Astrocyte-targeted gene delivery of interleukin 2 specifically increases brain-resident regulatory T cell numbers and protects against pathological neuroinflammation

Abstract

The ability of immune-modulating biologics to prevent and reverse pathology has transformed recent clinical practice. Full utility in the neuroinflammation space, however, requires identification of both effective targets for local immune modulation and a delivery system capable of crossing the blood–brain barrier. The recent identification and characterization of a small population of regulatory T (T_reg) cells resident in the brain presents one such potential therapeutic target. Here, we identified brain interleukin 2 (IL-2) levels as a limiting factor for brain-resident T_reg cells. We developed a gene-delivery approach for astrocytes, with a small-molecule on-switch to allow temporal control, and enhanced production in reactive astrocytes to spatially direct delivery to inflammatory sites. Mice with brain-specific IL-2 delivery were protected in traumatic brain injury, stroke and multiple sclerosis models, without impacting the peripheral immune system. These results validate brain-specific IL-2 gene delivery as effective protection against neuroinflammation, and provide a versatile platform for delivery of diverse biologics to neuroinflammatory patients.

Main

Acute central nervous system (CNS) trauma is the leading cause of death and disability for people under the age of 45 years¹. Although the causes of trauma are diverse, the common end result is substantial neuronal damage, or neuronal loss, in the affected region. This is thought to underlie the cognitive, sensorimotor function and personality changes typically seen in patients¹. To date, drug treatments adopting a ‘neuro-centric’ approach have failed to deliver notable clinical benefits for the treatment of CNS injury^1,2, indicating that this approach is too narrow. Acute CNS injury is now recognized as triggering a multicellular response, involving CNS-resident immune cells (microglia and astroglia) alongside infiltration of peripheral immune cells to the brain parenchyma³. While there is evidence to support a neuroprotective effect of immune activation during the initial CNS response, prolonged activation invariably becomes neurotoxic^3,4. The involvement of the immune system allows immune-modulating biologics to emerge as a key therapeutic option. However, adoption of immune-modulating biologics in the neuroinflammatory clinical space first requires identification of biologics with effective anti-inflammatory potential in the CNS, coupled with parallel development of delivery systems capable of crossing the blood–brain barrier.

IL-2 is a high-potential immune-modulating biologic, based on its capacity to support the survival and proliferation of T_reg cells. T_reg cells possess potent immunoregulatory capacity, and are common in the blood and secondary lymphoid organs, with a small population resident in the healthy CNS⁵. While the capacity of IL-2 supplementation to expand circulating T_reg cells and inhibit neuroinflammation has been well demonstrated, these effects can be attributed to T_reg cell function in secondary lymphoid organs. For example, more severe pathology is observed following systemic T_reg cell depletion in mouse models of neuroinflammation, such as the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS)^6,7, or models of stroke^8,9 and traumatic brain injury (TBI)¹⁰. In neuroinflammatory diseases, such as EAE, where T cells trigger the inflammatory cascade¹¹, T_reg cell depletion can enhance peripheral priming and infiltration of neuropathogenic T cells, regardless of any putative role for tissue-resident T_reg cells in the brain. Even in injury-driven neuroinflammation, such as stroke or TBI, the systemic T_reg cell depletion typically used to assess function also drives massive peripheral inflammation¹², with potential pathological consequences¹⁰. The involvement of CNS-resident T_reg cells, as opposed to peripheral-resident T_reg cells, in the control of neuroinflammatory pathology thus remains obscured. This unknown remains one of the key limitations in the clinical utility of IL-2 in the neurology space, with the need to define potential for CNS-based impact, as opposed to systemic effects.

The functional distinction between systemic and CNS-based IL-2 delivery is critical for any therapeutic exploitation in neuroinflammatory disease. Treatments that rely on systemic IL-2 provision to drive expansion of circulating T_reg cells as a mechanism to control CNS inflammation would cause parallel systemic immune suppression and are, therefore, unlikely to see wide adoption in the clinic. By contrast, CNS-specific increases in IL-2 could allow treatment of neuroinflammation without inducing peripheral immunosuppression. Here, we demonstrate the highly efficacious control of neuroinflammation by CNS-based IL-2, using a synthetic biological circuit to drive local production of IL-2 while leaving the peripheral immune system intact. Furthermore, we provide a solution to the biologic delivery problem for the brain, with an adeno-associated virus (AAV)-based therapeutic delivery system capable of providing exquisite temporal and spatial control over biologic production. The demonstrated neuroprotection in four independent neuroinflammatory models provides a clear pathway to clinical exploitation of brain-specific IL-2 gene delivery, and a platform for the delivery of diverse biologics, potentially suitable for broad classes of neuroinflammatory disease and injury.

Results

IL-2 in brain drives T_reg cell expansion and neuroprotection

The potent capacity of T_reg cells to prevent inflammation makes increased IL-2 expression (with its proven ability to expand the T_reg cell population¹³) an attractive therapeutic strategy for neuroinflammatory pathology. In peripheral organs, the main source of IL-2 is activated CD4⁺ conventional T (T_conv) cells. A negative feedback loop between T_reg cells and activated CD4⁺ T cells normally limits IL-2 provision, creating a stable T_reg cell niche¹⁴. In the brain, by contrast, IL-2 levels are ~tenfold lower than the serum (Fig. 1a), with the most common IL-2-producing cell type being neurons (Extended Data Fig. 1). As T_reg cells undergo elevated apoptosis during IL-2-starvation¹³, we sought to overcome IL-2 as a limiting factor using a transgenic model of IL-2 autocrine expression by T_reg cells (Fig. 1b). By effectively bypassing IL-2 silencing, Foxp3^Cre Rosa–IL-2 mice exhibit a profound expansion of peripheral T_reg cell numbers (Fig. 1c). Notably, however, expansion does not occur in the brain (Fig. 1c), with the expansionary effect of increased IL-2 production in the periphery primarily observed on T_reg cells of the circulating phenotype, rather than the brain-resident CD69⁺ population (Fig. 1d and Extended Data Fig. 2a). These results limit the practical utility of peripheral IL-2 dosing to treat neuroinflammatory pathology.