Systemic mRNA Therapy for the Treatment of Fabry Disease: Preclinical Studies in Wild-Type Mice, Fabry Mouse Model, and Wild-Type Non-human Primates.

Fabry disease is an X-linked lysosomal storage disease caused by loss of alpha galactosidase A (α-Gal A) activity and is characterized by progressive accumulation of globotriaosylceramide and its analogs in all cells and tissues. Although enzyme replacement therapy (ERT) is considered standard of care, the long-term effects of ERT on renal and cardiac manifestations remain uncertain and thus novel therapies are desirable. We herein report preclinical studies evaluating systemic messenger RNA (mRNA) encoding human α-Gal A in wild-type (WT) mice, α-Gal A-deficient mice, and WT non-human primates (NHPs). The pharmacokinetics and distribution of h-α-Gal A mRNA encoded protein in WT mice demonstrated prolonged half-lives of α-Gal A in tissues and plasma. Single intravenous administration of h-α-Gal A mRNA to Gla-deficient mice showed dose-dependent protein activity and substrate reduction. Moreover, long duration (up to 6 weeks) of substrate reductions in tissues and plasma were observed after a single injection. Furthermore, repeat i.v. administration of h-α-Gal A mRNA showed a sustained pharmacodynamic response and efficacy in Fabry mice model. Lastly, multiple administrations to non-human primates confirmed safety and translatability. Taken together, these studies across species demonstrate preclinical proof-of-concept of systemic mRNA therapy for the treatment of Fabry disease and this approach may be useful for other lysosomal storage disorders.

mRNA Therapy Improves Metabolic and Behavioral Abnormalities in a Murine Model of Citrin Deficiency.

Citrin deficiency is an autosomal recessive disorder caused by loss-of-function mutations in SLC25A13, encoding the liver-specific mitochondrial aspartate/glutamate transporter. It has a broad spectrum of clinical phenotypes, including life-threatening neurological complications. Conventional protein replacement therapy is not an option for these patients because of drug delivery hurdles, and current gene therapy approaches (e.g., AAV) have been hampered by immunogenicity and genotoxicity. Although dietary approaches have shown some benefits in managing citrin deficiency, the only curative treatment option for these patients is liver transplantation, which is high-risk and associated with long-term complications because of chronic immunosuppression. To develop a new class of therapy for citrin deficiency, codon-optimized mRNA encoding human citrin (hCitrin) was encapsulated in lipid nanoparticles (LNPs). We demonstrate the efficacy of hCitrin-mRNA-LNP therapy in cultured human cells and in a murine model of citrin deficiency that resembles the human condition. Of note, intravenous (i.v.) administration of the hCitrin-mRNA resulted in a significant reduction in (1) hepatic citrulline and blood ammonia levels following oral sucrose challenge and (2) sucrose aversion, hallmarks of hCitrin deficiency. In conclusion, mRNA-LNP therapy could have a significant therapeutic effect on the treatment of citrin deficiency and other mitochondrial enzymopathies with limited treatment options.

Dok-1 overexpression promotes development of γδ natural killer T cells.

In T cells, two members of the Dok family, Dok-1 and Dok-2, are predominantly expressed. Recent evidence suggests that they play a negative role in T-cell signaling. In order to define whether Dok proteins regulate T-cell development, we have generated transgenic mice overexpressing Dok-1 in thymocytes and peripheral T cells. We show that overexpression of Dok-1 retards the transition from the CD4(-) CD8(-) to CD4(+) CD8(+) stage. Moreover, there is a specific expansion of PLZF-expressing Vγ1.1(+) Vδ6.3(+) T cells. This subset of γδ T cells acquires innate characteristics including rapid IL-4 production following stimulation and requiring SLAM-associated adaptor protein (SAP) for their development. Moreover, Dok-1 overexpression promotes the generation of an innate-like CD8(+) T-cell population that expresses Eomesodermin. Altogether, these findings identify a novel role for Dok-1 in the regulation of thymic differentiation and in particular, in the development of PLZF(+) γδ T cells.

Differential role of NF-κB, ERK1/2 and AP-1 in modulating the immunoregulatory functions of bone marrow-derived dendritic cells from NOD mice.

Tolerogenic dendritic cells represent a promising immunotherapy in autoimmunity. However, the molecular mechanisms that drive tolerogenic DCs functions are not well understood. We used GM-CSF or GM-CSF+IL-4 to generate tolerogenic (GM/DCs) and immunogenic (IL-4/DCs) BMDCs from NOD mice, respectively. GM/DCs were resistant to maturation, produced large amounts of IL-10 but not IL-12p70. GM/DCs displayed a reduced capacity to activate diabetogenic CD8(+) T-cells and were efficient to induce Tregs expansion and conversion. LPS stimulation triggered ERK1/2 activation that was sustained in GM/DCs but not in IL-4/DCs. ERK1/2 and AP-1 were involved in IL-10 production in GM/DCs but not in their resistance to maturation. Supershift analysis showed that NF-κB DNA binding complex contains p52 and p65 in GM/DCs, whereas it contains p52, p65 and RelB in IL-4/DCs. ChIP experiments revealed that p65 was recruited to IL-10 promoter following LPS stimulation of GM/DCs whereas its binding to IL-12p35 promoter was abolished. Our results suggest that immunoregulatory functions of GM/DCs are differentially regulated by ERK1/2, AP-1 and NF-κB pathways.

Preclinical Immunogenicity and Efficacy of a Multiple Antigen-Presenting System (MAPSTM) SARS-CoV-2 Vaccine.

Despite the remarkable success of SARS-CoV-2 vaccines, the rise of variants, some of which are more resistant to the effects of vaccination, highlights the potential need for additional COVID-19 vaccines. We used the Multiple Antigen-Presenting System (MAPS) technology, in which proteins are presented on a polysaccharide polymer to induce antibody, Th1, Th17 and CD8+ T cell responses, to engineer a novel vaccine targeting SARS-CoV-2. This vaccine contains a fragment of the spike (S) protein receptor-binding domain (RBD) sequence of the original D614G strain and was used to immunize nonhuman primates (NHP) for assessment of immunological responses and protection against SARS-CoV-2 challenge. The SARS-CoV-2 MAPS vaccine generated robust neutralizing antibodies as well as Th1, Th17 and cytotoxic CD8 T-cell responses in NHPs. Furthermore, MAPS-immunized NHPs had significantly lower viral loads in the nasopharynx and lung compared to control animals. Taken together, these findings support the use of the MAPS platform to make a SARS-CoV-2 vaccine. The nature of the platform also could enable its use for the inclusion of different variants in a single vaccine.

IFN regulatory factors 4 and 8 expression in the NOD mouse.

Dendritic cells (DCs) contribute to islet inflammation and its progression to diabetes in NOD mouse model and human. DCs play a crucial role in the presentation of autoantigen and activation of diabetogenic T cells, and IRF4 and IRF8 are crucial genes involved in the development of DCs. We have therefore investigated the expression of these genes in splenic DCs during diabetes progression in NOD mice. We found that IRF4 expression was upregulated in splenocytes and in splenic CD11c(+) DCs of NOD mice as compared to BALB/c mice. In contrast, IRF8 gene expression was higher in splenocytes of NOD mice whereas its expression was similar in splenic CD11c(+) DCs of NOD and BALB/c mice. Importantly, levels of IRF4 and IRF8 expression were lower in tolerogenic bone marrow derived DCs (BMDCs) generated with GM-CSF as compared to immunogenic BMDCs generated with GM-CSF and IL-4. Analysis of splenic DCs subsets indicated that high expression of IRF4 was associated with increased levels of CD4(+)CD8α(-)IRF4(+)CD11c(+) DCs but not CD4(-)CD8α(+)IRF8(+)CD11c(+) DCs in NOD mice. Our results showed that IRF4 expression was up-regulated in NOD mice and correlated with the increased levels of CD4(+)CD8α(-) DCs, suggesting that IRF4 may be involved in abnormal DC functions in type 1 diabetes in NOD mice.

mRNA therapy restores euglycemia and prevents liver tumors in murine model of glycogen storage disease.

Glycogen Storage Disease 1a (GSD1a) is a rare, inherited metabolic disorder caused by deficiency of glucose 6-phosphatase (G6Pase-α). G6Pase-α is critical for maintaining interprandial euglycemia. GSD1a patients exhibit life-threatening hypoglycemia and long-term liver complications including hepatocellular adenomas (HCAs) and carcinomas (HCCs). There is no treatment for GSD1a and the current standard-of-care for managing hypoglycemia (Glycosade®/modified cornstarch) fails to prevent HCA/HCC risk. Therapeutic modalities such as enzyme replacement therapy and gene therapy are not ideal options for patients due to challenges in drug-delivery, efficacy, and safety. To develop a new treatment for GSD1a capable of addressing both the life-threatening hypoglycemia and HCA/HCC risk, we encapsulated engineered mRNAs encoding human G6Pase-α in lipid nanoparticles. We demonstrate the efficacy and safety of our approach in a preclinical murine model that phenotypically resembles the human condition, thus presenting a potential therapy that could have a significant therapeutic impact on the treatment of GSD1a.

Impact of mRNA chemistry and manufacturing process on innate immune activation.

Messenger RNA (mRNA) represents an attractive therapeutic modality for potentially a wide range of clinical indications but requires uridine chemistry modification and/or tuning of the production process to prevent activation of cellular innate immune sensors and a concomitant reduction in protein expression. To decipher the relative contributions of these factors on immune activation, here, we compared, in multiple cell and in vivo models, mRNA that encodes human erythropoietin incorporating either canonical uridine or N1-methyl-pseudouridine (1mΨ), synthesized by either a standard process shown to have double-stranded RNA (dsRNA) impurities or a modified process that yields a highly purified mRNA preparation. Our data demonstrate that the lowest stimulation of immune endpoints was with 1mΨ made by the modified process, while mRNA containing canonical uridine was immunostimulatory regardless of process. These findings confirm that uridine modification and the reduction of dsRNA impurities are both necessary and sufficient at controlling the immune-activating profile of therapeutic mRNA.

Accelerated Blood Clearance of Lipid Nanoparticles Entails a Biphasic Humoral Response of B-1 Followed by B-2 Lymphocytes to Distinct Antigenic Moieties.

Accelerated blood clearance (ABC) is a phenomenon in which certain pharmaceutical agents are rapidly cleared from the blood upon second and subsequent administrations. ABC has been observed for many lipid-delivery vehicles, including liposomes and lipid nanoparticles (LNP). Previous studies have demonstrated a role for humoral responses against the polyethylene glycol motifs in clearance, but significant gaps remain in our understanding of the mechanism of ABC, and strategies for limiting the impact of ABC in a clinical setting have been elusive. mRNA therapeutics have great promise, but require chronic administration in encapsulating delivery systems, of which LNP are the most clinically advanced. In this study, we investigate the mechanisms of ABC for mRNA-formulated LNP in vivo and in vitro. We present evidence that ABC of mRNA-formulated LNP is dramatic and proceeds rapidly, based on a previously unrecognized ability of LNP to directly activate B-1 lymphocytes, resulting in the production of antiphosphorylcholine IgM Abs in response to initial injection. Upon repeated injections, B-2 lymphocytes also become activated and generate a classic anti-polyethylene glycol adaptive humoral response. The ABC response to phosphorylcholine/LNP-encapsulated mRNA is therefore a combination of early B-1 lymphocyte and later B-2 lymphocyte responses.

Systemic Messenger RNA Therapy as a Treatment for Methylmalonic Acidemia.

Isolated methylmalonic acidemia/aciduria (MMA) is a devastating metabolic disorder with poor outcomes despite current medical treatments. Like other mitochondrial enzymopathies, enzyme replacement therapy (ERT) is not available, and although promising, AAV gene therapy can be limited by pre-existing immunity and has been associated with genotoxicity in mice. To develop a new class of therapy for MMA, we generated a pseudoU-modified codon-optimized mRNA encoding human methylmalonyl-CoA mutase (hMUT), the enzyme most frequently mutated in MMA, and encapsulated it into biodegradable lipid nanoparticles (LNPs). Intravenous (i.v.) administration of hMUT mRNA in two different mouse models of MMA resulted in a 75%-85% reduction in plasma methylmalonic acid and was associated with increased hMUT protein expression and activity in liver. Repeat dosing of hMUT mRNA reduced circulating metabolites and dramatically improved survival and weight gain. Additionally, repeat i.v. dosing did not increase markers of liver toxicity or inflammation in heterozygote MMA mice.

Thymic stromal lymphopoietin and thymic stromal lymphopoietin-conditioned dendritic cells induce regulatory T-cell differentiation and protection of NOD mice against diabetes.

OBJECTIVE: Autoimmune diabetes in the nonobese diabetic (NOD) mouse model results from a breakdown of T-cell tolerance caused by impaired tolerogenic dendritic cell development and regulatory T-cell (Treg) differentiation. Re-establishment of the Treg pool has been shown to confer T-cell tolerance and protection against diabetes. Here, we have investigated whether murine thymic stromal lymphopoietin (TSLP) re-established tolerogenic function of dendritic cells and induced differentiation and/or expansion of Tregs in NOD mice and protection against diabetes. RESEARCH DESIGN AND METHODS: We examined the phenotype of TSLP-conditioned bone marrow dendritic cells (TSLP-DCs) of NOD mice and their functions to induce noninflammatory Th2 response and differentiation of Tregs. The functional relevance of TSLP and TSLP-DCs to development of diabetes was also tested. RESULTS: Our results showed that bone marrow dendritic cells of NOD mice cultured in the presence of TSLP acquired signatures of tolerogenic dendritic cells, such as an absence of production of pro-inflammatory cytokines and a decreased expression of dendritic cell costimulatory molecules (CD80, CD86, and major histocompatibility complex class II) compared with LPS-treated dendritic cells. Furthermore, TSLP-DCs promoted noninflammatory Th2 response and induced the conversion of naïve T-cells into functional CD4(+)CD25(+)Foxp3(+) Tregs. We further showed that subcutaneous injections of TSLP for 6 days or a single intravenous injection of TSLP-DCs protected NOD mice against diabetes. CONCLUSIONS: Our study demonstrates that TSLP re-established a tolerogenic immune response in NOD mice and protects from diabetes, suggesting that TSLP may have a therapeutic potential for the treatment of type 1 diabetes.

Granulocyte-macrophage colony-stimulating factor prevents diabetes development in NOD mice by inducing tolerogenic dendritic cells that sustain the suppressive function of CD4+CD25+ regulatory T cells.

Autoimmune diabetes results from a breakdown of self-tolerance that leads to T cell-mediated beta-cell destruction. Abnormal maturation and other defects of dendritic cells (DCs) have been associated with the development of diabetes. Evidence is accumulating that self-tolerance can be restored and maintained by semimature DCs induced by GM-CSF. We have investigated whether GM-CSF is a valuable strategy to induce semimature DCs, thereby restoring and sustaining tolerance in NOD mice. We found that treatment of prediabetic NOD mice with GM-CSF provided protection against diabetes. The protection was associated with a marked increase in the number of tolerogenic immature splenic DCs and in the number of Foxp3+CD4+CD25+ regulatory T cells (Tregs). Activated DCs from GM-CSF-protected mice expressed lower levels of MHC class II and CD80/CD86 molecules, produced more IL-10 and were less effective in stimulating diabetogenic CD8+ T cells than DCs of PBS-treated NOD mice. Adoptive transfer experiments showed that splenocytes of GM-CSF-protected mice did not transfer diabetes into NOD.SCID recipients. Depletion of CD11c+ DCs before transfer released diabetogenic T cells from the suppressive effect of CD4+CD25+ Tregs, thereby promoting the development of diabetes. These results indicated that semimature DCs were required for the sustained suppressive function of CD4+CD25+ Tregs that were responsible for maintaining tolerance of diabetogenic T cells in NOD mice.

The role of endoplasmic reticulum-associated aminopeptidase 1 in immunity to infection and in cross-presentation.

Endoplasmic reticulum-associated aminopeptidase 1 (ERAP1) is involved in the final processing of endogenous peptides presented by MHC class I molecules to CTLs. We generated ERAP1-deficient mice and analyzed cytotoxic responses upon infection with three viruses, including lymphocytic choriomeningitis virus, which causes vigorous T cell activation and is controlled by CTLs. Despite pronounced effects on the presentation of selected epitopes, the in vivo cytotoxic response was altered for only one of several epitopes tested. Moreover, control of lymphocytic choriomeningitis virus was not impaired in the knockout mice. Thus, we conclude that lack of ERAP1 has little influence on antiviral immunohierarchies and antiviral immunity in the infections studied. We also focused on the role of ERAP1 in cross-presentation. We demonstrate that ERAP1 is required for efficient cross-presentation of cell-associated Ag and of OVA/anti-OVA immunocomplexes. Surprisingly, however, ERAP1 deficiency has no effect on cross-presentation of soluble OVA, suggesting that for soluble exogenous proteins, final processing may not take place in an environment containing active ERAP1.