A novel non-viral delivery method that enables efficient engineering of primary human T cells for ex vivo cell therapy applications.

BACKGROUND AIMS: Next-generation immune cell therapy products will require complex modifications using engineering technologies that can maintain high levels of cell functionality. Non-viral engineering methods have the potential to address limitations associated with viral vectors. However, while electroporation is the most widely used non-viral modality, concerns about its effects on cell functionality have led to the exploration of alternative approaches. Here the authors have examined the suitability of the Solupore non-viral delivery system for engineering primary human T cells for cell therapy applications. METHODS: The Solupore system was used to deliver messenger RNA (mRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) guide RNA ribonucleoprotein (RNP) cargos to T cells, and efficiency was measured by flow cytometry. Cell perturbation was assessed by immune gene expression profiling, including an electroporation comparator. In vitro and in vivo cytotoxicity of chimeric antigen receptor (CAR) T cells generated using the Solupore system was evaluated using a real-time cellular impedance assay and a Raji-luciferase mouse tumor model, respectively. RESULTS: Efficient transfection was demonstrated through delivery of mRNA and CRISPR CAS9 RNP cargos individually, simultaneously and sequentially using the Solupore system while consistently maintaining high levels of cell viability. Gene expression profiling revealed minimal alteration in immune gene expression, demonstrating the low level of perturbation experienced by the cells during this transfection process. By contrast, electroporation resulted in substantial changes in immune gene expression in T cells. CAR T cells generated using the Solupore system exhibited efficient cytotoxicity against target cancer cells in vitro and in vivo. CONCLUSIONS: The Solupore system is a non-viral means of simply, rapidly and efficiently delivering cargos to primary human immune cells with retention of high cell viability and functionality.

Network mapping of primary CD34+ cells by Ampliseq based whole transcriptome targeted resequencing identifies unexplored differentiation regulatory relationships.

With the exception of a few master transcription factors, regulators of neutrophil maturation are poorly annotated in the intermediate phenotypes between the granulocyte-macrophage progenitor (GMP) and the mature neutrophil phenotype. Additional challenges in identifying gene expression regulators in differentiation pathways relate to challenges wherein starting cell populations are heterogeneous in lineage potential and development, are spread across various states of quiescence, as well as sample quality and input limitations. These factors contribute to data variability make it difficult to draw simple regulatory inferences. In response we have applied a multi-omics approach using primary blood progenitor cells primed for homogeneous proliferation and granulocyte differentiation states which combines whole transcriptome resequencing (Ampliseq RNA) supported by droplet digital PCR (ddPCR) validation and mass spectrometry-based proteomics in a hypothesis-generation study of neutrophil differentiation pathways. Primary CD34+ cells isolated from human cord blood were first precultured in non-lineage driving medium to achieve an active, proliferating phenotype from which a neutrophil primed progenitor was isolated and cultured in neutrophil lineage supportive medium. Samples were then taken at 24-hour intervals over 9 days and analysed by Ampliseq RNA and mass spectrometry. The Ampliseq dataset depth, breadth and quality allowed for several unexplored transcriptional regulators and ncRNAs to be identified using a combinatorial approach of hierarchical clustering, enriched transcription factor binding motifs, and network mapping. Network mapping in particular increased comprehension of neutrophil differentiation regulatory relationships by implicating ARNT, NHLH1, PLAG1, and 6 non-coding RNAs associated with PU.1 regulation as cell-engineering targets with the potential to increase total neutrophil culture output. Overall, this study develops and demonstrates an effective new hypothesis generation methodology for transcriptome profiling during differentiation, thereby enabling identification of novel gene targets for editing interventions.

Shedding light: The importance of reverse transcription efficiency standards in data interpretation.

The RNA-to-cDNA conversion step in transcriptomics experiments is widely recognised as inefficient and variable, casting doubt on the ability to do quantitative transcriptomics analyses. Multiple studies have focused on ways to optimise this process, resulting in contradictory recommendations. Here we explore the problem of reverse transcription efficiency using digital PCR and the RT method's impact on subsequent data analysis. Using synthetic RNA standards, an example experiment is presented, outlining a method to (1) determine relevant efficiency and variability values and then to (2) incorporate this information into downstream analyses as a way to improve the accuracy of quantitative transcriptomics experiments.

Filling the void: allogeneic myeloid cells for transplantation.

PURPOSE OF REVIEW: The success of allogeneic haematopoietic stem and progenitor cell (HSPC) transplantations remains inconsistent. Umbilical cord blood (UCB) is a promising source of HSPCs for transplantation, but low cell yield hampers its widespread use. Multiple strategies are being developed to manipulate UCB to either increase HSPC content or enhance bone marrow homing upon transfusion. RECENT FINDINGS: Several ex-vivo manipulation protocols have increased engraftment success in recent phase I/II clinical trials. Additionally, by exploiting knowledge of the transcriptome, mature cells were dedifferentiated into induced haematopoietic stem cells capable of self-renewal and reconstitution of haematopoiesis in vivo. SUMMARY: UCB is a more readily available source of allogeneic transplant material compared with bone marrow and mobilized peripheral blood. However, the number of HSPCs in a graft is correlated to the rate and success of engraftment and UCB grafts typically contain 10 times less cells compared with bone marrow or mobilized peripheral blood grafts that contain around 1 × 108 CD34⁺ cells. Recently, research efforts have focused on increasing UCB engrafting cells in addition to the methods to accelerate engraftment or to provide transient protection and support until engraftment succeeds.