Professor of Hematology and Oncology, Director of the Medical Clinic for Hematology and Oncology, Charité - Universitätsmedizin Berlin
Breaking the Wall of HIV. How Transplantation of Uninfectable Stem Cells Leads to Cure
The day after the wall came down, I was woken already at five o’clock in the morning by hundreds of Trabis cruising in West Berlin.
I see only fourteen minutes; so I start immediately on the topic. After Bioinformatics, concerning drug assistance in HIV patients, I will present you quite another, and maybe, novel approach: namely, how to introduce cellular resistance against HIV. As you know, I am a haematologist and oncologist, and I am usually concerned with diagnosis, treatment, and cure of patients with Leukaemia and malignant Lymphomas. Exactly four years ago, we were confronted with another problem, namely with Leukaemia and HIV infection together in a patient. This was the time point when the story began.
I just want to shortly remind you of the HIV story: that is the HIV story. It is a story of an acquired immunodeficiency syndrome which appeared only three decades ago, and also I want to remind you that the retrovirus diagnosed as causative agent was described by Luc Montagnier and Francoise Barré-Sinoussi.
I also want to draw very shortly - concerning pandemic - your attention to the fact that we are dealing more and more with children, amounting now to more than two millions. Those children will have a life-long necessity of treatment with drugs, according to current knowledge.
Also I remind you that the target cell CD4, the helper cell of the immune system becomes depleted more and more during the infection, and later on we have to deal with opportunistic infection and typical malignant diseases. So, we feel if we would be able to protect these regulatory T-Cells we would get rid of all those problems.
There are many limitations to the drug treatment, namely toxicity of the drugs, no cure, viral resistance (we heard a lot about it before), costs of treatment, and no impact on global epidemic. So, we have no vaccine in sight at the moment, and new strategies are needed.
So, finally, I come to the situation where the patient approached our hospital; it was in July 2006.He was a 40-year-old patient with an HIV infection since 1996. He was on HAART treatment since 2002, and he had so far no AIDS defining diseases. But in spring 2006 he felt strong weakness, and in June 2006 he had a marked anaemia. On the day of hospitalisation, we made the diagnosis of an acute leukaemia by marrow puncture. We treated the patient by intensive polychemotherapy, and he went into remission. But we knew already at this moment that the leukaemia would come back one day. So we wanted to be prepared. We made a testing for possible stem cell donors, and we were surprised to find over 200 HLA identical donors in the worldwide registries. So at this moment we decided not to recline, but to reflect another typing.
I remember those days where we had intense and fruitful discussion also with the doctor in charge of the patient, Dr. Gero Hütter. We remembered the fact that virus is docked first at the cell target via the CD4 molecule, but thereafter he is taken up by the chemokine receptor CCR5. This chemokine receptor is taken like a ship by the virus to enter the cell via the membrane.
We also realised that some years ago, the crucial function of this receptor was described by the fact that around one percent of the Caucasian population is resistant by a double mutation of these chemokine receptor, in spite of multiple exposure. This deletion occurs in around one percent of the Caucasian population in a homozygous fashion. It is also very interesting to look to the geographic restriction of this deletion which occurs only in the Caucasian population, indicating that it occurred after the divergence of the populations all coming from Africa. This deletion dates back up to the Bronze Age, because it was demonstrated in Bronze skeletons 3,000 years ago.
We established a screening for the CCR5-Δ32 and from the registries we got the blood samples for testing, and in fact donor no. 61 we found to have a double mutation, double deletion for Δ32.
When the patient relapsed in January 2007, we were prepared. We treated the patient with a conditioning regimen, with high dose chemotherapy. We took the stem cells of the donor identified; we transplanted the patient, thereafter he was put on immunotherapy.
Here you see the course of the patient. On the left side you see the June 2006 diagnosis of the leukaemia. Here, namely in February 2007, was the transplantation. Here on the right side is October 2010. As you can also see, after allogeneic transplantation, already one month later the cells in blood and marrow were all of donor type: we call it chimerism. Also you can see that despite our stopping of the HAART treatment, exactly the day before the transplantation, we had no rebound of the HIV copies - they remained down. Also when we were looking for proviral DNA in various tissue specimens, for instance liver, gut, even brain, we never were able to find proviral DNA. Also we were very lucky to observe a rising of the CD4 count, and the patient has now a quite totally normal range of CD4 counts, indicating that there is no HIV infection active in this patient.
We also looked for the CCR5 expression in various tissues, for instance surrounding tissue of the gut; the gut is a very important organ, also for instance within the homosexual scenery. We could show that the T-Cells around the gut cryptae became CCR5 negative - no red staining. In contrast, macrophages, having also CD4, remained for some months positive for CCR5, but later on also converted to donor type. So, all the cells of hematopoietic origin of the patient are devoid of CCR5 and thus donor type.
We also exposed the cells of the patient to different strains and virus kinds, for instance R5 or X4 tropic. And before allotransplantation, the lymphocytes of the patients were susceptible for all virus material. The donor was not susceptible for the main and most relevant R5 HIV. And after allo- the patient was resistant against the virus.
We made also genotyping expression studies of virus type and coreceptor usage before transplantation, and we were able, for instance, to show that the virus material belonged to the group M-B. He mainly had the R5 tropic, CCR5 tropic virus material, but also a small minority of X4 virus.
Given the risk of allogeneic transplantation concerning toxicity and also the difficulties in finding HLA compatible and CCR5 negative donors, all people are now thinking to an autologous way to relay this method, and here you see a scheme for an autologous transfer of in vitro gene-modified stem cells. If you get the stem cell resistant against HIV, you can take it off here from the individual, make it resistant against HIV, you give it back in the patient, and you will see a selection of those transformed cells under the pressure of the HIV infection.
This indeed happens, as already has been shown. The best ways for engineering those stem cells, for example, by ribozymes against CCR5, or short hairpin RNA against CCR5, or the best way in my eyes, using zinc-finger nucleases, which cut the double helix at the gene region you want, for instance CCR5. This is certainly the best way to prepare HIV resistant stem cells. It has already been done just now in July 2010, published in a NOD/SCID mouse model transplanted with human hematopoietic stem cells- on one part made resistant against HIV by zinc-finger nucleases and the other part without. By this engineering, it was shown that the stem cell population holds on when the zinc-finger stem cells were used, and the HIV infection vanishes in the mouse. Those experiments were induced by our studies; we are happy about that, and a little bit proud, because those studies need lots of money, and this is available in California.
I come now to the conclusions. I think we have perhaps two main proof of principles shown by this. First of all, withdrawal of CCR5 leads to viral replication stop, and finally to eradication of HIV. The patient is now three and a half years off HIV. This, of course, is also a general example of inducing a secondary resistance against a microbial pathogen just by host target cell modification- never done before. This is also an example of how to deal with a so-far not curable infection.
What is still to do? We have to translate this approach into a better applicability, of cause, in a form of autologous cell modifications. We have to improve the gene therapy approaches of stem cell ex vivo to a large-scale procedure- so you can do it like an autologous blood transfusion. You take the blood out, transform it, and you give it back. This is, of course, a lot to do in the next years. Therefore, Rodin is helpful to think on it. In the mean time: 0:0.