ T cells in the liver, even when they were undetectable in blood and spleen, and their presence in the liver did not completely correlate with loss of OVA expression. In line with this observation, OVA-CD8+ T cells in the liver expressed multiple exhaustion markers PD1, 2B4, LAG3, TIM3, highly upregulated transcription factors TBet, STAT3, and BATF, but not TCF1, and did not express KLRG1 and CX3CR1 expression. All control and IS treated groups of animals developed NAb (IgM and IgG2c, equivalent to IgG1 in human) to the AAV8 vector, which precluded re-administration with the same AAV serotype (5X1012 vg/kg of AAV8-hFIX). In contrast, when IS therapies were initiated 3 weeks prior to AAV8 gene therapy (1X1011 vg/kg), we were successfully able to re-administer AAV8-hFIX (n=6-10) to 70% of α-CD20, 75% of α-CD20+rapa, and 80% of α-CD20+α-BAFF treated animals without transgene immunity development. Re-administration was unsuccessful in animals that did not receive immune modulation. We then tested pre-IS therapy with a clinically relevant primary administration dose (5X1012 vg/kg of AAV8-OVA). We were able to re-administer AAV8-hFIX to 44% of α-CD20, 88% of α-CD20+rapa, and 67% of α-CD20+α-BAFF treated animals. Mice that were precluded from re-dosing with AAV8-hFIX in the IS treatment groups had high circulating BAFF levels, which correlated with the development of α-AAV8 IgM NAbs. To validate this finding and mitigate α-AAV8 IgM NAbs, we extended α-BAFF injections for an additional 4 weeks in the α-CD20+α-BAFF group (n=10). Extending α-BAFF treatment prevented both BAFF and IgM production, allowing not only successful re-dosing in 100% of animals but also higher circulating FIX levels (12901 ± 686.7ng/mL) as compared to mice that only received AAV8-FIX (9725 ± 415.5 ng/mL). Finally, we tested the practicality of α-CD20+extended α-BAFF treatment in the event of delayed AAV8 re-dosing, once immune compartments have completely repopulated (~14 weeks based on flow cytometry analysis). We were successfully able to re-dose 100% of α-CD20+extended α-BAFF pre-IS treated mice with AAV8-FIX following a delayed re-administration schedule (15 weeks). FIX expression levels remained stable until the last timepoint tested (16 weeks post re-dosing). We conclude that timing and duration of IS, vector dose and transgene immunogenicity are important considerations in developing effective IS therapies. We propose that pre-IS therapy with αCD20+ extended αBAFF is an effective candidate to enable re-dosing with clinically relevant doses of AAV.

Immunotherapies for patients with food allergy have shown some success in limiting allergic responses. However, these approaches require lengthy protocols with repeated allergen dosing and patients can relapse following discontinuation of treatment. The purpose of this study was to test if a single dose of an adeno-associated virus (AAV) vector can safely prevent and treat egg allergy in a mouse model. AAV vectors expressing ovalbumin (OVA) under an ubiquitous or liver-specific promoter were injected prior to or after epicutaneous sensitization with OVA. Mice treated with either AAV8-OVA vector were completely protected from allergy sensitization. These animals had a significant reduction in anaphylaxis mediated by a reduction in OVA-specific IgE titers. In mice with established OVA allergy, allergic responses were mitigated only in mice treated with an AAV8-OVA vector expressing OVA from an ubiquitous promoter. In conclusion, an AAV vector with a liver-specific promoter was more effective for allergy prevention, but higher OVA levels were necessary for reducing symptoms in preexisting allergy. Overall, our AAV gene immunotherapy resulted in an expansion of OVA-specific FoxP3+ CD4+ T cells, an increase in the regulatory cytokine IL-10, and a reduction in the IgE promoting cytokine IL-13.

Hepatic gene transfer with adeno-associated viral (AAV) vectors shows much promise for the treatment of the X-linked bleeding disorder hemophilia B in multiple clinical trials. In an effort to further innovate this approach and to introduce alternative vector designs with potentially superior features into clinical development, we recently built a vector platform based on AAV serotype 3 because of its superior tropism for human hepatocytes. A vector genome with serotype-matched inverted terminal repeats expressing hyperactive human coagulation factor IX (FIX)-Padua was designed for clinical use that is optimized for translation using hepatocyte-specific codon-usage bias and is depleted of immune stimulatory CpG motifs. Here, this vector genome was packaged into AAV3 (T492V + S663V) capsid for hepatic gene transfer in non-human primates. FIX activity within or near the normal range was obtained at a low vector dose of 5 x 10(11) vector genomes/kg. Pre-existing neutralizing antibodies, however, completely or partially blocked hepatic gene transfer at that dose. No CD8(+) T cell response against capsid was observed. Antibodies against the human FIX transgene product formed at a 10-fold higher vector dose, albeit hepatic gene transfer was remarkably consistent, and sustained FIX activity in the normal range was nonetheless achieved in two of three animals for the 3-month duration of the study. These results support the use of this vector at low vector doses for gene therapy of hemophilia B in humans.

A 20-nt long sequence, termed the D-sequence, in the adeno-associated virus (AAV) inverted terminal repeat was observed to share a partial sequence homology with the X-box in the regulatory region of the human leukocyte antigen DRA (HLA-DRA) promoter of the human major histocompatibility complex class II (MHC-II) genes. The D-sequence was also shown to specifically interact with the regulatory factor binding to the X-box (RFX), binding of which to the X-box is a critical step in the MHC-II gene expression, suggesting that D-sequence might compete for RFX transcription factor binding, thereby suppressing expression from the MHC-II promoter. In DNA-mediated transfection experiments, using a reporter gene under the control of the HLA-DRA promoter, D-sequence oligonucleotides were found to inhibit expression of the reporter gene expression in HeLa and 293 cells by ∼93% and 96%, respectively. No inhibition was observed when nonspecific synthetic oligonucleotides were used. D-sequence oligonucleotides had no effect on expression from the cytomegalovirus immediate-early gene promoter. Interferon-γ-mediated activation of MHC-II gene expression was also inhibited by D-sequence oligonucleotides as well as after infection with either the wild-type AAV or transduction with recombinant AAV vectors. These studies suggest that the D-sequence-mediated downregulation of the MHC-II gene expression may be exploited toward the development of novel AAV vectors capable of dampening the host humoral response, which has important implication in the optimal use of these vectors in human gene therapy.

Although recombinant adeno-associated virus serotype 8 (AAV8) and serotype 5 (AAV5) vectors have shown efficacy in Phase 1 clinical trials for gene therapy of hemophilia B, it has become increasingly clear that these serotypes are not optimal for transducing primary human hepatocytes. We have previously reported that among the 10 most commonly used AAV serotypes, AAV serotype 3 (AAV3) vectors are the most efficient in transducing primary human hepatocytesin vitroas well as in "humanized" micein vivo, and suggested that AAV3 vectors expressing human coagulation factor IX (hFIX) may be a more efficient alternative for clinical gene therapy of hemophilia B. In the present study, we extended these findings to develop an AAV3 vector incorporating a compact yet powerful liver-directed promoter as well as optimized hFIX cDNA sequence inserted between two AAV3 inverted terminal repeats. When packaged into an AAV3 capsid, this vector yields therapeutic levels of hFIX in hemophilia B and in "humanized" micein vivo.Together, these studies have resulted in an AAV3 vector predicted to achieve clinical efficacy at reduced vector doses, without the need for immune-suppression, for clinical gene therapy of hemophilia B.

Hemophilia A is an inherited coagulation disorder resulting in the loss of functional clotting factor VIII (FVIII). Presently, the most effective treatment is prophylactic protein replacement therapy. However, this requires frequent life-long intravenous infusions of plasma derived or recombinant clotting factors and is not a cure. A major complication is the development of inhibitory antibodies that nullify the replacement factor. Immune tolerance induction (ITI) therapy to reverse inhibitors can last from months to years, requires daily or every other day infusions of supraphysiological levels of FVIII and is effective in only up to 70% of hemophilia A patients. Preclinical and recent clinical studies have shown that gene replacement therapy with AAV vectors can effectively cure hemophilia A patients. However, it is unclear how hemophilia patients with high risk inhibitor F8 mutations or with established inhibitors will respond to gene therapy, as these patients have been excluded from ongoing clinical trials. AAV8-coF8 gene transfer in naïve BALB/c-F8e16−/Y mice (BALB/c-HA) results in anti-FVIII IgG1 inhibitors following gene transfer, which can be prevented by transient immune modulation with anti-mCD20 (18B12) and oral rapamycin. We investigated if we could improve ITI in inhibitor positive mice by combining anti-mCD20 and rapamycin with AAV8-coF8 gene therapy. Our hypothesis was that continuous expression of FVIII protein from gene transfer compared to transient FVIII from weekly protein therapy, would enhance regulatory T cell induction and promote deletion of FVIII reactive B cells, following reconstitution. Mice that received anti-CD20 had a sharp decline in inhibitors, which corresponded to FVIII memory B (Bmem) cell deletion. Importantly, only mice receiving both anti-mCD20 and rapamycin failed to increase inhibitors following rechallenge with intravenous FVIII protein therapy. Our data show that B and T cell immune modulation complements AAV8-coF8 gene therapy in naïve and inhibitor positive hemophilia A mice and suggest that such protocols should be considered for AAV gene therapy in high risk or inhibitor positive hemophilia patients.