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Research Updates

2020 Update by Dr. Alice Bertaina

Dear Kruzn for a Kure Foundation,

First, we would like to kindly acknowledge the Foundation and all of those involved for supporting our research at Stanford University during 2020. Although this year has been challenging due to the COVID-19 pandemic, with your funding, we have advanced our project studying regulatory innate immune cells that may promote immune tolerance after abHaploHSCT (αβ T-cell/B-cell depleted haploidentical hematopoietic stem cell transplantation) in children with Schimke Immune Osseous Dysplasia (SIOD). Background: SIOD children urgently need HSCT to reconstitute the deficient immune system, as well as a kidney transplant to provide a healthy fully functional organ and a better quality of life. Three SIOD children were treated with abHaplo-HSCT and they subsequently received a kidney transplant from the same abHaplo-HSCT donor without longterm immunosuppressant treatment. This is a very innovative and efficacious strategy. Previous efforts to perform kidney transplant post-HSCT have been unable to completely eliminate the lifelong need of immunosuppressants, which negatively impact the quality of life of the patient. Importantly, this is the first time SIOD patients can go through an allogeneic HSCT without transplant-related complications. In fact, no patients died nor developed significative graft-versus-host disease or infections. Thus, we hypothesize that the presence of regulatory innate immune cells, such as gd T and Natural Killer (NK) regulatory subpopulations, especially in the first months post-abHaplo-HSCT in SIOD patients favors a tolerogenic environment that allows the solid organ to engraft and function. Understanding the underlying mechanisms of tolerance achieved in SIOD children will provide new clues to improve abHaplo-HSCT and to identify the best timing for solid organ transplants; a strategy that will translate to SIOD children and other pediatric patients currently waiting for an organ transplant.

 

Current research: In the immune monitoring data obtained from three SIOD children post-abHaplo-HSCT we observed high numbers of a NK cell subpopulation that may carry regulatory features. Within the first 90 days post-abHaplo-HSCT, the number of gd T cells is also abundant compared to other T cell populations and we believe that some of these cells can be regulatory. To date, the cell surface markers and the mechanisms of action of regulatory gd T and NK cells have not been fully elucidated. We attempted to identify gd T regs in three different healthy donors, however the gd T regs population was minimal to nonexistent, confirming the data available in literature. Thus, we successfully induced gd T cells in vitro to express regulatory protein markers using a cytokine cocktail. With the recent support of US$ 10.000,00 (ten thousand dollars) from Kruz for a Kure Foundation, we are currently developing functional assays to evaluate the ability of these cells to restrain T effector cell proliferation and to confidently characterize the regulatory potential of cytokine-induced gd T and NK subpopulations.

 

Perspective for 2021: Our goal is to proceed with single-cell experiments to characterize the phenotype of innate regulatory cells using CyTOF, a mass cytometry technique that assesses several cell surface and intracellular markers at the same time in a single-cell level. We will also use CITE-seq (single-cell RNA-seq combined with quantitative and qualitative information on cell surface proteins) to evaluate the molecular networks in gd T and NK regulatory cells. We anticipate to complete this task with an extra budget of US$ 65.000,00 (sixty-five thousand dollars). With a thorough investigation of these cells and regulatory networks, we will confidently analyze innate regulatory cells from the three SIOD patients in our cohort, and characterize their tolerogenic capacity. Once again, we thank Kruzn for a Kure Foundation for trusting in our research and for your incredible support to this project. With your continuous partnership, we will make several strides in finding the best strategies to cure SIOD and to translate our findings to other pediatric patients.

 

Priscila Ferreira Slepicka, Ph.D.

Dr. Alice Bertaina, MD, Ph.D. Postdoctoral Scholar Associate Professor in Pediatrics Inpatien

October 31, 2019    Update by Dr. David Lewis

Dear Kruzn for a Kure Foundation: 

I want to thank all of you and your community for your extraordinary support of Schimke Immuno-Osseous Dysplasia (SIOD) research at Stanford University for the last two-and-a-half years. Incredibly, you have given more than $1.4 million towards SIOD research. With this funding, we have been able to hire research staff who can dedicate their time to focus on SIOD and start new groundbreaking investigations into how we can treat SIOD. We are extremely grateful for your entrusting us with getting this novel research program off the ground. 

As you know, SIOD is due to mutations for the SMARCAL1 gene. Our goal is to develop therapies that address the multi-organ problems characteristic of the syndrome that are the result of SMARCAL1 deficiency, including decreased immune function, kidney failure, skeletal growth issues, and abnormal vascular function leading to migraines and seizures. 

 

Expanding our Team and Developing SIOD Treatments

Your funding has allowed our lab to jumpstart research into unraveling the mystery of how SMARCAL1 protein deficiency leads to SIOD. Because of your support, we have been able to grow our lab and accelerate our investigations. 

Over the last year, we’ve expanded our team, and Elizabeth Lippner, MD, PhD, has been joined by Postdoctoral Fellow Rebecca Saenz, MD, PhD; Senior Research Scientist Girija Dhamdhere, PhD; and Lab Science Research Professional, Vasavi Ramachandran, MS, to shed light on how a lack of SMARCAL1 protein causes the clinical problems of SIOD. 

Your gift in February of $150,000 to the Lucile Packard Foundation for Children’s Health for SIOD research allows us to hire two new researchers at the post-doctoral fellow or research scientist level. We are in the process of interviewing for both of these positions and hope to have one of these filled no later than the end of December 2019. 

Your funding over the last year supports our team’s time, purchasing of chemicals and supplies, and any costs related to carrying out investigations. Our studies could lead to developing new drugs or using ones already approved by the FDA or that have been tested in Phase III studies in human clinical trials (an approach also known as “repurposing” drugs). 

Armed with a diversity of skilled scientists, we have been undertaking these experiments: We are running a full profile of the types of mRNA in SIOD patient T cells compared to healthy T cells; we are also seeing if the structure of the DNA that encodes the genes differs in SIOD T cells compared to that of healthy controls; Dr. Saenz is using gene-editing technology in the lab to disrupt the SMARCAL1 gene of a healthy T cell to determine how this affects its function; Our team is engineering customized cell lines called induced pluripotent stem cells (IPSCs) from SIOD patients, including Kruz and Paizlee, so that we can ultimately study in lab how the disease affects many types of tissues, such as the podocytes of the kidney that are involved in making urine and cells that line the blood vessels and that appear to have abnormal function in SIOD. We have also now made EBV-transformed B cell lines from all four of the Davenport family. These cell lines will help expedite studies of the mechanism of disease in SMARCAL1 deficiency by allowing us to grow the cells quickly into large numbers. This limits the number of blood draws from all of you that are required for us to carry out our work. 

We plan to submit this work and Dr. Lippner’s discovery on short telomeres for publication by the end of this year. This is so important because it puts us in a strong position to apply for funding from the National Institutes of Health and other granting agencies and will also help establish a research network and collaborations with other scientists. 

 

Making Stem Cell Transplants Safe for SIOD Patients

At the moment, we feel that hematopoietic stem cell transplant (HSCT) is the best way to improve SIOD patients’ quality of life, but as you know in the past this procedure had a very high approximately 80% mortality rate (4 out of 5 children died within 3 months following the HSCT). However, the one 7-year old boy with SIOD who survived the procedure and shortly after HSCT received a kidney from his father has had a remarkable outcome – he is now 20 years post-transplant and is in generally good health. Based on this excellent outcome, we were able to convince Alice Bertaina, MD, PhD, a world-renowned expert in HSCT of children with primary immunodeficiency to treat Kruz and Paizlee. Dr. Bertaina has pioneered using haplo-HSCT in which one of the parents is used as a donor to their child 

Earlier this year, Kruz and Paizlee underwent HSCT using blood stem cells obtained from their parents. To the best of our knowledge, as we indicated above, only one patient has had a successful outcome previously. Your funding played an essential role in their carrying out HSCT for Kruz and Paizlee with a specialized protocol focusing on safety and rapid engraftment. 

Using your funds, Dr. Lippner learned that Kruz, Paizlee, and other SIOD patients have white blood cells with short telomeres at the ends of their chromosomes. This discovery informed Dr. Bertaina to use a lower dose of radiation and specialized chemotherapy to prepare Kruz and Paizlee for HSCT. 

Kruz did extremely well after the transplant with rapid engraftment of his stem cells and normalization of many of his white blood cell counts. As  Alice Bertaina, MD, PhD a result, Kruz successfully received his kidney transplant from Jessica in July 2019, months ahead of schedule. We look forward to watching Kyle’s stem cells fully engraft in Paizlee so that she can receive a kidney transplant from him hopefully by the end of this year. 

 

Thank You for Putting Your Faith in us.

There are so many reasons why the lab owes the success of the exciting research we are conducting to you. Because of your extraordinary efforts to raise awareness about SIOD, so many patient families have connected with us. We are going to see two new patients this year. 

I want to especially thank all the amazing donors in Muscle Shoals and elsewhere who support the Kruzn for a Kure Foundation. Jessica and Kyle, I remain awed by your commitment, tenacity, and strength in building this effort from scratch! It is all because you asked us to come up with a research program to try and develop new therapy for these kids. I am immensely grateful that you have put your faith in me and my lab and Dr. Bertaina and her lab to do this important work. 

David B. Lewis, MD

Professor of Pediatrics, Department of Pediatrics

Chief, Division of Allergy, Immunology, and Rheumatology

Member of the Program in Immunology and the Institute for Immunology, Transplantation, and Infectious Disease

Stanford University School of Medicine

June 23, 2019    Dr. David Lewis Update

I want to thank all of you for your extraordinary support of Schimke Immuno-Osseous Dysplasia (SIOD) research at Stanford University for the last two-and-a-half years.  We are extremely grateful for your entrusting us with getting this novel research program off the ground. This program, to the best of our knowledge, is the only of its kind focused on identifying disease mechanisms in SIOD and translating them into patient therapies.  

As you know, SIOD is due to mutations (changes in the DNA code) for the SMARACAL1 gene. These changes impair the function of the SMARCAL1 protein, which is essential for the cell to make a complete copy of its chromosome DNA before it divides into two daughter cells. Our ultimate goal is to develop therapies that bypass the problem with SMARCAL1 protein function that would benefit patients regardless of whether they have received or are able to receive a hematopoietic stem cell transplant (HSCT) and/or kidney transplant and the particular type(s) of SMARCAL1 gene mutations they have inherited.

As you know well, SIOD results in a number of distinct clinical problems including 1) decreased function of the T-cell immune system; 2) impaired the production of white blood cells by the bone marrow; 3) decreased kidney function resulting in the spilling of protein in the urine and, ultimately, kidney failure; 4) limited function of the growth plate of the bones involved in the growth of the skeleton in maintaining healthy joints, such as those of the hip; and 5) abnormal blood vessel function resulting in premature atherosclerosis and complications such as recurrent migraine headaches.

Currently, we feel that one major translational research priority is to define how impaired/absent SMARCAL1 protein function results in these diverse multi-organ problems characteristic of SIOD. Finding a common mechanism might allow us to develop a common therapy that bypasses these problems for all of the affected organ systems.  Our second major translational research priority is to identify how HSCT and kidney transplantation for SIOD can be performed more safely and effectively using recent technical advances for these procedures. A third research priority is to explore whether drug therapies that are far along in development or already FDA approved for other diseases (also called “repurposing”) could be used to treat some important clinical issues with SIOD patients.

HSCT, Bone Marrow Function, and the Telomere Biology of SIOD

On February 5 of this year, Kruz underwent an HSCT using blood stem cells obtained from Jessica. To the best of our knowledge, this is only the eighth SIOD patient who has undergone HSCT in the world of which four cases has been published in the medical literature. Of the previous seven cases, only one patient has had a successful outcome with HSCT. He received his HSCT at 7 years of age from an HLA-matched brother followed by a kidney transplant from his father; he is now 27 years old and doing well. Thanks to your support we were able to have him and his parents visit the research lab at Stanford, go over his medical history in detail, and obtain blood samples for research. This visit was very helpful in verifying that HSCT for SIOD leads to the normal function of the immune system following the transplant.  

We would also like to emphasize how your support of SIOD research was critically important for designing Kruz’s HSCT conditioning regimen, which makes room in the bone marrow for the donor hematopoietic stem cells to settle and carry out their function. Using your funds, Dr. Elizabeth Lippner in the lab discovered that Kruz, Paizlee, and several other SIOD patients have white blood cells with telomeres at the ends of their chromosomes that are much shorter than normal. Telomeres are special caps made of DNA and protein that are at the ends of all of the DNA chromosomes of the cell to prevent them from being damaged by normal cellular wear-and-tear. Previous work by other scientists using tissue culture cells that were manipulated to have decreased SMARCAL1 protein suggested that SMARCAL1 was involved in both the normal replication of both the main chromosome DNA as well as the telomere ends. Elizabeth was the first to show that the telomere length for blood cells from SIOD patients is very short.

This discovery changed how Dr. Alice Bertaina, decided to prepare Kruz for this HSCT:  She and others in the HSCT field know that patients who have short telomeres because of rare inherited genetic diseases other than SIOD are highly susceptible to bone marrow toxicity following standard HSCT conditioning regimens. To avoid this problem, Kruz received a combination of a low dose of radiation and special drugs for the preparation, an approach that has been used for other patients with telomere length defects. As you know, he did extremely well after the transplant with rapid engraftment of his HSCs and normalization of many of his white blood cell counts. His T cells are also, as expected, gradually increasing indicating that the HSCT is on track for correcting his immune problem. This approach will also be used for Paizlee as part of her upcoming HSCT.    

The discovery of short telomeres also provides an important insight as to why SIOD patients have reduced the production of red and white blood cells by their bone marrow since this is also a feature of genetic diseases other than SIOD that result in short telomeres.  In Kruz’s case, his bone marrow biopsy performed prior to the transplant confirmed that his bone marrow function was markedly reduced and, in fact, was more impaired than we predicted based on his white cell counts in the blood. As you know, Paizlee also has some signs of decreased bone marrow function. Based on Kruz’s response to HSCT, this decreased bone marrow function appears to normalize relatively quickly after the procedure. This linkage between short telomeres and reduced bone marrow function is also important because it raises the possibility that we might be able to treat it in SIOD using drugs that have been helpful for this problem in other telomere deficiency diseases. This would be particularly important for SIOD patients who for various reasons are not candidates for HSCT therapy.

As you know, our plan is for Kruz to also receive a kidney transplant from Jessica once his kidney transplant surgeons feel that he is well-nourished enough to have a speedy recovery after the procedure. We look forward to Paizlee undergoing the same approach of an initial HSCT followed by a renal transplant in which Kyle will be the donor for both. We appreciate how long a road it has been for your family to get to these important steps, and thank all of you for your perseverance and trust in the many doctors and health professionals who are involved in making this happen.

Defining How SMARCAL1 Deficiency Causes SIOD and Developing Clinical Intervention Strategies

SIOD is a complex and poorly understood the disease, and your funding has allowed the lab to jumpstart progress in identifying the underlying mechanisms of how reduced/absent SMARCAL1 protein has such a major adverse impact on diverse processes of the body. Although the discovery that the white blood cells of SIOD patients have shortened telomeres explains their reduced bone marrow function, short telomeres do not appear to explain SIOD’s T-cell immunodeficiency, decreased kidney function, blood vessel problems, and short stature: None of these other medical problems are commonly seen in patients who have genetic disorders that result in short telomeres but have normal SMARCAL1 gene and protein function.   

Thanks to your support, the lab was able to form a research team in Dr. Lippner was joined by  another post-doctoral fellow (Rebecca Saenz, M.D., Ph.D.), a senior research scientist (Girija Dhamdhere, Ph.D.), and a senior research technician (Vasavi Ramachandran, B.S.) to shed light on this mystery. Your funds have also been important for leveraging support for these individuals from other sources, such as grants from the National Institutes of Health and the Stanford Maternal and Child Health Research Institute so that they could focus on researching SIOD. You were recently able to make an additional gift to the Lucile Packard Foundation for Children’s Health for SIOD research and as a result, we will be hiring two new researchers to add to the SIOD team. The first of these will be Dr. Elodie Elkaim, M.D., who trained in one of the premier human immunology programs in the world (Hospital Neckers, Paris, France) under the direction of Dr. Cavazzana and who will be starting in August. We are also seeking another post-doctoral or research scientist to add to the team in order to keep building on our research momentum.

We believe that all of the major clinical problems observed in SIOD ultimately relate to the known role of the SMARCAL1 protein in normal DNA replication in preparation for cell division. Normally, a chromosome strand of DNA replicates in hundreds of little pieces simultaneously that are then stitched together so that there is an intact copy of the chromosome. We hypothesize that when the level of SMARCAL1 protein in the cell is markedly reduced or absent that certain regions (“pieces”) of the chromosomes are no longer effectively duplicated. We expect that this local “stalling” of replication leads to permanent changes in these pieces such that after they are stitched together the genes located in them have reduced function. Genes normally function by producing copies of their DNA sequence called messenger RNAs (mRNAs) that carry the information for the gene to be made into a protein, which actually carries out the gene function. In SIOD we believe that there are a set of genes with reduced mRNA and protein production that ultimately can be traced back to their location in pieces of the chromosome that were difficult to replicate and were structurally altered as a result. For example, we predict that decreased mRNA and protein for these genes in difficult to replicate pieces will account for SIOD’s T-cell dysfunction, kidney disease, blood vessel function abnormalities, and short stature/hip dysplasia.

To test these ideas, we are starting with studies of T cells in SIOD as these are readily obtained from blood samples. The team is determining the level of all of the mRNAs of T cells from Kruz and Paizlee and other SIOD patients compared to unaffected individuals.  Your funding is helping with the costs of our team’s time in isolating the T cells, the chemicals and procedures needed to doing the mRNA sequencing, which is called RNA-Seq and using costs of carrying out the sequencing using special machines at Stanford.

At the same time, we are collaborating with a gifted young scientist, Dr. Maya Kasowski, who is in the Stanford School of Medicine, Department of Pathology, who will determine if the structure of the chromosomes in SIOD T cells differs from those of healthy controls. These studies will use a technique called ATAC-Seq that was invented at Stanford and for which Dr. Kasowski is an expert. She will be looking for areas of the chromosomes that have a structure that makes it hard for the genes they contain to function normally. We anticipate the genes we find with reduced function in the RNA-Seq will be found in the regions that Dr. Kasowski identifies as having an abnormal structure.  

One major problem in SIOD research is that it is difficult to get enough cells from patients to be able to do large scale studies that are typically required for drug development. In an effort to move things faster, we are taking a number of approaches to determine if we can use cells that can be grown in large numbers in the lab for studies of disease mechanism and, ultimately, drug development. For example, Rebecca Saenz in the laboratory has used a technique for gene editing called CRISPR/Cas9 that allows us to take a normal T cell and disrupt its SMARCAL1 gene to determine how this affects the function of the T cell. This approach should improve our understanding of the normal function of SMARCAL1 and how reduced SMARCAL1 function might be overcome by drugs. Similarly, we are using a type of B lymphocyte found in the blood to make a special cell line that can be grown to very large numbers indefinitely. We are making these cell lines from Kruz, Paizlee, and other SIOD patients as well as healthy controls and will see if these can be used for studies of disease mechanism and drug development. Finally, we are making induced pluripotent stem cells (iPSCs) using blood cells from Paizlee and other SIOD patients as well as healthy controls. These iPSCs can be used to make cells that closely resemble T cells, kidney cells, blood vessel cells, and many other tissues. Again, the advantage of having the iPSC system is that we will be able to study SIOD disease mechanisms in many types of tissue and how they can be overcome much more practical than if we were to depend on actually obtaining these tissues from patients, such as from surgical biopsies

We have also been fortunate to have received advice on our research program from a number of Stanford scientists, including Drs. Steven Artandi, Karlene Cimprich, and Stephen Montgomery, who has been generous in providing their time and expertise for free. We plan to include some of these scientists as well as Dr. Kasowski in applying for grant support for joint research projects and programs to substantially increase SIOD translational research activity.     

We are confident that we will be able to submit most of this work for publication by the end of this year, and that this work along with the publication of the short telomere studies will put us in a strong position to apply for funding from the NIH and other granting agencies. In addition, pinpointing exactly which proteins are affected in SIOD may lead to potential therapeutic approaches to overcome the effects of these protein deficiencies.

Thank You for Putting Your Faith in Us

There are so many reasons why the lab owes the success of the exciting research we are conducting for you. Incredibly, you have given more than $1.3 million towards SIOD research. In addition, because of your extraordinary efforts to raise awareness about SIOD, we have heard from families not only in the United States (Oregon, San Diego, Texas, New Jersey, Michigan, and Nebraska) but also in France, Germany, Iran, and China. And it is purely the existence of the Kruzn for Kure Foundation that so many patient families have connected with us. I cannot overstate the impact your advocacy has made not only for our research but for the patient community in general. With such a rare disease it is so hard to get people connected and involved. As you know, we are updating a clinical and research registry of all SIOD patients that was originally started by Dr. Boerkoel. We believe that this registry is of major importance in establishing improved outcomes of patients with SIOD and are grateful for your support of this critical activity.  

I also want to especially thank all of the amazing donors in Muscle Shoals and elsewhere who support the Kruzn for a Kure Foundation. Jessica and Kyle, I’m awed by your commitment, tenacity, and strength in getting this all together! It’s all because you asked us what is necessary to find a way to help these kids and then went out and built the Foundation from scratch. I am immensely grateful that you have put your faith in me and my lab and Dr. Bertaina to do this important work.

March 1, 2018   Dr. David Lewis Update

As you are well aware, we know that what causes more than 95% of cases of SIOD is the inheritance of two mutated copies of the SMARCAL1 gene. (Dr. Boerkoel, MD, PhD, is the doctor and scientist who discovered that it was the defect in the SMARCAL1 gene that causes SIOD) However, the normal role of SMARCAL1 in the body and how deficient function of the SMARCAL1 protein causes the disease are not well understood. Much of our scientific research will focus on better understanding exactly how, and through what mechanisms, this mutation affects the body. One major breakthrough we had in the past year, which we accomplished using cells donated by Kruz and Paizlee, was to show for the first time that SIOD patients do in fact have shortened telomeres, as we had suspected. The telomere, if you remember, is like a special protein cap that fits on to the ends of the chromosomes, and keeps them from getting damaged when a cell divides and replicates all its genetic material. When telomeres are too short, there is a greater chance of damaging the chromosomes. Shortened telomeres have been associated with increased risk of cancer, and may also be a factor in other chronic diseases. This is a very important discovery in part because it links SIOD research to telomere research in general. Because defects in telomeres are responsible for a range of diseases, there are currently many scientists exploring this field, including possible ways to intervene and correct for shortened telomeres. I am confident that we will be able leverage some of this new research to help children like Kruz and Paizlee.

Now that we know that SIOD patients have shortened telomeres in one kind of cell – the T cells – we will start to explore whether or not other types of cells in their bodies have a similar defect. To do this we are going to capitalize on all the exciting new things we are now able to do with stem cells. Stem cells, as you may know, are a special kind of cell that have become key to scientific research because they are something like a “blank slate” cell, that can differentiate into many different types of specialized cells (such as a muscle cell, or a brain cell). Using stem cells from patients with SIOD, we will be able to re-create specialized cells and study how they behave, especially when compared to the same cells from a person without SIOD. This is a key tool in our arsenal. With it we will be able to zero in on an SIOD patient’s endothelial cells (the cells that line the insides of blood vessels), and hopefully understand how they work. This will be particularly important in trying to prevent some of the problems with blood vessels such as atherosclerosis (clogged arteries) and strokes, that as you know, SIOD patients face. I have also initiated a collaboration with an immunologist at UCSF, to investigate how the SMARCAL1 mutation affects T cells specifically. We know that SIOD patients have fewer T cells than healthy subjects, but we are trying to understand whether those patients’ T cells behave differently as well. This model of SIOD disease may allow us to determine if T cells can be used as an assay for treatments that reverse SIOD disease. In my discussions with Dr. Matt Might at UAB, he agrees that this might be good start also for screening purposes, and we will be talking with him regularly about our progress and to solicit advice.

Davis B. Lewis, MD