Objective
The overarching objective is to chart the molecular events triggered by LRH-1/NR5A2 activation in macrophages and dendritic cells that attenuate the pro-inflammatory phenotype and the subsequent impact on the global immune cell landscape and β cell regeneration via a wound healing trans-differentiation mechanism. The latter is indispensable for the development of a safe disease modifying therapy for T1DM using second-generation compounds such as BL003/ARD-008 that will also be evaluated herein.
Background Rationale
In a first round of JDRF funding (#17-2013-372), we were able to authenticate the safety and the therapeutic value of BL001-mediated LRH-1/NR5A2 activation in preventing and blunting development of hyperglycaemia in 3 preclinical mouse models of T1DM. This protection was conveyed by the induction of a favourable anti-inflammatory immune cell profile that promoted regeneration through alpha-to-beta cell conversion and increased beta cell survival. Consequently, we termed this process immune coupled regeneration which is reminiscent to wound healing. In a second round of JDRF funding (2-SRA-2019-837-S-B), we translated to humans our mouse data showing that activation of LRH-1/NR5A2 can endow an anti-inflammatory phenotype to monocytes-derived macrophages and dendritic cells obtained from T1DM individuals leading to suppression of killer T-cells. Regulatory T-cells, important to maintain self-tolerance were also stimulated by BL001. Importantly, BL001 was able to favor human islet engraftment and function in immune competent mice opening new venues for an immune tolerant rather than suppressive therapy for human transplantation using either organ donor- or induced pluripotent stem cell (iPSCs)-derived islets. The latter is of major relevance in view of recent transplantation clinical trial successes using either stem cells or iPSCs-derived islet-like cells in individuals with T1DM. The rationale of the current project is to develop an optimal and safe disease modifying therapy based on LRH-1/NR5A2-mediated immune couple regeneration without compromising the general immune response. To this end we propose to determine. 1) whether wound healing targets highlighted in our mice studies are also found in a human context, 2) the molecular and cellular events that relay the beneficial impact of BL001 on human macrophages and dendritic cells, 3) the impact of BL001 as well as immune cells on trans-differentiation of islet cells, 4) the net outcome of BL001 on mixed immune cell populations, alone or in combination with islet cells that recapitulate the in vivo condition, 5) whether age and gender alters the effect of BL001 and 6) the benefits of BL001 as well as of a second-generation agonist in long term transplantation studies using human islets as well as iPSCs-derived islet clusters.
Description of Project
Type 1 Diabetes Mellitus (T1DM) stems from the destruction of insulin-producing beta cells by one's own immune system resulting in hyperglycaemia. For the past several years we have claimed that an effective disease modifying therapy for T1DM must simultaneously attenuate/resolve (and not supress) the autoimmune attack which will in turn promote beta cell regeneration. We have coined this process as immune coupled regeneration which is reminiscent to wound healing. With the support of the JDRF, we have shown that activation of the nuclear receptor LRH-1 also known as NR5A2 using a small chemical compound denoted as BL001, developed ‘in house’, favors immune coupled regeneration in preclinical mouse models of T1DM reverting hyperglycemia. Glycaemia recovery correlated with the presence anti-, rather than pro, -inflammatory immune cells within the pancreas, reduced insulitis and restoration of the beta cell mass through alpha-to-beta trans-differentiation. Genetic analysis of trans-differentiating cells revealed the activation of genes involved in wound healing and cell development. More recently, these findings were translated to a human setting in which we demonstrate that BL001 can endow an anti-inflammatory phenotype to monocytes-derived macrophages and dendritic cells obtained from T1DM individuals leading to suppression of killer T-cells. Regulatory T-cells, important to maintain self-tolerance were also stimulated by BL001. BL001 was also able to favor human islet engraftment and function in immune competent mice opening new venues for an immune tolerant rather than suppressive therapy for human transplantation using either organ donor- or induced pluripotent stem cell (iPSCs)-derived islets. The latter is of major relevance in view of recent transplantation clinical trial successes using either stem cells or iPSCs-derived islet-like cells in individuals with T1DM. In the context, of developing a safe and efficacious disease modifying therapy based on BL001/LRH-1/NR5A2-mediated immune couple regeneration, we propose, in this project, to assess whether wound healing targets highlighted in mice are also found in a human context and to thoroughly characterize the molecular and cellular events that relay the beneficial impact of BL001 on human immune cells. We also propose to assess the global effect of BL001 on mixed immune cell populations, alone or in combination with islet cells that recapitulate the in vivo condition and determine whether age or gender may influence the therapeutic outcome of BL001. In parallel, the immune-coupled regeneration properties of a second-generation agonist will be evaluated in long term transplantation studies using human islets as well as iPSCs-derived islet clusters. Four objectives will be addressed: 1) Reconstruction of the wound healing trajectory coupled to alpha-to-β cells trans-differentiation in humans, 2) Define the contribution of individual and collective BL001/LRH-1-targeted immune cell subtypes in promoting an anti-inflammatory and islet cell survival/regeneration milieu and 3) Modeling of T1DM in vitro to assess BL001 empowerment to promote a holistic anti-inflammatory environment favoring beta cell survival and 4) Assess the therapeutic efficacy of BL001/BL003/ARD-008 on both human islets and iPSCs-derived islet organoids xenotransplantation, engraftment, maturation and function. The latter will open a new venue for a safe T1DM mono-drug immune coupled regeneration therapy that can also be implemented as an immuno-tolerizing therapy for islet transplantation.
Anticipated Outcome
As bi-hormonal (INSULIN/GLUCAGON) cells are detected in pancreas of T1DM donors, indicative of regeneration, we anticipate that similar wound healing and cell development targets identified in mice will also be detected in human pancreas. The latter will provide proof-of-concept that similar targets maybe induced by activation of LRH-1/NR5A2 in humans thereby promoting beta cell trans-regeneration. The repertoire of markers may be expanded to cover additional targets associated to wound healing and trans-regeneration in order to reconstruct as accurately as possible these coupled processes in humans. We also anticipate to confirm the implication of two intracellular organelles (the mitochondria and lysosome) in relaying the anti-inflammatory properties of BL001 to macrophages and dendritic cells derived from monocytes of individuals with T1DM. Furthermore, we foresee that BL001 will induce a global anti-inflammatory landscape to a mixed immune cell population which will mimic the in vivo effect of the compound. Based on preliminary results we also expect that human islets cultured in the presence of condition media derived from BL001-treated immune cells will exhibit alpha-to-beta cell trans-differentiation. Xenotransplantation experiments will provide proof-of-concept that BL001 as well as BL003/ARD-008-mediated activation of LRH-1/NR5A2 can enhance long-term engraftment, function and trans-regeneration of human islets as well as iPSCs-derived islets within an immune hostile and diabetic environment. The outcome of these studies will advance our understanding of immune coupled beta cell regeneration and this knowledge should accelerate development of an unprecedented mono drug therapy for T1DM.
Relevance to T1D
Despite advances in medical device technology and longer-acting insulin as well as strives in generating in vitro insulin-producing cells from various cell sources, there is still no robust therapy to either specifically dampen the immune attack or to substitute and protect beta cells that are lost in T1DM. Discovery of a single ‘druggable’ target with the property to promote regeneration of new insulin-producing cells through reprogramming of alpha cells with the assistance of tissue remodelling immune cells is highly significant to T1DM patients. This indispensable integrated non-mutually exclusive system may provide some clues on failure of clinical trial targeting solely either the immune system or islet regeneration in patients with T1DM. To date, such target remains elusive. LRH-1/NR5A2 may be the first target of its class with such properties and translation of its beneficial effects observed in mice to human is mandatory in order to move forward towards an unprecedented disease modifying T1DM therapy.