Forced degradation of cell-based medicinal products guided by flow imaging microscopy: Explorative studies with Jurkat cells.

Cell-based medicinal products (CBMPs) offer ground-breaking opportunities to treat diseases with limited or no therapeutic options. However, the intrinsic complexity of CBMPs results in great challenges with respect to analytical characterization and stability assessment. In our study, we submitted Jurkat cell suspensions to forced degradation studies mimicking conditions to which CBMPs might be exposed from procurement of cells to administration of the product. Flow imaging microscopy assisted by machine learning was applied for determination of cell viability and concentration, and quantification of debris particles. Additionally, orthogonal cell characterization techniques were used. Thawing of cells at 5 °C was detrimental to cell viability and resulted in high numbers of debris particles, in contrast to thawing at 37 °C or 20 °C which resulted in better stability. After freezing of cell suspensions at -18 °C in presence of dimethyl sulfoxide (DMSO), a DMSO concentration of 2.5% (v/v) showed low stabilizing properties, whereas 5% or 10% was protective. Horizontal shaking of cell suspensions did not affect cell viability, but led to a reduction in cell concentration. Fetal bovine serum (10% [v/v]) protected the cells during shaking. In conclusion, forced degradation studies with application of orthogonal analytical characterization methods allow for CBMP stability assessment and formulation screening.

Particulate impurities in cell-based medicinal products traced by flow imaging microscopy combined with deep learning for image analysis.

Cell-based medicinal products (CBMPs) are rapidly gaining importance in the treatment of life-threatening diseases. However, the analytical toolbox for characterization of CBMPs is limited. The aim of our study was to develop a method based on flow imaging microscopy (FIM) for the detection, quantification and characterization of subvisible particulate impurities in CBMPs. Image analysis was performed by using an image classification approach based on a convolutional neural network (CNN). Jurkat cells and Dynabeads were used in our study as a representation of cellular material and non-cellular particulate impurities, respectively. We demonstrate that FIM assisted with CNN is a powerful method for the detection and quantification of Dynabeads and cells with other process related impurities, such as cell agglomerates, cell-bead adducts and debris. By using CNN, we achieved a more than 50-fold lower misclassification rate compared with the use of output parameters from the FIM software. The limit of detection was ~15 000 beads/mL in the presence of ~500 000 cells/mL, making this approach suitable for the detection of these particulate impurities in CBMPs. In conclusion, CNN-assisted FIM is a powerful method for the detection and quantification of cells, Dynabeads and other subvisible process impurities potentially present in CBMPs.

Correction of murine Rag1 deficiency by self-inactivating lentiviral vector-mediated gene transfer.

Severe combined immunodeficiency (SCID) patients with an inactivating mutation in recombination activation gene 1 (RAG1) lack B and T cells due to the inability to rearrange immunoglobulin (Ig) and T-cell receptor (TCR) genes. Gene therapy is a valid treatment option for RAG-SCID patients, especially for patients lacking a suitable bone marrow donor, but developing such therapy has proven challenging. As a preclinical model for RAG-SCID, we used Rag1-/- mice and lentiviral self-inactivating (SIN) vectors harboring different internal elements to deliver native or codon-optimized human RAG1 sequences. Treatment resulted in the appearance of B and T cells in peripheral blood and developing B and T cells were detected in central lymphoid organs. Serum Ig levels and Ig and TCR Vß gene segment usage was comparable to wild-type (WT) controls, indicating that RAG-mediated rearrangement took place. Remarkably, relatively low frequencies of B cells produced WT levels of serum immunoglobulins. Upon stimulation of the TCR, corrected spleen cells proliferated and produced cytokines. In vivo challenge resulted in production of antigen-specific antibodies. No leukemia development as consequence of insertional mutagenesis was observed. The functional reconstitution of the B- as well as the T-cell compartment provides proof-of-principle for therapeutic RAG1 gene transfer in Rag1-/- mice using lentiviral SIN vectors.

Correction of B-cell development in Btk-deficient mice using lentiviral vectors with codon-optimized human BTK.

X-linked agammaglobulinemia (XLA) is the most common primary immunodeficiency (PID) in man and caused by mutations in the Bruton's tyrosine kinase (BTK) gene. XLA is characterized by a B-cell differentiation arrest in bone marrow, absence of mature B cells and immunoglobulins (Igs), and recurrent bacterial infections. We used self-inactivating lentiviral vectors expressing codon-optimized human BTK under the control of three different ubiquitous or B cell-specific promoters. Btk-/- mice engrafted with transduced cells showed correction of both precursor B-cell and peripheral B-cell development. Lentiviral vectors containing the wildtype BTK sequence did not correct the phenotype. All treated mice with codon-optimized BTK exhibited the recovery of B1 cells in the peritoneal cavity, and of serum IgM and IgG3 levels. Calcium mobilization responses upon B-cell receptor stimulation as well as in vivo responses to T cell-independent antigens were restored. Viral promoters overexpressing BTK >100-fold above normal resulted in erythro-myeloid proliferations independent of insertional mutagenesis. However, transplantation into secondary Btk-/- recipients using cellular promoters resulted in functional restoration of peripheral B cells and IgM levels, without any adverse effects. In conclusion, transduction of human BTK corrects B-cell development and antigen-specific antibody responses in Btk-/- mice, thus indicating the feasibility of lentiviral gene therapy for XLA, provided that BTK expression does not vastly exceed normal levels.

Sola dosis facit venenum. Leukemia in gene therapy trials: a question of vectors, inserts and dosage?

In clinical gene therapy trials for X-linked severe combined immunodeficiency, the development of leukemia has come up as a severe adverse effect. In all five cases, T-cell acute lymphoblastic leukemia (T-ALL) occurred as a direct consequence of insertional mutagenesis by the retrovirus used to deliver the therapeutic gene. Here, we review the mechanisms of insertional mutagenesis, the function of the Il2RG gene and the future developments in the field. New lentiviral and gamma retroviral vectors can significantly improve the safety profile of the tools used but still carry the risk of insertional mutagenesis, as shown in this issue of Leukemia. Finally, the unfortunate side effects of gene therapy have given more insight into the development of human T-ALL.

Ectopic retroviral expression of LMO2, but not IL2Rgamma, blocks human T-cell development from CD34+ cells: implications for leukemogenesis in gene therapy.

The occurrence of leukemia in a gene therapy trial for SCID-X1 has highlighted insertional mutagenesis as an adverse effect. Although retroviral integration near the T-cell acute lymphoblastic leukemia (T-ALL) oncogene LIM-only protein 2 (LMO2) appears to be a common event, it is unclear why LMO2 was preferentially targeted. We show that of classical T-ALL oncogenes, LMO2 is most highly transcribed in CD34+ progenitor cells. Upon stimulation with growth factors typically used in gene therapy protocols transcription of LMO2, LYL1, TAL1 and TAN1 is most prominent. Therefore, these oncogenes may be susceptible to viral integration. The interleukin-2 receptor gamma chain (IL2Rgamma), which is mutated in SCID-X1, has been proposed as a cooperating oncogene to LMO2. However, we found that overexpressing IL2Rgamma had no effect on T-cell development. In contrast, retroviral overexpression of LMO2 in CD34+ cells caused severe abnormalities in T-cell development, but B-cell and myeloid development remained unaffected. Our data help explain why LMO2 was preferentially targeted over many of the other known T-ALL oncogenes. Furthermore, during T-cell development retrovirus-mediated expression of IL2Rgamma may not be directly oncogenic. Instead, restoration of normal IL7-receptor signaling may allow progression of T-cell development to stages where ectopic LMO2 expression causes aberrant thymocyte growth.

T-, B- and NK-lymphoid, but not myeloid cells arise from human CD34(+)CD38(-)CD7(+) common lymphoid progenitors expressing lymphoid-specific genes.

Hematopoietic stem cells in the bone marrow (BM) give rise to all blood cells. According to the classic model of hematopoiesis, the differentiation paths leading to the myeloid and lymphoid lineages segregate early. A candidate 'common lymphoid progenitor' (CLP) has been isolated from CD34(+)CD38(-) human cord blood cells based on CD7 expression. Here, we confirm the B- and NK-differentiation potential of CD34(+)CD38(-)CD7(+) cells and show in addition that this population has strong capacity to differentiate into T cells. As CD34(+)CD38(-)CD7(+) cells are virtually devoid of myeloid differentiation potential, these cells represent true CLPs. To unravel the molecular mechanisms underlying lymphoid commitment, we performed genome-wide gene expression profiling on sorted CD34(+)CD38(-)CD7(+) and CD34(+)CD38(-)CD7(-) cells. Interestingly, lymphoid-affiliated genes were mainly upregulated in the CD7(+) population, while myeloid-specific genes were downregulated. This supports the hypothesis that lineage commitment is accompanied by the shutdown of inappropriate gene expression and the upregulation of lineage-specific genes. In addition, we identified several highly expressed genes that have not been described in hematopoiesis before.