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 Treatments for Hurler’s Syndrome


Mucopolysaccharidoses are a group of inherited diseases that result from loss-of-function mutations in those genes that encode enzymes that degrade long-chain sugar molecules. One of these mucopolysaccharidoses, Hurler syndrome, is a consequence of the inability to make a functional version of an enzyme called iduronidase. Without functional iduronidase, cells cannot degrade molecules called glycosaminoglycans (formerly called mucopolysaccharides), which are found in mucus and in fluid around the joints. The concentrations of these glycosaminoglycans increase and damage organs, including the heart. Symptoms can range from mild to severe.

From Kowalewski B et al. PNAS 2012;109:10310-10315. Heparan sulfate catabolism involving GlcNS3S structures. The scheme illustrates all nine different enzymatic activities required for the sequential catabolism of a NRE tetrasaccharide containing GlcNS3S. To expose the 3-O-sulfated residue at the terminus, the preceding uronic acid (iduronate 2-O-sulfate in this example) is modified sequentially by iduronate 2-sulfatase and iduronidase. Under normal conditions, the 3-O-sulfate then is removed from GlcNS3S by ARSG, thus generating the substrate for sulfamidase, which removes the N-sulfate group. Subsequently, another six different enzymes (plus again sulfamidase) have to act, which ultimately leads to a complete degradation of the chain. The loss of ARSG activity (MPS IIIE) leads to the accumulation of 3-O-sulfated ARSG substrate that cannot be acted upon by downstream catabolic enzymes. It should be noted that the 2-O-sulfation shown at the glucuronic acid (third residue) is relatively rare, which agrees with the finding that no pentasulfated trisaccharides were found as NRE structures (Fig. 3A). Scheme modified from Neufeld and Muenzer (6) according to findings from this work and from Lawrence et al.
From Kowalewski B et al. PNAS 2012;109:10310-10315.
Heparan sulfate catabolism involving GlcNS3S structures. The scheme illustrates all nine different enzymatic activities required for the sequential catabolism of a NRE tetrasaccharide containing GlcNS3S. To expose the 3-O-sulfated residue at the terminus, the preceding uronic acid (iduronate 2-O-sulfate in this example) is modified sequentially by iduronate 2-sulfatase and iduronidase. Under normal conditions, the 3-O-sulfate then is removed from GlcNS3S by ARSG, thus generating the substrate for sulfamidase, which removes the N-sulfate group. Subsequently, another six different enzymes (plus again sulfamidase) have to act, which ultimately leads to a complete degradation of the chain. The loss of ARSG activity (MPS IIIE) leads to the accumulation of 3-O-sulfated ARSG substrate that cannot be acted upon by downstream catabolic enzymes. It should be noted that the 2-O-sulfation shown at the glucuronic acid (third residue) is relatively rare, which agrees with the finding that no pentasulfated trisaccharides were found as NRE structures (Fig. 3A). Scheme modified from Neufeld and Muenzer according to findings from this work and from Lawrence et al.

Hurler syndrome is inherited, and both parents must pass the faulty gene to inherit Hurler syndrome.

The symptoms of Hurler syndrome usually appear between ages 3 and 8. Infants with severe Hurler syndrome appear normal at birth. Facial symptoms may become more noticeable during the first 2 years of life. The most common symptoms include abnormal bones in the spine, claw hand, cloudy corneas, deafness, halted growth, heart valve problems, joint disease (including stiffness),
Intellectual disability that gets worse over time, and thick, coarse facial features with a low nasal bridge.

Hurler’s syndrome appears in about 1 in 100,000 live births, and those afflicted with it normally die in their teens.

Treatments for Hurler Syndrome include “enzyme replacement,” which is very expensive. Enzyme replacement therapy utilizes genetic engineering to make large quantities of iduronidase, which is then administered to Hurler Syndrome patients. A second treatment is bone marrow transplantation, but this requires finding a good tissue match.

Sharon Byers from the University of Adelaide, Australia and her colleagues are genetically modifying adult stem cells (mesenchymal stem cells, specifically) to make large quantities of iduronidase. These modified stem cells are then infused into Hurler Syndrome patients. To date, these experimental treatments seem to be providing Hurler Syndrome patients some relief, but it is still early in the trial.

Matilda Jackson, a PhD candidate in Byers lab, described their trial in this manner: “We have turned adult stem cells into little ‘enzyme factories” by coupling them with a virus that makes them pump out high levels of the enzyme.” Matilda Jackson is a member of the School of Molecular and Biochemical Sciences and Adelaide University.

Dr. Jackson continued, “Those stem cells can then be injected into the blood where they move around the body and become liver or bone or brain or other cells and start producing the missing enzyme. They automatically migrate to areas of damage in the affected individual. So far in our laboratory studies we’ve measured improvements in brain function but we’ve yet to complete the analysis to determine if there are improvements in other organs.”

Sharon Byers, an affiliate senior lecturer in the School of Molecular and Biomedical Sciences, explained, “There are two current treatments for Hurler’s Syndrome: costly enzyme replacement therapy or bone marrow transplants which require a perfectly matched donor. And while they bring some improvements,, neither of these treatments prevents damage to the brain and bones because not enough enzyme reaches either of these tissues.”

Dr. Byers continued: “These stem cells, modified so that produce large quantities of the enzyme that people with Hurler’s Syndrome lack, offer great hope for a potential new therapy. If we can help reverse the disease symptoms, we could see these children able to perform normal tasks, giving them a better quality of life and increasing their life span.”

Stem Cell Gene Therapy For An Inherited Neurological Disease


Scientists at the University of Manchester in the United Kingdom have used stem cell gene therapy to treat a fatal genetic brain disease in mice. Sanfilippo is a fatal, inherited condition that causes progressive dementia in children. This particular treatment strategy could also be used to treat other types of neurological, inherited diseases. The Manchester team hopes to bring this strategy to a clinical trial within two years.

Sanfilippo afects one in 89,000 children in the United Kingdom and is an untreatable “mucopolysaccharide disease ” or MPS disease. MPS diseases involve an abnormal storage of mucopolysaccharides. This abnormal storage results from the absence of a specific enzyme. Without the enzyme, the breakdown process of mucopolysaccharides is incomplete. Partially broken down mucopolysaccharides accumulate in the body’s cells causing progressive damage. The storage process can affect appearance, development, and the function of various organs of the body. Each MPS disease is caused by the deficiency of a specific enzyme.

Patients with Sanfilippo are unable to degrade heparan sulfate. There are four different types of Sanfilippo, which is also called MPS type III. MPS type IIIA results from a deficiency in the enzyme N-sulfoglucosamine sulfohydrolase, MPS type IIIB lacks N-Acetylglucosaminidase, MPS type IIIC has an absence in Acetyl-CoA:alpha-glucosaminide-acetyltransferase, and MPS type IIID lacks N-acetylglucosamine 6-sulphatase. In all four forms of MPS III, excessive heparan sulphate storage occurs in the brain, leading to its progressive deterioration; the amount of heparan sulphate storage in other tissues influences the extent of physical symptoms. Children eventually lose the ability to walk and swallow.

Brian Bigger from the University of Manchester’s Institute of Human Development led this research into therapies for MPS type IIIA. According to Bigger, bone marrow transplants have been used to treat similar diseases (e.g., Hurler syndrome). In this case, gene therapy was used to introduce the missing enzyme into the transplanted cells. Unfortunately, this did not work terribly well because the white blood cells from the bone marrow did not make enough of the enzyme to treat the disease.

A fraction of the white blood cells made bone marrow are called monocytes, and some of the monocytes traffic to the brain to become microglia. Since microglia are made by hematopoietic stem cells (HSCs) in the bone marrow, genetic engineering of cultured HSCs should increase expression of the missing enzyme in microglia. In previous experiments, HSCs were engineered with viruses to express the missing enzyme, but this expression was poor in microglia.

To fix this problem, Bigger and his team increased enzyme expression in the engineered HSCs in bone marrow. They used a gene control region from the “pyruvate kinase” gene, which is a very highly expressed gene. This increased expression of the missing enzyme to five times the normal levels and to 11% of normal levels in the microglia cells in the brain. The enzyme

This type of treatment corrects the inflammation in the brain and completely corrects the hyperactivity behavior in mice with Sanfilippo. Bigger adds, “We now hope to work to a clinical trial in Manchester in 2015.”

Bigger and his colleagues are manufacturing a viral vector to deliver genetic material into cells for use in humans and they hope to use this in a clinical trial with patients at Central Manchester University Hospital NHS Foundation Trust by 2015.

This stem cell gene therapy approach was recently shown by Italian scientists to improve conditions in patients with a similar disease that affects the brain called metachromatic leukodystrophy. Bigger refined the vector used bythe Italian group.

According to Bigger, this approach might have the potential to treat several neurological genetic diseases.

Induced Pluripotent Stem Cell Model For Gaucher Disease Recapitulates Disease Pathology


In previous posts, I have discussed a group of inherited diseases known as lysosomal storage diseases. To briefly review, the garbage disposal of the cells is a small membrane-enclosed vesicle known as the lysosome. Lysosomes take up materials that need to be degrades and degrade them, and it does this by means of an array of enzymes called acid hydrolases. These enzymes are not active unless they are present inside the lysosome, since the lysosome acidifies its interior. Acid hydrolases are only active in an acid environment, and this prevents acid hydrolases from degrading the cell if they escape from the lysosome.

If the genes that encode acid hydrolases contain a loss-of-function mutation that causes them to produce nonfunctional proteins, then the lysosome will be unable to degrade particular molecules. Those undegraded molecules will build up to toxic levels and kill cells. These genetic diseases that result from nonfunctional lysosomal acid hydrolases are called lysosomal storage diseases. Fortunately, some new treatment strategies, such as enzyme replacement therapies and stem cell treatments have provided new hope for these diseases.

One particular lysosomal storage disease is called Gaucher disease and this disease results from mutations in the gene that encodes an enzyme called β-glucocerebrosidase. β-Glucocerebrosidase degrades a molecule called glucosylceramide, and in the absence of a functional copy of β-glucocerebrosidase, glucosylceramide builds up and accumulates especially in a type of white blood cell called a macrophage. Glucosylceramidase also tends to accumulate in the spleen, liver, kidneys, lungs, brain and bone marrow.

Babies that have Gaucher disease show enlarged spleens and livers. Their livers do not work properly and they also have bone problems, nervous system problems, swelling of their lymph nodes and their joints too, a bloated, swollen abdomen, a brownish tint to the skin, messed up blood work (too few red and white blood cells and platelets), and yellow fatty deposits on the white of the eye.

Gaucher disease is caused by a recessive mutation in a gene located on chromosome 1 and it equally affects both males and females. About 1 in 100 people in the United States are carriers of the most common type of Gaucher disease, but the carrier rate among Ashkenazi Jews is 8.9% while the birth incidence is 1 in 450.

Some forms of Gaucher disease can be treated by enzyme replacement treatment in which recombinant glucocerebrosidase (imiglucerase) is administered intravenously. Such treatments dramatically decrease liver and spleen size, reduce skeletal abnormalities, and reverse other manifestations of Gaucher disease. Unfortunately, this treatment is rather expensive (approximately US $200,000 per year for a single patient), has to be continued for life. Another treatment, Velaglucerase alfa, was approved by the Food and Drug Administration (FDA) as an alternative treatment in February 2010, and in May 2012 the FDA approved an additional treatment called Taliglucerase alfa, or Elelyso.

Is there a better way to treat this disease? To determine that, we need to know more about the disease, and that requires a better model for Gaucher disease. Fortunately, induced pluripotent stem cells (iPSCs) can provide such a model system, and some enterprising stem cell biologists have just made such a model system for Gaucher disease. Leelamma Panicker, and colleagues in the laboratory of Ricardo Feldman at the University of Maryland School of Medicine, in Baltimore, Maryland have made iPSCs from patients with Gaucher disease. These cells show many of the pathologies of Gaucher disease patients.

Panicker and her colleagues made human iPSCs from skin fibroblasts from patients with all three types of Gaucher disease. Gaucher disease comes in three different types, type 1, 2, and 3. Type I is a milder form of the disease and tends occur in Ashkenazi Jews, at 100 times the occurrence in the general populace, and the median age at diagnosis is 28 years of age. The life expectancy is mildly decreased, but there are no neurological symptoms. Type II is more severe and is characterized by neurological problems in small children. The prognosis is poor and most of these children die before the age of 3. Type III is moderately severe and occurs in Swedish patients from the Norrbotten region, and this disease is manifested somewhat later in life but most die before their 30th birthday.

The iPSCs made from Gaucher disease patients were differentiated in all kinds of cell types and tissues. They used the iPSCs to form a special type of tumor called teratomas in laboratory animals. Teratomas grow quickly and form a wild pastiche of tissues and cell types as they overgrow. Most usefully, the teratomas formed a whole wade of cell types that show pathologies in Gaucher disease. For example the teratomas differentiated into macrophages and nerve cells. The macrophages were able to gobble up materials responded to and were capable of responding to foreign molecules. This demonstrated that these macrophages are real macrophages.

The really cool part about these iPSC-derived cells is that they show many of the pathological hallmarks of Gaucher disease. First, the macrophages exhibited low levels of glucocerebrosidase enzymatic activity. They also accumulated glucosylceramide and their lysosomes worked very poorly to say the least. These macrophages were largely unable to digest red blood cells they had gobbled up. This is a feature of macrophages from patients with Gaucher disease. Even more interestingly, the speed with which each cultured macrophage population was able to digest red blood cells tightly correlated with the type of Gaucher disease that afflicted the patient from whom the original cells were isolated. Thus, the macrophages made from iPSCs derived from type I patients digested red blood cells the fastest of three lines, and those from type II patients digested them the slowest, if at all. To ensure that a lack of beta-glucosylceramidase was the problem with these cells, they Incubated the macrophages with recombinant beta-glucosylceramidase, and this completely reversed the delay in red blood cell digestion. if these macrophages were treated with a drug called isofagomine (Plicera), which is made by Amicus Therapeutics and has been approved as a treatment for Gaucher disease, the digestion of red blood cells was only partially restored. Isofagomine binds to beta-glucosylceramidase and locks it into a conformation that increases its activity by three-fold.

These experiments show that these cell types that were derived from iPSCs made from Gaucher’s disease patients effectively recapitulates the pathologic hallmarks of the disease.

According to senior author, Ricardo Feldman, associate professor of microbiology and immunology at the University of Maryland and a research scientist at the University of Maryland Center for Stem Cell Biology and Regenerative Medicine: “We are confident that this will allow us to test more drugs faster, more accurately, and more safely, bringing us closer to new treatments for Gaucher disease. Our findings have potential to help patients with other neurodegenerative diseases as well. For example, about 10 percent of Parkinson’s patients carry mutations in the recessive gene for Gaucher disease, making our research possible significant for Parkinson’s disease as well.”

This strategy will revolutionize the use of model systems to study and design and test drugs for genetic and metabolic diseases. This also demonstrate why Shinya Yamanaka’s Nobel Prize this year in Medicine for designing the technology that gave us iPSCs is so well deserved.