Advancement of CRISPR-Based Adipose Tissue Therapies for Type 2 Diabetes to Nonhuman Primates

Overview of This Focused Program:

This project addresses the severe problem of type 2 diabetes (T2D) in our general population and in our Veterans. T2D is the most common form of diabetes, affecting a remarkable 1 in 10 adults in the U.S. It is a chronic disease that causes many damaging complications, including blindness, amputations, kidney disease, and heart disease. Its toll on our healthcare system is also tremendous. In recent years, the U.S. spent over $250 billion for treatment of T2D and its complications or over $350 billion when loss of productivity due to inability to work is included. That amounts to over $1,000 a year for each American. The most alarming aspect of this statistic is that it is about 50% higher than it was in 2007, and if these trends continue, it will be catastrophic by 2050. Already, about 75,000 amputations of foot, toe or leg per year are performed in the U.S. based on wounds not healing due to diabetes.

One would think that curing T2D would be a simple matter of losing some excess weight, for example, with diet and exercise, since T2D is related to weight gain. But this is not always true. Many individuals develop T2D with only little weight gain, and losing those few extra pounds is almost impossible, especially as we get older. In India, even larger numbers of the population have T2D, despite the fact that most people are thin by our standards. We believe that the real cause of diabetes is not excessive overall adipose tissue, but the development of unhealthy forms of adipose tissue. Our proposal seeks to turn 'unhealthy' adipose tissue into its 'healthy' form that burns rather than stores fats, using very new methods that can isolate, expand, and change the adipose tissue into healthy therapeutic adipose tissue that can be implanted back into a patient in relatively small amounts.

Adipose tissue has many different and important functions. For example, fat is in our heels, providing important mechanical support for walking and running. Fat is also near our intestines, providing protection from microorganisms in our digestive system. The form of fat that is under our skin ('white') is useful for storing extra energy to use during fasting or exercise. But there is also a form of fat called 'beige' fat that keeps our heart and blood vessels warm, and also produces hormones that keep our metabolism healthy. Importantly, very little or no 'beige' fat can be found in people with T2D, suggesting that the absence of 'beige' adipocytes (the fat cells that make up fat) may be causing the disease. Importantly, 'beige' adipocytes have active genes that increase metabolism, burn fat, and produce healthy hormones, while 'white' adipocytes do not.

Scientists in this Focused Program project have shown that implanting relatively small amounts of 'beige' fat cells into fat fed obese mice alleviates diabetes, and they have extended this to human 'beige' fat cells that they can obtain and greatly expand from small samples of human adipose tissues. Implanting these human fat cells into 'humanized' mice that accept these transplants also alleviates diabetes. This suggests that 'beige' fat cells can themselves potentially be a therapeutic—a cell therapy for obesity and T2D. However, these successful mouse experiments need to be advanced to testing in nonhuman primates such as monkeys that are known to also become diabetic as they age and become somewhat overweight. This is the major overall goal of this Focused Program comprised of four interdependent projects. If this goal of succeeding in monkeys can be achieved here, it could lead to clinical trials of this adipocyte cell therapy in human subjects and potentially to realization as a therapy.

In order to attack this overarching challenge of alleviating T2D and obesity in monkeys by implanting small amounts of 'beige' adipocytes, we have assembled a world-class group of scientists with unique skills and demonstrated accomplishments. Each brings special skills and techniques that are complementary and synergistic in effect. Each scientist's team will drive experiments that will test specific hypotheses that are quite independent, but together have high chance of meeting the overarching challenge. They have already started working together in preliminary fashion and have come to realize the great potential of this approach:

1. Silvia Corvera, MD, Professor at the University of Massachusetts Medical School, has made the breakthrough discovery of being able to greatly multiply human 'beige' fat cells from small amounts of human adipose tissue samples. Her group demonstrated poof of principle that these cells when implanted into 'humanized' mice are able to alleviate diabetic conditions. She and her team will optimize methods to translate this technology to prediabetic and diabetic monkeys in collaborations with Projects 3 and 4 to test the therapeutic effects of 'beige' adipocytes that she produces. A U.S. Patent has been granted to the University of Massachusetts Medical School for her invention.

2. Michael Czech, PhD, Professor at the University of Massachusetts Medical School, has made the breakthrough discovery of a method to target specific genes in fat cells to make them more 'beige'-like, and more therapeutically effective. His group discovered a gene (denoted RIP140) that, when disrupted by a new application of the 'CRISPR' method his group developed, can convert 'white' adipocytes into 'beige' adipocytes. Importantly, he will optimize this 'beiging' process by targeting other genes known to be involved in 'beiging' with his team's methods. These results will be integrated into Projects 1, 3, and 4 to ultimately test the advantages of implanting genetically enhanced versions of adipocytes versus unmodified adipocytes into nonhuman primates. A U.S. Patent has been granted for his work on RIP140.

3. Kylie Kavanagh, DVM, MS, MPH, Associate Professor of Pathology at Wake Forest School of Medicine, has made the breakthrough discovery that a stress response pathway is a major potential therapeutic target in diabetes and has developed and characterized a large group of nonhuman primates for critical studies prior to clinical testing. Her group will meticulously characterize groups of these nonhuman primates for their prediabetic and diabetic conditions, determining their closeness to the human disease. She will collaborate with Projects 1 and 2 on the implants and will collaborate with Project 4 on the imaging of the 'beige' fat cells that are implanted. Critically, her group will assess the effects of the implanted 'beige' adipocytes in the monkeys and define the amount of therapeutic benefit derived from these implants.

4. Rosa Tamara Branca, PhD, Associate Professor of Physics and Astronomy, University of North Carolina at Chapel Hill has made the breakthrough discovery of a new method to enhance the imaging of 'beige' adipose tissue in mice and monkeys, and to 'see' the adipose tissue without any biopsies or invasive perturbation of the animals. She will further develop these techniques to enhance the ability to discern details of their capillaries and other features. Importantly, these techniques will be powerful in enhancing the assessments of implanted therapeutic adipose tissues in collaborations with Projects 1-3.

We believe bringing these four laboratory groups together in a concerted effort to link their interdependent projects has high potential to advance a novel breakthrough technology to treat obesity and T2D. The overall Focused Program goal of successful therapeutic implantation of 'beige' adipocytes into prediabetic and diabetic monkeys to alleviate T2D is a key milestone towards future clinical trials of this novel approach.

Project 1:

Project 1 addresses the severe problem of type 2 diabetes (T2D) and obesity in our general population and in our Veterans by taking advantage of (1) new discoveries in adipose tissue biology and (2) new methods to target specific genes to make adipose tissue healthy. Project 1 is based on the belief that the real cause of diabetes is not simply excessive overall adipose tissue, but the development of unhealthy forms of adipose tissue and loss of healthy adipose tissue. Our Project 1 proposal seeks to turn 'unhealthy' adipose tissue into its 'healthy' form that burns fat rather than stores fat, and secretes beneficial hormones that act on the liver, muscle, and other tissues to restore metabolic health.

Adipose tissues have many different and important functions, the most well-known being a storage depot for fat. But there is also a form of adipose tissue called 'beige' adipose tissue that keeps our heart and blood vessels warm, and also produces hormones that keep our metabolism healthy. Importantly, 'beige' fat is low or nonexistent in people with T2D, suggesting that the absence of 'beige' adipocytes (the fat cells that make up fat) may contribute to the cause of the disease. Importantly, 'beige' adipocytes express genes that increase metabolism, burn fat, and produce healthy hormones. Several years ago, Michael Czech and his colleagues discovered a gene (denoted RIP140) that controls conversion of white adipocytes that store fat into 'beige' adipocytes that burn fat. Thus, they reasoned that if one could find a method to target this gene in a safe way, it would be a powerful therapeutic strategy for alleviating obesity and T2DM by amplifying the 'beige' adipocytes.

Two recent scientific breakthroughs have now made this therapeutic concept tractable. First, the Corvera lab discovered a way to isolate and multiply adipocytes out of the body, some of which are beige, from small samples of human subcutaneous adipose tissue and showed that when implanted into mice alleviated diabetes. Second, the Czech lab discovered a way to selectively target the RIP140 gene in adipocytes with very high efficiency using methods referred to as 'CRISPR,' which have gained much press lately for other diseases as well. These advances enable these labs to access large numbers of human adipocytes to manipulate outside the body for the purpose of increasing their 'beige' properties.

Combining these two advances leads to an overarching transformative concept: Use the isolated human adipocytes that are multiplied in tissue culture to target genes like RIP140 to enhance the 'beige' type of adipocyte by the CRISPR method, then implant these adipocytes with increased 'beige' characteristics back into the same patient. Working under a DOD-funded grant that will expire next year, the Czech and Corvera labs have demonstrated proof of concept of this idea in mice. They succeeded in using the CRISPR method to target the RIP140 gene, increase the 'beige' nature of human adipocytes, and implant them into 'humanized' mice to show an improvement of sugar metabolism. This establishes a firm foundation for the proposed work for further advancement. A U.S. Patent was granted for the technology Czech invented for RIP140.

Project 1 will use the above advances as a springboard to investigate other candidate genes that may increase 'beiging' of adipocytes when targeted by the newly developed CRISPR methods. Once these other genes are tested for such activity, combinations of the best 'beiging' genes together with RIP140 will be targeted to obtain maximal 'beige' characteristics in tissue culture conditions. The goal of this first aim of Project 1 is thus to identify the optimal gene combinations to target in order to drive the adipocytes to the most promising therapeutic 'beige' condition. Once this is achieved in tissue culture, we will move to implantations in 'humanized' mice of the best genetically modified 'beige' adipocytes that we can prepare. When proof of principle is achieved in mice with these implantations of genetically modified 'beige' adipocytes, we will collaborate with Projects 2-4 in implantation of genetically modified monkey adipocytes into monkeys.

Thus, we plan to achieve the key milestone of demonstrating a therapeutic benefit of implanted, genetically enhanced 'beige' adipocytes in nonhuman primates to advance this technology to the clinic in future studies.

Project 2:

The overarching goal of this project is to create new therapies for type 2 diabetes (T2D) and its complications. Many studies in the past 10 years have convincingly showed that fat tissue plays a very important role in developing T2D. While becoming overweight is a risk factor, the most important aspect of adipose tissue is whether it is 'healthy' or 'unhealthy.' Individuals who can form healthy adipose tissue remain free of T2D even when overweight; in contrast, those who can't form healthy adipose tissue develop T2D sometimes after only relatively minor weight gain. Our laboratory seeks to understand the differences between 'healthy' and 'unhealthy' adipose tissue. One of the properties of 'healthy' adipose tissue is that is contains a type of cell (adipocyte) that can burn calories and can send signals to the rest of the body to improve metabolism.

These healthy adipocytes are called 'beige' or thermogenic or BAT adipocytes. While conducting experiments to understand the differences between 'healthy' and 'unhealthy' adipose tissue, we discovered a way to make many millions of 'brite'/thermogenic adipocytes from small amounts of adipose tissue and were awarded Patent No. US10093902B2 for this technology. One of the most exciting findings we then made was that these human adipocytes can be implanted into immunocompromised mice, where they are able to become 'healthy' thermogenic adipose tissue. Most interestingly, mice with new 'healthy' human adipose tissue have improved metabolism and are protected from getting T2D when fed a high-fat diet. We think that if this approach works for mice, it may work for people, and prevent development of T2D, or help control the disease even after it has appeared. Having T2D greatly affects quality of life of more than 1 in every 10 people in the general population, by causing liver, heart, and kidney disease, as well as blindness and amputations. Estimates in active military range from 7.4% in the Marine Corps to 11.4% in the Air Force. Further, 25% of eligible young adults do not qualify for military service due to being overweight. Therefore, new treatments are urgently needed, and using the person's own cells to treat T2D and its complications can be a very important advancement.

We have the opportunity to test this new idea in nonhuman primates (NHPs). Dr. Kavanagh is a world-expert in the study of Vervet monkeys, who, like people, develop obesity and T2D. She also studies the role of 'healthy' and 'unhealthy' adipose tissue and has collaborated with Dr. Branca to study the role of 'beige' thermogenic adipose tissue in the development of T2D in NHPs. Working with her group, we will be able to make 'brite'/thermogenic adipocytes from adipose tissue from NHPs. Moreover, we can compare which region of the body of NHPs is the best source of cells to make 'brite'/thermogenic adipocytes. We will then make 'brite'/thermogenic adipocytes from NHPs with prediabetes or diabetes and implant them into mice to determine to what extent they improve metabolism. Our goal then is to implant these 'brite'/thermogenic adipocytes back into the original NHP donors who have prediabetes or T2D. During the next 6 months, which is equivalent to 2 years in human life, we will see if the cells make 'healthy' adipose tissue in these donors and improve their metabolism. We will be able to 'see' the formation of new, adipose tissue in collaboration with Dr. Branca, who is a world expert in methods to examine thermogenic adipose tissue in live animals and humans.

We will then test yet another method, which is to improve the 'brite'/thermogenic adipocytes from NHPs by using genetic modifications. Dr. Czech has developed exciting, proven methods to alter genes in these cells to make them even more thermogenic. We will provide Dr. Czech with cells from NHPs, which he will improve using a technique called CRISPR-Cas9. Dr. Kavanagh will implant these into NHPs and monitor them in collaboration with Dr. Branca as described above. By itself, our project will provide new knowledge, translatable to humans, on the capacity of different depots to produce different types of adipocytes, and on the effects of prediabetes and T2D on the capacity to form healthy adipose tissue. Together with our collaborators, our work has the potential to usher a completely new class of therapies for T2D and its complications, which are ungently needed by our general population and military population in particular.

Project 3:

Project 3 addresses the health problems that relate to prediabetes and type 2 diabetes (T2D), which are diseases reaching epidemic proportions as the U.S. population becomes older and remains obese or overweight. In the general and Veteran populations, T2D rates approach 10% overall, and estimates even in active military range from 7.4% in the Marine Corps to 11.4% in the Air Force. Further, 25% of eligible young adults do not qualify for military service due to being overweight.

Our project aims to take new and exciting scientific findings about fat tissue and ensure that the biology, approaches, and therapy we are developing efficiently reverse T2D in a nonhuman primate (NHP; monkey) model of disease. We aim to demonstrate that a specialized type of fat tissue, brown fat (BAT), is present in healthy NHPs and specifically absent in unhealthy T2D NHPs. We believe that this BAT, rather than total fatness, determines healthiness, as 30% of human and nonhuman primates who are overweight or obese do not have T2D. However the reasons these healthy obese populations are protected from disease are currently unknown. BAT is a highly metabolically active tissue that interacts closely with the nerves and blood vessels, and secretes molecules that have positive effects on the metabolism of the whole body. These features are in contrast to the white adipose tissue, which accumulates in overweight and obese persons; however, Drs. Corvera and Czech have shown this tissue can include cells that can be 'driven' towards BAT in character, and thus be a potential source for increasing BAT amounts in people.

Mice and rats are excellent for discovery of new scientific directions for treating T2D. However, these rodents do not naturally develop T2D; they have very different metabolic rates and body locations for fat storage. They also naturally have much higher levels of BAT in their bodies; thus, we need to be sure that early studies completed in these animals have similar effects in more human-like animals (nonhuman primates or NHPs) and are safe. Rodent studies have shown us that transplanting self-generated BAT from laboratory manipulation of cells of an individual's own white storage-type fat reverses T2D and improves health. We also know that we can create these cells from NHPs, but we need to refine the amounts, the locations, and discover how long-lasting the effects of transplantation will be.

The hypotheses that our project will address include:

1) BAT will be present in healthy NHPs and absent in metabolically unhealthy NHPs, and we will be able to assess the amount and locations by advanced medical imaging.

2) We will confirm the BAT presence, locations, and tissue functions from surgically retrieved biopsies and laboratory assessments.

3) We will find that biopsies of storage (white) fat from under the skin will contain enough cells that have BAT-potential that we can grow an animal's own BAT to re-implant and see improvements in the same animal's risk for T2D through markers such as lowered blood glucose.

Dr. Kavanagh directs the research of a large colony of NHPs and studies them for age and obesity-related T2D development. Recently, Dr. Kavanagh identified that fatness and poor health do not always co-occur, as is the case in people, and characterized the cell types and cell functions that differentiate healthy and unhealthy obese NHPs. This population is uniquely well-suited for study, since they develop T2D naturally and we have already conducted preliminary studies to show the amounts and body locations of BAT similar to those seen in people.

Project 3 will use advances in cell therapy and medical imaging and provide a platform for ensuring proof of principle. Further, the access to BAT tissue sourced from NHPs, which is ethically not possible in human patients, will advance our knowledge about the cellular components, tissue structure, and function of this specialized organ in a model more relevant to human disease. In doing so, our Project 3 is the link that brings the rodent studies of BAT, and advanced imaging to identify this small but important tissue, together to (a) find BAT and relate it to disease, (b) create NHP BAT and characterize this self-generated fat transplant tissue, and (c) transplant the tissue in T2D NHPs to see how effective it is in reversing disease and for how long.

Project 4:

Project 4 addresses the issues associated with the lack of non-invasive imaging tools needed to detect and quantify BAT/beige fat in vivo.

Clinicians are increasingly utilizing non-invasive imaging techniques for both diagnostic purposes and for monitoring disease progression. Diabetes is no exception. In the past 10 years, it has become more and more clear that not all fat is created equal, and that the type of fat (white, brown, or beige) and its location (visceral or subcutaneous) can strongly influence one's risk of developing metabolic diseases. For example, while the amount of visceral white adipose tissue is a strong predictor of the risk of developing type 2 diabetes, brown and beige fat are known to protect obese subjects from the onset of metabolic diseases. For these reasons, significant effort is now placed on developing strategies to stimulate the growth of the good fats (brown and beige) that secrete hormones capable of restoring metabolic health.

While differentiation and quantification of visceral and subcutaneous white fat can be done relatively easily with the use of standard tomographic imaging approaches such as MRI and CT, detection and quantification of metabolically protective BAT/beige fat remain unmet needs. Current techniques are either too insensitive or lack the necessary specificity to differentiate these tissues from the more abundant white adipose tissue, especially in overweight and obese subjects. Our laboratory recently showed that the inert gas xenon can be used as a CT contrast agent for the detection and quantification of BAT of obese mouse phenotypes that cannot be detected by any other non-invasive imaging techniques.

To this end, under Aim 1 we will assess the ability of xenon-enhanced CT (XeCT) to detect beige fat in mice. Specifically, XeCT and other clinically relevant imaging modalities (MRI, PET and ultrasound) will be used on the same mouse to identify and quantify imaging biomarkers associated with white adipose tissue browning, while histology will be used to assess ground truth. These studies will enable us to make a head-to-head comparison of effect sizes associated with the different beige fat imaging biomarkers, enabling us to determine which of the clinically relevant imaging modalities is best poised to detect beige fat and the browning process in vivo.

In the second aim, we will refine the current XeCT imaging protocol, used to detect BAT, to detect beige fat and beige adipocyte transplants in vivo. To improve the sensitivity of xenon-enhanced CT for detecting beige fat, we will adopt a dual-energy CT approach, which will allow a single CT scan to identify and quantify xenon uptake in tissue. The use of dual-energy as opposed to the current single-energy protocol will eliminate issues associated with subject's motion between enhanced and non-enhanced CT scans. Further, the subject's radiation exposure would be reduced to half, enabling longitudinal XeCT studies needed to assess treatment efficacy.

At the completion of Project 4, we expect to have completely characterized and quantified different imaging biomarkers associated with the browning process and to have a XeCT imaging protocol that will enable researchers around the world to better phenotype research subjects based on their BAT/beige morphological and functional appearance, to understand the biology of beige fat and its relevance in human metabolic disease, and to develop and assess the efficacy of new treatments that target this tissue to combat obesity and type 2 diabetes.