Abstract A challenge of “once-and-done” adeno associated virus (AAV)-based gene therapy is the inability to modulate the level of therapeutic protein expression post-administration. Herein, we demonstrate the utility of an adenosine deaminase acting on RNA (ADAR) - mediated gene switch to control AAV-delivered gene expression. Using a premature termination codon (PTC) in the human Factor IX (hFIX) transgene, we established an ON switch, where expression of hFIX is contingent on rescuing the PTC mutation via RNA editing. In vitro and in vivo studies demonstrated silencing of the hFIX transgene by the PTC mutation and induction of protein expression by administration of an ADAR-recruiting trigger RNA. Mice transduced with a hepatotropic AAV capsid encoding an ApoE-hAAT hFIX-PTC transgene expression cassette showed a dose-dependent response between the levels of LNP-delivered trigger RNA and the amount of plasma hFIX expression achieved. We observed predictable and reproducible levels of hFIX expression upon multiple rounds of RNA editing and demonstrated that this system can achieve clinically relevant levels of hFIX. This work suggests that ADAR-mediated RNA editing may be a valuable tool for tunable expression of therapeutic transgenes in applied gene therapies.

Adeno-associated viral (AAV) vector-based gene therapy is gaining foothold as treatment for genetic neurological diseases with encouraging clinical results. Nonetheless, dose-dependent adverse events have emerged in recent clinical trials through mechanisms that remain unclear. We have modelled here the impact of AAV transduction in cell models of the human central nervous system (CNS), taking advantage of induced pluripotent stem cells. Our work uncovers vector-induced innate immune mechanisms that contribute to cell death. While empty AAV capsids were well tolerated, the AAV genome triggered p53-dependent DNA damage responses across CNS cell types followed by the induction of inflammatory responses. In addition, transgene expression led to MAVS-dependent activation of type I interferon responses. Formation of DNA damage foci in neurons and gliosis were confirmed in murine striatum upon intraparenchymal AAV injection. Transduction-induced cell death and gliosis could be prevented by inhibiting p53 or by acting downstream on STING- or IL-1R-mediated responses. Together, our work identifies innate immune mechanisms of vector sensing in the CNS that can potentially contribute to AAV-associated neurotoxicity.

Crigler-Najjar syndrome is an ultra-rare monogenic recessive liver disease caused by UGT1A1 gene mutations. Complete UGT1A1 deficiency results in severe unconjugated hyperbilirubinemia in newborns that, if not treated, may lead to brain damage and death. Treatment is based on intensive phototherapy, but its efficacy decreases with age, rendering liver transplantation the only curative option. Adeno-associated virus (AAV)-mediated gene therapy has shown long-term correction in adult patients, but loss of viral DNA and therapeutic efficacy are expected in younger patients associated with liver growth. Effective vector re-administration is hindered by anti-AAV neutralizing antibodies generated during the first administration. Here, we investigated AAV vector re-administration by modulating the immune response with rapamycin-loaded nanoparticles (ImmTOR) in Gunn rats (Ugt1a-'-) and Ugt1a-'- mice. We administered a liver-specific AAV8 vector expressing a codon-optimized hUGT1A1 cDNA (1.0E11 vg/ kg) in P25-P28 mutant animals and, upon loss of efficacy after 3 to 5 weeks, a higher second dose (1.0E12 or 5.0E12 vg/kg) was given. ImmTOR co-administration reduced anti-AAV neutralizing antibodies and immunoglobulin Gs generation in male animals of both models allowing effective re-dosing, underscored by a significant and long-term decrease in plasma bilirubin, although efficacy was affected by low-titer residual anti-AAV antibodies suggesting that re-administration in patients may require combination with other methods.

Introduction Hemophilia A (HA) is managed with factor (F)VIII replacement, or non-factor replacement therapy such as emicizumab. More recently, gene therapies have been under investigation for HA. These use a modified viral vector to deliver a functional FVIII gene to liver cells, aiming to safely provide long-term, stable FVIII expression to prevent bleeding. Dirloctocogene samoparvovec (SPK-8011), an investigational gene therapy, uses a modified adeno-associated viral vector designed to achieve clinically meaningful and sustained FVIII expression at the lowest possible dose. The ongoing Phase I/II trial (NCT03003533/NCT03432520) is evaluating the safety and efficacy of dirloctocogene samoparvovec, assessed by FVIII activity levels and patient-reported bleeding events.

Maple syrup urine disease (MSUD) is rare autosomal recessive metabolic disorder caused by the dysfunction of the mitochondrial branched-chain 2-ketoacid dehydrogenase (BCKD) enzyme complex leading to massive accumulation of branched-chain amino acids and 2-keto acids. MSUD management, based on a life-long strict protein restriction with nontoxic amino acids oral supplementation represents an unmet need as it is associated with a poor quality of life, and does not fully protect from acute life-threatening decompensations or long-term neuropsychiatric complications. Orthotopic liver transplantation is a beneficial therapeutic option, which shows that restoration of only a fraction of whole-body BCKD enzyme activity is therapeutic. MSUD is thus an ideal target for gene therapy. We and others have tested AAV gene therapy in mice for two of the three genes involved in MSUD, BCKDHA and DBT. In this study, we developed a similar approach for the third MSUD gene, BCKDHB. We performed the first characterization of a Bckdhb(-/-) mouse model, which recapitulates the severe human phenotype of MSUD with early-neonatal symptoms leading to death during the first week of life with massive accumulation of MSUD biomarkers. Based on our previous experience in Bckdha(-/-) mice, we designed a transgene carrying the human BCKDHB gene under the control of a ubiquitous EF1 alpha promoter, encapsidated in an AAV8 capsid. Injection in neonatal Bckdhb(-/-) mice at 10(14) vg/kg achieved long-term rescue of the severe MSUD phenotype of Bckdhb(-/-) mice. These data further validate the efficacy of gene therapy for MSUD opening perspectives towards clinical translation.

Abstract Adeno-associated viral (AAV) vector-based gene therapy is gaining foothold as a treatment option for a variety of genetic neurological diseases with encouraging clinical results. Nonetheless, dose-dependent toxicities and severe adverse events have emerged in recent clinical trials through mechanisms that remain unclear. We have modelled here the impact of AAV transduction in the context of cell models of the human central nervous system (CNS), taking advantage of induced pluripotent stem cell-based technologies. Our work uncovers vector-induced cell-intrinsic innate immune mechanisms that contribute to apoptosis in 2D and 3D models. While empty AAV capsids were well tolerated, the AAV genome triggered p53-dependent DNA damage responses across CNS cell types followed by induction of IL-1R- and STING-dependent inflammatory responses. In addition, transgene expression led to MAVS-dependent signaling and activation of type I interferon (IFN) responses. Cell-intrinsic and paracrine apoptosis onset could be prevented by inhibiting p53 or acting downstream of STING- and IL-1R-mediated responses. Activation of DNA damage, type I IFN and CNS inflammation were confirmed in vivo, in a mouse model. Together, our work identifies the cell-autonomous innate immune mechanisms of vector DNA sensing that can potentially contribute to AAV-associated neurotoxicity.

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.

As the clinical experience in adeno-associated viral (AAV) vector-based gene therapies is expanding, the necessity to better understand and control the host immune responses is also increasing. Immunogenicity of AAV vectors in humans has been linked to several limitations of the platform, including lack of efficacy due to antibody-mediated neutralization, tissue inflammation, loss of transgene expression, and in some cases, complement activation and acute toxicities. Nevertheless, significant knowledge gaps remain in our understanding of the mechanisms of immune responses to AAV gene therapies, further hampered by the failure of preclinical animal models to recapitulate clinical findings. In this review, we focus on the current knowledge regarding immune responses, spanning from innate immunity to humoral and adaptive responses, triggered by AAV vectors and how they can be mitigated for safer, durable, and more effective gene therapies.

OBJECTIVE:Pompe disease (PD) is caused by deficiency of the lysosomal enzyme acid α-glucosidase (GAA), leading to progressive glycogen accumulation and severe myopathy with progressive muscle weakness. In the Infantile-Onset PD (IOPD), death generally occurs <1 year of age. There is no cure for IOPD. Mouse models of PD do not completely reproduce human IOPD severity. Our main objective was to generate the first IOPD rat model to assess an innovative muscle-directed adeno-associated viral (AAV) vector-mediated gene therapy.METHODS:PD rats were generated by CRISPR/Cas9 technology. The novel highly myotropic bioengineered capsid AAVMYO3 and an optimized muscle-specific promoter in conjunction with a transcriptional cis-regulatory element were used to achieve robust Gaa expression in the entire muscular system. Several metabolic, molecular, histopathological, and functional parameters were measured.RESULTS:PD rats showed early-onset widespread glycogen accumulation, hepato- and cardiomegaly, decreased body and tissue weight, severe impaired muscle function and decreased survival, closely resembling human IOPD. Treatment with AAVMYO3-Gaa vectors resulted in widespread expression of Gaa in muscle throughout the body, normalizing glycogen storage pathology, restoring muscle mass and strength, counteracting cardiomegaly and normalizing survival rate.CONCLUSIONS:This gene therapy holds great potential to treat glycogen metabolism alterations in IOPD. Moreover, the AAV-mediated approach may be exploited for other inherited muscle diseases, which also are limited by the inefficient widespread delivery of therapeutic transgenes throughout the muscular system.

novel molecular imprinting technique is used to map and identify four antigen-bindingfragments (Fab) sequences of Adeno-Associated Virus (AAV) - neutralising antibodiescapable of binding to AAV. The presented proof-of-principle study demonstrates thatsynthesised Fab peptides, identified by molecular imprinting, can prevent AAV8binding to antibodies. A study shows the opportunity to use Fab peptides specific toAAV as a formulation for improving the efficiency of gene therapy by preventing theimmune response.

Pompe disease (PD) is a neuromuscular disorder caused by acid α-glucosidase (GAA) deficiency. Reduced GAA activity leads to pathological glycogen accumulation in cardiac and skeletal muscles responsible for severe heart impairment, respiratory defects, and muscle weakness. Enzyme replacement therapy with recombinant human GAA (rhGAA) is the standard-of-care treatment for PD, however, its efficacy is limited due to poor uptake in muscle and the development of an immune response. Multiple clinical trials are ongoing in PD with adeno-associated virus (AAV) vectors based on liver- and muscle-targeting. Current gene therapy approaches are limited by liver proliferation, poor muscle targeting, and the potential immune response to the hGAA transgene. To generate a treatment tailored to infantile-onset PD, we took advantage of a novel AAV capsid able to increase skeletal muscle targeting compared to AAV9 while reducing liver overload. When combined with a liver-muscle tandem promoter (LiMP), and despite the extensive liver-detargeting, this vector had a limited immune response to the hGAA transgene. This combination of capsid and promoter with improved muscle expression and specificity allowed for glycogen clearance in cardiac and skeletal muscles of Gaa-/- adult mice. In neonate Gaa-/- , complete rescue of glycogen content and muscle strength was observed 6 months after AAV vector injection. Our work highlights the importance of residual liver expression to control the immune response toward a potentially immunogenic transgene expressed in muscle. In conclusion, the demonstration of the efficacy of a muscle-specific AAV capsid-promoter combination for the full rescue of PD manifestation in both neonate and adult Gaa-/- provides a potential therapeutic avenue for the infantile-onset form of this devastating disease.

BACKGROUND:Patients with the Crigler-Najjar syndrome lack the enzyme uridine diphosphoglucuronate glucuronosyltransferase 1A1 (UGT1A1), the absence of which leads to severe unconjugated hyperbilirubinemia that can cause irreversible neurologic injury and death. Prolonged, daily phototherapy partially controls the jaundice, but the only definitive cure is liver transplantation. METHODS:We report the results of the dose-escalation portion of a phase 1-2 study evaluating the safety and efficacy of a single intravenous infusion of an adeno-associated virus serotype 8 vector encoding UGT1A1 in patients with the Crigler-Najjar syndrome that was being treated with phototherapy. Five patients received a single infusion of the gene construct (GNT0003): two received 2×1012 vector genomes (vg) per kilogram of body weight, and three received 5×1012 vg per kilogram. The primary end points were measures of safety and efficacy; efficacy was defined as a serum bilirubin level of 300 μmol per liter or lower measured at 17 weeks, 1 week after discontinuation of phototherapy. RESULTS:No serious adverse events were reported. The most common adverse events were headache and alterations in liver-enzyme levels. Alanine aminotransferase increased to levels above the upper limit of the normal range in four patients, a finding potentially related to an immune response against the infused vector; these patients were treated with a course of glucocorticoids. By week 16, serum bilirubin levels in patients who received the lower dose of GNT0003 exceeded 300 μmol per liter. The patients who received the higher dose had bilirubin levels below 300 μmol per liter in the absence of phototherapy at the end of follow-up (mean [±SD] baseline bilirubin level, 351±56 μmol per liter; mean level at the final follow-up visit [week 78 in two patients and week 80 in the other], 149±33 μmol per liter). CONCLUSIONS:No serious adverse events were reported in patients treated with the gene-therapy vector GNT0003 in this small study. Patients who received the higher dose had a decrease in bilirubin levels and were not receiving phototherapy at least 78 weeks after vector administration. (Funded by Genethon and others; ClinicalTrials.gov number, NCT03466463.).