I just received this from Pat Girondi. I have to say that Dr Sadelain continues to amaze me.
Thalassemia and Gene Therapy
Professor Lucio Luzzatto
Definitive therapy of thalassemia has been the goal of haematologists and the hope of patients for a long time. Thalassemia is an inherited disease; and the commonest form in Mediterranean countries and in many other parts of the world is caused by genetic mutations, which we have known in detail for some time, in the beta globin gene (one of the genes essential to make hemoglobin). Therefore a logical approach to treatment is the correction of the genetic abnormality: that is, modifying some of the patient’s blood forming cells (the stem cells) so that the normal beta globin gene will now function. This approach goes under the name of gene therapy, and I can say first-hand that thalassemia was one of the very first diseases for which gene therapy was considered: so much so that a first attempt was made in two patients as early as 1980. That attempt was well-meaning, but ill-conceived, carried out almost in secret, and of no benefit to those two patients.
In the subsequent two decades an enormous amount of research was carried out in laboratories in many countries, in order to work out how genes could be effectively introduced or transferred into cells, in ways that would then allow them to function normally. It was found that, at least for blood cells, the best way to do the transfer was to use certain special viruses, after having them thoroughly disabled – meaning they cannot cause disease. Because the virus is loaded with the gene we want to transfer it is like a vehicle with a cargo, and in jargon it is called a vector. For bone marrow cells the best vectors are called retrovirus or lentivirus, because either of these is able to become part and parcel of the DNA, or genome, of the cell it has entered. Thus, when the cell divides or replicates, the gene which has been introduced by the vector replicates too; and that way not only the original cell is corrected, but all the billions of cells that for years will derive from it will be corrected as well. It was further discovered that it is necessary to introduce, along with the beta globin gene, special bits of DNA absolutely essential for that gene to function normally for a long time.
It took years to understand all of this, and to develop the technology required to implement what was understood. Moreover, responsible scientists will not attempt to treat patients until they have made sure that the treatment works in the lab and in experimental animals, and that all possible safety devices have been incorporated in the treatment protocol. In year 2000 Michel Sadelain (Memorial Sloan-Kettering Cancer Center, New York) was able to publish with his team a landmark paper in Nature, reporting that for the first time gene therapy of thalassemia, using a unique vector that he had constructed, had been carried out successfully and reproducibly in mice.
During the next ten years Sadelain’s group has worked incessantly towards the gene therapy for patients with thalassemia, aiming to further improving the quality of the vector, and particularly to making sure that one can obtain from patients a sufficient number of stem cells for efficient gene transfer. A formally approved clinical trial having this purpose has been completed.
In 2010 a new development hit the news. A group of workers in Paris, led by Philippe Leboulch, has reported, again in Nature, that a patient with transfusion-dependent thalassemia was treated with gene therapy. In order to understand the implications it seems appropriate to summarize the case history as it was reported. The patient is a young man who since the age of 3 had been on regular blood transfusion and had been splenectomized at the age of 6. By 2007, aged 18, the patient required 2-3 packed red cell units every month; and he was receiving iron chelation with desferal. At that time, after his bone marrow cells were harvested, the patient was treated with 4 daily doses of busulfex, a chemiotherapeutic agent, in the aim to eliminate in large part the patient’s remaing bone marrow. In the meantime, an aliquot of what had been harvested was cultured for 7 days with a lentiviral vector – very similar to that used by Sadelain – and then returned to the patient.
There were no clinical side effects, and for about one year the patient still required blood transfusion. Subsequently his hemoglobin increased to a steady state level of 9-10 G/dl, and blood transfusion was no longer required. Most significantly, about one-third of his hemoglobin is Hb A (the normal adult haemoglobin, that he did not have at all before treatment). Clearly to date the therapeutic procedure appears to have been a success; and it is important to clear the ground from two objections that have been raised. First, the patient did not have ‘classical’ thalassemia, but a special from of it called Hb E-thalassaemia, common in Sri Lanka and in South East Asia. I think this objection is irrelevant, because Hb E is itself a form of thalassemia, and the clinical picture of Hb E-thalassemia ranges from thalassemia major to thalassemia intermedia: in this case, it was clinically undistinguishable from thalassemia major. Second, after recovery from a treatment with bulsufex it is expected that there may be an improvement in the production of red cells, particularly of those that have fetal hemoglobin (Hb F): this may have helped the patient’s recovery, but it does in no way explain the production of hemoglobin A (one-third of the total), which is unquestionably a highly beneficial result of gene therapy.
However, there are two reasons for concern. First, it is exceptional that a publication on a novel therapy reports on one single patient: since reliability and reproducibility are of the essence in the clinic, we need more before we can say this form of treatment is established. The second reason is a little more complicated. As stated above, the vector becomes part of the cell genome (in jargon we say it inserts into it): but the way the procedure was carried out, we have no control as to where it integrates. The human genome is like a vast landscape (its DNA is 3 billion bases long), and the vector lands somewhere: it may be in a part of the genome where there are few active genes (like landing in a desert), or it may land in a part that is busy with many active genes. It happens that in one of the stem cells of this patient the insertion has taken place very near a gene (called in jargon HMGA2), that has something to do with benign tumors. Moreover, it happens that of all the cells in which insertion has taken place, this particularly cell is the one that has been most successful in re-populating the patient’s bone marrow (it makes up less than 10% of it, but over one-half of all cells in which there was a gene insertion), suggesting that perhaps the insertion neighbor, HMGA2, has favored growth. Although this can cause concern, the remarkable clinical improvement of this patient may result in part precisely from this site of insertion: at any rate, it is gratifying that 4 years after the procedure he is well and leading an active life, as I learnt recently from a kind personal communication of Philippe Leboulch.
At this point the thalassemia community is more than entitled to ask: where do we go from here? To give an account of the facts, as I have done so far, is easier than to answer this question: but let me try. First, there are now better vectors than we ever had before; second, thanks to the trial conducted at the Sloan Kettering that I have mentioned earlier, we know that in thalassemia patients there are enough stem cells to receive a good vector. In order to make the procedure as safe as possible there are at least two possible approaches. (1) To select those stem cells in which the beta globin gene has inserted in certain sites and not others. In this respect, Michel Sadelain has just published a paper in Nature Biotechnology, in which he has introduced an entirely novel concept: to identify ‘safe harbors’ for the inserted gene. (2) Instead of inserting the normal beta globin gene more or less at random, to replace neatly the thalassemia gene with the normal gene in the exact site where it belongs. This has become conceivable only recently thanks to a technology called the induced pluripotent stem cells (iPSC): this approach would take a bit more space to explain and considerably more time to become a form of human therapy, but personally I hope very much that somebody will take up the challenge.
In conclusion, I feel that the thalassemia community is justified in holding a renewed hope that gene therapy will work. When it comes to timing, we must be more than sympathetic to the impatience of the patients, but I feel personally unable to predict when this new form of therapy will become generally available. Experience from the past has taught us, and I have given some examples, that to jump the gun is not a good idea. I know that the Sadelain Team are planning a carefully designed clinical trial, not in one patient but in a significant number of patients, and I know they want to start as soon as possible.
I have written this account upon request of Patrick Girondi, Chairman of the Board of Errant Gene Therapeutics (EGT). I am taking this opportunity to pay tribute to Pat, and also to Angela Iacono, President of the Giambrone Italian Thalassemia Foundation, Sigma Tau, TIF, CFT, Cooley’s Anemia International, the Cooley’s Anemia Foundation and Maria Chironna, Vice President and Scientific Advisor of the Orphan Dream Foundation for their unrelenting commitment over many years to the development of new approaches to the treatment of human disease, particularly thalassemia.
Lucio Luzzatto
Firenze, December 13, 2011.
Lucio Luzzatto is a hematologist who has conducted clinical and research work in Nigeria (University of Ibadan), UK (Hammersmith Hospital), USA (Sloan-Kettering) and Italy (CNR- Napoli, University of Genova and University of Firenze). Lucio Luzzatto has in the PubMed database 350 publications, 22 of which regard thalassemia, the first of which in 1964. Currently he is Scientific Director of the Istituto Toscano Tumori and Honorary Professor of Haematology, University of Florence, Firenze, Italy.