Incorporating TLR9-inhibitory sequences in the AAV vector genome inhibits immunogenicity and enhances transgene expression in multiple animal models.

Decades ago, Friedmann and Roblin postulated several barriers to gene therapy, including tissue targeting, delivery across the blood–brain barrier (BBB), and host immune responses. These issues remain pertinent till today. Since then, several advances have been made in elucidating structures of adeno-associated virus (AAV) serotypes, antibody epitopes, and ways to modify antibody-binding sites. AAVs capsid has also been engineered to re-direct tissue tropism, reduce ubiquitination, and promote passage across the BBB. Furthermore, the use of high(er) dose recombinant AAV (rAAV) has been accompanied by a better understanding of immune responses in both experimental animals and early clinical trials, and novel work is being performed to modulate the immune response. While the immune responses to rAAV remains a major challenge in translating experimental drugs to approved medicine, and will likely require more than a single solution, we now better understand the hurdles to formulate and test experimental solutions to surmount them.

Recently, adeno-associated virus (AAV)-mediated gene therapies have attracted clinical interest for treating neurodegenerative diseases including spinal muscular atrophy (SMA), Canavan disease (CD), Parkinson’s disease (PD), and Friedreich’s ataxia (FA). The influx of clinical findings led to the first approved gene therapy for neurodegenerative disorders in 2019 and highlighted new safety concerns for patients. Large doses of systemically administered AAV stimulate host immune responses, resulting in anti-capsid and anti-transgene immunity with implications for transgene expression, treatment longevity, and patient safety. Delivering lower doses directly to the central nervous system (CNS) is a promising alternative, resulting in higher transgene expression with decreased immune responses. However, neuroinflammatory responses after CNS-targeted delivery of AAV are a critical concern. Reported signs of AAV-associated neuroinflammation in preclinical studies include dorsal root ganglion (DRG) and spinal cord pathology with mononuclear cell infiltration. In this review, we discuss ways to manage neuroinflammation, including choice of AAV capsid serotypes, CNS-targeting routes of delivery, genetic modifications to the vector and/or transgene, and adding immunosuppressive strategies to clinical protocols. As additional gene therapies for neurodegenerative diseases enter clinics, tracking biomarkers of neuroinflammation will be important for understanding the impact immune reactions can have on treatment safety and efficacy.

Recombinant adeno-associated virus–mediated (rAAV-mediated) gene delivery can efficiently target muscle tissues to serve as “biofactories” for secreted proteins in prophylactic and therapeutic scenarios. Nevertheless, efficient rAAV-mediated gene delivery is often limited by host immune responses against the transgene product. The development of strategies to prevent antitransgene immunity is therefore crucial. The use of endogenous microRNA-mediated (miRNA-mediated) regulation to detarget transgene expression from antigen-presenting cells (APCs) has shown promise for reducing immunogenicity. However, the mechanisms underlying miRNA-mediated modulation of antitransgene immunity by APC detargeting are not fully understood. Using the highly immunogenic ovalbumin (OVA) protein as a proxy for foreign antigens, we show that rAAV vectors containing miR142-binding sites efficiently repress costimulatory signals in DCs, significantly blunt the cytotoxic T cell response, allow for sustained transgene expression in skeletal myoblasts, and attenuate clearance of transduced muscle cells in mice. Furthermore, the blunting of humoral immunity against circulating OVA correlates with detargeting of OVA expression from APCs. This demonstrates that incorporating APC-specific miRNA-binding sites into rAAV vectors provides an effective strategy for reducing transgene-specific immune response. This approach holds promise for clinical applications where the safe and efficient delivery of a prophylactic or therapeutic protein is desired.

Chronic inflammation is a constitutive component of many age-related diseases, including age-related macular degeneration (AMD). Here, we identified interleukin-1 receptor–associated kinase M (IRAK-M) as a key immunoregulator in retinal pigment epithelium (RPE) that declines during the aging process. Rare genetic variants ofIRAK3, which encodes IRAK-M, were associated with an increased likelihood of developing AMD. In human samples and mouse models, IRAK-M abundance in the RPE declined with advancing age or exposure to oxidative stress and was further reduced in AMD.Irak3-knockout mice exhibited an increased incidence of outer retinal degeneration at earlier ages, which was further exacerbated by oxidative stressors. The absence of IRAK-M led to a disruption in RPE cell homeostasis, characterized by compromised mitochondrial function, cellular senescence, and aberrant cytokine production. IRAK-M overexpression protected RPE cells against oxidative or immune stressors. Subretinal delivery of adeno-associated virus (AAV)–expressing humanIRAK3rescued light-induced outer retinal degeneration in wild-type mice and attenuated age-related spontaneous retinal degeneration inIrak3-knockout mice. Our data show that replenishment of IRAK-M in the RPE may redress dysregulated pro-inflammatory processes in AMD, suggesting a potential treatment for retinal degeneration.