Stem Cell Gene Therapy for Sickle Cell Disease Moves Toward Clinical Trials

UCLA stem cell researchers and “gene jockeys” have successfully proven the efficacy of using genetically engineered hematopoietic (blood cell-making) stem cells from a patient’s own bone marrow to treat sickle-cell disease (SCD).

In a study led by Donald Kohn, professor of pediatrics and microbiology at the UCLA Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, an “anti-sickling” gene was introduced into the hematopoietic stem cells (HSCs) from patients with SCD. Because the HSCs divide continuously throughout the life of the individual, all the blood cells they make will possess the anti-sickling gene and will therefore no sickle. This breakthrough gene therapy technique is scheduled to begin clinical trials by early 2014.

SCD results from a specific mutation in the beta-globin gene. Beta-globin is one of the two proteins that makes the multisubunit protein hemoglobin. Hemoglobin is a four-subunit protein that ferries oxygen from the lungs to the tissues and carbon dioxide from the tissues to the lungs. It is tightly packed into each red blood cell.

The structure of hemoglobin, each subunit is in a different color.
The structure of hemoglobin, each subunit is in a different color.

Hemoglobin has a very high affinity for oxygen when oxygen concentrations are high, but a low affinity for oxygen when oxygen concentrations are low. Therefore, hemoglobin does a very good job of binding oxygen when it is in the lungs, where oxygen is plentiful, and a very good job of releasing oxygen in the tissues, where oxygen is not nearly as plentiful. This adaptive ability displayed by hemoglobin is the result of cooperativity between the four polypeptide chains that compose hemoglobin. Two of these polypeptide chains are alpha-globin proteins and the other two a beta globin proteins. Hemoglobin acts as though it is an alpha-beta dimer, or as though it is composed of two copies of an alpha-globin.beta-globin pair. The interactions between these polypeptide chains and the movement of the hemoglobin subunits relative to each other creates the biochemical properties of hemoglobin that are so remarkable.

A mutation in the beta-globin gene that substitutes a valine residue where there should be a glutamic acid residue (position number 6), creates a surface on the outside of the beta-globin subunit that does not like water, and when oxygen concentrations drops, the mutant hemoglobin molecule changes shape and this new water-hating surface becomes a site for protein polymerization.

Sickle cell hemoglobin

The mutant hemoglobin molecules for long, stiff chains that deform the red blood cells into a quarter moon-shaped structure that clogs capillaries.

Sickle cell RBCs


This new therapy seeks to correct the genetic mutation by inserting into the genome of the HSC that makes the abnormal red blood cells a gene for beta-globin that encodes a normal version of beta globin rather than a version of it that causes sickle-cell disease.  By introducing those engineered HSCs back into the bone marrow of the SCD patient, the engineered HSCs will make normal red blood cells that do not undergo sickling under conditions of low oxygen concentration.

Dr. Kohn noted that the results from his research group “demonstrate that our technique of lentiviral transduction is capable of efficient transfer and consistent expression of an effective anti-sickling beta-globnin gene in human SCD bone marrow progenitor cells, which improved the physiologic parameters of the resulting red blood cells.” Dr. Kohn’s statement may lead the reader to believe that this was done in a human patient, but that is not the case.  All this work was done in culture and in laboratory animals.

Kohn and his co-workers showed that in laboratory experiments, genetically engineered HSCs from SCD patients produced new non-sickled blood cells at a rate that would effectively allow SCD patients to show significant clinical improvement.  These new red blood cells also survived longer than those made by the nonengineered SCD HSCs.  The in vitro success of this technique has convinced the US Food and Drug Administration to grant Kohn the right to conduct clinical trials in SCD patients by early next year.

SCD affects more than 90,000 patients in the US, but it most affects people of sub-saharan African descent.  As stated before, the mutation that causes SCD produces red blood cells that are stiff, long, and get stuck in the tiny blood vessels known as capillaries that feed organs.  SCD causes multi-organ dysfunction and failure and can lead to death.


Treatment of SCD include bone marrow transplants, but immunological rejection of such transplants remains a perennial problem.   The success rate of bone marrow transplants is low and it is typically restricted to those patients with very severe disease who are on the verge of dying.

If Kohn’s clinical trials are successful, this stem cell-based treatment will hopefully become the gold standard for treatment of patients with SCD.  One potential problem with this technique is the use of lentiviral vectors to introduce a new gene into the HSCs.  Because lentiviruses are retroviruses, they insert their DNA into the genome of the host cells.  Such insertions can produce mutations, and it will be incumbent on Kohn and his colleagues to carefully screen each transformed HSC line to ensure that the insertion is not problematic and that the transformed cells are not sick or potentially tumorous.  However, such a vector is necessary in order to ensure permanent residence of the newly-introduced gene.

Even with these caveats, Kohn’s SCD treatment should go forward, and we wish all the best to Dr. Kohn, his team, and to the patients treated in this trial.

Stem Cell Treatments for Retinitis Pigmentosa Inch Toward Clinical Trials

Retinitis Pigmentosa or RP is the most common form of inherited blindness. There are many different genes involved in the onset of RP. Molecular defects in more than 40 different genes can cause “isolated RP” and defects in more than 50 different genes can cause “syndromic RP.” Not only are there a host of different genes involved in RP, two patients with exactly the same molecular lesion can have a type of RP that differs substantially in its presentation.

The retina at the back of the eye is composed of two thick layers known as the inner neural retina and the outer pigmented retina. The neural retina consists of an outer layer of photoreceptors that are connected to an inner layer of bipolar cells. The bipolar cells connect with ganglion cells that have axons that extend to the optic nerve. The photoreceptor cells have their tips embedded in the pigmented retina, and the pigmented retina maintain and nourish the photoreceptors.

Pigmented Retina

If the pigmented retina does not function properly, then the effects are most profoundly displayed in the photoreceptors. Photoreceptors respond to light and the constant exposure to light causes the photoreceptors to take a beating. The byproducts of all that light-induced damage accumulates at the tips of the photoreceptors cells, and these rubbish-filled tips are taken a gulped down by the cells of the pigmented retina. The pigmented retina cells degrade the damaged byproducts and recycle the precursor molecules. Without properly functioning pigmented retina cells, the photoceptors cells accumulate toxic light damage and then eventually die. Photoreceptor cell death is the end product of RP, and it results in blindness.

There is no cure for RP, and the treatments available are very hit-and-miss. For this reason, cell therapies have been examined in a variety of animal models of RP, which, in many cases, closely mimic the human disease to some degree.

Two different experimental treatments, one with induced pluripotent stem cells (iPSCs) and another with gene therapy have produced long-term improvement in visual function in mice with RP. These studies have been conducted at the Columbia University Medical Center (CUMC).

Stephen Tsang, associate professor of pathology, cell biology and ophthalmology who led both studies commented: “While these therapies still need to be refined, the results are highly encouraging. We’ve never seen this type of improvement in retinal function in mouse models of RP. We hope we may finally have something to offer patients with this form of vision loss.”

In one study, CUMC researchers tested the long-term safety and efficacy of iPSC grafts into the pigmented retina to restore visual function in a mouse model of RP. The mice were injected with undifferentiated iPSCs when they were five years old, and the cells differentiated into retinal pigmented epithelial (RPE) cells and integrated into the retinas. None of the mice that received these transplantations developed tumors over their lifetimes.

To test the effects of the implanted cells on the vision of the mice, Tsang’s group used electrophysiological measurements of the retina. In RP mice, as they become blind, the electrophysiology of the retina becomes rather abnormal, but in these mice implanted with the iPSCs, the electrophysiology of their retinas were not only normal, but stayed normal for a long period of time.

According to Tsang: “This is the first evidence of lifelong neuronal recovery in an animal model using stem cell transplants, with vision improvement persisting throughout the lifespan.”

In 2011, the FDA approved clinical trials of embryonic stem cell (ESC) transplants for the treatment of macular degeneration, but this treatment requires the application of drugs that suppress the immune system. Such drugs have rather nasty side effects.

“Our study focused on patient-specific iPS cells, which offer a compelling alternative,” Tsang said. “The iPS cells can provide a potentially unlimited supply of cells for functional rescue and optimization. Also, since they would come from a patient’s own body, immunosuppression would not be necessary to prevent rejection after transplantation.”

Theoretically, iPSC transplants, could also be used to treat age-related macular degeneration, which is the leading cause of vision loss in older adults.

In a second approach to treating RP, CUMC scientists tested a gene therapy protocol in RP mice. A specific type of RP that results from mutations in a PDE6alpha gene was used as a model system for gene therapy protocol. This particular type of RP is rather common in humans. The CUMC scientists injected a virus into one of the eyes of afflicted mice. This virus was engineered to express the PDE6alpha gene when it entered cells. Because this virus is the AAV or adenovirus-associated virus, it only spreads in the presence of adenovirus. Without a helper adenovirus in the retina, the engineered virus particles will infect the cells they initially contact, but they will not produce a productive infection. However, ferry the genes inside them to the cell they initially infect. This the engineered AAV particles are excellent vehicles for getting genes inside cells without causing an infection.

Examination of the mice six months later, the photoreceptors in the AAV-treated eyes were healthy and these eyes were able to see, but the uninjected eyes were unable to see and their photoreceptors were mostly dead.

Again Tsang commented: “These results provide support that RP due to PDE6alpha deficiency in humans is also likely to be treatable by gene therapy.”

CUMC and its teaching-hospital affiliate, New York-Presbyterian Hospital are part of an international consortium that was recently formed to bring this PDE6A gene therapy to patients. Pending FDA approval, clinical trials could begin within a year.

See  Li, Y., Tsai, Y.T., Hsu, C.W., Erol, D., Yang, J., Wu, W.H., Davis, R.J., Egli, D., and Tsang, S.H. Long-term safety and efficacy of human induced pluripotent stem cell (iPS) grafts in a preclinical model of retinitis pigmentosa. Mol Med. 2012 Aug 9. doi: 10.2119/molmed. 2012.00242. [Epub ahead of print] (2012).

Wert KJ, Davis RJ, Sancho-Pelluz J, Nishina PM, Tsang S.H. Gene therapy provides long-term visual function in a pre-clinical model of retinitis pigmentosa. Hum. Mol. Genet. (2012) doi: 10.1093/hmg/dds46

Scientists Remove Extra Chromosome 21 from the Cells of Down Syndrome Patient

University of Washington researchers have done something seemingly impossible: they have removed the extra copy of chromosome 21 in cells taken from a patient with Down syndrome. This gene therapy technique targets only the extra genetic material in the cell, and scientists were able to successfully remove the extra chromosome 21 without damaging the integrity of the rest of the chromosomes present in the nucleus.

The first reaction to this news is to shout, “there’s a cure for Down Syndrome!” Unfortunately that is not the case. However, it might be a way to treat Down Syndrome patients who have blood cancers. Down syndrome patients are at increased risk for leukemia, and this technique, pioneered by Dr. David Russell and his colleagues is meant to fix the errant bone marrow cells in culture and then reintroduce the fixed cells back into the patient.

Dr. Russell explained: “We are certainly not proposing that the method we describe would lead to a treatment for Down syndrome. What we are looking at is the possibility that medical scientists could create cell therapies for some of the blood-forming disorders that accompany Down syndrome.” Dr. Russell is from the University of Washington’s Department of Medicine.

This technique works on cultured cells grown in a laboratory. The cells are infected with an engineered virus that inserts into the extra chromosome. Then the cells are grown under conditions that kill all cells with the viral DNA. Only those cells that spontaneously lose the extra copy of chromosome 21 survive the culture conditions.

This protocol could potentially treat Down syndrome patients with leukemia with genetically-modified stem cells that are derived from their own cells, but lack the extra chromosome. Stem cells could be taken from the bone marrow of the patients, the doctors could remove the extra chromosome, and then the healthy cells could then be grown and transplanted back into the bone marrow of the patient. This same technique could also be used for leukemia patients whose bone marrow cells have an extra chromosome, but do not have Down syndrome.

This is great news for those with Down syndrome and for all those who live with any kind of trisomy. Also, since gene therapy can introduce new defects into the patient’s DNA, this technique could potentially remove unwanted extra bits of DNA without adversely affecting other chromosomes. This is certainly a major achievement.