Bone marrow transplant and gene therapy as treatments for leukodystrophies


In this article we will first describe the procedures of bone marrow transplant and gene therapy, before describing the applications and results obtained in leukodystrophies.


The goal of bone marrow transplant is to replace the bone marrow and blood cells of a sick patient with those taken from a healthy person. The goal of gene therapy is to repair mutated cells of a patient by introducing a normal version of the gene involved in the disease. There are possibilities to repair a gene in the true sense of the term (notably with the CRISPR / Cas9 technique), but they are not yet used in the clinic.

Bone marrow transplant

The "allogeneic" bone marrow transplant, that is to say from a donor, consists of replacing the defective cells of the bone marrow of a sick person with those of a healthy donor.

Bone marrow and hematopoietic stem cells.

The bone marrow is a diffuse organ located inside the long bones. It is a factory in which are made billions of blood cells that are generated there and multiply there. Three main families of cells circulate in the blood: the red blood cells responsible for the transport of oxygen, the white blood cells (polynuclear cells, lymphocytes and monocytes) which guard against infections and platelets responsible for preventing haemorrhages by activating coagulation.

The bone marrow makes these different elements from special cells, called hematopoietic stem cells. These cells, called undifferentiated cells, are like a canvas that can be used to make what the body needs. These cells are indeed capable of multiplying and differentiating into red blood cells, white blood cells or platelets. Red blood cells and platelets have a relatively long half-life (120 days for red and 10 days for platelets). The polynuclear cells and the monocytes, on the other hand, have a shorter half-life and are rapidly renewed. If necessary, the reserve is mobilized and the production of the cells intensifies. Bone marrow is vital to life.

The different stages

The different sources of hematopoietic stem cells: stem cells are present in the marrow, peripheral blood or in the umbilical cord of newborns, also called placental blood.

The choice of the donor: the donor may be a brother or a sister, not affected, with an identical human leukocyte antigen (HLA) system. It may also be an unrelated, anonymous donor sought in national and international registries. Current chances, given the existence of 13 million donors worldwide, are 40-50% to find a compatible donor for a patient waiting for transplant. There are also cord blood registers that increase the probability of finding a compatible donor to at least 80%.

Donor sample collection: in the case of bone marrow donation, the specimen is taken by bone punctures of the donor's iliac crests (pelvic bones) under general anesthesia, allowing the removal of a sufficient quantity of cells for normal recovery of the bone marrow of the recipient after the transplant. The amount required is of the order of 10 ml / kg of recipient weight.

When the sample is collected from peripheral blood, it is necessary to use a drug (a hematopoietic growth factor) to force the exit of stem cells from the bone marrow to the peripheral blood where they can be harvested by cytapheresis and then isolated. Cytapheresis is based on the principle that blood cells do not have the same weight and therefore can be separated by centrifugation. Pretreatment with a growth factor can cause donor bone pain, and headache that will disappear quickly upon discontinuation of the treatment.

The placental blood is the blood of the newborn. Naturally, the blood of newborns contains a fairly large amount of hematopoietic stem cells. However, part of the newborn blood (placental side) is not used for by the newborn and can therefore be taken just after the birth of the child and used as a graft of hematopoietic stem cells. The fact of the matter is that placental blood is a globally more tolerant graft than a bone marrow graft. Thus, it is possible to use placental blood grafts which are not totally HLA identical to the patient. This is very important since we will be able to graft patients who need a transplant but do not have an identical HLA donor, either in their siblings or on the different registries of volunteers for the donation of bone marrow. The pitfall of this type of graft is the relatively small amount of cells injected which explains the longer duration of aplasia and that the initial indication was to small children. Solutions to this limitation are being developed either by manipulating the graft or by using 2 grafts.

Conditioning of the recipient: before the patient can receive the donor cells, the bone marrow cells of the patient must be destroyed by chemotherapy. The adverse effects are digestive disorders (nausea, vomiting, diarrhea), ulcers in the oral cavity, transient loss of hair. Treatments are provided to prevent and treat these effects.

The transplant of the graft consists of a simple transfusion of stem cells to the recipient. The injected cells will circulate and then implant into the bone marrow. The two weeks following the transfusion are tricky because the patient no longer has stem cells and can no longer manufacture blood cells, especially the new white blood cells needed to defend against infections and platelets needed to prevent bleeding; And this last until the stem cells taken from the donor reproduce. The precautions of asepsis (sterile chamber) are then indispensable. It is a difficult time to pass, physically because the patient is tired and psychologically because the wait is long and agonizing.


As with any graft, even with a compatible donor, the risk of rejection exists. The patient's immune cells that remain in the organs, especially lymphocytes, can attack the graft (rejection), as well as the graft cells can attack the patient (graft versus host disease). These complications can sometimes lead to death.

For all these reasons, the development of alternative treatments to bone marrow transplant is desirable. In this sense, gene therapy if made possible, appears as an interesting alternative solution. Using the patient's own cells avoids the risk of rejection and graft-versus-host disease.

Results of clinical trials testing bone marrow transplant applied to leukodystrophies

Between 1999 and 2011, 152 studies were published worldwide, describing bone marrow transplant trials in 689 patients with leukodystrophy (adrenoleukodystrophy, metachromatic leukodystrophy and Krabbe leukodystrophy).

The results indicate a benefit of marrow transplantation in patients with little or no symptoms. Indeed, a retrospective study of 2007 shows that the 5-year survival of patients with cerebral adrenoleukodystrophy who have received a bone marrow transplant is close to 95%, compared with only 54% in patients who have not been transplanted. Five years later, symptoms were stable in 53% of transplant patients, compared with only 6% in ungrafted patients.

The treatment is however not beneficial for patients who have already developed more important symptoms. Bone marrow transplantation is only recommended for patients with little or no symptoms and for whom a donor is available.

Gene Therapy

Some technical notions

Gene therapy involves repairing the mutated cells of a patient. To repair the cells, two methods are possible:

  1. either the hematopoietic stem cells are taken from the patient and repaired in the laboratory by adding a good copy of the gene responsible for the disease and the corrected cells are reinjected into the patient after having performed the same chemotherapy as for a graft of bone marrow with a donor: this is called ex vivo gene therapy based on the transplantation of hematopoietic stem cells.
  2. either a viral vector (now most often an AAV, a small non-pathogenic DNA virus) is injected directly into the patient's blood or brain, which will penetrate the mutated cells of the brain and repair these cells by adding a normal version of the gene in question: this is called in vivo gene therapy.


The gene therapy vectors

These are viruses. We still have not found better yet. Their ability to "infect" (penetrate) cells with genetic material is used. But the genetic material of the virus is replaced by the gene of the disease to be treated. These viruses cannot multiply and spread. They are called "non-infectious". There are two main types of viral vectors used for gene therapy today. The lentiviral vectors used for the gene therapy of hematopoietic stem cells, which enable the therapeutic gene to be placed in the chromosomes, in the middle of the other genes. And the AAV vectors which make it possible to put the therapeutic gene in the form of a DNA ring (as is the case for the genetic material of the bacteria) in the nucleus of the cells. In the first case, the therapeutic gene is transmitted to all daughter cells of the corrected cells, if they divide, and to all the cells derived from them. In the second case, the therapeutic gene is transmitted only to 50% of the daughter cells, and if the corrected cells divide a lot, the therapeutic gene ends up being lost.

Application to leukodystrophies

Gene therapy of hematopoietic stem cells is limited to only three leukodystrophies: adrenoleukodystrophy (ALD), metachromatic leukodystrophy (MLD) and eventually Krabbe disease. After hematopoietic stem cell transplantation and bone marrow reconstruction, a subpopulation of specialized cells will be directed to the brain and transformed into cells called microglial cells. In the case of metachromatic leukodystrophy and Krabbe disease, microglial cells will produce the normal lysosomal enzyme (ARSA in metachromatic leukodystrophy and GALC in Krabbe disease). The enzyme produced can be recaptured by other uncorrected cells, especially oligodendrocytes. Adrenoleukodystrophy is not due to the deficiency of an enzyme but to a protein that remains in the cells and participates in the degradation of very long chain fatty acids. In adrenoleukodystrophy, transplantation of hematopoietic stem cells from a donor or gene therapy of hematopoietic stem cells will only repairs microglial cells in the brain, not the oligodendrocytes. This is however sufficient to stop the process of cerebral demyelination.

For all other leukodystrophies, the therapeutic gene must be placed directly in the brain cells: oligodendrocytes, astrocytes or both. A gene therapy vector must therefore be capable of penetrating into these cells whether the vector is injected directly into the brain or intravenously. For certain forms of leukodystrophy (Pelizaeus-Merzbacher disease due to duplications of the PLP gene, Alexander's disease due to mutations in the GFAP gene, which give the mutated cells a new function), it is not necessary to put a copy of the normal gene but to introduce genetic material (most often derived from an RNA) that can decrease mutated gene expression.

The choice of vectors and the mode of administration

The choice of the vectors used to carry the therapeutic gene depends on the type of cell that is to be corrected and therefore on the type of leukodystrophy. In the case of gene therapy using bone marrow stem cells, there is no choice but to use a lentivirus. But this technic puts the therapeutic gene anywhere in the chromosomes, anywhere in the middle of other genes. There is thus a risk of disrupting the normal cycle of growth of the bone marrow cells and, with other added factors, of inducing a leukemia. However, no such complication has been observed in more than 30 treated patients. The vector must be capable of transporting a large therapeutic gene, if the target gene is large. This is the case of lentiviruses, but not AAV vectors. When injected intravenously, the vector of the gene therapy must be able to pass the blood-brain barrier to reach the brain. This is the case for some types of AAV, but not all AAVs, and lentiviruses do not pass the blood-brain barrier, or very poorly. The vector can be injected directly into the brain or even into the cerebrospinal fluid (as for a lumbar puncture). But the diffusion of the vectors from the sites of injection, to penetrate into the brain tissue, is low, which necessitates multiple intracerebral injections. This increases the risk of causing an intracerebral hematoma. For intravenous and intrathecal injections (in the cerebrospinal fluid), it is necessary to determine the amount of virus to be injected, to verify that the virus is not toxic to the other organs, because it is necessary to inject a very large quantity of it.

The blood-brain barrier

Because it is so important to the functioning of the body as a whole, the brain is well protected. It bathes in a liquid and is encapsulated in the skull. The contact between blood and cells is close in all organs. But the brain is an exception. A barrier exists that controls entry and exit like a border post. It is the blood-brain barrier, made up of the cells of the cerebral vessels, astrocytes and specialized cells called pericytes. And although it is there to protect the brain, this barrier also limits access to treatments developed by doctors.

In the case of intravenous gene therapy applied to leukodystrophy, the passage of the blood-brain barrier represents a major challenge. Researchers are working to modify the envelope of viruses to allow them to pass this blood-brain barrier.

Results of clinical trials testing gene therapy applied to leukodystrophies

A total of 42 children (21 ALDs, 20 MLDs, 13 Canavan disease) participated in clinical trials of gene therapy.

·In adrenoleukodystrophy (ALD)

The first gene therapy attempts for adrenoleukodystrophy, funded greatly by ELA, were carried out by the team of Pr. Aubourg in four patients with cerebral adrenoleukodystrophy for which a compatible donor was not available. They showed that the therapy was feasible and well tolerated. Indeed, for the first time in the world, hematopoietic stem cells taken from patients with cerebral adrenoleukodystrophy were modified ex-vivo to correct the genetic defect responsible for the disease, before being reinjected to the patients. This innovative gene therapy approach allowed to stop the progression of the disease, 14 to 16 months after the injection in 3 of the 4 patients treated, with similar results in terms of efficacy than the bone marrow transplantation of hematopoietic stem cells.

This treatment by gene therapy is currently being tested (Phase II / III) in 17 other patients and will be extended to other children. As for the transplantation of hematopoietic stem cells with a donor, it can only be offered in patients with adrenoleukodystrophy who do not have advanced symptoms of the cerebral form of the disease and when no sibling is compatible to perform a bone marrow transplant.

·In metachromatic leukodystrophy (MLD)

Clinical trials evaluating gene therapy were conducted by Dr. Biffi's team in Italy on 20 patients with metachromatic leukodystrophy. They showed that treatment was well tolerated by patients. The first results also show that a clinical benefit is possible in patients treated at the pre-symptomatic stage, several months before the first motor symptoms appear. It is not clear that efficacy is observed in patients already having symptoms, such as after a hematopoietic stem cell transplant. This is probably because the disease is evolving too fast and it takes at least 12 months before a sufficient number of corrected microglial cells are generated allowing a clinical effect.

·In Canavan disease

No clear therapeutic benefit was observed. It must be said, however, that this old attempt was carried out with a type of AAV vector that was not very effective and that the patients treated had a very advanced form of their disease.


With more than twenty-five years of follow-up, the results indicate a benefit of bone marrow transplantation in patients with adrenoleukodystrophy, in children with late forms of metachromatic leukodystrophy (juvenile forms) and Krabbe disease, when the transplant is done at birth for early forms, or in the late forms of onset of the disease. In all cases, the results indicate that the graft has no effect in children with obvious symptoms of the disease. It only works at the very beginning of the disease, in practice mainly when patients do not yet have obvious symptoms.


Gene therapy trials in patients with adrenoleukodystrophy and metachromatic leukodystrophy are truly hopeful. But again these treatments concern only children treated at the very beginning of their disease or even better when they are without symptoms. A gene therapy trial, funded by ELA, and based on intracerebral injection of AAV vector is underway in metachromatic leukodystrophy (Pr. Aubourg). For Krabbe disease, studies are still in the preclinical stage, in mice, dogs or monkeys. For all other leukodystrophies, a specific gene therapy strategy should be developed. All leukodystrophies will not be treatable by gene therapy.

The first steps towards gene therapy in the treatment of leukodystrophy were largely achieved through donations to ELA to support leukodystrophy research.