Autologous, lentivirus‐modified, T‐rapa cell “micropharmacies” for lysosomal storage disorders

Abstract

T cells are the current choice for many cell therapy applications. They are relatively easy to access, expand in culture, and genetically modify. Rapamycin-conditioning ex vivo reprograms T cells, increasing their memory properties and capacity for survival, while reducing inflammatory potential and the amount of preparative conditioning required for engraftment. Rapamycinconditioned T cells have been tested in patients and deemed to be safe to administer in numerous settings, with reduced occurrence of infusion-related adverse events. We demonstrate that ex vivo lentivirus-modified, rapamycin-conditioned CD4 + T cells can also act as next-generation cellular delivery vehicles-that is, "micropharmacies"-to disseminate corrective enzymes for multiple lysosomal storage disorders. We evaluated the therapeutic potential of this treatment platform for Fabry, Gaucher, Farber, and Pompe diseases in vitro and in vivo. For example, such micropharmacies expressing a-galactosidase A for treatment of Fabry disease were transplanted in mice where they provided functional enzyme in key affected tissues such as kidney and heart, facilitating clearance of pathogenic substrate after a single administration.

Identification of a human hematopoietic stem cell subset that retains memory of inflammatory stress

“…Approximately 300,000 CD45+CD34-CD19-CD33-were isolated by FACS as described above from cryopreserved xenograft bone marrow to enrich T cells and deplete B, myeloid and progenitor cells. Cells were seeded in X-VIVO 20 media supplemented with 5% human AB serum and 30U/mL hIL-2 and stimulated with anti-CD3/CD28 Dynabeads (0.5 beads per cell) ( 85 ). Live cells were counted by trypan blue exclusion on a hemocytometer, and maintained in culture for 8 days before harvesting.…”

Neurological Disease Modeling Using Pluripotent and Multipotent Stem Cells: A Key Step Towards Understanding and Treating Mucopolysaccharidoses

“…In those two cases, however, the cells which are collected need to be genetically modified ex vivo to a normal function. Currently, those approaches are under clinical trial for a few LSDs [26][27][28][29][30]. Regardless of the HSCT type, in terms of procedure, its principle is simple: first, the patient needs to receive some type of therapy that will inhibit the immune system (to prevent rejection); then the modified cells are injected in the patient.…”

Engineering Memory T Cells as a platform for Long-Term Enzyme Replacement Therapy in Lysosomal Storage Disorders

“…Precision gene modification of primary human T cells has thus far focused largely on expressing chimeric antigen receptors (CAR-T) as anti-cancer therapeutics, yet this technology has applications outside the field of cancer immunotherapy, and the tools that have been developed by that field can easily be leveraged for novel T cell-based protein delivery systems. [21][22][23][24][25][26][27] The CRISPR-Cas9 system mediates precise insertion of transgenes by the induction of a targeted double-stranded DNA break (DSB) along with a DNA template for homology directed repair (HDR). 21,28,29 Some recent studies have reported results with T cells engineered to produce and secrete various proteins via transposon or lentiviral methods.…”

“…Precision gene modification of primary human T cells has thus far focused largely on expressing chimeric antigen receptors (CAR-T) as anti-cancer therapeutics, yet this technology has applications outside the field of cancer immunotherapy, and the tools that have been developed by that field can easily be leveraged for novel T cell-based protein delivery systems. 21–27…”

“…21,28,29 Some recent studies have reported results with T cells engineered to produce and secrete various proteins via transposon or lentiviral methods. 27,30 However, to date, no therapies have explored T cells precision-engineered at a safe harbor locus as an enzyme delivery platform for enzymopathies.…”