Periodic Reporting for period 1 - MABank (Creation of a GLP bank of immune-privileged, immortal mesoangioblasts to treat monogenic, recessive diseases of muscle and connective tissue)
Reporting period: 2023-09-01 to 2025-02-28
Stem cells led to effective cures for diseases of blood and epithelia but now face the problem of the extremely high costs of production (up to 2M$ for a single produt). This will make such therapies unsustainable for NHS. Through the ongoing ERC ADG 884952-UniMab, we are completing the development of immortal, immune-privileged mesoangioblasts (Mabs: vessel associated progenitors) thanks to genome editing that removed HLA and the expression of proteins that induce immune tolerance. In this PoC project we studied the feasibility of producing and storing a large quantity of universal Mabs through a bank of universal donor Mabs from healthy donors, ready to be corrected for the specific genetic mutation and stored in specific working banks (one for each disease) ready to be injected into the patient following the motto “one serves many”. However, we found that Mabs already express two of these tolerogenic proteins when grown in our medium. This will allow us to put in a single vector both the tolerogenic and therapeutic gene(s), thus reducing risks and shifting the project to the direct production of several working banks. The results obtained confirmed the feasibility of this strategy and allow the future business exploitation of the MABANK technology, by creating a company with a GMP biobank that may operate internally and make cells available to clinicians and Biotech companies.
Cell and gene therapy has revolutionized medicine, providing treatments and in some case a cure for previously incurable and devastating genetic diseases. As mentioned, the costs associated to these treatments are prohibitive to the point that several life-saving therapies risk to be withdrawn from the market for their cost. In the case of autologous ex vivo gene therapy (most successful therapies so far) an extremely expensive medicinal product is produced for one patient only. Thus, we developed universal donor cells, invisible to the immune system, that can become an “off the shelf product” available for all patients with the same disease. This required the creation of clinical-grade level cell banks where “ready to go” medicinal products would be stored until needed. This means that with the approximately the same cost needed to treat one patient we could produce enough cells to treat 100 patients or more, with a corresponding reduction of costs, making these therapies affordable for the national health systems or the health insurances.
Cell and gene therapy has revolutionized medicine, providing treatments and in some case a cure for previously incurable and devastating genetic diseases. As mentioned, the costs associated to these treatments are prohibitive to the point that several life-saving therapies risk to be withdrawn from the market for their cost. In the case of autologous ex vivo gene therapy (most successful therapies so far) an extremely expensive medicinal product is produced for one patient only. Thus, we developed universal donor cells, invisible to the immune system, that can become an “off the shelf product” available for all patients with the same disease. This required the creation of clinical-grade level cell banks where “ready to go” medicinal products would be stored until needed. This means that with the approximately the same cost needed to treat one patient we could produce enough cells to treat 100 patients or more, with a corresponding reduction of costs, making these therapies affordable for the national health systems or the health insurances.
During the funded period we have developed a prototype GMP-like bank following the original plan of a “master bank” composed of immune-privileged healthy mesoangioblasts. These cells would have been later engineered with lentivectors expressing either the full-length cDNA of a small gene (e.g. -sarcoglycan) or a small nuclear RNA engineered to skip a specific exon of large genes such as dystrophin or dysferlin. Each of the transduced cell population would constitute a specific “working bank” for a given monogenic recessive disease of muscle. This was made possible by the development of a novel, proprietary tissue culture medium that promotes unlimited proliferation without affecting differentiation.
Cells were first subjected to genome edited with CRISPR-Cas9 to eliminate simultaneously -2 micoglobulin (B2M) and Class II Common Trans-activator (CIICT). Editing efficacy was about 50% which led us to counter select remaining positive cells with magnetic beads loaded with antibodies for B2M and the CII HLA expressed by the original cell population. This two-step procedure leads to an enrichment of Double Knock Out (DKO) cells > 99%. Edited cells proliferate at the same rate, differentiate like control non edited cells and express a normal karyotype. However, cells that do not express any HLA in theory should be eliminated by human NK cells. To overcome this problem, we generated a novel 3rd generation SIN, bidirectional lentivector, expressing from one arm PDL1 and CD20 and from the other CD20 (suicide gene) and HLA-E fused to B2M, to prevent killing by NK cells. However, we noticed that, when grown in our proprietary Unimedium, cells spontaneously express PDL-1 and CD47, thus making its lenti-mediated over-expression unnecessary. In this way there will be space in the vector to insert the therapeutic agent (U7 snRNA x skip exon 51 in this case) and reduce the viral transduction to 1 only. The advantage of this modification of the strategy mainly resides in reducing to half the risk of insertional mutagenesis. Therefore, this would eliminate the need of a single master bank as each cell population, transduced with a different viral vector would constitute a unique working bank, specific for a given monogenic, recessive of the solid mesoderm or for a specific mutation, in case of large genes.
Assuming the need of 1 billion cells for each infusion. A 250ml bag can store a maximum of 72ml. Considering a freezing density of 14x10^6/ml, there would be a dose of 1x10^9 cells in a single bag. 500 bags would allow to treat 100 patients for five consecutive infusions or more patients with less infusions, depending on the protocol. Of course, fewer bags would be needed for less frequent mutations in the dystrophin gene or for rarer forms of muscular dystrophy.
Cells were first subjected to genome edited with CRISPR-Cas9 to eliminate simultaneously -2 micoglobulin (B2M) and Class II Common Trans-activator (CIICT). Editing efficacy was about 50% which led us to counter select remaining positive cells with magnetic beads loaded with antibodies for B2M and the CII HLA expressed by the original cell population. This two-step procedure leads to an enrichment of Double Knock Out (DKO) cells > 99%. Edited cells proliferate at the same rate, differentiate like control non edited cells and express a normal karyotype. However, cells that do not express any HLA in theory should be eliminated by human NK cells. To overcome this problem, we generated a novel 3rd generation SIN, bidirectional lentivector, expressing from one arm PDL1 and CD20 and from the other CD20 (suicide gene) and HLA-E fused to B2M, to prevent killing by NK cells. However, we noticed that, when grown in our proprietary Unimedium, cells spontaneously express PDL-1 and CD47, thus making its lenti-mediated over-expression unnecessary. In this way there will be space in the vector to insert the therapeutic agent (U7 snRNA x skip exon 51 in this case) and reduce the viral transduction to 1 only. The advantage of this modification of the strategy mainly resides in reducing to half the risk of insertional mutagenesis. Therefore, this would eliminate the need of a single master bank as each cell population, transduced with a different viral vector would constitute a unique working bank, specific for a given monogenic, recessive of the solid mesoderm or for a specific mutation, in case of large genes.
Assuming the need of 1 billion cells for each infusion. A 250ml bag can store a maximum of 72ml. Considering a freezing density of 14x10^6/ml, there would be a dose of 1x10^9 cells in a single bag. 500 bags would allow to treat 100 patients for five consecutive infusions or more patients with less infusions, depending on the protocol. Of course, fewer bags would be needed for less frequent mutations in the dystrophin gene or for rarer forms of muscular dystrophy.
The potential impact of this project is huge and potentially game changing for advanced therapies in general. First, once reached the GMP production, we will be able to produce a single ATMP that will be available for all patients affected by Duchenne Muscular Dystrophy with a specific mutation. This step alone would dramatically cut the costs, making this approach affordable for National Health Systems. Then muscular dystrophies whose mutations affect small genes, like many Limb-Girdle Muscular Dystrophies, could be treated with a single ATMP (i.e. the over-expressed full-length cDNA) for all patients. Finally, this approach may be extended to many genetic diseases affecting solid mesoderm. The banks, after IP protection, would be made available to the biomedical international community, and to do this we are planning to create a spin off from our Institution. We are already engaging with regulatory bodies and patients’ associations to plan together in details all the remaining step to make this a consolidated therapy for a variety of genetic diseases.