Thrombotic microangiopathy (TMA) has been reported as an uncommon but severe adverse effect of recombinant adeno-associated virus (rAAV)-based gene therapy. Here, we describe in detail the occurrences of clinically evident TMA following infusion of the rAAV9-based therapy fordadistrogene movaparvovec (1 or 3 × 1014 vg/kg) in 22 participants with Duchenne muscular dystrophy (DMD). Three participants experienced clinical evidence of TMA. Platelet levels rapidly decreased 7-10 days post infusion, together with C3 and C4 depletion and elevated C5b-9 after ∼7 days. Peak vector blood concentration was observed for 4-7 days, and type 1 interferon cytokine levels increased within 2-7 days. Each affected participant was hospitalized and received supportive care and anti-complement therapy. Anti-AAV9 immunoglobulin M (IgM) and IgG were detected within days post infusion, and participants with TMA demonstrated a particularly rapid rise of neutralizing antibodies and total antibody to AAV9 in the first 1-2 weeks post infusion. Limited early time points from other participants were not assessed for complement activation. With appropriate monitoring of early time points following AAV exposure, TMA can be successfully managed. However, these early events should be considered related to the benefit-risk profile when using rAAV-based gene therapy for the treatment of DMD. ClinicalTrials.gov identifier: NCT03362502.

Cell-mediated immune (CMI) responses to adeno-associated virus (AAV) can lead to tissue damage and loss of therapeutic transgene expression. Identifying robust biomarkers and mechanisms of CMI can aid clinical practice and advancement of AAV gene therapies. The present work evaluated peripheral blood mononuclear cells (PBMC) from non-human primates (NHP) before and after immunization with adenovirus 5 encoding AAV9 capsid antigen. PBMC were stimulated ex vivo with AAV9 capsid peptides to evaluate CMI responses by interferon (IFN)-γ ELISpot, intracellular cytokines/activation markers, secreted cytokines, and RNAseq. AAV peptide stimulation produced a robust IFNγ ELISpot 11 days after immunization and ≈ 4 years after cryopreservation. Flow cytometry revealed increased IFNγ, interleukin (IL)-2, or tumor necrosis factor (TNF)-positive T-cells. Increases in secreted CXCR3 ligands (IP-10, I-TAC) were detected. Robust changes and correlations to ELISpot responses were revealed by RNAseq, including IFNγ, IP-10, and I-TAC, many downstream transcripts, and several IFN-independent pathways. These data from AAV-immunized NHP identify biomarkers that could serve as robust and sensitive supplements/alternatives to ELISpot for early detection of CMI responses. Assessment of these biomarkers in non-clinical and clinical studies is a critical next step to determine the translation of this work to administration of a therapeutic AAV vector.

Clinical trials for Duchenne muscular dystrophy (DMD) are assessing the therapeutic efficacy of systemically delivered adenoassociated virus (AAV) carrying a modified DMD transgene. High vector doses (>1E14 vg/kg) are needed to globally transduce skeletal muscles; however, such doses trigger immunerelated adverse events. Mitigating these immune responses is crucial for widespread application of AAV-based therapies. We used single-cell RNA sequencing and T cell receptor (TCR) sequencing on peripheral blood mononuclear cells from five participants prior to, and after, dosing. One subject in the high-dose cohort experienced thrombotic microangiopathy (TMA). Few changes in cell frequencies occurred after treatment; however, differential gene expression demonstrated induction of interferon response genes in most T cell types. T cell clonotype and clumping analysis showed the expansion or appearance of groups of related TCR sequences in the post-treatment samples. Three of these expanded clumps could be assigned to prior human herpesvirus infections, two of which were present in the participant that exhibited TMA. These data provide insight on the mechanistic basis of human immune-AAV interactions and lay a foundation for improved understanding of why TMA arises in some patients and not others.

Treatment of monogenetic disorders using vectors based on adeno-associated virus (AAV) is an area of intense interest. AAV is non-pathogenic human virus, and preexisting capsid antibodies are prevalent in the population posing a challenge to the safety and efficacy of AAV-mediated gene therapies. In this study, we investigated the risk of AAV-mediated complement activation when sera from a cohort of human donors were exposed to AAV9 capsid. Seropositive donor sera carrying neutralizing antibodies from a previous environmental exposure activated complement when admixed with AAV9 capsids and complement activation was associated with donors who had higher levels of anti-AAV IgG1 antibodies. These findings were consistent with mass spectrometry analysis that identified increased binding of immunoglobulins and complement factors when AAV9 capsids were admixed with seropositive sera. Finally, complement activation was abrogated after IgG-depletion using affinity columns or serum pretreatment with an IgG degrading enzyme. Overall, these results demonstrate an important role of preexisting neutralizing antibodies in activating complement; a risk that can be mitigated by using adequate immunosuppression strategies when dosing seropositive patients with vector.

Recombinant adeno-associated viruses (AAVs) have emerged as promising vectors for human gene therapy, but some variants have induced severe toxicity in Rhesus monkeys and piglets following high-dose intravenous (IV) administration. To characterize biodistribution, transduction, and toxicity among common preclinical species, an AAV9 neurotropic variant expressing the survival motor neuron 1 (SMN1) transgene (AAV-PHP.B-CBh-SMN1) was administered by IV bolus injection to Wistar Han rats and cynomolgus monkeys at doses of 2 × 1013, 5 × 1013, or 1 × 1014 vg/kg. A dose-dependent degeneration/necrosis of neurons without clinical manifestations occurred in dorsal root ganglia (DRGs) and sympathetic thoracic ganglia in rats, while liver injury was not observed in rats. In monkeys, one male at 5 × 1013 vg/kg was found dead on day 4. Clinical pathology data on days 3 and/or 4 at all doses suggested liver dysfunction and coagulation disorders, which led to study termination. Histologic evaluation of the liver in monkeys showed hepatocyte degeneration and necrosis without inflammatory cell infiltrates or intravascular thrombi, suggesting that hepatocyte injury is a direct effect of the vector following hepatocyte transduction. In situ hybridization demonstrated a dose-dependent expression of SMN1 transgene mRNA in the cytoplasm and DNA in the nucleus of periportal to panlobular hepatocytes, while quantitative polymerase chain reaction confirmed the dose-dependent presence of SMN1 transgene mRNA and DNA in monkeys. Monkeys produced a much greater amount of transgene mRNA compared with rats. In DRGs, neuronal degeneration/necrosis and accompanying findings were observed in monkeys as early as 4 days after test article administration. The present results show sensory neuron toxicity following IV delivery of AAV vectors at high doses with an early onset in Macaca fascicularis and after 1 month in rats, and suggest adding the autonomic system in the watch list for preclinical and clinical studies. Our data also suggest that the rat may be useful for evaluating the potential DRG toxicity of AAV vectors, while acute hepatic toxicity associated with coagulation disorders appears to be highly species-dependent.

Abstract The anti-tumor necrosis factor (TNF) antibodies, infliximab and adalimumab are efficacious in Crohn’s disease (CD) patients but their relevant mechanism(s) of action (MOA) are not fully elucidated. Neutralization of TNF activity is a key efficacy mechanism, but may not be the only relevant MOA for efficacy in CD. Fc receptor-mediated dampening of the immune response has been proposed as one of the alternative hypotheses. Two FcγR3A-158 polymorphisms (158V and 158F) have been shown to influence binding affinity of IgG1 antibodies, and CD patients with V/V genotypes have increased biological responses to infliximab. Previous work by others showed that in a mixed lymphocyte reaction (MLR), infliximab and adalimumab induced formation of immunosuppressive macrophages in an Fc region-dependent manner and that these macrophages inhibited proliferation of activated T cells. We ran MLRs with FcγR3A genotype-matched donor pairs to determine if Fc receptor genotype influenced the anti-inflammatory response by infliximab and adalimumab. Our results showed that regulatory macrophages were induced regardless of FcγR3A genotype but that decreases in T cell proliferation were more pronounced with V/V donor pairs than with V/F or F/F genotypes. In conclusion, our work suggests that infliximab and adalimumab may be more effective at inducing an anti-inflammatory state in individuals with the 158V polymorphism of FcγR3A.

Linezolid has a wide spectrum of activity against gram-positive organisms, but no clinical activity against gram-negative organisms. In a phase 3 study comparing linezolid with vancomycin in patients with intravascular catheter-related infections, a mortality imbalance occurred in patients with gram-negative bacterial infections. The hypothesis that this imbalance might be related to immunosuppressive effects of linezolid (impeding the ability of phagocytic cells to respond to gram-negative infections) was tested using in vitro and ex vivo approaches. Human whole blood was incubated with linezolid in vitro. Phagocytosis of Escherichia coli (E.coli) particles, cytokine (interleukin-1 receptor antagonist [IL-1ra], IL-8) secretion and mRNA synthesis by neutrophils in response to lipopolysaccharide (LPS) stimulation, were measured. Subsequently, in an ex vivo study, linezolid or placebo was administered to healthy subjects before measurements of phagocytosis of E. coli particles by neutrophils and monocytes, and cytokine (IL-1ra, IL-1β, IL-6, IL-8, and tumor necrosis factor-α) secretion by total white blood cells in response to LPS stimulation. Linezolid and placebo group comparisons were based on individual baseline-corrected values. Linezolid had no effect on phagocytosis of E.coli particles by neutrophils or monocytes in vitro or ex vivo. Small to moderate decreases in IL-1ra and IL-8 secretions from neutrophils were observed at high linezolid concentrations (≥16.5 μg/mL) in vitro, but cytokine gene expression was not affected. Linezolid had no significant effect on secretion of 5 cytokines evaluated ex vivo. There is no strong evidence suggesting that linezolid inhibits phagocytic cell functions in response to gram-negative pathogensat clinically relevant concentrations.

The mononuclear phagocyte system (MPS) which provides protection against infection is made up of phagocytic cells that engulf and digest bacteria or other foreign substances. Suppression of the MPS may lead to decreased clearance of pathogenic microbes. Drug delivery systems and immunomodulatory therapeutics that target phagocytes have a potential to inhibit MPS function. Available methods to measure inhibition of MPS function use uptake of radioactively-labeled cells or labor-intensive semi-quantitative histologic techniques. The objective of this work was to develop a non-radioactive quantitative method to measure MPS function in vivo by administering heat-killed E. coli conjugated to a pH-sensitive fluorescent dye (Bioparticles(®)). Fluorescence of the Bioparticles(®) is increased at low pH when they are in phagocytic lysosomes. The amount of Bioparticles(®) phagocytosed by MPS organs in rats was determined by measuring fluorescence intensity in livers and spleens ex vivo using an IVIS(®) Spectrum Pre-clinical In Vivo Imaging System. Phagocytosis of the particles by peripheral blood neutrophils was measured by flow cytometry. To assess method sensitivity, compounds likely to suppress the MPS [clodronate-containing liposomes, carboxylate-modified latex particles, maleic vinyl ether (MVE) polymer] were administered to rats prior to injection of the Bioparticles(®). The E. coli particles consistently co-localized with macrophage markers in the liver but not in the spleen. All of the compounds tested decreased phagocytosis in the liver, but had no consistent effects on phagocytic activity in the spleen. In addition, administration of clodronate liposomes and MVE polymer increased the percentage of peripheral blood neutrophils that phagocytosed the Bioparticles(®). In conclusion, an in vivo rat model was developed that measures phagocytosis of E. coli particles in the liver and may be used to assess the impact of test compounds on MPS function. Still, the detection of inhibition of splenic macrophage function will require further assay development.