Reactivation of fetal hemoglobin (HbF) is being pursued as a treatment strategy for hemoglobinopathies. Here, we evaluated the therapeutic potential of hematopoietic stem and progenitor cells (HSPCs) edited with the CRISPR-Cas9 nuclease platform to recapitulate naturally occurring mutations identified in individuals who express increased amounts of HbF, a condition known as hereditary persistence of HbF. CRISPR-Cas9 treatment and transplantation of HSPCs purified on the basis of surface expression of the CD34 receptor in a nonhuman primate (NHP) autologous transplantation model resulted in up to 30% engraftment of gene-edited cells for >1 year. Edited cells effectively and stably reactivated HbF, as evidenced by up to 18% HbF-expressing erythrocytes in peripheral blood. Similar results were obtained by editing highly enriched stem cells, defined by the markers CD34+CD90+CD45RA−, allowing for a 10-fold reduction in the number of transplanted target cells, thus considerably reducing the need for editing reagents. The frequency of engrafted, gene-edited cells persisting in vivo using this approach may be sufficient to ameliorate the phenotype for a number of genetic diseases.

Collagen fiber alignment in the tumor microenvironment directs migration, a process called contact guidance, enhancing the efficiency of cancer invasion and metastasis. 3D systems that assess contact guidance by locally orienting collagen fiber alignment are lacking. Furthermore, cell type differences and the role of extracellular matrix stiffness in tuning contact guidance fidelity are not well characterized. In this paper rotational alignment of collagen fibers is used as a 3D contact guidance cue to illuminate cell type differences and the role of extracellular matrix stiffness in guiding cell migration along aligned fibers of collagen. This local alignment offers a simple approach by which to couple collagen alignment with gradients in other directional cues in devices such as microfluidic chambers.

Hematopoietic stem cell (HSC) gene therapy has the potential to cure many genetic, malignant, and infectious diseases. We have shown in a nonhuman primate gene therapy and transplantation model that the CD34 + CD90 + cell fraction was exclusively responsible for multilineage engraftment and hematopoietic reconstitution. In this study, we show the translational potential of this HSC-enriched CD34 subset for lentivirus-mediated gene therapy. Alternative HSC enrichment strategies include the purification of CD133 + cells or CD38 low/– subsets of CD34 + cells from human blood products. We directly compared these strategies to the isolation of CD90 + cells using a good manufacturing practice (GMP) grade flow-sorting protocol with clinical applicability. We show that CD90 + cell selection results in about 30-fold fewer target cells in comparison to CD133 + or CD38 low/– CD34 + hematopoietic stem and progenitor cell (HSPC) subsets without compromising the engraftment potential in vivo . Single-cell RNA sequencing confirmed nearly complete depletion of lineage-committed progenitor cells in CD90 + fractions compared to alternative selections. Importantly, lentiviral transduction efficiency in purified CD90 + cells resulted in up to 3-fold higher levels of engrafted gene-modified blood cells. These studies should have important implications for the manufacturing of patient-specific HSC gene therapy and gene-engineered cell products.

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BackgroundChimeric antigen receptor (CAR) T-cell therapies have demonstrated transformational outcomes in the treatment of B-cell malignancies, but their widespread use is hindered by technical and logistical challenges associated with ex vivo cell manufacturing. To overcome these challenges, we developed VivoVec, a lentiviral vector-based platform for in vivo engineering of T cells. UB-VV100, a VivoVec clinical candidate for the treatment of B-cell malignancies, displays an anti-CD3 single-chain variable fragment (scFv) on the surface and delivers a genetic payload that encodes a second-generation CD19-targeted CAR along with a rapamycin-activated cytokine receptor (RACR) system designed to overcome the need for lymphodepleting chemotherapy in supporting successful CAR T-cell expansion and persistence. In the presence of exogenous rapamycin, non-transduced immune cells are suppressed, while the RACR system in transduced cells converts rapamycin binding to an interleukin (IL)-2/IL-15 signal to promote proliferation.MethodsUB-VV100 was administered to peripheral blood mononuclear cells (PBMCs) from healthy donors and from patients with B-cell malignancy without additional stimulation. Cultures were assessed for CAR T-cell transduction and function. Biodistribution was evaluated in CD34-humanized mice and in canines. In vivo efficacy was evaluated against normal B cells in CD34-humanized mice and against systemic tumor xenografts in PBMC-humanized mice.ResultsIn vitro, administration of UB-VV100 resulted in dose-dependent and anti-CD3 scFv-dependent T-cell activation and CAR T-cell transduction. The resulting CAR T cells exhibited selective expansion in rapamycin and antigen-dependent activity against malignant B-cell targets. In humanized mouse and canine studies, UB-VV100 demonstrated a favorable biodistribution profile, with transduction events limited to the immune compartment after intranodal or intraperitoneal administration. Administration of UB-VV100 to humanized mice engrafted with B-cell tumors resulted in CAR T-cell transduction, expansion, and elimination of systemic malignancy.ConclusionsThese findings demonstrate that UB-VV100 generates functional CAR T cells in vivo, which could expand patient access to CAR T technology in both hematological and solid tumors without the need for ex vivo cell manufacturing.