Objective
Our long-term objective is to develop new safe drugs able to stop autoimmunity and restore a sufficient number of insulin-producing cells for patients with T1D to provide optimal glycemic control and make exogenous insulin useless.
We have developed two new, bifunctional, intelligent drugs (PS02 and PS03) that specifically recognize mouse and human β cells in vivo and induce insulin-producing cells to multiply and defend themselves from autoimmunity. We will soon start the clinical development of these drugs to evaluate their efficacy in clinical trials. However, because we aim to test these drugs in humans for the first time, it is essential to be sure that they are safe and effective in relevant models and understand which possible toxicities can be to eventually mitigate them by changing the formulation. We have already tested the drug that increases the number of insulin-producing cells (PS02) using human islets transplanted in mice. We observed that this drug effectively restores the normal glucose level in humanized mice by increasing the number of human β cells. We did not see any effects in all other tissues and cells. These effects and observations are highly relevant since we used human tissues in mice. Unfortunately, because of the length of the experiment lasting more than a year, we tested the efficacy of the immune-protective drug (PS03) using only an unoptimized formulation in mouse models. Nevertheless, this unoptimized formulation was able to block diabetes in 50% of the mice. The new optimized formulation is more effective and easier to produce, and we are confident it will be more effective.
Given these promising results and to move the clinical development fast and without obstacles, our goals in this project are a) check if these drugs can “alarm” and be recognized by the immune system, b) determine if they activate other activities of genetic programs, not identified by the bio-informatic analysis, in the β cells, c) assess if the effects of long-term drug administration on the function of insulin-producing cells and other tissues, and d) determine the efficacy of PS03 in relevant mouse model and in vitro settings that uses only human cells.
The successful completion of the proposed experiments is of paramount importance. These experiments will not only reveal any potential, unexpected toxicities of the new drugs but also allow us to further enhance the formulation of these drugs to mitigate any such side effects. Moreover, the results of these experiments will enable us to design the clinical trials and laboratory assays more effectively, ensuring a comprehensive evaluation of the efficacy and any unforeseen toxicities of these drugs.
Background Rationale
In the last decades, many molecular mechanisms that regulate the number of insulin-producing cells and those that regulate the immune system to avoid autoimmunity have been identified (although not fully clarified). The main problem is that these mechanisms also regulate the homeostasis of other cell types (e.g., those that do not produce insulin) and the immune response against viruses, bacteria, and tumors not yet detectable by doctors. As such, the modulation of these mechanisms in the whole body creates significant safety concerns because they can affect other tissues, promote the aberrant proliferation of tissues, expose the patient to infection, and increase the risk of tumors. To overcome these concerns, we have developed intelligent RNA molecules that can specifically recognize the insulin-producing cells and specifically provide the instructions to avoid the immune attack and increase β cell number. Importantly, this new class of drugs called bifunctional RNA therapeutics is naturally disintegrated a few days after delivering the instructions to the β cells. This provides an important safety element to these drugs because they do not modulate β cell biology for a lifetime but, instead, only for the time necessary to make β cell duplicate and to induce exhaustion (a state of chronic fatigue in which immune cells cannot work) on only the immune cells that cause T1D. Among the many mechanisms and genes involved in β cell proliferation and the regulation of autoimmunity, we chose two that have relevant and proven clinical implications in humans.
To generate PS02, we chose to inhibit a gene (p57kip2) that blocks cell proliferation. Notably, spontaneous mutation in this gene caused hyperinsulinism of infancy, a genetic disease associated with an aberrantly high number of insulin-producing cells, extremely elevated levels of insulin, and dangerously low blood glucose levels. Hyperinsulinism of infancy can thus be considered the opposite of diabetes. While mutations in p57kip2 are sufficient to induce β cell proliferation during infancy, in adults, high glucose levels (such as those frequently reached by patients with T1D) are also needed to increase β cell number. This high-glycemia-dependent mechanism is an important intrinsic safeguard of our strategy because, in adolescents and adults with T1D, β cells will not proliferate once normal glucose levels are maintained, making the possibility of overdosing PS02 unlikely.
To control autoimmunity, we created PS03, and we chose to instruct β cells to produce a protein called PDL1. Various tissues use this protein to protect themselves from autoimmunity. It became very popular when scientists discovered that tumors hijack this protein to escape the attack by the immune system. Indeed, some drugs (PDL1 antagonists) used in cancer treatment block this protein and allow the immune system to recognize and kill the tumor cells. Doctors noticed that some patients taking PDL1 antagonists against their cancer effectively destroyed the tumors but also developed T1 diabetes. These clinical observations suggested that β cells in these patients learned to protect themselves from autoimmunity and that, once the anti-cancer treatment blocked PDL1 action, the immune system rapidly destroyed the tumor alongside the β cells.
Because of these clinical observations, we developed PS02 and PS03 to modulate p57kip2 and PDL1 only in β cells, thereby protecting and increasing the number of insulin-producing cells while leaving other cell types and tissues untouched.
Description of Project
Type 1 diabetes (T1D) is an autoimmune disease in which the immune system recognizes and destroys the insulin-producing cells of the patient. This destruction is not sudden, but, instead, it is gradual, and an important number of insulin-producing cells (called β cells) are still present even years after diagnosis. However, their number is insufficient to provide sufficient insulin for the body's needs. Thus, it is crucial to develop treatments to increase β cell number and defend them from immune attack. The problem is that the mechanisms that regulate the capacity of β cell cells to proliferate are also present in other cells and tissues. Thus, stimulating these mechanisms in the whole body induces other tissues to proliferate, increasing the risks of hypertropia or cancer.
Similarly, the mechanisms that block cell recognition by the immune system and the immune attack are the same ones that viruses, microbes, and tumors use to avoid their destruction. Thereby, the activation of these pathways in the whole body can facilitate infection and tumors. To induce β cell proliferation and protection and prevent these problems, we have developed two new smart drugs using advanced RNA technology. These drugs have a part that is a vehicle able to recognize and deliver the "package" only to β cells. The package contains commands that instruct β cells to multiply and defend themselves from immune attack. Importantly, the proliferative commands spontaneously self-destroy after a while to avoid the risks that β cells proliferate too much. Similarly, the defense that β cells erect to shield themselves from immune attack induces exhaustion only on the attackers (the immune cells that cause diabetes) but leaves the immune cells that fight the infections and tumors untouched. The results are that, after some time, the cells that attack the immune cells are tired, unable to kill, and eventually commit suicide. Because of the novelty of these drugs, this project will evaluate their safety, identify unforeseen risks using preclinical models, and further optimize their formulation to mitigate eventual side effects. Furthermore, in collaboration with the City of Hope and the University of Pennsylvania, we will use new techniques to evaluate the efficacy of the immune-protective drug in systems that use human cells to assess in vitro the interaction between the immune system and the β cells. Successful completion of this project will allow us to define the formulation, understand the risks associated with our new intelligent drugs, and move forward with the processes needed to evaluate their safety and efficacy in humans.
Anticipated Outcome
We developed PS02 and PS03 to restore a sufficient number of insulin-producing cells and restrain autoimmunity in patients with T1D. These drugs represent a new class of therapeutics with the potential to revolutionize how we treat diabetes. These drugs appear safe and extremely precise in their mechanisms of action and β cell specificity in our in vivo and in vitro supporting data and computer simulation. However, since they are entirely new therapeutics different from everything tested to date in T1D, it is necessary to perform extensive tests to ensure and identify unforeseen side effects. We designed and will employ a comprehensive series of assays that should reveal toxicities, off-target activities, and immune reactivities not detectable by standard methods or computer simulations. We will also use state-of-the-art assays using human insulin-producing cells and human T cells as well as human living pancreatic tissues that will allow us to test PS03 efficacy before moving into clinical trials. The proposed research can produce two possible outcomes:
1) The first potential outcome of our research is the confirmation of PS02 and PS03 safety, even under extreme conditions. In this scenario, we can expedite the clinical development, FDA approval for clinical testing, clinical experimentation, and FDA approval for the general use of these drugs.
2) we will identify and pinpoint some toxicities, off-target specificities, and immunogenicities not seen with standard methodologies. In this case, we will have the opportunity to mitigate or prevent these undesired side effects by slightly changing PS02 and PS03 formulations or optimizing the dose or administration route during clinical development and experimentation. Additionally, identifying potential side effects allows for a better design of clinical trials and patients' selection to avoid complications or side effects that may affect a subset of patients. For example, a phase 2 clinical trial testing a new anti-coagulant using reagents similar to ours but formulated with a molecule called PEG (that we do not use), failed in phase 2 clinical trials. Indeed, a few patients who had a previous sensitization to the PEG molecule developed severe allergic reactions, urging the scientists to stop the trial and halting any further development of those promising drugs. Testing those reagents before clinical experimentation, as we proposed for ours, would have allowed them to avoid those allergic issues by either not using PEG or testing patients for sensitivity to PEG before enrolment.
Additionally, evaluating PS03 efficacy using two independent experimental settings that use human tissues and cells will allow us to partially de-risk the investment in time, energy, and money needed to develop new, safe drugs.
Finally, we will try to validate a blood test that may predict PS03 efficacy. If this assay works as expected, we may design a similar one to be used during clinical experimentation. A similar blood test could identify early those patients who respond to therapy and those who do not, allowing for precise and personalized medicine.
Relevance to T1D
Functional insulin-producing cells are detectable in patients with T1D even years after diagnosis. Thus, treatments that increase β cell number while halting autoimmunity can potentially cure T1D.
We developed two new intelligent RNA therapeutics, PS02 and PS03, that have the potential to accomplish these goals. If the results from this project confirm their safety and efficacy, we envision that PS03, the drug with the potential to halt autoimmunity, may be used as monotherapy on those patients positive for multiple autoantibodies to prevent diabetes onsets. Instead, patients with already clinically relevant T1D but still detectable c-peptide will benefit from a combination therapy of PS02 to increase the number of insulin-producing cells and PS03 to block autoimmunity.
Patients with undetectable c-peptide may not benefit from our therapeutic alone. Still, they may need the transplant of allogenic islets, pancreas, or, hopefully soon, islets produced in the laboratory. These patients will take immunosuppressive drugs to prevent immune rejection, and, unfortunately, their new insulin-producing cells will eventually die despite immunosuppression, resulting in the need for exogenous insulin. If we are successful, these patients will benefit from PS02 treatment because it should increase the number of insulin-producing cells from the transplant.
In summary, PS02 and PS03 may benefit, alone or in combination with other treatments, a large proportion of patients with T1D and may, in association with prognostic tests, prevent T1D onset in the future. Therefore, if successful, the strategy we are proposing is relevant to T1D.