Chimeric antigen receptor (CAR) T-cell therapy has produced remarkable anti-tumor responses in patients with B-cell malignancies. However, clonal kinetics and transcriptional programs that regulate ...the fate of CAR-T cells after infusion remain poorly understood. Here we perform TCRB sequencing, integration site analysis, and single-cell RNA sequencing (scRNA-seq) to profile CD8
CAR-T cells from infusion products (IPs) and blood of patients undergoing CD19 CAR-T immunotherapy. TCRB sequencing shows that clonal diversity of CAR-T cells is highest in the IPs and declines following infusion. We observe clones that display distinct patterns of clonal kinetics, making variable contributions to the CAR-T cell pool after infusion. Although integration site does not appear to be a key driver of clonal kinetics, scRNA-seq demonstrates that clones that expand after infusion mainly originate from infused clusters with higher expression of cytotoxicity and proliferation genes. Thus, we uncover transcriptional programs associated with CAR-T cell behavior after infusion.
Summary
Genome editing therapies represent a significant advancement in next‐generation, precision medicine for the management of haematological diseases, and CRISPR/Cas9 has to date been the most ...successful implementation platform. From discovery in bacteria and archaea over three decades ago, through intensive basic research and pre‐clinical development phases involving the modification of therapeutically relevant cell types, CRISPR/Cas9 genome editing is now being investigated in ongoing clinic trials. Despite the widespread enthusiasm brought by this new technology, significant challenges remain before genome editing can be routinely recommended and implemented in the clinic. These include risks of genotoxicity resulting from off‐target DNA cleavage or chromosomal rearrangement, and suboptimal efficacy of homology‐directed repair editing strategies, which thus limit therapeutic options. Practical hurdles such as high costs and inaccessibility to patients outside specialised centres must also be addressed. Future improvements in this rapidly developing field should circumvent current limitations with novel editing platforms and with the simplification of clinical protocols using in vivo delivery of editing reagents.
Extending Gene Medicines to All in Need McCune, Joseph M; Kiem, Hans-Peter
The New England journal of medicine,
05/2024, Letnik:
390, Številka:
18
Journal Article
Ex vivo CRISPR gene editing in haematopoietic stem and progenitor cells has opened potential treatment modalities for numerous diseases. The current process uses electroporation, sometimes followed ...by virus transduction. While this complex manipulation has resulted in high levels of gene editing at some genetic loci, cellular toxicity was observed. We have developed a CRISPR nanoformulation based on colloidal gold nanoparticles with a unique loading design capable of cellular entry without the need for electroporation or viruses. This highly monodispersed nanoformulation avoids lysosomal entrapment and localizes to the nucleus in primary human blood progenitors without toxicity. Nanoformulation-mediated gene editing is efficient and sustained with different CRISPR nucleases at multiple loci of therapeutic interest. The engraftment kinetics of nanoformulation-treated primary cells in humanized mice are better relative to those of non-treated cells, with no differences in differentiation. Here we demonstrate non-toxic delivery of the entire CRISPR payload into primary human blood progenitors.
Despite the success of antiretroviral therapy (ART) for people living with HIV, lifelong treatment is required and there is no cure. HIV can integrate in the host genome and persist for the life span ...of the infected cell. These latently infected cells are not recognized as foreign because they are largely transcriptionally silent, but contain replication-competent virus that drives resurgence of the infection once ART is stopped. With a combination of immune activators, neutralizing antibodies, and therapeutic vaccines, some nonhuman primate models have been cured, providing optimism for these approaches now being evaluated in human clinical trials. In vivo delivery of gene-editing tools to either target the virus, boost immunity or protect cells from infection, also holds promise for future HIV cure strategies. In this Review, we discuss advances related to HIV cure in the last 5 years, highlight remaining knowledge gaps and identify priority areas for research for the next 5 years.
The small number of hematopoietic stem and progenitor cells in cord blood units limits their widespread use in human transplant protocols. We identified a family of chemically related small molecules ...that stimulates the expansion ex vivo of human cord blood cells capable of reconstituting human hematopoiesis for at least 6 months in immunocompromised mice. The potent activity of these newly identified compounds, UM171 being the prototype, is independent of suppression of the aryl hydrocarbon receptor, which targets cells with more-limited regenerative potential. The properties of UM171 make it a potential candidate for hematopoietic stem cell transplantation and gene therapy.
Bringing gene therapy to where it’s needed Radtke, Stefan; Kiem, Hans-Peter
Trends in molecular medicine,
March 2022, 2022-03-00, 20220301, Letnik:
28, Številka:
3
Journal Article
Recenzirano
Gene editing allows the precise modification of cells to correct genetic defects or enhance immunotherapies. A limitation is the delivery of this technology to specific cells or organs. Recently, ...Banskota et al. reported the use of virus-like particles (VLPs) loaded with gene-editing agents for gene therapy delivery directly inside the body.
The preclinical development of hematopoietic stem cell (HSC) gene therapy/editing and transplantation protocols is frequently performed in large animal models such as nonhuman primates (NHPs). ...Similarity in physiology, size, and life expectation as well as cross-reactivity of most reagents and medications allows for the development of treatment strategies with rapid translation to clinical applications. Especially after the adverse events of HSC gene therapy observed in the late 1990s, the ability to perform autologous transplants and follow the animals long-term make the NHP a very attractive model to test the efficiency, feasibility, and safety of new HSC-mediated gene-transfer/editing and transplantation approaches.This protocol describes a method to phenotypically characterize functionally distinct NHP HSPC subsets within specimens or stem cell products from three different NHP species. Procedures are based on the flow-cytometric assessment of cell surface markers that are cross-reactive in between human and NHP to allow for immediate clinical translation. This protocol has been successfully used for the quality control of enriched, cultured, and gene-modified NHP CD34
hematopoietic stem and progenitor cells (HSPCs) as well as sort-purified CD34 subsets for transplantation in the pig-tailed, cynomolgus, and rhesus macaque. It further allows the longitudinal assessment of primary specimens taken during the long-term follow-up post-transplantation in order to monitor homing, engraftment, and reconstitution of the bone marrow stem cell compartment.
There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein ...binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection.
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•Meta-analysis of human, non-human primate, and mouse single-cell RNA-seq datasets for putative SARS-CoV-2 targets•Type II pneumocytes, nasal secretory cells, and absorptive enterocytes are ACE2+TMPRSS2+•Interferon and influenza increase ACE2 in human nasal epithelia and lung tissue•Mouse Ace2 is not upregulated by interferon, raising implications for disease modeling
Analysis of single-cell RNA-seq datasets from human, non-human primate, and mouse barrier tissues identifies putative cellular targets of SARS-CoV-2 on the basis of ACE2 and TMPRSS2 expression. ACE2 represents a previously unappreciated interferon-stimulated gene in human, but not mouse, epithelial tissues, identifying anti-viral induction of a host tissue-protective mechanism, but also a potential means for viral exploitation of the host response.
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