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[00:00:05] Dr Alessandra Biffi: So, good morning and thank you for joining us for this webinar, which is focused on the role of microglia and the implications for the treatment of patients with MLD. Before getting started, I just remind you there is no screenshot policy and please ask your questions at any time via text. These are my disclosures in the field and these are the learning objectives for today. I will talk to you about microglia, which is a specialized population of macrophage- like cells in the CNS. We will review the role and the involvement of microglia in promoting or limiting the pathogenesis of lysosomal diseases and of MLD in particular, and then we will explore the potential for the management and treatment of neurometabolic diseases based on microglia. So, as I briefly mentioned, microglia cells are the tissue- resident macrophages of the central nervous system. They are cells of hematopoietic lineages and can be distinguished from other tissue- resident macrophages because of their embryonic origin, their maintenance, and their immunophenotype. In particular, microglia precursors arise in the yolk sacs and then they enter the developing embryo and colonize the cephalic mesenchyme and then differentiate into microglia. So, an origin that is unique for this population as compared to the other immune elements. The other key property of microglia is the fact that their pool remains stable in the healthy brain by means of proliferation and cell renewal and programmed cell death. So, there's not an incoming population in physiological conditions. They are cells with a variety of morphological and immunological phenotypes. In particular, during development, they undergo a stepwise program and they change their shape and morphology from an amoeboid one into a ramified microglia in the adult brain. These cells have a key role in the central nervous system. They participate in the surveillance, development, homeostasis, and protection and functional integrity of the CNS. They act as key players in synaptic pruning, synaptic plasticity, and they obviously surveil and patrol the central nervous system also in pathological conditions. Indeed, microglia are involved in many CNS disorders. They can be detrimental or essential in the pathogenesis and in the disease progression according to the disease and the stage of their involvement. MLD. In the setting of neurometabolic diseases, lysosomal storage disorders and in particular MLD in the microglia play a key and central role. You know that the pathogenesis in these disorders begins with the deficiency in the activity of a lysosomal hydrolase that determines accumulation of undegraded compounds, and this leads to impaired lysosomal functions, impaired autophagy, and accumulation of dysfunctional organelles mitochondria that lead to protein aggregates, to oxidative stress, to organelle destabilization, rupture, tissue inflammation, and cell death. At many of these steps, microglia indeed play a key role, and many of these steps also occur within microglia the lysosome a key therapeutic element but also a key target for treating neurometabolic diseases such as metabromatic leukodystrophy. Because of this, a lot of the efforts have been placed in the setting of exploiting microglia as a therapeutic tool for these conditions. And the key element in this concept has been the idea of replacing activating and metabolically impaired microglia with a healthy population, a safe population that could replace their function and ideally promote tissue repair and prevent tissue damage. The way by which many of us, including myself, have approached this concept has been based on the evidence that a population of cells in the CNS that resembles microglia and recapitulates some of their function can be generated starting from bone marrow precursors.
[00:04:57] Dr Alessandra Biffi: Indeed, what the experience that started more than 30 years ago of allogeneic hematopoietic cell transplant has told us is that by transplanting HLA- compatible hematopoietic stem cells from a donor, ideally not a carrier we can reconstitute into the recipient multiple blood lineages and we can also reconstitute in general the population of progeny cells in the central nervous system that can indeed recapitulate many of the functions of microglia. And we call these cells microglia functionally equivalent because these cells of transplant and donor origin can modulate and reduce tissue damage and neuroinflammation into the pathological brain. They can restore scavenging and homeostatic functions of microglia, replacing the metabolically incompetent and inactivated cells, and they could eventually also become a local source of functional proteins in the central nervous system that as you can imagine in the case of lysosomal disorders is of particular relevance. Based on this rationale, as I said, we have more than 30 years of experience and attempt of exploiting the transplantation of allogeneic hematopoietic stem cells for the treatment of lysosomal diseases and here you have a list of candidate lysosomal diseases in which hematopoietic cell transplantation from compatible donors has been performed . The result of all these experiences which you see are ongoing now has allowed us to identify two classes of lysosomal diseases, the first one being the group of LSDs which can benefit in a clear and robust manner from the allogeneic transplant. And these diseases for sure comprise Mucopolysaccharidosis type I H, the Hurler syndrome, particularly when the patients are younger than 2.5 years old, cerebral X- linked adrenal echolystrophy, again in patients who have a low score, which is relatively low, so with a limited disease burden, and in some variants, globolic cell leukodystrophy. On the other side, we identified diseases in which there's weak or very poor evidence of benefit derived from the allogeneic transplant procedures, and these diseases for sure include metatraumatic leukodystrophy. The reason why allogeneic hematopoietic cell transplantation did not provide overt and robust evidence of benefit in these kinds of disorders, such as MLD, has to be considered related to the timing and the extend of donor's engraftment in the brain which is considered to be in the time frame that you see in this graph.
[00:08:03] Dr Alessandra Biffi: In particular, what we know at the moment is that the timing for achieving sufficient engraftment of the transplant is usually comprised in between 12 and 18 months post- transplant, and in this time, usually we observe a rapid progression of severe neurodegenerative lysosomal disorders, such as MLD. In this graph you can see a demonstration of the age and the timing of progression of the neuromotor impairment in MLD patients, and as you can see, the early onset forms, such as late infantile and early juvenile MLD, have a rapid decline of function in a time frame which is much shorter than what's required by the allogeneic transplant to efficiently colonize the central nervous system so the progression of neurodegeneration exceeds the time for benefit associated with the allogeneic transplant procedure.
[00:08:59] Dr Alessandra Biffi: In order to address this limitation, not many years ago my group decided to invest on the opportunity to augment the therapeutic potential of allogeneic stem cell transplantation by gene transfer and we developed at that time the first protocols, first in mice and then in patients from using hematopoietic stem gene therapy as a way to pass them in time of benefiting the CNS of LSD patients and in particular of MLD patients . Starting from the rationale that the transplatation of using genetically modified hematopoetic cells would reproduce what we observe in an allogeneic transplant setting which is a re constitution of multiple blood lineages by the transplanted cells including the populations resulting to the CNS of the microglia functioning equivalent cells. And this allows us by needs of gene transfer to increase the therapeutic potential associated to the generation of the microglia functioning equivalent cells right from the transplant by enhancing their ability to produce and secrete lysosomal enzymes. So to compensate as you know cross- correction is a key mechanism allowing us to propose this kind of approach to patients affected by lysosomal diseases and metachromatic leukodystrophy Basically we want to compensate the ability of the CNS engrafted and find compatible cells to produce and release arylsulfatase A in our case and thus compansate the possibility and the potential for cross- correction. We initially tested these concepts in the animal models of several lysosomal diseases starting from the animal model of metachromatic leukodystrophy. And we clearly demonstrate that the patient gene transfer into gene-modified stem cells allows for sustained enzyme production by the brain infiltrating myeloid cells derived from the transplant and allows for more rapid and robust therapeutic enzyme delivery to the CNS by the infiltrated cells, by the microglia fuctioning cells derived from the transplant. And this has resulted in the rationale for building a clinical development plan that allowed us to activate the clinical trial of HSC gene therapy for MLD patients of which we will likely discuss in an upcoming webinar but which resulted in a robust and dramatic therapeutic benefit that as I said we will discuss in the future. But to clearly demonstrate the rationale and the hypothesis I presented to you so that the transplantation of genetically engineered autologous hematopoietic stem cells in a recipient who received an adequate conditioning regimen allowing and powering the turnover of brain microglia cells with the transplant progeny population, could result in prevention of disease manifestations to the central nervous system because of active enzyme delivery to the CNS. In the cerebrospinal fluid of treated patients, we could document a robust increase of arylsulfatase A activity, demonstrating that indeed progeny cells of the transplant engrafted in the brain prevented motor and cognitive deterioration that as you know characterized the most severe forms of this disease. This was clearly important proof that the concept that I described to you, so the ability of the transplant to populate the brain and the ability of gene transfer to increase the therapeutic potential of HSC transplantation. However, also in this setting that was optimized for powering the enzyme delivery to the brain and for anticipating the time of benefit, again also in this setting, the benefit was mostly restricted to patients treated in a very early stage of the disease. Thus, indicating that despite amelioration of our therapeutic approach, still time of engraftment in the CNS and time of enzyme delivery to the CNS could be limiting, and that we might need to work further at the level of the bench in order to anticipate the timing and extent of engineered engraftment in the brain for being able to benefit rapidly progressive diseases such as infantile disease or neurodegenerative forms of cerebral neurodegeneration, but also to benefit patients who come to our attention when already symptomatic. And there are a number of ways by which we can address this aspect and this phenomenon. I just wanted to focus in my few remaining slides on these ways that we are exploiting and that we are optimising in the laboratory
[00:13:56] Dr Alessandra Biffi: The first way is to enrich the population to be transplanted in hematopoietic stem and progenitor cells, because of the clinical evidences we and many others accumulated on the ability of the most immaculate populations within the hematopoietic stem cell pool to successfully go to the central nervous system and give rise to a microglia- like functional progeny. Secondly, we can and we should work on admistering to the recipients of the transplant an adequate preconditing that could allow target in microglia progenitors.
[00:14:33] Dr Alessandra Biffi: As I mentioned at the beginning, microglia cells have a unique profile of being able to self- sustain because of the ability to self- renew, and many laboratories are investigating the existence and the characterisation of microglia progenitors that could be residing in the central nervous system that need to be properly targeted by conditioning in order to allow the opportunity for engraftment of transplant derived cells progeny cells in the central nervous system. And there are many groups that are working on this and many targeted protocols that are being developed for brain conditioning. There are some protocols that are based on already available drugs that have been FDA approved for other purposes. For example the ones mentioned here on the bottom of the table, based on PLXs that indeed are able to actively kill microglia, including cells possibly able to secrete chemical and that could be exploited for these purposes. But obviously a lot of of expectation and hope is based on our ability to identify microglia progenitors and target them through specific and potentially antibody- targeted approaches. The other way we are exploring in the laboratory is, in order to include and improve the engraftment of the transplanted cell in the central nervous system is the strategy of direct delivery of the microglia population into the central nervous system. We have, in particular, exploited and developed towards the clinics two routes of administration that could be of potential relevance for MLD. This route has been extensively described in our laboratory not only in terms of efficiency but also in terms of feasibility and safery.Indeed, it allows a faster and more robust engraftment of the transplanted cell progeny in the CNS and powers their differentiation towards a microglia- like phenotype. And more recently, we have developed and generated relevant data on the possibility of transplanting hematopoietic stem and progenitor cells also in the intrathecal lumbar space. That could be more clinically feasible and similarly favor the engraftment of the transplant at the level of the central nervous system.
[00:17:04] Dr Alessandra Biffi: Additional approaches we are developing in order to fasten and also to render more specific our gene therapy strategy based on the hematopoietic stem cells and myeloid turnover include the use and the development of microglia-specific promoters that could also sense neuroinflammation and the disease setting and therefore adequate the amount and the extent of protein production and enzyme delivery according to the disease setting, but also strategies based on gene editing that could allow us targeted gene additions that could not only allow us to achieve targeted and regulated expression of the therapeutic gene but also again to favor the engraftment of the transplanted cells and their progeny in the central nervous system.
[00:17:52] Dr Alessandra Biffi: These are developments that are at the moment being conducted in the bench of our laboratories and the laboratories of many other investigators and we hope in the future could ameliorate our ability to develop HSC therapy approaches for metabolic patients and in particular allow us better target the disease, the progression of the disease and address the needs of symptomatic patients.Thank you. This is the presentation I prepared for you. I would be happy to now address your question. If you have any, please provide them via the text messages. I read here, recovery of healthy function is dependent on how advanced the signs of MLD are. Will it just work in pre- symptomatic patients? Yes, this is exactly the point we were covering. At the moment, what we know is that the extent of benefit is absolutely dependent on the disease burden at the time of treatment. And this is a key variable that has to be somehow evaluated according to the disease variant we are looking at.
[00:19:28] Dr Alessandra Biffi: And this is particularly true and relevant whenever we look at the early onset forms of the disease, whose progression is very rapid, but it could be less impactful if we look at the late onset forms in which the progression of the disease could allow us for more time for the engraftment of the transplanted cells in the CNS and the enzyme delivery to the CNS. So we need to look at this concept according to the kind of phenotype we are willing to address and to treat and according to the time frame we are approaching.
[00:20:04] Dr Alessandra Biffi: So we don't have yet data, it's being experimented, but I would assume that we could have some more room for treatment and benefit, also in early symptomatic patients, as far as we look at the later onset form, whose progression could be a little bit less rapid. I see another question.
[00:20:28] Dr Alessandra Biffi: Does cross- correction work for MLD and non- MPSs at any certain MLD? Yes, cross- correction is a mechanism that was described in the 80s by Elisabeth Neufeld, and that is working for all lysosomal enzymes because of their nature, they are secreted, 40% of the amount that every cell produces is secreted in the extracellular space. And because of the mannose-6-phosphate residue they have, they could be taken up by mannose-6-phosphate receptor and other receptors in a biophysiological manner. So cross- correction is valid for MLD, for all MPSs and for all lysosomal enzymes.
[00:21:14] Dr Alessandra Biffi: Do MLV patients not show a positive response to treatment in the trial? Yes, the majority of the patients who have been treated in the trial had a favourable response in terms of either complete prevention of disease progression or significant reduction of the expected progression of the disease. There are very, very few limited cases in whom we did not see a robust benefit and are those patients who were treated with the greater burden of the disease according to their disease state. So obviously in pre- symptomatic late infantile patients, benefit was robust. In a few patients who were treated with late infantile phenotype or early juvenile phenotype in symptomatic stage benefit was less robust according to the rationale I just presented to you. Okay, I see that we don't have any further questions. I thank the audience, all of you, for your kind attention and I hope this was informative and helpful for you all. Thank you so much. Bye- bye.