Objective
The overarching aim of this proposal is to use genome-editing techniques to generate stem-cell-derived beta cells that avoid immune rejection upon transplantation. Specifically, we aim to:
I. Define the optimal protocol for human stem cell differentiation into pancreatic beta cell organoids and establish tools to facilitate beta cell detection in downstream analysis.
II. Develop methods to prevent silencing of immune ‘evasion genes’ during stem cell differentiation into beta cells.
III. Assess the ability of gene-edited beta cells to regulate blood glucose levels in immunocompromised mice compared to their unedited counterpart.
IV. Assess the ability of gene-edited beta cells to avoid immune attack, survive long-term, and functionally secrete human c-peptide in healthy and autoimmune mouse models.
Background Rationale
Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing pancreatic beta cells are destroyed by the patients’ own immune system. This lack of functional beta cells leaves patients dependent on a life-long insulin therapy to regulate blood glucose levels. Even the most advanced insulin delivery systems are technically difficult to use and place a significant burden on the patient’s quality of life, especially in children and adolescents. Although better glucose monitoring and insulin delivery systems have improved patients’ ability to reduce harmful fluctuations in blood glucose levels, the risk of serious secondary complications remains high.
The transplantation of pancreatic beta cells from organ donors is currently the only therapy that can result in extended insulin independence in T1D patients, highlighting the potential of cell replacement therapy as a long-term, biological cure for the condition. However, donor scarcity and the need for lifelong immune suppression to prevent rejection of grafted cells prevent this approach from wide and affordable implementation.
It is now possible to generate functional pancreatic beta cells from pluripotent stem cells (PSC) and they have emerged as an attractive alternative to donor beta cells as they can be generated in large quantities in the laboratory. Yet, this strategy can only be efficacious if the grafted cells are protected from immune assault. While tissue derived from patient-specific PSCs may normally avoid immune graft rejection, the autoimmune environment of T1D places an additional threat to grafted beta cells that must be suppressed or avoided. Targeting these pathways by pharmacological means can have serious side effects, especially when applied over long periods of time.
Studies in mice and humans have identified several key genes that are involved in the immune detection and destruction of grafted cells. These genes are either necessary for immune cells to be able to detect grafted cells or conversely their expression can inhibit the activation of immune cells. Recent advancements in the field of genome editing now allow for the genetic modification of stem cells. This new technology, called CRISPR/Cas9, has opened up the possibility of eliminating or inserting genes in a very efficient and accurate manner.
In this project we will build upon our previous studies showing that alteration of certain ‘evasion genes’ allows human stem cell-derived beta cells to evade immune attack in healthy mice with unaltered immune systems which are highly primed to target foreign invaders. We will determine the optimal protocol to derive beta cells from gene-edited induced pluripotent stem cells, establish methods to avoid silencing of the immune ‘evasion genes’ in beta cells, and evaluate the long-term survival and functionality of gene-edited beta cells in immune competent mice.
Description of Project
Type 1 diabetes (T1D) is a chronic disease defined by the autoimmune destruction of insulin-producing pancreatic beta cells, leaving patients dependent on life-long insulin therapy to regulate blood glucose levels. The transplantation of pancreatic beta cells from organ donors is currently the only therapy that can result in extended insulin independence in T1D patients. Stem cell technology offers the potential to generate pancreatic beta cells in the laboratory that can be used to replace those lost to the autoimmune attack as a long-term, biological cure for the condition. Our team has developed highly effective methods for generating functional beta cells from stem cells. However, the main obstacle that remains for the development of stem-cell-based therapies for T1D is the need for protecting grafted cells from immune rejection upon transplantation. Recent advancements in CRISPR genome editing technology allow efficient DNA modification of stem cells, which opens up the possibility of genetically “camouflaging” cells from the immune system by eliminating genes that are responsible for the immunological targeting of transplanted cells. Preliminary studies from our labs have used gene editing in human stem cells to eliminate sets of genes that are involved in triggering an immune response and enhance sets that are known to protect cells from immune attack. When these edited stem cells were transplanted into healthy mice with fully functional immune systems, the cells were able to evade immune attack. The ability of the cells to survive in a different species shows that they are robust and likely to evade attack under the most stringent of conditions. To our knowledge, these are the most promising results ever reported in engineering cells that are “camouflaged” from the immune system and suggest that this strategy could be effective in patients. In unpublished work, the NYSCF lab has generated beta cells from these “camouflaged” stem cells and shown that this combination of genes is effective at protecting beta cells from immune attack when transplanted into both healthy and diabetic mice. However, it remains to be seen whether these beta cells can properly function and regulate glucose levels in these animals. In this project, we will determine the most effective protocol for producing beta cells from “camouflaged” stem cells, develop methods to prevent silencing of immune ‘evasion genes’ during differentiation, and test beta cells’ ability to evade immune attack in healthy mice. Altogether, the goal of this project is to devise robust methodologies to produce stem cell-derived beta cells that stably express immune evasion factors, and function and survive long-term in immunocompetent mice – laying the groundwork for the development of cell replacement therapies for T1D patients.
Anticipated Outcome
We predict that by establishing the optimal conditions to differentiate beta cells from “camouflaged” human induced pluripotent stem cells, we will be able to generate beta cells that exhibit extended survival and increased functionality upon transplantation into immunocompetent mice. These experiments could serve as a basis for the development of a universal stem cell product that can be used for cell replacement therapies in T1D patients.
Relevance to T1D
T1D affects 1.25 million Americans, and this number is expected to increase to 5 million by 2050. Since the discovery of insulin in the early 1920s, the sum of all technological advances in diabetes care has allowed for more convenient, precise, and accurate dosing of this life-saving drug. Yet a persistent problem is that recombinant insulin therapy, regardless of the delivery method, is costly, burdensome, and inherently imprecise. Even groundbreaking automated delivery systems that purport to relieve the mental burden of T1D on patients by eliminating the need for constant dosing decisions are subject to the same limiting factors and risks of high costs, accessibility, and malfunction.
A “biological cure” — that is, a solution for diabetes that restores healthy insulin production and eliminates the need for glucose monitoring, insulin therapy, and dietary restriction — is the holy grail of T1D therapeutics. Beta cell replacement therapy has shown to be effective at restoring normal blood glucose levels in the relatively small population of patients that have received a donor transplant. There are, however, two main limitations to this therapy: the scarce supply of donor islets and the need for lifelong immune suppression to protect the grafted allogeneic islets. Replacing cadaver islets with stem-cell-derived beta cells resolves the issue of tissue availability as these cells can be produced in large quantities in the laboratory. This leaves immune protection of the grafted cells as the single biggest technical obstacle for the implementation of this therapy. The herein proposed research aims at taking a decisive step towards solving this fundamental problem by using state-of-the-art genome editing tools to systematically modify human pluripotent stem cells such that their derivative beta cells become tolerant to the body’s immune system. Perhaps most importantly, the development of an immune-tolerant stem cell product that can be used for replacement therapies across patients would make this therapeutic option economically viable for the wider T1D population.