Lipid nanoparticles outperform electroporation in mRNA-based CAR T cell engineering.

Engineered T cells expressing chimeric antigen receptors (CARs) have been proven as efficacious therapies against selected hematological malignancies. However, the approved CAR T cell therapeutics strictly rely on viral transduction, a time- and cost-intensive procedure with possible safety issues. Therefore, the direct transfer of in vitro transcribed CAR-mRNA into T cells is pursued as a promising strategy for CAR T cell engineering. Electroporation (EP) is currently used as mRNA delivery method for the generation of CAR T cells in clinical trials but achieving only poor anti-tumor responses. Here, lipid nanoparticles (LNPs) were examined for ex vivo CAR-mRNA delivery and compared with EP. LNP-CAR T cells showed a significantly prolonged efficacy in vitro in comparison with EP-CAR T cells as a result of extended CAR-mRNA persistence and CAR expression, attributed to a different delivery mechanism with less cytotoxicity and slower CAR T cell proliferation. Moreover, CAR expression and in vitro functionality of mRNA-LNP-derived CAR T cells were comparable to stably transduced CAR T cells but were less exhausted. These results show that LNPs outperform EP and underline the great potential of mRNA-LNP delivery for ex vivo CAR T cell modification as next-generation transient approach for clinical studies.

Comparison of two lab-scale protocols for enhanced mRNA-based CAR-T cell generation and functionality.

Process development for transferring lab-scale research workflows to automated manufacturing procedures is critical for chimeric antigen receptor (CAR)-T cell therapies. Therefore, the key factor for cell viability, expansion, modification, and functionality is the optimal combination of medium and T cell activator as well as their regulatory compliance for later manufacturing under Good Manufacturing Practice (GMP). In this study, we compared two protocols for CAR-mRNA-modified T cell generation using our current lab-scale process, analyzed all mentioned parameters, and evaluated the protocols' potential for upscaling and process development of mRNA-based CAR-T cell therapies.

Humanized mouse model: Hematopoietic stemcell transplantation and tracking using short tandem repeat technology.

INTRODUCTION: Models of mice carrying a human immune system, so-called humanized mice, are used increasingly as preclinical models to bridge the gap between model organisms and human beings. Challenges of the humanized mouse model include finding suitable sources for human hematopoietic stem cells (HSC) and reaching sufficient engraftment of these cells in immunocompromised mice. METHODS: In this study, we compared the use of CD34+ HSC from cord blood (CB) vs HSC from adult mobilized peripheral blood. Furthermore, we developed a simple and highly specific test for donor identification in humanized mice by applying the detection method of short tandem repeats (STR). RESULTS: It was found that, in vitro, CB-derived and adult HSC show comparable purity, viability, and differentiation potential in colony-forming unit assays. However, in vivo, CB-derived HSC engrafted to a significantly higher extent in NOD.Cg-Prkdcscid IL2rγtm1Wjl /SzJ (NSG) mice than adult HSC. Increasing the cell dose of adult HSC or using fresh cells without cryopreservation did not improve the engraftment rate. Interestingly, when using adult HSC, the percentage of human cells in the bone marrow was significantly higher than that in the peripheral blood. Using the STR-based test, we were able to identify and distinguish human cells from different donors in humanized mice and in a humanized allogeneic transplantation model. CONCLUSION: From these findings, we conclude that adult mobilized HSC are less suitable for generating a humanized immune system in mice than CB-derived cells.

Dynamics of the DEAD-box ATPase Prp5 RecA-like domains provide a conformational switch during spliceosome assembly.

The DEAD-box family of proteins are ATP-dependent, RNA-binding proteins implicated in many aspects of RNA metabolism. Pre-mRNA splicing in eukaryotes requires three DEAD-box ATPases (Prp5, Prp28 and Sub2), the molecular mechanisms of which are poorly understood. Here, we use single molecule FRET (smFRET) to study the conformational dynamics of yeast Prp5. Prp5 is essential for stable association of the U2 snRNP with the intron branch site (BS) sequence during spliceosome assembly. Our data show that the Prp5 RecA-like domains undergo a large conformational rearrangement only in response to binding of both ATP and RNA. Mutations in Prp5 impact the fidelity of BS recognition and change the conformational dynamics of the RecA-like domains. We propose that BS recognition during spliceosome assembly involves a set of coordinated conformational switches among U2 snRNP components. Spontaneous toggling of Prp5 into a stable, open conformation may be important for its release from U2 and to prevent competition between Prp5 re-binding and subsequent steps in spliceosome assembly.

Conformational dynamics of stem II of the U2 snRNA.

The spliceosome undergoes dramatic changes in both small nuclear RNA (snRNA) composition and structure during assembly and pre-mRNA splicing. It has been previously proposed that the U2 snRNA adopts two conformations within the stem II region: stem IIa or stem IIc. Dynamic rearrangement of stem IIa into IIc and vice versa is necessary for proper progression of the spliceosome through assembly and catalysis. How this conformational transition is regulated is unclear; although, proteins such as Cus2p and the helicase Prp5p have been implicated in this process. We have used single-molecule Förster resonance energy transfer (smFRET) to study U2 stem II toggling between stem IIa and IIc. Structural interconversion of the RNA was spontaneous and did not require the presence of a helicase; however, both Mg(2+) and Cus2p promote formation of stem IIa. Destabilization of stem IIa by a G53A mutation in the RNA promotes stem IIc formation and inhibits conformational switching of the RNA by both Mg(2+) and Cus2p. Transitioning to stem IIa can be restored using Cus2p mutations that suppress G53A phenotypes in vivo. We propose that during spliceosome assembly, Cus2p and Mg(2+) may work together to promote stem IIa formation. During catalysis the spliceosome could then toggle stem II with the aid of Mg(2+) or with the use of functionally equivalent protein interactions. As noted in previous studies, the Mg(2+) toggling we observe parallels previous observations of U2/U6 and Prp8p RNase H domain Mg(2+)-dependent conformational changes. Together these data suggest that multiple components of the spliceosome may have evolved to switch between conformations corresponding to open or closed active sites with the aid of metal and protein cofactors.