Objective
Type 1 diabetes (T1D) is a chronic autoimmune disease in which the immune system attacks and destroys the pancreatic beta cells that produce insulin. Without insulin, blood sugar rises uncontrollably, leading to serious complications such as heart disease, kidney failure, nerve damage, and blindness. Insulin injections are lifesaving but do not stop the immune attack or restore normal immune balance. The ultimate goal of this research is to retrain the immune system to tolerate the body’s own cells and prevent the progression of T1D.
The objective of this project is to develop an mRNA-based epigenetic therapy that temporarily silences a key immune gene, TYK2 (Tyrosine Kinase 2), to calm autoimmune inflammation and restore immune tolerance. Instead of permanently altering DNA or suppressing the entire immune system, this therapy will deliver messenger RNA (mRNA) that encodes a gene-silencing protein directly into immune cells. The approach is reversible, precise, and designed to target only the pathways that drive the autoimmune attack.
TYK2 is a signaling protein that amplifies inflammatory responses in immune cells. When overactive, it contributes to the activation of immune pathways that destroy pancreatic beta cells. Genetic studies have shown that people who naturally carry mild TYK2 loss-of-function mutations are protected against several autoimmune diseases, including T1D, while remaining resistant to infection. This makes TYK2 an ideal target for fine-tuned immune regulation.
The therapeutic system proposed in this project combines three technologies. First, an mRNA molecule will encode dCas9-KRAB, a programmable “epigenetic silencer” that can turn down specific genes without cutting DNA. Second, a guide RNA will direct dCas9-KRAB to the TYK2 gene, reducing its expression and the inflammatory signaling it controls. Third, the therapy will be delivered using lipid nanoparticles (LNPs), tiny fat-like particles that protect the mRNA and carry it into the right immune cells, such as macrophages and dendritic cells. These cells play a critical role in regulating immune balance, making them ideal targets for this approach.
The research will pursue three Specific Aims:
Aim 1: Design and validate mRNA constructs that encode dCas9-KRAB together with TYK2-targeting guide RNAs. These constructs will be tested in cultured immune cells to confirm their ability to silence TYK2 expression and reduce inflammatory cytokines such as IL-6 and IFN-β.
Aim 2: Develop and optimize lipid nanoparticle formulations that deliver mRNA efficiently to immune cells. The team will test different combinations of ionizable lipids and helper components to identify formulations that maximize uptake and gene-silencing activity in macrophages and dendritic cells while minimizing off-target delivery.
Aim 3: Evaluate the optimized mRNA-LNP therapy in Non-Obese Diabetic (NOD) mice, a well-established model of T1D. The study will measure TYK2 suppression, inflammatory markers, and glucose control, and determine whether the therapy protects beta cells and delays or prevents diabetes onset.
The short-term objectives of this project are to demonstrate that TYK2 can be selectively silenced in immune cells using mRNA-based delivery and to show that this reduces autoimmune inflammation in preclinical models. These experiments will establish the feasibility of using mRNA as a platform for transient and reversible immune modulation.
The long-term objectives are to build a modular therapeutic platform that can be adapted to other autoimmune diseases by simply changing the target gene. The ultimate vision is to translate this technology into safe, personalized treatments that restore immune tolerance without lifelong immunosuppression. In the context of T1D, such a therapy could prevent the onset of disease in high-risk individuals or preserve residual beta-cell function in those newly diagnosed.
Background Rationale
Type 1 diabetes (T1D) is an autoimmune disorder in which the immune system destroys the pancreatic beta cells responsible for producing insulin. The loss of these cells results in lifelong insulin dependence and a high risk of complications such as heart disease, kidney failure, and neuropathy. Despite major progress in glucose management and insulin delivery, no therapy has yet succeeded in halting or reversing the autoimmune attack that drives the disease. The inability to restore immune tolerance remains the greatest barrier to a lasting cure.
In a healthy immune system, self-reactive immune cells are either eliminated or controlled by mechanisms that maintain tolerance. In T1D, this balance breaks down. Antigen-presenting cells such as macrophages and dendritic cells become hyperactive, releasing cytokines that recruit and activate T cells against self-antigens in pancreatic tissue. Over time, chronic inflammation leads to irreversible destruction of insulin-producing beta cells. Current treatments that target the immune system, including broad immunosuppressants and monoclonal antibodies, reduce inflammation only temporarily and often compromise the body’s ability to fight infection. A more precise therapeutic strategy is required—one that selectively regulates the molecular pathways responsible for autoimmunity without weakening overall immunity.
Advances in human genetics have identified molecular targets that contribute to autoimmune susceptibility. Among these, TYK2 (Tyrosine Kinase 2) has emerged as a particularly compelling candidate. TYK2 is a signaling enzyme involved in the JAK-STAT pathway, which mediates cellular responses to interferons and interleukins. When overactivated, TYK2 amplifies inflammatory signaling cascades that sustain autoimmune reactions. Importantly, individuals who naturally carry partial loss-of-function mutations in TYK2 are protected from several autoimmune diseases, including T1D, multiple sclerosis, and systemic lupus erythematosus. These individuals maintain normal immune defense, demonstrating that controlled TYK2 inhibition can achieve disease protection without compromising immunity. This genetic evidence provides a strong biological rationale for therapeutic targeting of TYK2 in T1D.
Conventional approaches to suppress TYK2 signaling, such as small-molecule inhibitors, often affect multiple JAK family kinases, leading to undesirable side effects. Gene-editing technologies, while more specific, permanently alter the genome and raise significant safety and ethical concerns. To overcome these challenges, the proposed research introduces an mRNA-based epigenetic silencing approach that achieves selective, reversible suppression of TYK2 expression at the transcriptional level.
This strategy employs messenger RNA encoding dCas9-KRAB, a deactivated Cas9 enzyme fused to the KRAB repressor domain. The protein functions as a transcriptional regulator rather than a DNA-cutting enzyme. When guided by a specific RNA sequence that targets the TYK2 promoter, it binds to the gene and recruits chromatin-modifying complexes that silence transcription. This process is epigenetic, meaning it regulates gene activity without altering the DNA sequence itself. Because the effect is mediated by mRNA, gene repression is transient and naturally reversible, offering a level of safety and control not achievable with permanent genome editing.
A key challenge in realizing this concept is the delivery of mRNA to the correct immune cell populations. The project addresses this through the design of lipid nanoparticles (LNPs), nanoscale carriers composed of biocompatible lipids. LNPs protect mRNA from degradation and promote efficient cellular uptake. Their composition can be adjusted to favor uptake by specific immune cells. The proposed work will optimize LNP formulations to achieve high transfection efficiency in macrophages and dendritic cells, which are central regulators of inflammation and antigen presentation in T1D.
Description of Project
Type 1 diabetes (T1D) is an autoimmune disease in which the body’s immune system mistakenly destroys the insulin-producing beta cells in the pancreas. Without insulin, the body cannot properly control blood sugar levels, leading to lifelong dependence on insulin injections and a high risk of serious complications such as heart disease, kidney failure, and blindness. While insulin therapy is lifesaving, it does not address the underlying immune dysfunction. The central goal of T1D research is to stop the immune attack itself and restore immune tolerance, allowing the body to regulate its own blood sugar naturally.
This project aims to develop a new kind of RNA-based medicine that can retrain immune cells at the genetic level to restore tolerance in T1D. Instead of using drugs that broadly suppress the immune system, this approach targets a specific gene that drives inflammation and immune attack: TYK2 (Tyrosine Kinase 2). TYK2 helps control how immune cells respond to inflammatory signals. Genetic studies have shown that people who naturally carry mild loss-of-function mutations in TYK2 are protected from autoimmune diseases, including T1D, while maintaining normal immunity against infections. This discovery makes TYK2 a promising target for precise and reversible immune reprogramming.
The proposed therapy uses messenger RNA (mRNA), the same type of molecule that powers COVID-19 vaccines, but with a very different purpose. Instead of instructing cells to make a viral protein, the mRNA in this project will produce a programmable gene-silencing tool called dCas9-KRAB. This molecule works like a genetic dimmer switch, reducing the expression of specific genes without permanently altering DNA. When combined with a short guide RNA that directs it to TYK2, the dCas9-KRAB system can temporarily silence TYK2 activity in immune cells. This process is called epigenetic silencing, meaning that it regulates gene function without changing the genetic code. By silencing TYK2 in key immune cells, the therapy aims to reduce inflammation and prevent the immune system from attacking pancreatic beta cells.
A key part of the project involves developing a safe and effective way to deliver the mRNA into immune cells. For this purpose, the research will use lipid nanoparticles (LNPs), tiny fat-based particles that protect the mRNA and carry it into target cells. LNPs are already proven safe and effective in humans and can be chemically tuned to reach specific tissues. The team will optimize LNP formulations that efficiently deliver the therapeutic mRNA into immune cells involved in T1D, such as macrophages and dendritic cells, which are responsible for controlling inflammation and tolerance.
The project will advance through three main stages. First, the researchers will design and test mRNA molecules that encode the dCas9-KRAB silencing machinery along with TYK2-specific guide RNAs. These constructs will be tested in immune cells to confirm that they effectively reduce TYK2 expression and inflammatory signaling. Second, the optimized mRNA will be packaged into different LNP formulations and tested for delivery efficiency and functional silencing in relevant immune cell types. Finally, the best-performing formulation will be evaluated in NOD mice, a well-established animal model of T1D, to determine whether temporary TYK2 silencing can delay or prevent disease onset. The team will monitor blood glucose, immune-cell activity, and pancreatic tissue health to assess the therapeutic outcome.
If successful, this project will provide the first demonstration of an mRNA-based epigenetic immunotherapy for autoimmune diseases. Unlike traditional gene editing, this approach does not permanently modify DNA, making it reversible and potentially safer. The outcome of this work could lay the foundation for a new class of personalized, safe, and durable therapies for restoring immune balance and improving the quality of life for people living with T1D.
Anticipated Outcome
This project aims to generate both scientific insight and practical innovation in the development of mRNA-based therapies for autoimmune diseases. By the end of the study period, the research is expected to provide clear experimental evidence that transient, mRNA-mediated epigenetic silencing of the TYK2 gene can reduce inflammation and restore immune balance in Type 1 diabetes (T1D). The anticipated outcomes will be evaluated at molecular, cellular, and organismal levels to determine feasibility, safety, and translational potential.
The first anticipated outcome is demonstration of successful epigenetic repression of TYK2 in immune cells using synthetic mRNA. The study will confirm that mRNA encoding the transcriptional regulator dCas9-KRAB can be efficiently delivered into macrophages and dendritic cells to silence TYK2 expression. Quantitative analysis will measure the extent and duration of gene suppression, the reduction of downstream signaling activity, and normalization of cytokine release. These findings will show that transient mRNA-driven epigenetic modulation can precisely control an inflammatory pathway implicated in T1D without permanent DNA alteration.
The second major outcome is the optimization of lipid nanoparticles (LNPs) for immune-cell–specific mRNA delivery. By systematically varying lipid composition, charge, and particle size, the study will identify LNP formulations that achieve high transfection efficiency with minimal toxicity. Successful formulations will demonstrate selective uptake by macrophages and dendritic cells while avoiding non-target tissues. Establishing this relationship between formulation parameters and immune-cell tropism will create a design framework for future immune-targeted RNA therapeutics.
At the cellular and functional level, the anticipated outcome is restoration of immune tolerance through reduced inflammatory signaling. TYK2 silencing is expected to suppress key immune-activation pathways, lowering production of cytokines such as IL-6, IFN-β, and IL-12p70. Flow cytometry and immune profiling will verify a shift toward less inflammatory cell states. These results will support the concept that targeted repression of one key gene can reprogram immune behavior toward a tolerogenic profile, rather than requiring broad immunosuppression.
In animal studies using Non-Obese Diabetic (NOD) mice, the expected outcome is protection of pancreatic beta cells and delay in the onset of hyperglycemia. Treated mice are anticipated to exhibit lower systemic inflammation, improved glucose regulation, and preservation of islet structure. Histological and molecular analyses will demonstrate that transient TYK2 inhibition can achieve therapeutic benefit without long-term toxicity or off-target effects. This outcome will provide the first in vivo evidence that temporary, mRNA-guided gene silencing can alter autoimmune disease progression.
Beyond experimental validation, the project will yield a generalizable mRNA-LNP platform for epigenetic modulation of gene expression. The findings will define the key parameters that determine silencing strength, delivery efficiency, and reversibility. This knowledge will be valuable for adapting the technology to other immune genes relevant to autoimmune and inflammatory diseases. The resulting platform could serve as a versatile tool for fine-tuning immune responses in diverse pathological settings.
In the broader context, the research will also produce quantitative benchmarks for the duration, magnitude, and safety profile of mRNA-based gene repression. These data will guide future translational studies and support the design of early-stage clinical candidates. By integrating molecular, cellular, and animal-level validation, the study will provide a comprehensive demonstration that transient, epigenetic control of immune signaling is both feasible and effective.
The ultimate anticipated outcome is to establish a new paradigm for treating autoimmune diseases: one that uses temporary gene regulation to restore immune equilibrium rather than permanent genetic modification or systemic immunosuppression. The successful completion of this project will mark a critical step toward developing precision RNA medicines capable of reprogramming immune function safely, reversibly, and specifically.
Relevance to T1D
Type 1 diabetes (T1D) is a life-long autoimmune disease that develops when the body’s immune system attacks the pancreatic beta cells responsible for producing insulin. Once these cells are destroyed, blood glucose control becomes dependent on external insulin administration. Although modern insulin delivery systems have improved glucose management, they do not address the autoimmune process that drives disease progression. This persistent immune attack makes T1D fundamentally different from other forms of diabetes and underscores the urgent need for treatments that target its immunological cause rather than its symptoms.
The proposal is directly relevant to this need because it seeks to restore immune tolerance by correcting a specific signaling abnormality known to trigger autoimmune activation. The project targets TYK2 (Tyrosine Kinase 2), a signaling enzyme that regulates multiple inflammatory pathways, including type I interferon and interleukin signaling. These pathways are hyperactivated in the immune cells of patients with T1D, contributing to chronic inflammation and destruction of pancreatic tissue. Genetic studies have identified TYK2 as a risk gene for several autoimmune diseases, including T1D, and have shown that individuals with naturally reduced TYK2 activity are protected from autoimmunity without experiencing immune deficiency. Replicating this protective state in a controlled and reversible manner represents a rational therapeutic strategy for T1D.
The proposed approach introduces messenger RNA (mRNA) therapeutics as a platform for precise modulation of TYK2 expression. mRNA can be engineered to produce a regulatory protein, dCas9-KRAB, that silences the TYK2 gene epigenetically—altering gene activity without modifying the DNA sequence. This form of temporary repression mimics the protective effect of genetic variants while avoiding the risks associated with permanent gene editing. Because mRNA naturally degrades after a defined period, the intervention is self-limiting and adaptable to individual treatment schedules. For T1D, this provides a safe, reversible way to reduce inflammatory signaling during critical windows of immune dysregulation, such as the early stages of beta-cell loss.
The project is also relevant to T1D because it focuses on immune-cell–specific delivery. The therapy will employ lipid nanoparticles (LNPs), a clinically proven system for delivering RNA medicines. LNPs will be optimized to target macrophages and dendritic cells, the immune populations that coordinate antigen presentation and cytokine signaling in the pancreas. These cells are central to the initiation and propagation of autoimmune responses in T1D. By reducing TYK2 activity in these cells, the therapy is expected to dampen inflammatory signaling and decrease the activation of autoreactive T cells that destroy beta cells. This targeted modulation of upstream immune responses has the potential to preserve remaining insulin-producing cells and delay or prevent disease onset.
Relevance extends to both prevention and intervention. In individuals at genetic or autoantibody-defined risk of developing T1D, transient TYK2 silencing could reduce inflammatory activation and maintain immune tolerance before beta-cell destruction becomes irreversible. In patients with established disease, the same approach could protect residual beta-cell mass, improve insulin production, and stabilize glucose control. By targeting an upstream regulator of inflammation, this research addresses a critical bottleneck in T1D therapy—how to reset immune balance without suppressing normal defense mechanisms.
From a translational perspective, the proposal aligns with the growing emphasis in T1D research on immune reprogramming and disease modification. It builds directly on human genetic evidence, integrates proven mRNA and nanoparticle technologies, and focuses on mechanisms central to the pathogenesis of T1D. The expected results will clarify how transient, gene-specific modulation of immune signaling can alter disease trajectory and create a foundation for precision RNA medicines tailored to autoimmune conditions driven by similar pathways.