Stem-Cell Gene Therapy for Sickle Cell Disease


Donald Kohn, a professor of pediatrics and microbiology, immunology and molecular genetics in the UCLA College of Letters and Science, and his colleagues, have successfully established the means to cure sickle-cell disease. This strategy uses hematopoietic (blood-producing) stem cells from the bone marrow of patients with sickle-cell disease in order to treat the disease itself.

This approach provides a revolutionary alternative to current treatments, since it creates self-renewing, normal blood cells by inserting a gene that abrogates the sickling properties into hematopoietic stem cells. With this technique, there is no need to identify a matched donor, and therefore, patients avoid the risk of their bodies rejecting donor cells.

During the clinical trial, the anti-sickling hematopoietic stem cells will be transplanted back into patients’ bone marrow to increase the population of “corrected” cells that make red blood cells that don’t sickle. Kohn will hopefully begin enrolling patients in the trial within three months. The first subject will be enrolled and observed for safety for six months. The second subject will then be enrolled and observed for safety for three months. If evaluations show that no problems have arisen, the study will continue with two more subjects and another evaluation, until a total of six subjects have been enrolled.

Sickle cell disease, which affects more than 90,000 individuals in the U.S., is seen primarily in people of sub-Saharan African descent. It is caused by an inherited mutation in the beta-globin gene that transforms normal-shaped red blood cells, which are round and pliable, into rigid, sickle-shaped cells. Normal red blood cells are able to pass easily through the tiniest blood vessels (capillaries) and carry oxygen to organs like the lungs, liver and kidneys. However, sickled cells get stuck in the capillaries, depriving the organs of oxygen, which can lead to organ dysfunction and failure.

Current treatments include transplanting patients with hematopoietic stem cells from a donor. This is a potential cure for the disease, but due to the serious risks of rejection, only a small number of patients have undergone this procedure, and it is usually restricted to children with severe symptoms.

“Patients with sickle-cell disease have had few therapeutic options,” Kohn said. “With this award, we will initiate a clinical trial that we hope will become a treatment for patients with this devastating disease.”

Finding for this work comes from new grants to researchers at UCLA’s Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, which total nearly $21 million.  These grants were announced Dec. 12 at a meeting of the California Institute of Regenerative Medicine (CIRM) Citizen’s Oversight Committee.  They are apart of the state agency’s Disease Team Therapy Development III initiative.

Using Human Induced Pluripotent Stem Cells to Study Diamond Blackfan Anemia


Diamond-Blackfan Anemia or DBA results from mutations in a gene on chromosome 19 (in most cases). Mutations in the ribosomal protein S19 affects the ability of blood cells to make protein and causes low numbers of red blood cells. DBA patients are dependent on blood transfusions, but some are cured, to some extent at least, by bone marrow transplants. Unfortunately, some DBA patients have severe side effects from bone marrow transplants, which means that bone marrow transplants are not a panacea for all DBA patients.

Fortunately, Michell J. Weiss and his colleagues at the Children’s Hospital of the Philadelphia (CHOP) have used human induced pluripotent stem cells (iPSCs) to study DBA at the molecular level and even develop the beginnings of a cure for DBA patients. Weiss collaborated with Monica Bessler, Philip Mason, and Deborah French, all of whom work at CHOP.

Remember that red blood cells are made inside the bone marrow of the patient by hematopoietic stem cells (HSCs). HSCs divide to renew themselves, and to produce a daughter cell that will differentiate into one of several different types of blood cells. As a kind of gee-wiz number, a healthy adult person will produce approximately 10[11]–10[12] (100 billion to 1 trillion) new blood cells are produced daily in order to maintain steady state levels in the peripheral circulation.

In DBA patients, the bone marrow is empty of red blood cells. In order to get a better idea why, Weiss and his team isolated fibroblasts from the skin of DBA patients, and used genetic engineering techniques to convert them into iPSCs. When Weiss and his group tried to differentiate these iPSCs derived from DBA patients into red blood cells, they were not able to make normal red blood cells. However, Weiss and his colleagues used different genetic engineering techniques to fix the mutation in these iPSCs. After fixing the mutation, these cells could be differentiated into red blood cells. This experiment showed that it is possible to repair a patient’s defective cells.

This is a proof-of-principle experiment and there are many hurdles to overcome before this type of experiment can be done in the clinic to DBA patients. However, these iPSCs can play a vital role in deciphering some of the mysteries surrounding this disease. For example, two family members may have exactly the same mutation, but only one of them shows the disease whereas the other does not. Since iPSCs are specific to the patient from whom they were made, Weiss and his group hope to compare the molecular differences between them and understand the difference in expression of this disease.

Also, these cells offer a long-lasting model system for testing new drugs or gene modifications that may offer new treatments that are personalized to individual patients.

Weiss and his research group used this same technology to test drugs for the often aggressive childhood leukemia, JMML or Juvenile Myelomonocytic Leukemia. Once again, iPSCs were made from JMML patients and differentiated into myeloid cells, which divided uncontrollably just as the original myeloid cells from JMML patients.

Weiss and his colleagues used these cells to test two drugs, both of which are active against JMML. One of them is an inhibitor of the MEK kinase that was quite active against these cells. This illustrates how iPSCs can be used to test personalized treatment regimes for patients.

The stem cell core facility at CHOP is also in the process of making iPCS lines for several inherited diseases: dyskeratosis congenita, congenital dyserythropoietic anemia, thrombocytopenia absent radii, Glanzmann’s thrombasthenia, and Hermansku-Pudlak syndrome.

The even longer term goal is the use these lines to specifically study the behavior of such cells in culture and under certain conditions, test various drugs on them, and to develop treatment strategies on them as well.

Stem Cells to Make Red Blood Cells and Platelets in Culture


A collaborative study between Boston University School of Public Health and researchers at Boston Medical Center has used induced pluripotent stem cells to make unlimited numbers of human red blood cells and platelets in culture.

This finding could potentially reduce the need for blood donations to treat patients who require blood transfusions. Such research could also help researchers examine fresh and new therapeutic targets in order to treat blood diseases such a sickle-cell anemia.

The lead scientist on this project was George Murphy, assistant professor of medicine at Boston University School of Medicine and co-director of the Center for Regenerative Medicine at Boston University. Murphy’s main collaborator was David Sherr, professor of environmental health at Boston University School of Medicine and the Boston University School of Public Health.

Induced pluripotent stem cells or iPSCs are made from adult cells by applying genetic engineering technology to the adult cells that introduces genes into them. The introduction of four specific genes de-differentiates the adult cells into pluripotent stem cells that can, potentially, differentiate into any adult cell type. This makes iPSCs powerful tools for research and potential therapeutic agents for regenerative medicine.

In this study, Murphy and others used iPSCs from the CreM iPS Cell Bank and exposed them to a battery of different growth factors in order to push them to differentiate into different adult cell types. They were looking for the precise cocktail to differentiate iPSCs into red blood cells, since they wanted to further study red blood cell development in detail.

One group of compounds given to the set of iPSCs were molecules that activate “aryl hydrocarbon” receptors. Aryl hydrocarbon receptors (AHRs) play important roles in the expansion of hematopoietic stem cells, which make blood cells, since antagonism of AHRs promotes expansion of hematopoietic stem cells (see AE Boitano et al.,Science 10 September 2010: Vol. 329 no. 5997 pp. 1345-1348). In this case, however, Murphy and his colleagues observed a dramatic increase in the production of functional red blood cells and platelets in a short period of time. THis suggests that the ARH is important for normal blood cell development.

Aryl Hydrocarbon Receptor
Aryl Hydrocarbon Receptor

“This finding has enabled us to overcome a major hurdle in terms of being able to produce enough of these cells to have a potential therapeutic impact both in the lab and, down the line, in patients,” said Murphy. “Additionally, our work suggests that AHR has a very important biological function in how blood cells form in the body.”

“Patient-specific red blood cells and platelets derived from iPSC cells, which would solve problems related to immunogenicity and contamination, could potentially be used therapeutically and decrease the anticipated shortage and the need for blood donation,” added Murphy.

iPS-derived cells have tremendous potential as model systems in which scientists can test and develop new treatments for disease, given that such diseases can be constructed in the laboratory. These iPSC-derived red blood cells could be used by malaria researchers, and IPSC-derived platelets could be used to explore cardiovascular disease and treatments for blood clotting disorders.

Because my mother died from myelodysplasia, this finding has some personal interest to me. Mom had a difficult blood type to match, since she had the Bombay blood type (H). Finding blood for her was a major tour de force, and as she received blood that was less and less well matched to her body, she suffered the ravages of poorly matched blood. A treatment of red blood cells made from IPSCs derived from her own cells might have extended her life and even improved her quality of life in her later years.

I look forward to this research eventually culminating in clinical trials.

Engineered T Cells Help a Child Get Rid of Leukemia


Pediatric oncologists from The Children’s Hospital of Pennsylvania (CHOP) and collaborating scientists from the University of Pennsylvania (UPenn) have used genetic engineering techniques to reprogram T lymphocytes from a young cancer patient’s blood. This reprogramming drove the T cells to attack the child’s leukemia, and, to date, has completely cured the child of leukemia.

Stephan Grupp, a pediatric oncologist from CHOP, is part of a clinical trial that tests cell therapy for adult chronic lymphocytic leukemia (CLL). CLL is the most common type of leukemia in adults and usually occurs during or after middle age and only rarely occurs in children.

As regular readers of this blog are aware, the bone marrow contains a stem cell population called hematopoietic stem cells. While this stem cell population is not a homogeneous one, these stem cells divide to renew themselves and replenish all the blood cells that we lose each day. When the hematopoietic stem cells divide, they renew themselves and give rise to either a myeloid or lymphoid progenitor cells. Myeloid progenitors differentiate into one of three types of mature blood cells: 1) red blood cells, which carry oxygen and the other substances to all tissues in the body; 2) white blood cells that fight infection and disease; 3) platelets that form blood clots to stop bleeding. Lymphoid progenitors become lymphoblast cells which then differentiate to become one of three cell types: 1) B lymphocytes, which make antibodies to fight infection; 2) T lymphocytes that help B lymphocytes to make antibodies to fight infection; 3) natural killer cells that attack cancer cells and viruses.

Hematopoietic stem cells

CHOP’s Stephen Grupp and Carl June, of the Perelman School of Medicine at the Univ. of Pennsylvania, lead this research group. Together, they have presented new data at the American Society of Hematology annual meeting in Atlanta that shows nine of 12 patients with advanced leukemias in the clinical trial, including two children, who responded to treatment with their newly engineered cells. This treatment strategy uses an engineered T lymphocyte that Grupp and June call “CTL019 cells.” By reprogramming the T cells to specifically attack this aggressive form of leukemia, some of these patients showed a complete remission of their leukemias.

Of the nine patients who responded to CTL019 treatment, one was a 7-year-old patient who suffered from acute lymphoblastic leukemia (ALL). Grupp and Penn colleagues adapted their treatment to combat ALL, which is the most common type of childhood leukemia and the most common childhood cancer. Although physicians cure roughly 85 percent of ALL cases, the remaining 15 percent of such cases stubbornly resist treatment.

Grupp’s research builds on his ongoing collaboration with scientists from UPenn. These UPenn researchers developed modified T cells as a treatment for B-cell leukemias. T cells are at the center of the immune response. T lymphocytes recognize and attack invading foreign invaders, but cancer cells slip under their surveillance net because they are so similar to normal cells. CAR T cells, which stands for “chimeric antigen receptor T cells” are engineered to specifically detect and target cancerous B cells. Since the B cells are the cancerous cells in the case of certain leukemias, such as ALL and CLL, CAR T cells can purge the body of these cancers rather effectively.

On the surface of B cells is a protein called CD19. By raising high-affinity antibodies to CD19 and then physically attaching those antibodies to T cells, UPenn researchers invented a kind of guided missile that detects and destroys B cells and B-cell leukemias.

When Grupp and his crew used CLT019 in his pediatric patients, they found that the engineered T cell was very active, but it caused an undesirable side effect called cytokine release syndrome. The child became very ill and was admitted to the intensive care unit. However, Grupp and his team counteracted these toxic side effects by using two 2 drugs that suppress the immune response and these thwarted the overactive immune response and rapidly relieved the child’s treatment-related symptoms. An added bonus was that these drugs had no effect on the engineered T cells, which still destroyed leukemia cells until the cows came home. These results were so effective, that this clinical approach is now being successfully incorporated into CTL019 treatments for adults as well.

The CHOP/UPenn team reported on early results of this clinical trial in adult chronic lymphocytic leukemia (CLL) patients in August of 2011. In their seven-year-old patient, they engineered her own T cells to attack her aggressive form of childhood leukemia. Without this treatment, she faced grim prospects once her cancer relapsed after conventional treatment. However, with this innovative CTL019 experimental therapy, the bioengineered T cells multiplied rapidly in her body and destroyed the leukemia cells. After her CTL019 treatment, the child’s doctors found that she had no evidence of cancer.

According to Grupp: “These engineered T cells have proven to be active in B cell leukemia in adults. We are excited to see that the CTL019 approach may be effective in untreatable cases of pediatric ALL as well. Our hope is that these results will lead to widely available treatments for high-risk B cell leukemia and lymphoma, and perhaps other cancers in the future.”

Susan Rheingold, one of the leaders in the Children’s Hospital program for children with relapsed leukemia added: “This type of pioneering research addresses the importance of timing when considering experimental therapies for relapsed patients. To ensure newly relapsed patients with refractory leukemia meet criteria for options like CTL019, we must begin exploring these innovative approaches earlier than ever before. Having the conversation with families earlier provides them more treatment options to offer the best possible outcome.”

In August 2012, the biotechnology company Novartis acquired exclusive rights from UPenn to CART-19, the therapy that was the subject of this clinical trial and which is now known as CTL019.