Gene-Specific Models and Therapies for Type One Diabetes

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Gene-Specific Models and Therapies for Type One Diabetes

jeremy_racine_lab Project Researcher: Jeremy Racine, Ph.D. – The Jackson Laboratory

Project Description

We like to think of type 1 diabetes as a disease that has one all-encompassing cure, but because multiple genetic factors can contribute to disease susceptibility, it is likely to be more involved than just one simple fix. As researchers studying the disease, we have been limited to using a model with one genetic profile – the non-obese diabetic (NOD) mouse. This model doesn’t represent the genetic diversity of children and adults with type 1 diabetes. Development of a suite of mice that express human diabetes-relevant gene variants in various combinations will allow scientists to develop therapies for specific genetics. In this way, we will be better suited to find the necessary cures for different genetic profiles that make up the disease collectively called type 1 diabetes.
Overall, this project has two goals. First, the project aims to generate a new mouse model with a genetic “blank slate” for insertion of relevant human HLA gene variants that are linked to diabetes development. Second, the project will test a therapy that has been specifically designed for a diabetes susceptible gene variation known as HLA-A*0201 (A2.1).

Project Details

More than 200,000 children and 1 million adults have type 1 diabetes (T1D). T1D is a disease in which the immune system kills the insulin-producing cells of the pancreas. Much of our knowledge on the biology of the disease comes from the “non-obese diabetic (NOD)” mouse model. Many of the genes that contribute to the disease in the NOD mouse have been similarly identified in children with diabetes.

With T1D incidence on the rise in children (21% increase from 2001-2009), it’s essential to develop therapies for children at risk for or recently diagnosed with the disease. We tend to think of T1D as one disease, needing one cure, when in reality multiple genetic traits can give rise to the same symptoms we collectively call T1D. Our current pre-clinical mouse model, the NOD mouse, is a single genetic profile that doesn’t represent the diverse nature of the diabetic patient population. Even a therapy that effectively prevents progression to diabetes or reverses it in the NOD mouse is unlikely to similarly work for children with a wide variety of different genetic traits.

My goal is to make new mouse models that better represent the genetic diversity of children with diabetes in order to provide improved platforms for testing trait-specific potential therapies. Many genes contribute to diabetes development, but none more so than the genes known as the human leukocyte antigens (HLA). Certain variants of these genes, of which there are Class I and Class II varieties, can confer risk for or protection from the disease. If we could create mouse models in which the mouse versions of these genes are replaced with different human variants, we would then have a suite of new models that are specific for genetic traits found in diabetic children. With these new models, we could design therapies tailored for specific genetic profiles, like the vaccine-like therapy described below.

While Class II HLA variants in humans and NOD mice are clearly important contributors to T1D development, the cells that ultimately kill the insulin producing cells require particular Class I gene variants to be present. In fact, complete loss of the mouse Class I variants (through disruption of an associated gene, β2m, that is required for expression of the Class I molecules) prevents diabetes development in the NOD mouse. Combining the mutation to β2m with the human Class I variant HLA-A*0201 (HLA-A2.1) in NOD mice created a model in which therapies could be developed specifically for patients with this particular Class I variation. The new model allowed for the identification of the exact targets (peptides) against which the immune cells respond in patients that carry this Class I variant.

The overall goal of this project is two-fold: 1) Generate a next generation blank-slate model for developing therapies tailored to specific combinatorial genetic characteristics; 2) Further develop a “peptide-vaccination” therapy designed for the specific genetic variant HLA-A2.1.

The first generation model, as well as two related models specific for additional Class I genetic variations, all, rely on the indirect mutation to β2m in order to remove corresponding mouse molecules. Unfortunately, this mutation makes these models ineffective for developing antibody-based therapies. The next generation of these mouse models will accomplish two things: 1) They will not rely on this indirect mutation to β2m, and 2) they will allow for a combination of human Class I and Class II variants linked to diabetic children. I have recently succeeded in removing the two mouse Class I genes directly in the NOD mouse, which accomplishes the first goal to no longer rely on the β2m mutation to prevent expression of the mouse Class I molecules. This proposal aims to address the 2nd goal of these next generation models by directly removing the Class II gene found in NOD mice. In this way, a new “blank slate” mouse model will be created, in which Class I or Class II HLA found in diabetic children can be added back to the mouse in any combination, based on specific patient profiles. As an example, with this mouse, therapies can be devised based upon a profile of HLA-A2.1+ paired with one of several relevant Class II variants already available. Future modifications to this new model would also allow the injection of cells isolated from the blood of diabetic children, in order to directly pre-test the therapy on the specific cell populations found in those specific children.

While working on developing these next generation mouse models, we have devised a candidate therapy utilizing the first generation HLA-A2.1 model. Having identified the specific peptides toward which the immune system responds in HLA-A2.1+ individuals, we have devised a “vaccination-like” therapy in which these peptides are coupled to a small “microsphere.” Delivery of the therapy protects mice from diabetes when initiated at a young age. Exactly how this method re-educates the immune system still needs to be determined. It also needs to be determined if this therapy is effective in a more clinically relevant treatment window. Work on this specific therapy will help inform the development of similar therapies for other specific targets to be identified for children carrying different Class I variants.

Gene-Specific Models and Therapies for Type 1 Diabetes Slideshare

About Me

I was a biochemistry major at Cornell University and had every intention of eventually applying to medical school. After graduation, I worked for three years as a research technician in a genetics laboratory at Harvard Partners Center for Genetics and Genomics/Brigham & Women’s Hospital and realized that I wanted to approach medicine from the side of research and development. With this in mind, I made the decision to attend the Irell & Manella Graduate School of Biological Sciences at the Beckman Research Institute of the City of Hope in Duarte, CA in the fall of 2007. I did my thesis work in the laboratory of Dr. Defu Zeng. After graduation in 2013, I stayed with Dr. Zeng for an additional year before joining Dr. Dave Serreze at The Jackson Laboratory, in Bar Harbor, Maine.

My thesis project focused on using bone marrow transplantation as a curative therapy for type 1 diabetes. During my seven years with Dr. Zeng, as both a graduate student and a postdoctoral fellow, I studied how a non-autoimmune-prone donor’s immune system could re-educate the immune system of a diabetes-prone mouse. Towards the end of my tenure with Dr. Zeng, I realized that in order to develop therapies for autoimmune diseases like type 1 diabetes, we needed better models than the standard mouse models with which we were working. In order to develop new models, however, I needed additional training. With this in mind, I decided to join the laboratory of Dr. Dave Serreze at The Jackson Laboratory, as he had started to develop the first generation of mice carrying specific human gene variants linked to diabetes in children, and he was the ideal mentor for this next phase of my training.

My research goal is to design a suite of mouse models that express various combinations of clinically relevant human genes that contribute to diabetes development. In this way, children with a specific combination of traits could have their own corresponding mouse model in which to develop and validate trait-specific therapies.
When I am not working in the laboratory, I spend time with my wife, Toni, renovating the 100-year-old house we share with our four cats: Monkey, Winkin, Blinkin, and Nod.

Project Updates

 

Final Update

In part through the contributions of the DRC, I have now published a manuscript in Diabetes https://doi.org/10.2337/db17-1467 detailing the creation of several new mouse models for type I diabetes (T1D) research and therapy development. Genes encoding what are called major histocompatibility complex (MHC) proteins (designated HLA in humans) are the primary contributors to T1D development in both humans and the NOD mouse model.  Using a genetic engineering technique known as CRISPR/Cas9, I have eliminated the murine MHC encoding genes in NOD mice and replaced them with human counterparts called HLA-A2 and B39 hypothesized to be important in T1D development. The HLA-A2 genetic variant is found in a sizeable portion of Caucasian type I diabetic patients. HLA-B39, while being a rare variant, has been associated with patients that have extreme early onset T1D.  Production of these genetically engineered NOD mice allows them to be used to develop and therapies tailored specifically to patients expressing these HLA molecules. Work is currently ongoing using these mice to test a therapy tailored to HLA-A2 since this molecule characterizes a preponderance of T1D patients.

Another CRISPR generated ND mouse detailed in the above manuscript will allow for the expression in the absence of murine counterparts of combinations of HLA molecules contributing to T1D development in humans. Additionally, these models are being further engineered to allow the future transplantation of immune cells from diabetic patients in order to test drugs in mouse-“test-tubes” before attempting their use in patient populations.

While it is disappointing that the therapy did not translate from the old model to the new, I think that points to the utility of these new model systems and their potential to improve the translation of therapies from bench to clinic. I’ve already had requests for the next step model I am currently developing from this work, that would allow the transplantation of immune cells from diabetic patients. I have also provided all the mouse models described in my Diabetes paper to The Jackson Laboratory repository, so they will soon be available to the entire Diabetes research community.

Update on 3-14-18

In part through the contributions of the DRC, I have now published a manuscript in Diabetes https://doi.org/10.2337/db17-1467 detailing the creation of several new mouse models for type I diabetes (T1D) research and therapy development. Genes encoding what are called major histocompatibility complex (MHC) proteins (designated HLA in humans) are the primary contributors to T1D development in both humans and the NOD mouse model.  Using a genetic engineering technique known as CRISPR/Cas9, I have eliminated the murine MHC encoding genes in NOD mice and replaced them with human counterparts called HLA-A2 and B39 hypothesized to be important in T1D development. The HLA-A2 genetic variant is found in a sizeable portion of Caucasian type I diabetic patients. HLA-B39, while being a rare variant, has been associated with patients that have extreme early onset T1D.  Production of these genetically engineered NOD mice allows them to be used to develop and therapies tailored specifically to patients expressing these HLA molecules. Work is currently ongoing using these mice to test a therapy tailored to HLA-A2 since this molecule characterizes a preponderance of T1D patients.

Another CRISPR generated ND mouse detailed in the above manuscript will allow for the expression in the absence of murine counterparts of combinations of HLA molecules contributing to T1D development in humans. Additionally, these models are being further engineered to allow the future transplantation of immune cells from diabetic patients in order to test drugs in mouse-“test-tubes” before attempting their use in patient populations.

Update on 10-10-17

Just prior to initiation of this project, I successfully removed the two MHC Class I genes found in NOD mice. I proposed to target the Class II variant found in these NOD.cMHC1-/- mice in order to generate a new “blank slate” mouse model (NOD.cMHC-/-) to allow the introduction of Class I or Class II HLA combinations prevalent in diabetic patient populations. I have now successfully generated this mouse and am in the final process of characterizing it and plan on submitting a manuscript in the coming month(s) so that the entire diabetic research field has access to this new model.  Additionally, I have shared this mouse with a collaborator who is in the process of moving these new mutations into an immunodeficient mouse model (NSG mice) so that human blood cell populations can be transferred into these mice and potential immune therapies can be tested. This novel immunodeficient version will have utility far beyond the field of diabetes.  The next step in this project is to introduce two HLA Class I variants found in subsets of diabetic patients, either HLA-A2.1 or our newly characterized-in-mice HLA-B*39:06.  Once these genes have been introduced, HLA Class II variants will then be introduced into these two new models.

While working on developing this next generation mouse model, I proposed to begin testing a  “vaccination-like” therapy in which insulin and IGRP peptides are coupled to small “microspheres.” Recently completed analyses in the first-generation mouse model (NOD.β2m-/-.HLA-A2.1) indicates that when injections are initiated just prior to diabetes onset, the combination therapy with all four peptides drastically reduced progression to diabetes. Treatment with the two insulin peptides was partially effective, and treatment with the two IGRP peptides was no better than controls.  Additionally, preliminary experiments indicate that short-term therapy (3 injections) is close to providing some protection compared to controls, and additional cohorts are now being followed to determine if there is in fact a statistical significance in protection. Studies using the next generation mouse model NOD.cMHC1-/-.HLA-2.1 are now underway to compare the findings to the results with the first-generation model.  This next generation model provides us the opportunity to determine whether this therapy shows efficacy after the appearance of anti-insulin autoantibodies, or whether it has to be initiated in as-risk individuals prior to the appearance of these antibodies in circulation. The next step in this project is to characterize the mechanisms of disease protection and to determine whether anti-insulin autoantibody status prior to initiation of treatment affects efficacy of the therapy.

Update on 5/19/17

This project has two aims, both of which have been initiated. The first aim of this project is to create new models that carry specific genetic traits found in the type 1 diabetes patient population so that therapies are designed with more specificity. To accomplish this, the mouse model currently used in research has to be prepared to receive these new genetic traits by first removing the mouse genes for these traits. In particular, there are three genes that have to be removed before we can start to introduce some of these genetic traits from diabetic patient populations. Two of those genes have already been removed, and work on the 3rd gene has been initiated and several candidate models are currently being tested. The next step of this project will be to determine the best of these new models with which to start introducing traits from diabetic patient populations.

The second aim of this project is to develop a therapy designed specifically for one trait found in a sizable portion of diabetic patients, specifically those patients carrying the genetic allele HLA-A*0201. This therapy, which is a vaccine-like approach, has already shown promise in a proof-of-principle 1st generation mouse model carrying this genetic trait. Work has been initiated to repeat these results in a 2nd generation model carrying this trait. This 2nd generation model will allow the fine tuning (based on specific blood markers) the necessary timing of the first dose of this “vaccine”. Additionally, I plan on determining whether this therapy can be used over a short time frame (<3 months), or if it will require periodic injections to maintain protection for diabetes development. Finally, I will be determining how specifically this therapy is re-educating the immune system in order to make improvements for this therapy, and to educate the future design of similar therapies for other genetic traits.

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