Alain Fischer on gene therapy and immune deficiency
What happened to the children treated with gene therapy?
Alain Fischer, M.D., Ph.D., joins Amit Agrawal, Ph.D., and Julia Majewska, Ph.D., to revisit gene therapy for X-linked severe combined immunodeficiency, the lessons of long-term follow-up and the responsibilities of translating research into treatment.
Recorded · Published on YouTube · 1 hour 8 minutes · English
A genetic defect can interrupt the development of an immune system. Fischer explains how understanding the common gamma chain helped researchers design gene therapy for X-linked severe combined immunodeficiency, or X-SCID. He describes lasting T-cell benefits alongside incomplete immune correction and leukemia complications, and explains how those complications drove changes in vector design.
Why was the common gamma chain important?
This receptor component participates in several cytokine signaling pathways. Fischer explains how loss of those signals can block T-cell and natural killer cell development, and how the biology helped identify a route to restoring T-cell immunity.
Did the early treatment restore every part of immunity?
No. Fischer describes sustained T-cell reconstitution, while B-cell function and natural killer cell counts remained impaired in the early unconditioned treatment. Some patients continued to need immunoglobulin replacement. The outcomes cannot be summarized as complete restoration of the immune system.
What did the leukemia complications change?
The early integrating vectors could activate cancer-associated genes. Fischer describes the investigation of those events and the removal of the vector's enhancer activity in later designs. The historical clinical reports below document both immune benefits and the serious safety problem; later designs should not be described as universally risk-free.
What does this history tell us about immune aging?
Fischer distinguishes treating a defined immune deficiency from enhancing immunity beyond normal physiology. He raises the possibility of harmful consequences and treats intervention in normal immune aging as an unresolved question, rather than a clinical recommendation.
These historical reports describe earlier cohorts and follow-up periods. The longer follow-up discussed in the recording is Fischer's account at the time of the discussion, rather than a result established by those earlier papers. The conversation is for scientific education.
This transcript follows the published YouTube edit. Caption wording and speaker labels were compared with the original Riverside transcript, with corrections supplied by Amit Agrawal. Timestamps link to the published recording. Joint labels identify brief overlapping exchanges. Unresolved words are marked [unclear]; an unresolved name is marked [name unclear].
For much of modern medicine, we have treated disease by acting around the defect. We block a pathway. We replace a missing molecule. We suppress inflammation. We support the body when one part fails. But today we are asking a different question. How do we repair a living system from within? The immune system is different because it has to be built before it can defend. It has to develop, remember and patrol. If one essential instruction is missing early enough, the system doesn't merely become weaker. Major part of its architecture failed to form. This is why rare inborn errors of immunity are so powerful scientifically. They are not small diseases just because they affect few patients. They are precise experiments in human biology. A single genetic defect in a child can reveal mechanisms that keep the rest of us healthy and principles that matter far beyond the rare disease itself. Gene therapy changed what medicine could attempt. It asked whether we could stop treating only the consequences of an immune system that failed to develop and instead restore the instruction that the system needed in the first place. But this story is not a simple miracle because there are living cells. They do not simply deliver a drug effect. They expand, persist and become part of the patient. That is what makes the therapy powerful and also what makes safety redesign long-term follow-up and trust so important. A field becomes mature when it learns not only from its success but from the consequences of pushing it into the unknown. So today the question is not only how one of the first
definitive clinical successes of gene therapy worked. The larger question is what does this story teach us about the future of experimental medicine? How does clinical observation become biology? How does biology become therapy? How does the therapy earn the right to be trusted? That is the question at the heart of today's Burstcast. Please join me in welcoming Professor Alain Fischer. And to begin the dialogue, my colleague Julia Majewska. Julia, please.
Immune system is a complex network of diverse sets of immune cells which need to communicate between each other to support the function. One means of communication is via cytokines. Cytokines bind to the receptor and drive signaling pathway. They drive survival of immune cells proliferation and their differentiation. One of the central cytokine is IL-2 which supports function of T cells and NK cells. But here comes the puzzle. Deficiency of IL-2 cytokine doesn't cause severe immunodeficiency. The depletion of NK and T cells. However, mutation of only one chain of IL-2 receptor drives dramatic immunodeficiency. Here is our great pleasure to have with us professor Alain Fischer who will explain how understanding molecular mechanism of the defective cytokine signaling paved the way for the first clinical gene therapy for primary immunodeficiency X-linked severe combined immunodeficiency. We are very happy to have with us you professor Alain and to begin with we would like to know how your journey to answer this question how did it start where was the field when you started to work on this and what were the questions that you were asking
okay thank you so I would say in the 60s at the time T and B lymphocytes were identified as separate lymphocytes. So it was recognized by colleagues that there were very rarely young patients, young children who had no T lymphocytes in their blood. Some of them also had no B lymphocytes in their blood. So these diseases were named severe combined immunodeficiencies. severe because they are causing death within the first months of life because of overwhelming infections. So that was the time the 60s then it was recognized that one of these form of severe combined immunodeficiency was X-linked that is the indicating that the gene was on the X chromosome and boys were affected. So then came the 90s where several groups in the world tried to clone the gene and it happened that a Japanese group cloned the gene encoding the gamma chain of the IL-2 receptor that is shown here on this slide where it is associated to bind IL-2 with high affinity with an alpha and beta subunit and we had mapped the gene or the locus for the disease exactly in the same region where the IL-2R gamma gene is placed. So this led to the discovery by Warren Leonard groups that indeed mutations of that gene or the IL-2 receptor gamma encoding gene was the cause of X-linked SCID. But of course as you said it came as a surprise because it was not expected that defective signaling from interleukin-2 should lead to an absence of lymphocytes more specifically
of T lymphocytes as well as NK lymphocytes. So something didn't go didn't match in that study. So the but this discrepancy actually was extremely fruitful because it led to understand that this IL-2 receptor gamma protein is not only part of the IL-2 receptor but as renamed as gamma C for gamma common is also a member of the receptor for other cytokines as shown here namely the receptor for interleukin-4 interleukin-7 interleukin-9 interleukin-15 and interleukin-21. So now we have a more complex situation where a monogenic disease affecting impairing the expression of the common gamma chain is leading to six different cytokine signaling defect. So the next question came immediately is okay among these six cytokines what is or what are the cytokine or the cytokines that are critical for T lymphocyte differentiation and NK lymphocyte differentiation. So mostly based on work performed in mice, knockout mice, we did Jim Di Santo did the gamma C knockout gene model and then looked in detail what was going on in the thymus. It was understood that actually what is driving T cell differentiation and the very early steps in T cell development the so-called double negative T cell differentiation stage in the thymus is interleukin-7 so interleukin-7 is produced by stromal cells in different tissues including the thymus and interleukin-7 is delivering a signal for survival and proliferation secondary inducing differentiation of T cells and in parallel it was shown that interleukin-15 is doing the same job more or less for NK lymphocytes. So based on that now at that time this was the mid-90s approximately it was understood that severe
combined immunodeficiency caused by mutations in the gene encoding the common gamma chain led to an early block in T and NK cell differentiation because of the defective respective defective signaling of interleukin-7 and interleukin-15 and in parallel it was shown by Warren Leonard was as well as by Notarangelo that there was a phenocopy in humans of X-linked SCID but with autosomal recessive inheritance and this phenocopy was caused by mutations in another gene that is the gene encoding the kinase JAK3 and this led to understand that signaling from these cytokines through the common gamma chain was mediated at least in part by the kinase JAK3 and so if either the common gamma chain or the JAK3 kinase is absent then the signaling is interrupted and this is leading to both the block in NK and T differentiation. So at that time we were able to understand quite precisely the pathophysiology of this disease with this early block in lymphocyte differentiation for T and NK cells at a step that is just immediately before the first massive wave of cell proliferation of T cell precursors in the thymus.
it was designed was it designed this way that the common gamma chain is needed for signaling for multiple cytokines? was it duplication event or what what is the beneficial is it
Since it has been selected during evolution of mammals obviously it has to be beneficial and of course there are multiple functions that are linked linked to this cytokine receptor. So interleukin-4 for instance as well as interleukin-21 are important for B cell function a little bit for B cell development and B cell function. By the way, interleukin-7 is also involved in B cell differentiation. But apparently in humans, it's it is not required unlike in mice since patients with IL-7 receptor deficiency they do have B cells normal. They are not functioning properly but they do have B cells. So there are multiple functions associated with these receptors. So obviously the the selectivity and the specificity of these different cytokine receptors is mediated by the associated subunits. So for IL-2 receptor alpha and beta for IL-4 receptor alpha and so on and so on. So the specificity comes with the other subunit or subunits. But then in terms of signaling as far as signaling is associated with the common gamma chain it goes in all of the cases with JAK3 whereas the JAK1 the other kinase JAK1 plays a role more in association with the other subunit of the receptor.
Yeah, I mean the signaling downstreams based on these STAT molecules that are inducing expression of a set of genes is shared. So the question is just this is the in the context where cells are stimulated that comes the difference. Of course if you stimulate B cells or T cells you don't get the same message and the same function but the architecture of the biochemical pathway is the same.
yes they are not competing since the binding does their function is is a consequence of the receptor globally that is is the second or even the third in some cases subunit that drive specificity. So there is no really compet competitiveness and the common gamma chain is expressed in a way that allows for the cells that express se several of these receptors. So they are able to express both say IL-2 and IL-7 receptor or other combinations depending on the cells and the cell activation status. So there is no real competition.
So Alain, the defect we see in IL-2 receptor signaling is it coming because of the genetic reason present in the parents or it's a stochastic like event that happens when the baby in the
no it was it's usually transmitted by the gene from that is mutated already in the mother but from time to time there are new mutations that occurs we know in all embryos so that's classical genetic genetic observations that for for in the case of the gamma C two-thirds of mutations are inherited from the mother and one-third are de novo mutations present in either the oocyte or very early stage of the or so in the study of
that disease we observed a very special case of a patient who inherited the mutation of that gene but the phenotype with which he presented was very different. So this guy knew, this was a six-year-old boy. He had T cells. He had no NK cells, but he had T cells approximately half of normal. They all had a memory T cell phenotype. There were zero naive T cell and he had a mild immunodeficiency. So very still an immunodeficiency, but an immunodeficiency that is much much milder than the usual one as observed in newborns with severe combined immunodeficiency. But so there was a discrepancy with the fact that he inherited a mutation that should have caused him to have no T cells and the fact that he had T cells and it was checked that the T cells were his own T cells not from a twin or whatever could have happened and not the mother maternal T cells. So actually what was found in this in this patient and now it was also observed in others later on is that reverse event so a somatic rescue of the mutation occurred by chance in some T cell precursor enabling one or few of the T cell precursors as shown here on this slide to express now on a normal common gamma chain. So explaining why in the periphery the patient had T cells as I said half of normal memory T cells and we looked at the repertoire of the diversity of the T cell receptor at least of the beta chain of the T cell receptor and it
was found that it was not monoclonal it but it was not normal approximately this child had 1% of the number of T cell receptor V beta subchains of a normal individual of the same age. So this led to understand what is shown here is that because by chance one cell it's likely it's only one cell among the T cell precursor had this fortunate event of somatic reversion of the mutation. He had a missense mutation in in the genome encoding gamma C. This now this unique T cell became a became able to survive first to proliferate in the presence of interleukin-7 to divide extensively prior to the stage of T cell rearrangement. So the these events occurred in the thymus generating so some T cells not as many as a normal individual but still one cell likely generated approximately 1% of the repertoire. Here they are only memory cells because it is proposed that this event occurred only once and that the cells that were generated they all underwent for some time in the life of the child they underwent an immune response to a given antigen.
So this can le lead lead to the estimation that in order to get approximately this level of diversity the cells had to divide in the thymus prior to T cell receptor rearrangement at least 11 times more if you consider that most that many cells actually in the thymus in the thymus do die because of negative selection and so on. So this demonstrates in a kind of natural experiment that T cell precursor based on signaling through the interleukin-7 receptor and the common gamma chain can proliferate tremendously in the early stage of T cell development. So in the clinically speaking it as I said it led for the patient to have a milder immunodeficiency since he had T cells un unlike patients where such events do not occur and we considered that actually what happened in this child is a kind of so-called natural gene therapy by chance he had this correction that occurred that partially protected him from lethal infections. So because we made that observation in
the 90s, we thought okay if this may occur from time to time naturally, we may try to engineer it in a more effective way and that could be that could offer a possibility to treat that disease by correcting the expression of the common gamma C protein in T cell precursors and perhaps other lymphocyte precursors by using vectors and this is of course the principle of gene therapy. So this was initiated first in in vitro experiments in animal models which worked showing that for instance gamma knockout mice can have its immunodeficiency corrected by gene transfer of the common gamma chain into hematopoietic progenitor cells. So based on this data we proposed in the end of the 90s next slide please we proposed clinical protocol for for gene therapy. So the how the protocol is it's pretty simple in the principle. So bone marrow cells are harvested from the child. The cells that are immature expressing the CD34 molecule at the surface were selected because they contain the stem cells and the progenitor cells. They were cultured for one day to have them proliferating because this is necessary for the action of the gamma retroviral vector and so the cells were infected with a gamma retrovirus that had in its genetic material the cDNA encoding the common gamma chain. So the at that time the protocol was made in a way that there were three cycles of infections in three days and then the infected cells were injected back to the patient. So after a fourth day by intravenous route. So the clinical protocol was
proposed to patients who had no donor for bone marrow transplantation which is an alternative therapy but at least at that time didn't work very well if there was no matched donor. See this was done without any chemotherapy. So the stem cells in the bone marrow were not destroyed. The idea was to be as less toxic as possible and to observe hopefully the differentiation of progenitor cells into T cells and perhaps NK cells. So at the cell level as shown on this cartoon. So you have the vector the virus. Of course this is not at scale but so the RNA for the retrovirus is once in the cell retrotranscribed into DNA and this DNA is able in cycling cells to be in contact with chromosomes and to integrate somewhere in the in the genome. This is not controlled it could happen in many places. So then the gene is there there is a promoter in that case it was the promoter enhancer of the retrovirus itself. that is present in the long terminal repeat of the virus and so this is inducing the transcription of the messenger RNA the translation into the protein and the expression of the protein on the cell surface and the restoration of function. So this was the principle of that gene therapy. So a first trial was performed in Paris between 1999 and 2002 and actually soon after the British group did something similar in London.
refer here yes to CD34 cells which were used for this gene therapy do we have understanding how many of true long-term HSCs do we need to have for correct the gene copy of this receptor to have clinical benefit
so this was analyzed posteriorly by looking at the integration site of the retro of the V of the vector into the peripheral T cells found in the patients post therapy 5 10 20 years later it's possible to determine exactly to map by by sequencing where these integration occurred which chromosome which place exactly so and it was found in this trial in the absence of chemotherapy and so on that the number of sites was of unique sites of different sites was in the range of 50 to 1,000. So, and these guys, they all had, as you will see on one of the slides, normal or close to normal T cell count. So this is telling us that with as low as say a few hundred of progenitor T cells you can reconstitute a full a fully or one a full number of pool of T cells and secondly as you will see later al also a fully diversified pool of of T cells.
So based on this capacity of T cell precursors to proliferate very intensively once they receive the interleukin-7 signal in the thymus very few T cells are T cell precursors excuse me are sufficient to develop into a full diversity and quantity of T cells in the body. one of the things we learned from that study
and how the situation looks like if it would be donor derived CD34 cells so not patient not patients own hematopoietic stem/progenitor cells but allogeneic
allogeneic we don't know exactly because this has not been determined precisely but I don't it shouldn't be different in principle well providing that there is no immune conflict that is graft-versus-host disease in principle I don't see why there should be differences but this has not been determined in the context of allogeneic transplantation
you made a brave decision there no chemotherapy just trusting the corrected to win inside the body what made you believe that it would work like what gave you the confidence that
because because we knew from allogeneic transplantation that for SCID patients this can be done with some success in the absence of chemotherapy. So this this was observed earlier. So this was the basis not to give chemo. Today we will do it differently because as you will see only T cells were corrected and we would like to have a bit more better and we can I would say today so 25 years later we do control better the usage of chemotherapy so that such patients would receive also chemotherapy to have not only T cells but also B cells NK
26:35 · Long-term follow-up and remaining immune deficits
cells corrected. So this is for the patients both from London and Paris. There were 20 patients collectively treated and we have now a follow-up that extends up to 27 years for the first patient treated. So overall the 18 of these patients are alive but more significantly 17 are alive with some correction of the immunodeficiency at least for T cells and this is stable over time but it's not perfect since the B cells are not reconstituted only the T cells so some of not all but two-thirds of the patients as written do require immunoglobulin replacement therapy but apart from that and apart from a complication I will mention that occurred in some of the patients they have normal quality of life and interestingly enough this looks to be stable for more than 25 years. So these babies who they were babies at the time of treatment they are now young adults living normally.
Super cool. This is super cool. This you followed them for 25 years such a I think it's rare follow in the medical science like other than the survival like what is the other other positive effect you have noticed on these patients?
The positive effects are that they are c they cope with infections. I mean for instance all of them they had COVID as everyone basically and none of them had severe severe COVID very benign or even asymptomatic case indicating that their T cell functions to control COVID was there and same with other
oh yeah they don't make well they makes up a little bit of antibodies depends on the patients but some of them do not make antibodies well enough because their B cells still lack the expression of the common gamma chain So they do not properly react to stimulation with interleukin-21 or and interleukin-4 so explaining why that some of them require immunoglobulin replacement therapy.
work partially I would say depend dep again it's variable from one patient to another but it may not work in terms of antibody production it works in terms of T cell stimulation that's for sure they do develop antigen specific memory T cells CD4, CD8 with the different functions Th1 Th2 Th17 cytotoxic all of these things they have but they don't make well antibodies. So you will see the T cell counts. These are the patients treated in Paris. So every single line corresponds to the T cell counts of the patients over the year in this trial. So starting from zero. So as you can see at the beginning there was number of there is quite a variability but later on they have t cell counts that are close to or within the normal range indicated by the dotted line. so they were all T cells it was checked all of the T cells did carry the gamma C transgene but when looking at other leukocytes like B lymphocytes monocytes neutrophils very very few not zero but very few something like 0.1% did express the transgene indicating that no stem cells were transduced but rather progenitor cells and NK cell counts were low indicating ing that NK cell development that occurred early after treatment did not persist unlike the persistence of the T cells as you can see. Yeah.
there are two hypothesis here one is that I mean based on what is known that the T cell lifespan is much much longer than the NK cell lifespan so we know that T cells can persist for years even for 20 50 memory T cells can be there and perhaps also it's possible that the flux the rate of T cell production may be much higher than the flux of NK cell production but this is not so clear but at least in terms of lifespan this is quite clear so the cells won't produce they are there but actually for life whether whereas for NK cells they may be there only for a few months and Once you have there is no longer any progenitor cells left there won't be any production of NK cells
right but so I mean if we would assume that also true hematopoietic stem cells were being transduced but we see this effect so but we see this bias towards T cells no so could it be that the transduction is mostly happening on stem/progenitor cells that are more primed to
there are really no evidence at least after two to three years following treatment that they were persisting transduced bona fide stem cells. I think the cells that were transduced were most likely I would say hematopoietic progenitor cells that were able to migrate to the thymus and differentiate into T cells and transiently in the bone marrow into NK cells and that's it in order to get all of the lineages as it was done much much later by US group this occurs provided that niches were emptied in the bone marrow by chemotherapy. In that case, one observed not only the development of T cells but also of NK cells and B cells as well as a few myeloid cells too. But we unfortunately
33:23 · Leukemia complications and vector redesign
observed in five cases five patients after two to 15 years post treatment the occurrence of a form of T cell acute lymphoblastic leukemia that was fatal in one case and but could be cured in the four others pretty easily actually with very even corticosteroids were enough to get rid of the blasts and those patients are alive and still have T cells functioning that's so that is the chemotherapy given for the treatment of the ALL did not destroy the transduced stem cells or progenitor cell so the question immediately rose obviously why did it occur and actually what
was decided to interrupt as as soon as the first case was observed. so but all of the patients had been treated the 10 patients had been treated it was decided to stop the trial and to understand what's going on and see from trying to understand the mechanisms what could be done to prevent this complication. So after some years it appeared clearly what happened is that the vector so the MFG so-called MFG vector here in shown in red is integr I mentioned is integrated everywhere in the genome but it is not random it integrates there are two two points one it integrates within genes or either in introns as shown here of a given gene or in the promoter this is the first characteristic and the second characteristic is that this integration occurs in genes that are highly transcribed in the cells being infected with the virus and among the highly transcript transcribed genes in bone marrow progenitor cells is the gene named LMO2 is an very important gene in early steps of hematopoietic development and of course of T cell development. So what happened you can see in four patients there was an integration in a clone that occurred either for P9 in the promoter or P5 also in the promoter P4 in intron one and P10 in intron 2. In the fifth patient, there was an integration in a gene involved in cell cycling CCND2. And in all of these five cases the what appear as very clear is
that the long-term long terminal repeat of the ve of the vector which has an enhancer activity was able to transactivate the gene in which the integration occurs and LMO2 is a known oncogene, CCND2 too so that the abnormal aberrant expression constant expression of LMO2, CCND2 was the primary cause of the occurrence of this T cell acute lymphoblastic leukemia. so once this was understood it was proposed to modify the vector in a way that the enhancer present in the long terminal repeat was deleted and instead in order to get expression of the IL-2 receptor I'm sorry because the letters are a bit mixed up here on the bottom part of this representation of the vector. So the now we have a new vector where the enhancer is gone. So there is no enhancing activity but there is a promoter has been added. So we have the new vector where the enhance so the enhancer has been deleted and instead a promoter which is the promoter of the short form of the elongation factor one alpha was added to trigger expression of the IL2RG or the gamma C gene. So these are the so-called self-inactivating vector. So this was tested and a new trial was performed together with colleagues in UK and US with such a vector. Nothing was changed but this the vector and if you click again please. So 13 patients were enrolled and today 11 of them are alive with the benefit as in the first trial in 10 of them. So basically the reconstitution of the T cell pool was the same
but the very important difference is that there were no direct toxicity of the vector although one patient unfortunately died from cancer but which is unrelated to therapy. This was a epithelial cancer with no integration of the vector. So this is just bad luck. but the important point here is that indeed there were no longer leukemic events. so this confirmed on the clinical base basis excuse me that indeed this was this transactivation of an oncogene by the enhancer of the of the vector that was causing these leukemic events.
Yeah, it I mean it was in all of not there were not so many trials but a few other trials where such vectors were used. There was a trial in Germany for another immunodeficiency called Wiskott-Aldrich syndrome that was performed a bit later. Another trial performed in Switzerland for another immunodeficiency chronic granulomatous disease where they used similar vectors and actually they observe the leukemia events. So this is clear confirming several trials that the mechanism the cause of these events is link is cause is the consequence of using such vectors. Of course, such vectors are no longer used and well today gamma retroviruses are replaced by lentiviruses that are more effective. But in the these lentiviruses, the pre the same principle is also taken into account that is these are self-inactivating vectors with no enhancer activity and an internal and a promoter is used to get expression of the transgene whatever the transgene is. So we learned a lot from from these observations as made in the year approximately 2002 to 2010
We'll discuss a little bit gene editing tools but for severe combined immunodeficiency we may consider gene editing but I'm not sure this is going to be an an advantage compared to gene addition. I mean gene editing as I will show you a little bit later. I think it's more interesting to treat diseases where we want the gene to be at its right place under physiological regulation for a gene like the common gamma C gene that is
40:56 · T cells and NK cells decades after treatment
expressed continuously in lymphocytes doesn't matter that much. Now we have pictures and this is one example of how lymphocytes of these patients look in the blood. So say more than 25 years post gene therapy. So this is the picture for the first patient treated. I think this was taken exactly 26 years postgene therapy. So this is a detailed analysis of all of the subsets of these cells and other cells. And if you compare so P1 to a control there is not much difference. They are very similar with one important exception which is now circled. These are the NK cells. As you can see in P1 there are very few NK cells. Whereas in the control at the same place you see the NK cells. But as far as the different T cell subsets are concerned they are there mostly the all of the central memory effector memory and so on and so on. I will not detail them but they are basically similar in terms of global phenotype compared to a control of the same age. So and they have gamma delta t cells they have NKT cells they have regulatory T cells. So everything is there. So in terms of T cells we have there is I would say quite stable reconstitution of the T cell pool and its different subsets and effective subsets.
Based on this observation where we don't see where we see still deficiency in NK cells in this patients what about gene therapy which would give selective advantage survival advantage for NK like both T and NK cells but to boost the number of NK cells in this patient so that they would have
for sure of course it would be better that these patients could also have NK cells and this is why today this is on with chemotherapy to have the genuine stem cells to be transduced but still interestingly enough these guys so there are 17 of them who don't have NK cells at the age of 20 25 doesn't seem that there there is any problem they go and actually the same picture is has been observed following non conditioned allogeneic transplantation for SCID patients and here there there is a follow-up extending to 50 years or some cases in the US in France and other places and again it doesn't seem that the NK cell deficiency helps so which suggests that at least in this context there is a redundancy between NK cell function I would say T cell functions so another now
consideration we previously discussed a little bit but I think it's important to rephrase it I mentioned that based on the integration sites the integration sites in the of the vector is shown in this slide by the small square on on the on the line that represents the genome. So we know from the analysis of T cells in the periphery that approximately 50 to 1,000 T cell precursors were effectively transduced and were able to generate T cells. So very few precursors but these precursors expanded massively before undergoing in the thymus rearrangements and leading actually as we observed to a fully diversified cell repertoire and the number of T cells that is normal or close to normal. So actually in gene therapy what was observed is a recapitulation of what was observed in the patient with the somatic genetic rescue the reversion of the of of the mutation and confirming the extremely potent capacity of early t precursors to expand in the presence of interleukin-7. So this is really impressive. So actually a gene therapy that is poorly efficient because correction say of a few hundred cells this is not much today one one does much better with the present presently available vectors but at that time with the vectors we used poorly efficient gene therapy still was sufficient to fully correct the T cell pool and T cell functions which is interesting to notice
well there is the deficiency in we know in humans SCID caused by interleukin-7 receptor deficiency so these guys don't have T cells they have NK cells but they don't have T cells so there hasn't been any gene therapy of that disease up to now so I can't tell you for sure but it's likely that I would predict that if We would perform gene therapy of IL-7 receptor. We will see the same.
Okay. And next slide. So based on this observation for X-linked SCID gamma deficiency today including the work of several groups in UK, in Holland, in the US and in Paris and Italy. there are six well for the SCID there are four I would say four SCID that are treated for which it has been shown that it's possible or clinical trials have been performed showing efficacy the first one was adenosine deaminase deficiency ADA that is causing a block in development of all T cells, not only T and NKs so the group in Italy as well in the UK And in the US they shown that the very same strategy was efficious efficacious excuse me to correct the immunodeficiency and if performed with chemotherapy reconstituting all the lineages. Then we have done one study for one form of what we call a V(D)J recombination deficiency that is the deficiency in the Artemis protein and it looks to work well. Another group did the same in the US and there is an ongoing study for RAG1 gene mutation performed in Holland is also with positive results. So the same principle the same technology in principle being used is efficient to correct the immunodeficiency associated with these different form of SCID. Next slide please.
Okay. So you already alluded to it. for the future are we going to use still gene therapy based on gene addition or could we consider that the usage of the CRISPR-Cas systems or genome editing will be the solution. So the answer is not as simple there are three strategies actually there are more but three strategies basically that can be considered with the CRISPR-Cas9 system. So first of all the top one which is the classic CRISPR Cas9 system where you use the Cas9 to induce at a very precise place in the in the genome break into the two strands of the DNA and this is occurring precisely because the Cas9 here in blue is guided by an complementary RNA so leading to the break and if you provide the cells with a matrix for recombination in order to correct to fix a mutation. For instance, this is the pink piece shown on the bottom. You can in principle induce what is shown on the last line of this first part that is the full correction of the mutation.
It occurs in some cells not very efficiently in stem cells because this is based on homologous re combination and non-dividing cells do not develop do not use are unable to use homologous re combination but on top of that there are events that we do not wish to see that is insertions deletions so imprecise re recombination which make the procedure not efficient. as perhaps could be even harmful if if the indels lead to who knows activation of oncogene. So this strategy cannot be used basically to correct an genetic defect including an immunodeficiency but in the last 10 years David Liu in Boston developed two strategies that may overcome this limitation. The first one is base editing. So what is base editing? So this is a strategy that does not lead to a break double strand break in the DNA but only in a single strand break. So which has as a good the positive consequence the fact that the non-homologous end joining DNA repair pathway is not triggered and this is this pathway that is causing the insertion deletions and the different things we do not want into the DNA. So how does it work? So the Cas9 is modified so that only one strand of the DNA is broken. The other one is not affected. so still there is a Cas9 so-called nickase Cas9. There is still of course an an RNA to guide the Cas9 but on addition to the Cas9 is linked what is shown here in purple and other enzyme which is a deaminase that was
modified nucleotide and may transform. If this is a deaminase affecting cytidine, it will lead to modified a C and then CG into a TA. And if it if it is an adenine deaminase, it will lead to transform an AT into a GC. So this is very interesting to correct single nucleotide mut mutation and actually that started to be used with some success and probably there is most in most of the cases a precise conversion without all of these insertions, deletions but so this strategy is very interesting but it is limited to correct point mutations single nucleotide mutations. So then the next strategy is prime editing slightly more complex where still with the Cas9 that is not inducing the DNA DNA strand breaks is still guided by a messenger RNA but on the messenger RNA there are two other pieces there is the purple stretch which is complementary to the genome the gene sequence and this is a stretch then once wishes to see integrated as shown. into the genome. So it could correct a mutation and the stretch can be up to 100 to 200 nucleotides large.
So it can correct a number of mutations found in in nature. But of course this is not sufficient because the purple piece is RNA not DNA. So in order to get have a DNA one should add shown in in pale pink on the on the cartoon an enzyme that is a reverse transcriptase that will be able to specifically retrotranscribe this purple piece. This is a bit more complicated than that but this is the principle. So the purple RNA stretch is retrotranscribed into DNA and can be inserted in the nicked strand of the DNA and reconstitute a given sequence that could be a way to correct genetic lesion. This has not been done for SCID for the reasons I've mentioned earlier. We don't really need that for the SCID. But for instance, it has been done now in a couple of patients with chronic granulomatous disease. this is affecting neutrophils with full correction of the mutation. So this is very still preliminary but it is very encouraging to think that base editing and prime editing actually generally speaking broadly speaking could be used to treat a number of genetic diseases and including of course primary immunodeficiencies. The summary of that is that there is no DNA breaks in these two instances. So likely safer and more efficacious. So this is really encouraging for the future and today is done ex vivo but perhaps later perhaps in one day it will be possible even to do it in vivo by targeting hematopoietic stem cells but this is
not that easy and next slide please. If I put a conclusion I don't remember let's go to the next there's no next conclusion. Okay. So the conclusion is without a slide is to see that following something like a bit more than 25 years of experience we moved from understanding the pathophysiology of severe combined immunodeficiencies. Understanding the fact that in some cases there is extremely strong selective advantage provided by expression of the transgene to be able to generate a number of T cells. The basis of the first successes of of gene therapy. We learned that with the first generation of vectors, the retroviral vectors used, they were not fully safe since in some cases they could induce leukemias. So we learned that this could be this risk could be excuse me suppressed by modifying engineering the vectors so that there is no enhancer any longer and this has led to the treatment of several form of severe combined immunodeficiencies and actually a few other immunodeficiencies and that for the future these new genome editing tools specifically base and prime editing could be used especially to treat diseases where we want the gene to be exactly at the place it has to be that is in the physiological locus to be regul regulated properly because if not this will lead to inflammation actually at least has been experimentally shown but this is very promising for such diseases to consider these
so as you said this has been more than 30 years of clinical research and trials successful clinical trials for gene therapy on hematopoietic stem progenitor precursor cells for hematological diseases. What whether could we use the lesson learned for nonhematological diseases?
Well, first of all, it was used to treat other hematological diseases like thalassemia and sickle cell disease where it doesn't work fully but it works. It can improve the situation of such patients. Of course, then you have to introduce the gene encoding the beta chain of hemoglobin and you have to use a system that triggers expression in erythroid precursors. But this is feasible. It has also been used to get expression of enzyme that are deficient in metabolic diseases. Expression of enzyme notably in macrophages with clinical benefit for diseases like mucopolysaccharidosis or leukodystrophies. So outside of the field of hematopoietic stem cells there has been advances in gene therapy but based on another strategy which is in vivo usage of adeno associated vectors to correct any type of cells. the advantage is that these vector the vectors do not integrate into the genome. So there is no risk of insertional mutagenesis. The disadvantage is that they are not replicated if cells divide. So they can be used only to target cells that are not dividing or poorly dividing like hepatocytes, retina cells, brain muscles. So some successes have been observed up to now in the treatment of hemophilia A and B in some form of inherited deafness inherited blindness in the treatment of a neuromuscular disease named spinal amyotrophy with the interesting observation that such vectors one of these AAV can cross the blood-brain barrier and so reach neurons and there is also there has been some successes in
treating some genetic liver diseases. So there are today perhaps something like 25 genetic diseases for which either strategy retroviral or based on AAV vectors have been used to correct to pro the genetic defect and provide a clearcut benefit for the patients.
It's very interesting how how gene therapy on hematopoietic precursor cells can also lead to benefit of neurodegenerative disease. So adrenoleukodystrophy which is neurodegenerative disease due to accumulation of fatty acids and could you comment on this?
Yeah, this is based on the fact that monocytes do can can move into well of course in all tissues leading to macrophage differentiation including in the brain into the microglia. So locally you have the production of the right enzyme that can be for depending on the disease can be captured by other cells and thus being able to eliminate the substances like mucopolysaccharides in one case or lipids in another that accumulate and cause the disease. So this is the mechanism by which hematopoietic stem cell gene therapy can be helpful to treat some not many but some of these neurodegenerative diseases.
It's also a very interesting lesson that we learned I think less than 10 years ago. HIV patients who developed blood cancers and were treated with the were treated with anti- cancer therapy including bone marrow transplantation and it happened that bone marrow transplantation was from donors which had mutation in CCR5 gene which made their cells resistant for HIV entry. Yes. So these patients were treated for their blood cancer but as a bonus they were also treated for for acquired immunodeficiency syndrome.
Alain, I would like as we are now looking towards gene therapy with more precise genome editing, what is the important lesson from this research journey and its ups and downs that the clinicians and the funding agencies should shouldn't forget.
Yeah. I mean the I mean new therapies that this is never a linear progression. we are learning from from clinical trials and improving the way it is performed. This is I think this happened in many fields of medicine and today of course this the tools available open the capacity of potentially treat quite a number of genetic diseases of course diseases of the immune system but many many others in different fields of medicine not talking about the CAR T cells that are also based on gene therapy by the way. So there are multiple possibilities to develop gene therapies which is very encouraging. the difficulties are that it requires for all of the diseases some preclinical studies that are pretty expensive to perform and it's not completely sure that it will be economically viable to develop the many therapies that are potentially there because of the complexity of the system and to get to go up to drug approval. So this is potential limitation
1:03:50 · Immune aging and the limits of enhancement
for example if I'm I remember correctly elephant has more perforin copies comparing to humans and that is that some people are correlating it with the elephant living longer comparing to us. So, so I would like to know your opinion. Let's say if we have more perforin copy in our body so that will improve the NK and CD8 function
I would be cautious to go up beyond physiology because there may be we don't know but they may be harmful consequences. Maybe if we have cytotoxic cells that are more efficient than observing the physiology, we may have immunopathology caused by this cytotoxic cells in the context of infections for instance. So I will not go into the direction.
well first animals are not humans. You may there may be a lot of differences. I think they are aging is an extremely complex process with multiple parameters. so I'm not sure I will engage too much in such procedures. That's my personal opinion.
And how do you think like when the way our immune system is changing with age how what impacts it it is having on our physiology and disease occurrence such as cancer or v virus
obviously there are lots of changes because many immune cells become senescent there is evolution involution of the thymus there is maybe from time to time clonal hematopoiesis with activated monocytes for instance that may lead to pathology from atherosclerosis to leukemia. So there are many many aspects of of aging of the immune system but still it's interesting to see that people over the age of 80 can still be immunized. I mean let's say take the COVID example and still not as with a vaccine that is not as effective as in younger people but still effective showing that even at relatively old age we still have an immune system that works. We know it works less well but still it works. I'm quite impressed to see that. And on top of that, as discussed earlier, we have memory cells that can persist for 50 years, 60 years, maybe 100 years. , making the people of course they are more susceptible to infections, to cancer, the old the elderly people, but still have some relatively good defense. I think this is quite amazing such a complex process whether it can can can this process be ameliorated I think in people who are who have an immunodeficiency say people above the age of 50 or 60 an immunodeficiency that is significantly more pronounced than the majority of people then of course those guys have to be considered and see what can be done for Can should we improve the decline of the immune system that is the physiological decline? I'm not sure because then I think there may be many unwanted
Professor Fischer, thank you. This conversation shows shows that gene therapy is not only about technical achievement. It is a long-term responsibility to patients, to families, and to the future of the field. Thank you for helping us to understand the science, the clinical courage and the responsibility behind this work. Thank you.
