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RAG2-SCID is a primary immunodeficiency caused by mutations in Recombination-activating gene 2 (RAG2), a gene intimately involved in the process of lymphocyte maturation and function. ex-vivo manipulation of a patient’s own hematopoietic stem and progenitor cells (HSPCs) using CRISPR-Cas9/rAAV6 gene editing could provide a therapeutic alternative to the only current treatment, allogeneic hematopoietic stem cell transplantation (HSCT). Here we show an innovative RAG2 correction strategy that replaces the entire endogenous coding sequence (CDS) for the purpose of preserving the critical endogenous spatiotemporal gene regulation and locus architecture. Expression of the corrective transgene leads to successful development into CD3+TCRαβ+ and CD3+TCRγδ+ T cells and promotes the establishment of highly diverse TRB and TRG repertoires in an in-vitro T-cell differentiation platform. Thus, our proof-of-concept study holds promise for safer gene therapy techniques of tightly regulated genes. RAG2-SCID is a primary immunodeficiency caused by mutations in Recombination-activating gene 2 (RAG2). Here the authors report a RAG2 correction strategy that replaces the entire endogenous coding sequence (CDS) to maintain the endogenous spatiotemporal gene regulation and locus architecture.
Rochester Institute of Technology researchers are improving non-invasive treatment options for degenerative disc disease, an ailment that impacts 3 million adults yearly in the U.S., according to the Mayo Clinic.
Scientists at Leipzig University, in collaboration with colleagues at Vilnius University in Lithuania, have developed a new method to measure the smallest twists and torques of molecules within milliseconds.
Vaccine boosting modifies CAR T cell metabolism and promotes crosstalk between CAR
T cells and endogenous immunity to elicit and sustain antigen spreading, thereby effectively
treating tumors with antigen heterogeneity.
Molecular routes to metastatic dissemination are critical determinants of aggressive cancers. Through in vivo CRISPR–Cas9 genome editing, we generated somatic mosaic genetically engineered models that faithfully recapitulate metastatic renal tumors. Disruption of 9p21 locus is an evolutionary driver to systemic disease through the rapid acquisition of complex karyotypes in cancer cells. Cross-species analysis revealed that recurrent patterns of copy number variations, including 21q loss and dysregulation of the interferon pathway, are major drivers of metastatic potential. In vitro and in vivo genomic engineering, leveraging loss-of-function studies, along with a model of partial trisomy of chromosome 21q, demonstrated a dosage-dependent effect of the interferon receptor genes cluster as an adaptive mechanism to deleterious chromosomal instability in metastatic progression. This work provides critical knowledge on drivers of renal cell carcinoma progression and defines the primary role of interferon signaling in constraining the propagation of aneuploid clones in cancer evolution. Using genetically engineered models, Genovese and colleagues study patterns of convergent evolution in renal cancer, and pinpoint dysregulation of interferon signaling as a means of adaptation to chromosomal instability in metastatic progression.
Three young patients with relapsed T-cell leukemia have now been treated with base-edited T-cells, as part of a 'bench-to-bedside' collaboration between UCL and Great Ormond Street Hospital for Children (GOSH).
The most common form of genetic heart disease is hypertrophic cardiomyopathy (HCM), which is caused by variants in cardiac sarcomeric genes and leads to abnormal heart muscle thickening. Complications of HCM include heart failure, arrhythmia and sudden cardiac death. The dominant-negative c.1208G>A (p.R403Q) pathogenic variant (PV) in β-myosin (MYH7) is a common and well-studied PV that leads to increased cardiac contractility and HCM onset. In this study we identify an adenine base editor and single-guide RNA system that can efficiently correct this human PV with minimal bystander editing and off-target editing at selected sites. We show that delivery of base editing components rescues pathological manifestations of HCM in induced pluripotent stem cell cardiomyocytes derived from patients with HCM and in a humanized mouse model of HCM. Our findings demonstrate the potential of base editing to treat inherited cardiac diseases and prompt the further development of adenine base editor-based therapies to correct monogenic variants causing cardiac disease. Adenine base editing successfully corrected a MYH7 pathogenic variant that causes hypertrophic cardiomyopathy in human cardiomyocytes and a mouse model of the disease, highlighting the potential of the approach to correct monogenic variants causing cardiac disease.
Chimeric antigen receptors (CARs) and synthetic Notch (synNotch) receptors are engineered cell-surface receptors that sense a target antigen and respond by activating T cell receptor signaling or a customized gene program, respectively. Here, to expand the targeting capabilities of these receptors, we develop “universal” receptor systems for which receptor specificity can be directed post-translationally via covalent attachment of a co-administered antibody bearing a benzylguanine (BG) motif. A SNAPtag self-labeling enzyme is genetically fused to the receptor and reacts with BG-conjugated antibodies for covalent assembly, programming antigen recognition. We demonstrate that activation of SNAP-CAR and SNAP-synNotch receptors can be successfully targeted by clinically relevant BG-conjugated antibodies, including anti-tumor activity of SNAP-CAR T cells in vivo in a human tumor xenograft mouse model. Finally, we develop a mathematical model to better define the parameters affecting universal receptor signaling. SNAP receptors provide a powerful strategy to post-translationally reprogram the targeting specificity of engineered cells. Chimeric antigen receptors (CARs) and synthetic Notch (synNotch) receptors are promising platforms for cell-based immunotherapies. Here, the authors develop highly programmable versions of these receptors that can be universally targeted to antigens of interest through covalent enzyme chemistry.
Adoptive transfer of genetically engineered chimeric antigen receptor (CAR) T cells is becoming a promising treatment option for hematological malignancies. However, T cell immunotherapies have mostly failed in individuals with solid tumors. Here, with a CRISPR–Cas9 pooled library, we performed an in vivo targeted loss-of-function screen and identified ST3 β-galactoside α-2,3-sialyltransferase 1 (ST3GAL1) as a negative regulator of the cancer-specific migration of CAR T cells. Analysis of glycosylated proteins revealed that CD18 is a major effector of ST3GAL1 in activated CD8+ T cells. ST3GAL1-mediated glycosylation induces the spontaneous nonspecific tissue sequestration of T cells by altering lymphocyte function-associated antigen-1 (LFA-1) endocytic recycling. Engineered CAR T cells with enhanced expression of βII-spectrin, a central LFA-1-associated cytoskeleton molecule, reversed ST3GAL1-mediated nonspecific T cell migration and reduced tumor growth in mice by improving tumor-specific homing of CAR T cells. These findings identify the ST3GAL1–βII-spectrin axis as a major cell-intrinsic program for cancer-targeting CAR T cell migration and as a promising strategy for effective T cell immunotherapy. CAR T cell success requires targeting tumors, but these cells can get trapped in other tissues, such as in the lungs, where they can cause pathology. Here, the authors use a loss-of-function CRISPR screen to identify regulators of CAR T cell tumor trafficking and engineer CAR T cells accordingly to overcome this limitation.
The expanding applications of nonviral genomic medicines in the lung remain restricted by delivery challenges. Here, leveraging a high-throughput platform, we synthesize and screen a combinatorial library of biodegradable ionizable lipids to build inhalable delivery vehicles for messenger RNA and CRISPR–Cas9 gene editors. Lead lipid nanoparticles are amenable for repeated intratracheal dosing and could achieve efficient gene editing in lung epithelium, providing avenues for gene therapy of congenital lung diseases. A high-throughput screen improves lipid nanoparticle delivery of gene editors in the lung.
Researchers from Utah State University and Germany’s Helmholtz Institute for RNA-based Infection Research demonstrate that upon recognition of an RNA target, Cas12a2 cleaves all the other nucleic acids present, destroying the bacterial cell and preventing bacteriophage from replicating.
The genetic mutation underlying a severe immune system deficiency can be corrected
by adenine base editing of human patient stem cells, restoring the ability of these
cells to develop into functional T cells in model systems, and paving the path for
future treatment of CD3δ SCID in patients.
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The gene editing technique CRISPR/Cas9 has allowed researchers to make precise and impactful changes to an organism's DNA to fix mutations that cause genetic disease.
A broad new strategy could hold hope for treating virtually all blood cancers with CAR T cell therapy, which is currently approved for five subtypes of blood cancer.
In the context of relapsed and refractory childhood pre-B cell acute lymphoblastic leukemia (R/R B-ALL), CD19-targeting chimeric antigen receptor (CAR)-T cells often induce durable remissions, which requires the persistence of CAR-T cells. In this study, we systematically analyzed CD19 CAR-T cells of 10 children with R/R B-ALL enrolled in the CARPALL trial via high-throughput single-cell gene expression and T cell receptor sequencing of infusion products and serial blood and bone marrow samples up to 5 years after infusion. We show that long-lived CAR-T cells developed a CD4/CD8 double-negative phenotype with an exhausted-like memory state and distinct transcriptional signature. This persistence signature was dominant among circulating CAR-T cells in all children with a long-lived treatment response for which sequencing data were sufficient (4/4, 100%). The signature was also present across T cell subsets and clonotypes, indicating that persisting CAR-T cells converge transcriptionally. This persistence signature was also detected in two adult patients with chronic lymphocytic leukemia with decade-long remissions who received a different CD19 CAR-T cell product. Examination of single T cell transcriptomes from a wide range of healthy and diseased tissues across children and adults indicated that the persistence signature may be specific to long-lived CAR-T cells. These findings raise the possibility that a universal transcriptional signature of clinically effective, persistent CD19 CAR-T cells exists. In children with relapsed or refractory B cell acute lymphoblastic leukemia and in complete remission after CD19 CAR-T cell therapy, long-lived CAR-T cells express a persistence gene signature that is also present in persistent CD19 CAR-T cells from adults with chronic lymphocytic leukemia.
Induced pluripotent stem (iPS) cells have a great impact on biology and medicine, and they are expected to improve regenerative medicine.
Gene therapy that alters hemoglobin genes may be an answer to curing sickle cell disease (SCD) and beta thalassemia. These two common life-threatening anemias afflict millions of individuals across the globe. Scientists at St. Jude Children's Research Hospital and the Broad Institute of MIT and Harvard used a next-generation genome editing technology, adenosine base editing, to restart fetal hemoglobin expression in SCD patient cells. The approach raised the expression of fetal hemoglobin to higher, more stable, and more uniform levels than other genome editing technologies that use CRISPR/Cas9 nuclease in human hematopoietic stem cells. The findings were published in Nature Genetics.
Vα24-invariant natural killer T cells (NKTs) have anti-tumor properties that can be enhanced by chimeric antigen receptors (CARs). Here we report updated interim results from the first-in-human phase 1 evaluation of autologous NKTs co-expressing a GD2-specific CAR with interleukin 15 (IL15) (GD2-CAR.15) in 12 children with neuroblastoma (NB). The primary objectives were safety and determination of maximum tolerated dose (MTD). The anti-tumor activity of GD2-CAR.15 NKTs was assessed as a secondary objective. Immune response evaluation was an additional objective. No dose-limiting toxicities occurred; one patient experienced grade 2 cytokine release syndrome that was resolved by tocilizumab. The MTD was not reached. The objective response rate was 25% (3/12), including two partial responses and one complete response. The frequency of CD62L+NKTs in products correlated with CAR-NKT expansion in patients and was higher in responders (n = 5; objective response or stable disease with reduction in tumor burden) than non-responders (n = 7). BTG1 (BTG anti-proliferation factor 1) expression was upregulated in peripheral GD2-CAR.15 NKTs and is a key driver of hyporesponsiveness in exhausted NKT and T cells. GD2-CAR.15 NKTs with BTG1 knockdown eliminated metastatic NB in a mouse model. We conclude that GD2-CAR.15 NKTs are safe and can mediate objective responses in patients with NB. Additionally, their anti-tumor activity may be enhanced by targeting BTG1. ClinicalTrials.gov registration: NCT03294954 . In updated results from a phase 1 trial of GD2-specific CAR-NKT cells in patients with neuroblastoma, no dose-limiting toxicities were observed across multiple dose levels; the maximum tolerated dose was not reached; and there was evidence of anti-tumor activity.
Antibiotic treatments have detrimental effects on the microbiome and lead to antibiotic resistance. To develop a phage therapy against a diverse range of clinically relevant Escherichia coli, we screened a library of 162 wild-type (WT) phages, identifying eight phages with broad coverage of E. coli, complementary binding to bacterial surface receptors, and the capability to stably carry inserted cargo. Selected phages were engineered with tail fibers and CRISPR–Cas machinery to specifically target E. coli. We show that engineered phages target bacteria in biofilms, reduce the emergence of phage-tolerant E. coli and out-compete their ancestral WT phages in coculture experiments. A combination of the four most complementary bacteriophages, called SNIPR001, is well tolerated in both mouse models and minipigs and reduces E. coli load in the mouse gut better than its constituent components separately. SNIPR001 is in clinical development to selectively kill E. coli, which may cause fatal infections in hematological cancer patients. Phage engineered with tail fibers and CRISPR–Cas reduce Escherichia coli load in animals.
CRISPR-Cas9 is widely used to edit the genome by studying genes of interest and modifying disease-associated genes.
Decades of oncologic clinical use have demonstrated that cancer immunotherapy provides
unprecedented therapeutic benefits. Tragically, only a minority of patients respond
to existing immunotherapies. RNA lipid nanoparticles have recently emerged as modular
tools for immune stimulation. Here, we discuss advancements in RNA-based cancer immunotherapies
and opportunities for improvement.
Chimeric antigen receptor T-cell (CAR-T) therapy has transformed cancer treatment, yet relatively few studies have investigated the impact of the therapy on longitudinal patient quality of life – an aspect of care that often suffers from receiving traditional intensive cancer medications, such as chemotherapy.
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Severe combined immunodeficiency represents rare monogenic disorders with defects in humoral and cellular immunity. RAG genes code for proteins that initiate lymphoid-specific V(D)J recombination, essential for T and B lymphocyte maturation. As a result, individuals carrying pathogenic RAG variants are either completely devoid of, or possess far fewer, B and T cells. In a recent study, researchers described a CRISPR-Cas9-based genome-editing approach to replace the entire respective RAG2 coding sequences (CDS) with a corrective transgene in a 34-positive (CD34+) hematopoietic stem and progenitor cell (HSPC) differentiation cluster. Two recombinant adeno-associated virus serotype 6 (rAAV6) vectors were synthesized to integrate a green fluorescent protein expression cassette and a bovine growth hormone polyA sequence into CD34+ HSPCs after delivery of a single modified RAG2 guide RNA or the Cas9 ribonucleoprotein complex. The researchers' strategy completely replaced RAG2 CDS to treat autosomal recessive immunity disorders. The method induced expression of RAG2 coding DNA without exceeding endogenous RAG2 expression levels. Correction donors promoted the successful recombination and differentiation of V(D)J into CD3 + TCRγδ + and CD3 + TCRαβ + T cells.