Preclinical proof of concept for VivoVec, a lentiviral-based platform for in vivo CAR T-cell engineering.

Background: Chimeric 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.

Stochastic fate decisions of HSCs after transplantation: early contribution, symmetric expansion, and pool formation.

Hematopoietic stem cells (HSCs) are assumed to be rare, infrequently dividing, long-lived cells not involved in immediate recovery after transplantation. Here, we performed unprecedented high-density clonal tracking in nonhuman primates and found long-term persisting HSC clones to actively contribute during early neutrophil recovery, and to be the main source of blood production as early as 50 days after transplantation. Most surprisingly, we observed a rapid decline in the number of unique HSC clones, while persisting HSCs expanded, undergoing symmetric divisions to create identical siblings and formed clonal pools ex vivo as well as in vivo.

Efficient polymer nanoparticle-mediated delivery of gene editing reagents into human hematopoietic stem and progenitor cells.

Clinical applications of hematopoietic stem cell (HSC) gene editing are limited due to their complex and expensive logistics. HSC editing is commonly performed ex vivo using electroporation and requires good manufacturing practice (GMP) facilities, similar to bone marrow transplant centers. In vivo gene editing could overcome this limitation; however, electroporation is unsuitable for systemic in vivo applications to HSCs.

AMD3100 redosing fails to repeatedly mobilize hematopoietic stem cells in the nonhuman primate and humanized mouse.

AMD3100 (plerixafor) is a vital component of many clinical and preclinical transplant protocols, facilitating harvest of hematopoietic stem and progenitor cells through mobilization into the peripheral blood circulation. Repeat mobilization with AMD3100 is also necessary for many patients with suboptimal first stem cell collection or those requiring repeat transplantation. In this study we investigated the mobilization efficacy of repeated AMD3100 dosages in the nonhuman primate and humanized mouse models.

Isolation of a Highly Purified HSC-enriched CD34CD90CD45RA Cell Subset for Allogeneic Transplantation in the Nonhuman Primate Large-animal Model.

Unlabelled: Allogeneic hematopoietic stem cell transplantation (allo-HCT) is a common treatment for patients suffering from different hematological disorders. Allo-HCT in combination with hematopoietic stem cell (HSC) gene therapy is considered a promising treatment option for millions of patients with HIV+ and acute myeloid leukemia. Most currently available HSC gene therapy approaches target CD34-enriched cell fractions, a heterogeneous mix of mostly progenitor cells and only very few HSCs with long-term multilineage engraftment potential.

Purification of Human CD34CD90 HSCs Reduces Target Cell Population and Improves Lentiviral Transduction for Gene Therapy.

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 CD34CD90 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.

Therapeutically relevant engraftment of a CRISPR-Cas9-edited HSC-enriched population with HbF reactivation in nonhuman primates.

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.

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells.

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, underlies the only known cure of human immunodeficiency virus (HIV-1) infection, and shows immense promise in the treatment of genetic diseases such as beta thalassemia. Our group has developed a protocol to model HSPC gene therapy in nonhuman primates (NHPs), allowing scientists to optimize many of the same reagents and techniques that are applied in the clinic. Here, we describe methods for purifying CD34 HSPCs and long-term persisting hematopoietic stem cell (HSC) subsets from primed bone marrow (BM).

Contact guidance diversity in rotationally aligned collagen matrices.

Unlabelled: Cancer cell metastasis is responsible for approximately 90% of deaths related to cancer. The migration of cancer cells away from the primary tumor and into healthy tissue is driven in part by contact guidance, or directed migration in response to aligned extracellular matrix. While contact guidance has been a focus of many studies, much of this research has explored environments that present 2D contact guidance structures.