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Kidney organoids are an innovative tool in transplantation research. The aim of the present study was to investigate whether kidney organoids are susceptible for allo-immune attack and whether they can be used as a model to study allo-immunity in kidney transplantation. Human induced pluripotent stem cell-derived kidney organoids were co-cultured with human peripheral blood mononuclear cells (PBMC), which resulted in invasion of allogeneic T-cells around nephron structures and macrophages in the stromal cell compartment of the organoids. This process was associated with the induction of fibrosis. Subcutaneous implantation of kidney organoids in immune-deficient mice followed by adoptive transfer of human PBMC led to the invasion of diverse T-cell subsets. Single cell transcriptomic analysis revealed that stromal cells in the organoids upregulated expression of immune response genes upon immune cell invasion. Moreover, immune regulatory PD-L1 protein was elevated in epithelial cells while genes related to nephron differentiation and function were downregulated. This study characterized the interaction between immune cells and kidney organoids, which will advance the use of kidney organoids for transplantation research.

The use of genetic engineering to generate point mutations in induced pluripotent stem cells (iPSCs) is essential for studying a specific genetic effect in an isogenic background. We demonstrate that a combination of p53 inhibition and pro-survival small molecules achieves a homologous recombination rate higher than 90% using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in human iPSCs. Our protocol reduces the effort and time required to create isogenic lines.

CRISPR-Cas9 genome editing often induces unintended, large genomic rearrangements, posing potential safety risks. However, there are no methods for mitigating these risks. Using long-read individual-molecule sequencing (IDMseq), we found the microhomology-mediated end joining (MMEJ) DNA repair pathway plays a predominant role in Cas9-induced large deletions (LDs). We targeted MMEJ-associated genes genetically and/or pharmacologically and analyzed Cas9-induced LDs at multiple gene loci using flow cytometry and long-read sequencing. Reducing POLQ levels or activity significantly decreases LDs, while depleting or overexpressing RPA increases or reduces LD frequency, respectively. Interestingly, small-molecule inhibition of POLQ and delivery of recombinant RPA proteins also dramatically promote homology-directed repair (HDR) at multiple disease-relevant gene loci in human pluripotent stem cells and hematopoietic progenitor cells. Our findings reveal the contrasting roles of RPA and POLQ in Cas9-induced LD and HDR, suggesting new strategies for safer and more precise genome editing. The online version contains supplementary material available at 10.1186/s12915-024-01896-z.

Allogeneic cell therapies, such as those involving macrophages or Natural Killer (NK) cells, are of increasing interest for cancer immunotherapy. However, the current techniques for genetically modifying these cell types using lenti- or gamma-retroviral vectors present challenges, such as required cell pre-activation and inefficiency in transduction, which hinder the assessment of preclinical efficacy and clinical translation. In our study, we describe a novel lentiviral pseudotype based on the Koala Retrovirus (KoRV) envelope protein, which we identified based on homology to existing pseudotypes used in cell therapy. Unlike other pseudotyped viral vectors, this KoRV-based envelope demonstrates remarkable efficiency in transducing freshly isolated primary human NK cells directly from blood, as well as freshly obtained monocytes, which were differentiated to M1 macrophages as well as B cells from multiple donors, achieving up to 80% reporter gene expression within three days post-transduction. Importantly, KoRV-based transduction does not compromise the expression of crucial immune cell receptors, nor does it impair immune cell functionality, including NK cell viability, proliferation, cytotoxicity as well as phagocytosis of differentiated macrophages. Preserving immune cell functionality is pivotal for the success of cell-based therapeutics in treating various malignancies. By achieving high transduction rates of freshly isolated immune cells before expansion, our approach enables a streamlined and cost-effective automated production of off-the-shelf cell therapeutics, requiring fewer viral particles and less manufacturing steps. This breakthrough holds the potential to significantly reduce the time and resources required for producing e.g. NK cell therapeutics, expediting their availability to patients in need. Subject terms: Genetic transduction, Tumour immunology, Immunotherapy, Genetic vectors, Innate immune cells

In preclinical studies on the T-cell engager MP0533, the authors show that targeting multiple tumor-associated antigens may lead to better selectivity and efficacy in eliminating leukemic stem cells and blasts, representing a promising therapeutic strategy for AML.

Succinic Semialdehyde Dehydrogenase Deficiency (SSADHD) is an ultra-rare autosomal recessive neurometabolic disorder caused by ALDH5A1 mutations presenting with autism and epilepsy. Here, we report the generation and characterization of human induced pluripotent stem cells (hiPSCs) derived from fibroblasts of three unrelated SSADHD patients – one female and two males with the CRISPR-corrected isogenic controls. These individuals are clinically diagnosed and are being followed in a longitudinal clinical study.