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.
