Human Stem Cell Gene Therapy Appears Safe and Effective


Two recent studies in the journal Science have reported the outcome of virally-mediated gene correction in hematopoietic stem cells (HSCs) to treat human patients. These studies may usher in a new era of safe and effective gene therapy. These exciting new clinical findings both come from the laboratory of Luigi Naldini at the San Raffaele Scientific Institute, Milan, Italy. The first experiment examined the treatment of metachromatic leukodystrophy (MLD), which is caused by mutations in the arylsulfatase A (ARSA) gene, and the second, investigated treatments for Wiskott-Aldrich syndrome (WAS), which is caused by mutations in the gene that encodes WASP.

MLD is one of several diseases that affects the lysosome; a structure in cells that acts as the garbage disposal of the cell. So called “lysosomal storage diseases” result from the inability of cells to degrade molecules that come to the lysosome for degradation. Without the ability to degrade these molecules, they build up to toxic levels and produce progressive motor and cognitive impairment and death within a few years of the onset of symptoms.

To treat MLD, workers in Naldini’s laboratory isolated blood-making stem cells from the bone marrow of three pre-symptomatic MLD patients (MLD01, 02 and 03). These stem cells were infected with genetically engineered viruses that encoded the human ARSA gene. After expanding these stem cells in culture, they were re-introduced into the MLD patients after those same patients had their resident bone marrow wiped out. The expression of the ARSA gene in the reconstituted bone marrow was greater than 10 fold the levels measured in healthy controls and there were no signs of blood cancers or other maladies. One month after the transplant, the implanted cells showed very high-level and stable engraftment. Between 45%-80% of cells isolated and grown from bone marrow samples harbored the fixed ARSA gene. AS expected, the levels of the ARSA protein rose to above-normal levels in therapeutically relevant blood cells and above normal levels of ARSA protein were isolated from hematopoietic cells after one month and cerebrospinal fluid (CSF) one to two years after transfusion. This is remarkable when you consider that one year before, no ARSA was seen. This shows that the implanted cells and their progeny properly homed to the right places in the body. The patient evaluations at time points beyond the expected age of disease onset was even more exciting, since these treat patients showed normal, continuous motor and cognitive development compared to their siblings who had MLD, but were untreated. The sibling of the patient designated “MLD01” was wheelchair-bound and unable to support their head and trunk at 39 months, but excitingly, after treatment, patient MLD01 was able to stand, walk and run at 39 months of age and showed signs of continuous motor and cognitive development. Lastly, and perhaps most importantly, there was no evidence of implanted cells becoming cancerous, even though they underwent self-renewal, like all good stem cells. This is the first report of an MLD patient at 39 months displaying such positive clinical features.

The second study treated WAS, which is an inherited disease that affects the immune system and leads to infections, abnormal platelets, scaly skin (eczema), blood tumors, and autoimmunity. In this second study, blood-making stem cells were collected from three patients infected with genetically engineered viruses that expressed the WASP gene. These stem cells were then reinfused intravenously (~11 million cells ) three days after collection. Blood tests and bone marrow biopsies showed evidence of robust engraftment of gene-corrected cells in bone marrow and peripheral blood up to 30 months later. WASP expression increased with time in most blood cells. Although serious adverse infectious events occurred in two patients, overall clinical improvement resulted in reduced disease severities in all patients. None of the three patients demonstrated signs of blood cancers and the platelet counts rose, but, unfortunately, not to normal levels. Again, no evidence for adverse effects were observed.

Simply put, these authors have presented a strategy for ex vivo gene correction in HSCs for inherited disorders which works and appears safe in comparison to previous strategies. Long-term analyses will undoubtedly need to be intensely scrutinized, but this research surely represents a huge step forward in the safe treatment of these and similar genetic disorders.

Stem Cell Transplants for Non-Hodgkin’s Lymphoma


In patients with aggressive non-Hodgkin’s lymphoma, early stem cell transplants do not improve the overall survival in high-risk patients, but are beneficial in those patients who are at the highest risk.

Lymphomas are cancers of the lymphocytes, which are a specific group of white blood cells. A particular type of lymphoma known as Non-Hodgkin’s lymphoma is more common than the other general type of lymphoma — Hodgkin lymphoma. There are several different subtypes of non-Hodgkin’s lymphoma. The most common non-Hodgkin’s lymphoma subtypes include diffuse large B-cell lymphoma and follicular lymphoma.

The symptoms of non-Hodgkin’s lymphoma include Non-Hodgkin’s lymphoma symptoms may include: swollen lymph nodes in the neck, armpits or groin, swelling of the abdomen and abdominal pain, Chest pain, coughing or trouble breathing, fatigue (tiredness), fever, night sweats, and weight loss.

The usual treatment for aggressive non-Hodgkin’s lymphoma is a combination of four different chemotherapeutic agents designated as “CHOP,” which stands for Cyclophosphamide (alkylating agent that rituximabdamages DNA), Hydroxydaunorubicin (also called doxorubicin or Adriamycin, also a DNA-damaging agent), Oncovin (vincristine, which binds to microtubules and prevents cells from dividing duplicating by binding to the protein tubulin), and Prednisone or prednisolone (corticosteroids). Recently, many oncologists are adding Rituximab to this drug regimen (but only if the lymphoma is of B-cell origin). Rituximab is a monoclonal antibody that binds to the surface of B-lymphocytes (the very cells that have become cancerous) and facilitates their destruction. This new five-drug regimen, R-CHOP, can drive many patients into remission. However, some relapse and go on to receive stem cell transplants.

This present study, which was directed by Patrick Stiff from the Loyola University Medical Center’s Cardinal Bernardin Cancer Center, was designed to determine if an early stem cell transplant before the patient relapsed increase patient survival. This study examined patients from 40 different clinical sites in the United States and Canada.

397 patients who were in defined groups of high risk or intermediate-high risk of relapsing. After initial chemotherapy treatment, those patients who responded to treatment were randomly assigned to receive an autologous stem cell transplant (125 patients) or to a control group (128 patients) who received three additional cycles of the R-CHOP regimen.

After two years, 69 percent of the transplantation patients had no disease progression, compared with 55 percent of the control group. This is a statistically significant difference, but the two-year survival rates in the transplantation group was 74 percent versus 71 percent in the control group, which was not statistically significant. However, patients in the control group who relapsed were later offered stem cell transplants, which is probably why the differences are not statistically significant.

However, mining the data further reveals something even more interesting. While the stem cell transplants did not improve overall survival among the entire group of high-risk and high-intermediate risk patients, the high-risk patients as an isolated subset rather clearly received a remission and survival benefit from the early stem cell transplants. The two-year survival rate was 82 percent in the stem cell transplant group and 64 percent in the control group, which is statistically significant.

Patrick Stiff and his colleagues concluded: “Early transplantation and late transplantation achieve roughly equivalent overall survival in the combined risk groups.” However, “early transplantation appears to be beneficial for the small group of patients presenting with high-risk disease.”

Stiff hopes that this finding will “trigger discussions between such patients and their physicians as to the feasibility of doing early transplants.”

Patients who receives doses of their own stem cells (so-called autologous stem cell transplants), can tolerate very high doses of chemotherapy and/or radiation. This high-dose treatment kills off many cancer cells, but it also destroys the patient’s immune system. Therefore, prior to the treatment, stem cells are removed from the blood or bone marrow of he patient and infused back into the patient. These stem cells then form a new immune set of immune cells that replace the ones destroyed by the chemotherapy.

Previous studies have shown that patients who undergo autologous stem cell transplants have a higher risk of developing secondary cancers that are caused by the chemotherapy or the radiation. However, this new study did not find a statistically significant difference (11 percent in the control group and 12 percent in the stem cell transplant group) in secondary tumor formation between the two groups.

Stiff and his crew are continuing to crunch the numbers and mine the data. “As years go by, there may be additional analysis that may help fine-tune the results so that we will be able to more carefully and concisely define any potential benefit,” said Stiff.

See Patrick J. Stiff, et al, New England Journal of Medicine 2013; 369(18):1681.

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.

Expanding Blood-Making Stem Cells for Use in Patients


John Dick is a senior scientist at the University Health Network’s McEwen Centre for Regenerative Medicine and a professor at the University of Toronto. He is also the senior investigator for a study that includes a collaboration between Canadian and Italian stem cell scientists that examined ways to expand human blood stem cells for human use.

A new master control gene was identified in this study that, when manipulated, could increase stem cell production.

In the words of Dick, “For the first time in human blood stem cells, we have established that a new class of non-coding RNA called miRNA represents a new tactic for manipulating these cells, which opens the door to expanding them for therapeutic uses.”

In 2011, Dick’s research group published a landmark paper in which he and his colleagues succeeded in isolating “CD49f+” cells. Just one of these CD49f+ cells could reconstitute an entire blood-cell making system in bone marrow. It has been known for some time that the population of blood cell-making stem cells in bone marrow is rather heterogeneous, and some cells have tremendous regenerative capacities, but others shown only slight regenerative abilities. DIck’s group isolated bone marrow stem cells that could replenish the whole blood-making system of a laboratory animal (see Notta, et al., Science 333, 218-221).

Dick has also pioneered the field of cancer stem cells when his lab identified leukemia stem cells in 1994 (Lapidot T, et al., Nature. 367, 645-8.) and colon cancer cells in 2007 (O’Brien CA, et al. Nature. 445, 106-10).

The lead author of this study, Eric Lechman, recounted his laboratory work with a master control gene known as microRNA 126 or miR-126. THis small RNA normally silences the expression of many genes, and thus keeps stem cells in a quiescent, dormant state. His strategy in working with miR-126 was to introduce new binding sites into the cell for miR-126 in order to lower the concentration of free miR-126 inside the cell. To do this, he infected stem cells with a genetically engineered virus that was loaded with miR-126 binding sites. The results were remarkable.

According to Lechman, “The virus acted like a sponge and mopped up the specific miRNA in the cells. This enabled the expression of normally expressed genes to become prominent, after which we observed a long-term expansion of the blood stem cells without exhaustion or malignant transformation.”

Given the difficult many labs have has growing sufficient quantities of blood stem cells in the laboratory, this finding could completely revolutionize blood stem cell research and clinical treatments with these stem cells.

According to Dick, “We’ve shown that if you remove the miRNA you can expand the stem cells while keeping their identity intact. That’s the key to long-term stem cell expansion for use in patients.”

Reducing the Incidence of a Deadly Side Effect of Bone Marrow Transplants in Mice


Bone marrow transplants save the lives of leukemia, but they have one risky drawback and that is “graft-versus-host disease.” Graft-versus-host disease (GVHD) results when immune cells in the donor’s bone marrow attack the tissues and cells of the recipient’s body as foreign. Almost half of bone marrow transplant recipients develop graft-versus-host disease (GVHD), the main organs affected are the skin, liver and gut. Obviously, finding a way to quell or even prevent GVHD would be a boon for bone marrow transplantations.

By utilizing a mouse model, researchers at Washington University School of Medicine in St. Louis have managed to reduce the risk of GVHD from bone marrow transplants. Since bone marrow transplants are the only available curative treatment when leukemia returns, decreasing the risk of GVHD is the first step to improving the prognosis of leukemia patients.

The main strategy behind decreasing the effects of GVHD is to direct immune cells from the donor’s bone marrow away from healthy tissue and lead them to their intended purpose, which is to kill cancer cells.

“This is the first example of reducing graft-versus-host disease not by killing the T-cells, but simply by altering how they circulate and traffic,” says John F. DiPersio, MD, PhD, the Virginia E. and Sam J. Golman Professor of Medicine at Barnes-Jewish Hospital and Washington University School of Medicine. “Donor T-cells do good things in terms of eliminating the recipient’s leukemia, but they can also attack normal tissues leading to death in a number of patients. The goal is to minimize graft-versus-host disease, while maintaining the therapeutic graft-versus-leukemia effect.”

By working in a mouse model, Jaebok Choi, PhD, research assistant professor of medicine, showed that if he eliminated or blocked a particular protein known as the interferon gamma receptor on donor T-cells, these cell were unable to migrate to critical organs such as the intestines. However, these same T-cells were still capable of killing leukemia cells.

“The fact that blocking the interferon gamma receptor can redirect donor T-cells away from the gastrointestinal tract, at least in mice, is very exciting because graft-versus-host disease in the gut results in most of the deaths after stem cell transplant,” DiPersio says. “People can tolerate graft-versus-host disease of the skin. But in the GI tract, it causes relentless diarrhea and severe infections due to gut bacteria leaking into the blood, which can result in severe toxicity, reduction in the quality of life or even death in some patients.”

Interferon gamma has, for some time, been known to play a vital role in inflammation. The signal transduction pathway that works downstream of the receptor is just now being better understood. It is this signal transduction pathway downstream of the receptor that is responsible for activating the T-cells so that they cause GVHD. The signaling cascade initiated when interferon gamma binds its receptor activates molecules known as JAK kinases, followed by another protein called “STAT,” and finally a protein called CXCR3. CXCR3 mediates the trafficking of donor T-cells to the GI tract and other target organs.

Deleting the interferon gamma receptor from donor T-cells steers them away from target organs. This, however, leads to a second question: “Could the same result be observed by inhibiting some of the other molecules that act downstream of the interferon gamma receptor?” To address this question, Choi knocked out CXCR3 and discovered that such a knock out reduced graft-versus-host disease, but did not completely wipe it out.

“There are probably additional downstream targets of interferon gamma receptor signaling other than JAKs, STATs and CXCR3 that are responsible for T-cell trafficking to the GI tract and other target organs,” DiPersio says. “We’re trying to figure out what those are.”

This worked beautifully in mice, but could it work in humans? To make these data more relevant to human biology, Choi and DiPersio used drugs known to block JAK kinases in human cells. These drugs are presently approved by the Food and Drug Administration to treat myelofibrosis, which is a pre-leukemic condition in which bone marrow is replaced with fibrous tissue. Ruxolitinib and pacritinib are two such drugs and Choi and DiPersio showed that treating mice with either of these two drugs could mimic the protective effect of deleting the interferon gamma receptor. The JAK inhibitors definitely redirect the donor T-cells away from target organs and reducing graft-versus-host disease in leukemic mice. Unfortunately, they have yet to determine if these drugs preserve the anti-leukemia effect of these T-cells.

“The proof-of-principle behind these experiments is the exciting part,” DiPersio says. “If you can change where the T-cells go as opposed to killing them, you prevent the life-threatening complications and maintain the clinical benefit of the transplant.”

Umbilical Cord Stem Cells and Cancer


The umbilical cord blood stem cells have been used to treat cancer patients whose bone marrow tissues have been wiped out by radiation or chemotherapeutic treatments. Several clinical trials have addresses the capacity of umbilical cord blood to reconstitute the bone marrow of cancer patients.

The first set of clinical trials have examined the use of umbilical cord blood in children. Gluckman and her colleagues reported the use of umbilical cord blood to treat children who had suffered from a variety of blood maladies. 74 patients were treated with umbilical cord blood. 63% of the patients survived one year after the procedure, and the rate of graft-versus-host disease (GVHD) was only 9%. Now this study showed that umbilical cord blood could be used to reconstitute the bone marrow, but how well does it work compared to bone marrow transplants?

To answer this question, Rocha and his colleagues compared kids who had received umbilical cord blood transplants with those who had received bone marrow transplants. 113 cord blood transplant patients were compared to 2052 bone marrow transplant recipients. In this study, the umbilical cord blood recipients took longer to have their bone marrow reconstituted, but the rate of graft-versus-host disease was lower. The survival rate of the two groups three years after the procedure was also about the same (64% for the umbilical cord blood recipients and 66% for the bone marrow recipients). Thus, umbilical cord blood seemed to work as well as bone marrow when it came to reconstituting the bone marrow.

Since the rates of GVH disease were so low, could umbilical cord blood that was not properly tissue matched to the recipient also work? The answer was yes. Once again Gluckman and her colleagues showed that the rate of GVH disease was rather low, and the rate of recovery in a group of 65 patients was quite high (87%). Such a treatment with unmatched bone marrow would be a disaster, since GVH disease would almost certainly result from such a treatment. The results of Gluckman’s small study were confirmed by a much larger study by Rubinstein and others in 1998.

Can cord blood be used to treat adults with similar maladies? Clinical studies have confirmed that the answer is yes. Survival rates from a host of clinical trials have ranged from 15%-70%, but clearly adults can benefit from umbilical cord blood transplantations. Once again, the rates of GVH disease were lower in umbilical cord blood recipients when compared to bone marrow recipients, but once again, the time required for bone marrow recovery was greater.

In Minnesota, Wagner and his colleagues pioneered the use of “double umbilical cord blood grafts” in which umbilical cord blood is taken from two different babies to treat an adult patient. This overcomes the limited volume and cell numbers in an umbilical collection from a single donor. These are only used for patients who are very ill, but studies have shown that patients who have received double umbilical cord blood grafts have a ten-fold lower decrease in the risk of relapse of blood cancers.

Thus over the past two decades, umbilical cord blood transplants have become rather attractive sources of material to reconstitute bone marrow. Although low cell numbers are still a chronic problem with them, the ability to culture and expand these cells in culture may give a new life to this useful treatment.