Gene Editing Technology Research Articles

The contribution of a novel PHEX gene mutation to X-linked hypophosphatemic rickets: a case report and an analysis of the gene mutation dosage effect in a rat model.

A Chinese family was identified to have two patients with rickets, an adult female and a male child (proband), both exhibiting signs related to X-linked hypophosphatemic rickets (XLH). Gene sequencing analysis revealed a deletion of adenine at position 1985 (c.1985delA) in the PHEX-encoding gene. To investigate the relationship between this mutation and the pathogenicity of XLH, as well as analyze the effects of different dosages of PHEX gene mutations on clinical phenotypes, we developed a rat model carrying the PHEX deletion mutation. The CRISPR/Cas9 gene editing technology was employed to construct the rat model with the PHEX gene mutation (c.1985delA). Through reproductive procedures, five genotypes of rats were obtained: female wild type (X/X), female heterozygous (-/X), female homozygous wild type (-/-), male wild type (X/Y), and male hemizygous (-/Y). The rats with different genotypes underwent analysis of growth, serum biochemical parameters, and bone microstructure. The results demonstrated the successful generation of a stable rat model inheriting the PHEX gene mutation. Compared to the wild-type rats, the mutant rats displayed delayed growth, shorter femurs, and significantly reduced bone mass. Among the female rats, the homozygous individuals exhibited the smallest body size, decreased bone mass, shortest femur length, and severe deformities. Moreover, the mutant rats showed significantly lower blood phosphorus concentration, elevated levels of FGF23 and alkaline phosphatase, and increased expression of phosphorus regulators. In conclusion, the XLH rat model with the PHEX gene mutation dosage demonstrated its impact on growth and development, serum biochemical parameters, and femoral morphology.

CRISPR/Cas9 in the treatment of -thalassemia

-thalassemia is a congenital hemolytic anemia, a class of hemoglobinopathies caused by a common single-gene recessive condition in which the synthesis of the chain is either totally or partially blocked. About 50% of patients have symptoms within the first three months of life. At birth, the disease is often asymptomatic, and it mostly gets worse during infancy. If left untreated, the disease usually claims its victims by the time they are five years old since its severity rises with age. Currently, there is no cure for this disease, and the main treatment is blood transfusion. With the continuous progress of medical level, gene editing technology is developing rapidly, among which CRISPR/Cas system as a gene editing technology has been applied in the research of single gene genetic disease treatment, and CRISPR/Cas9 has been widely used in the research of treating geopathic anemia because of its high efficiency, low cost, and high specificity, for example, repairing pluripotent stem cells HBB genes, inducing fetal hemoglobin expression and inhibition of HBA gene expression. This review methodically explains the development of CRISPR/Cas9 through its creation and mode of operation, various research hypotheses tested in animal studies and clinical trials, as well as analyses of safety concerns.

Exploration of the application of gene editing technology in the treatment of colorectal cancer, taking CRISPR technology as an example for editing FAT4 genes

Colorectal cancer, the fourth most common cancer in the world, is the most common digestive tract tumor. At present, conservative treatment is still the main treatment method for colorectal cancer in clinical practice. Among the pathogenic factors of colorectal cancer, genetic factors need to be emphasized. Currently, many studies have focused on pathological studies of related gene mutations, mostly focusing on genes such as p53 and APC, and their molecular biological reactions have been thoroughly studied. However, research on other pathogenic genes is still lacking, and there is limited research on specific treatment methods. With the birth of CRISPR-Cas9 technology, applying of gene editing technology in the field of cancer has made breakthrough progress. This article will consider combining gene editing technology with related gene mutations in colorectal cancer, attempting to explore the possibility of applying gene editing technology in the field of cancer treatment, proposing feasibility for new treatment plans for colorectal cancer, and also providing new treatment ideas for future cancer treatment research.

Construction of tumour disease models based on CRISPR/Cas and iPSC technology

Cancer, which is defined as malignant tumours and neoplasms, is one of the most concerned diseases in the world. According to the WHO website, almost 10 million of people lost their lives due to cancer in 2020. The complexity of tumorigenesis and cancer development, which have not been explored enough, lead to its high morbidity and mortality. Therefore, it is in an urgent need to establish stable and reliable tumour disease models to study the complicated pathogenesis, hence developing novel therapies and drugs. As the third generation of gene-editing technology, CRISPR/Cas system has the advantages of high suitability to all types of model forms owing to the simplicity and convenience of operation. It can efficiently construct tumour disease models for verifying and screening related targets. iPSC, as a stem cell technology, can provide a new cell source for CRISPR/Cas system-based tumour disease modelling. It can be the basic of establishing disease models in specific areas like brain tumours. This review introduced the advantages of CRISPR/Cas gene editing and iPSC technology in establishing disease models and the combined application strategies of the two technologies through the cases of tumour disease modelling in the past five years. It also explored the modelling strategies and model categories adopted in the popular cancer field.

Targeted therapies for non-small cell lung cancer based on CRISPR-Cas9 technology

Lung cancer is the leading cause of cancer death worldwide, with non-small cell lung cancer (NSCLC) being the largest subgroup and having very low overall survival rates. This paper will briefly describe the pathogenic mechanism of NSCLC and analyze the pros and cons of several treatments that have been put into the clinic as well as the prospects and potential risks of new gene therapy approaches. Based on these approaches, this article will provide researchable directions for the treatment of NSCLC using CRISPR-Cas9 gene editing technology. The findings of the paper show that targeted drugs rely on the signaling pathway that acts on EGFR to treat cancer, with fewer but still present side effects than conventional therapies. Immunotherapy, which uses a patients own immune recognition to destroy cancer cells, is subject to problems such as an overactive immune response. CRISPR-Cas9 gene editing technology, which starts with the mutated gene that causes cancer and is intended to eradicate the cancer at its source, is underdeveloped, has no proven clinical protocols and carries the risk of incorrect editing. Nowadays, as cancer strikes globally, research on cancer treatment is of great significance in saving lives, ending cancer, individualizing treatment, promoting scientific progress and fostering socio-economic development.

Development and characterization of a robust in vitro disease cell line to study tauopathies

AbstractBackgroundTauopathies, such as frontotemporal dementia (FTD) and Alzheimer’s dimentia (AD), are neurodegenerative diseases characterized by the pathological aggregation of paired helical filaments (PHFs) or neurofibrillary tangles (NFTs) within neurons and glia, leading to cell death1. PHFs and NFTs are formed by aggregation of hyperphosphorylated tau1. Mutations in the microtubule‐associated protein tau (MAPT) gene result in tauopathies. Moreover, FTD is a common clinical syndrome of 4 repeat (4R) tauopathies, as defined by aggregation of tau protein isoforms with four microtubule binding domains2. Here, we aimed to develop and characterize a physiologically relevant and robust in vitro FTD model, to aid the future development of FTD disease therapeutics.MethodUsing CRISPR‐Cas9 gene editing technology, familial mutations MAPT P301S and N279K underlying AD/FTD3 were engineered into an iPSC line that carries the opti‐oxTM technology and can rapidly be reprogrammed into glutamatergic neurons4. By means of immunocytochemistry we characterized b‐amyloid oligomer treated neurons, and neurons with the MAPT mutations to assess tau hyperphosphorylation.ResultThe CRISPR‐edited MAPT neurons can mature and show a healthy morphology. b‐amyloid oligomers (dose dependently) induce tau hyperphosphorylation in wild type glutamatergic neurons. Total tau expression is reduced in MAPT P301S homozygous and N279K heterozygous cells when compared to wild type cells. The MAPT P301S cells also show hyperphosphorylation for p‐tau 217, p‐tau202/205, and p‐tau404, and the N279K cells show hyperphosphorylation for p‐tau202/205, and p‐tau404.ConclusionThe elevated p‐tau to total tau ratio measured by the immunocytochemistry assay indicates the potential of at least two cell lines as possible disease models to aid future research into developing AD/FTD disease therapeutics.

Bioinformatics pipeline to guide post‐GWAS studies in Alzheimer’s: a new catalogue of disease candidate short structural variants

AbstractBackgroundShort structural variants (SSVs) including indels are common in the human genome and impact disease risk. The role of SSVs in late onset Alzheimer’s disease (LOAD) has been understudied. Previously we developed a bioinformatics pipeline that characterizes and prioritizes candidate regulatory SNPs in enhancers located in LOAD‐GWAS regions. Here we developed a bioinformatics pipeline of SSVs within LOAD‐GWAS regions to prioritize regulatory SSVs based on the strength of their predicted effect on transcription factor (TFs) binding sites. The impact of the genes and TFs prioritized by the pipeline are assessed in two LOAD single cell rna‐seq datasets.MethodsThe pipeline utilized publicly available functional genomics data sources. Primarily, candidate cis‐regulatory elements (cCREs) from ENCODE and publicly available single‐cell RNA‐seq data from LOAD patient samples from human prefrontal cortex brain samples obtained from UCI MIND’s Alzheimer’s Disease Research Center (ADRC) and ROSMAP. For TF binding analysis we employed motifs from MotifDb. In addition, we used various bioinformatics software including motifbreakR.ResultsWe catalogued 1230 proximal CTCF‐bound candidate cis‐regulatory elements in LOAD‐GWAS regions, 912 showed epigenetic evidence in relevant brain tissue. We catalogued 426 indels in these cCREs. These indels disrupted 391 TFs, 362 of these had snRNA‐seq data from LOAD samples. Of note, TF motifs within the APOE‐TOMM40, SPI1 and MS4A2 regions were significantly disrupted by the candidate regulatory indels. Amongst these TFs are RUNX3, SPI1 and SMAD3. Interestingly, these significant findings with the APOE‐TOMM40, SPI1 and MS4A2 regions are consistent with our prior results for SNPs.ConclusionsThis study provides an analytical framework to catalogue noncoding indel variation in cis‐regulatory elements located in LOAD‐GWAS loci and characterize their likelihood to perturb TF binding. The approach integrates multiple data types to prioritize genes and variants for validation experiments using disease models and gene editing technologies.

Gene Editing Technologies and Programming: CRISPR-Cas9 Implementation with Python

Gene editing technologies, particularly CRISPR-Cas9, have revolutionized biotechnology by enabling precise modifications in the genetic code. This paper explores the integration of programming, specifically Python, in CRISPR-Cas9 implementation. Python's computational capabilities are harnessed to address challenges in CRISPR-Cas9, focusing on off-target effects and delivery mechanisms. The research provides a comprehensive examination of CRISPR-Cas9, introduces Python as a vital tool, and details its implementation to enhance precision and efficiency. Case studies demonstrate Python's superiority in diverse applications, including gene therapy and agriculture. Machine learning integration minimizes errors, and ethical considerations are discussed. The findings highlight the transformative potential of CRISPR-Cas9 with Python, shaping the future of genetic engineering across disciplines. This paper establishes Python as a driving force in advancing CRISPR-Cas9 implementations, ensuring a future defined by the synergy between biology and code.

Overcoming T Cell Exhaustion in Tumor Microenvironment via Immune Checkpoint Modulation with Nano-Delivery Systems for Enhanced Immunotherapy.

Immune checkpoint blockade (ICB) therapy for tumors has arisen in growing interest. However, the low response rate of tumors to ICB is mainly attributed to the inhibitory infiltration of immune cells in the tumor microenvironment (TME). Despite the promising benefits of ICB, the therapeutic effects of antibodies are dependent on a high dose and long-term usage in the clinic, thereby leading to immune-related adverse effects. Accordingly, ICB combined with nano-delivery systems could be used to overcome T cell exhaustion, which reduces the side effects and the usage of antibodies with higher response rates in patients. In this review, the authors aim to overcome T cell exhaustion in TME via immune checkpoint modulation with nano-delivery systems for enhanced immunotherapy. Several strategies are summarized to combine ICB and nano-delivery systems to further enhance immunotherapy: a) expressing immune checkpoint on the surface of nano-delivery systems; b) loading immune checkpoint inhibitors into nano-delivery systems; c) loading gene-editing technology into nano-delivery systems; and d) nano-delivery systems mediated immune checkpoint modulation. Taken together, ICB combined with nano-delivery systems might be a promising strategy to overcome T cell exhaustion in TME for enhanced immunotherapy.

CRISPR: POTENTIAL USES AND CHALLENGES

CRISPR Cas is a genome editing tool, that has the potential to revolutionize the medical world. As a cheap and simple alternative to other gene editing technologies, it has grabbed scientists attention and is being actively studied. Its uses range from the removal of disorders caused by genetic mutations to developing rapid diagnostic tests. As well as a potential cure for cancer.This paper aims to understand CRISPRs potential along with the challenges it may face while providing a brief explanation of its mechanism.

Futuristic Novel Therapeutic Approaches in the Treatment of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by systemic inflammation and joint destruction, leading to significant morbidity and reduced quality of life. Although significant progress has been made in the management of RA over the past few decades, many patients still fail to respond adequately to currently available therapies. This article aims to review the current landscape of RA treatment and explore potential novel therapeutic approaches that hold promise for the future. Advances in our understanding of the underlying pathogenesis of the disease have led to the identification of new targets and the development of innovative treatment strategies. This review focuses on emerging therapies including small molecule inhibitors, targeted biologics, cell-based therapies, and gene editing technologies that have shown potential in preclinical and early clinical trials. Additionally, we discuss the challenges and opportunities associated with the use of these new approaches in the treatment of RA. By elucidating the future of novel therapeutic approaches, this article provides insights that can guide clinicians and researchers in their efforts to improve outcomes for patients with RA.

Application of gene editing technology to DNA digital data storage

While the archival digital storage industry is approaching its physical limits, demand is increasing significantly, so alternatives are emerging. The modern world is in dire need of durable, scalable and economical alternative storage media. Deoxyribonucleic acid (DNA), a promising storage medium, offers superior information durability, capacity and energy consumption, making it a promising candidate for long-term data storage. However, the design and realization of DNA digital data storage face many problems, but gene editing technology, as a technology that makes modifications to genes directly from the molecular level, provides a breakthrough in solving these problems. In this paper, I show some methods for designing DNA digital data storage based on gene editing technology. The method utilizes gene editing technology to modify DNA molecules to improve their storage capacity and stability. At the same time, this paper also introduces the application cases of gene editing technology in DNA bio storage devices and looks forward to its future development.

Application of CRISPR gene editing technology in cystic fibrosis treatment

CRISPR-Cas9 gene editing technology is to identify the target genome sequence through artificially designed sgRNA, guide Cas9 protease, so that it can effectively cut the DNA double strand, form a double strand break, repair after damage to lead to gene knockout or knock-in, and finally achieve the purpose of modifying genomic DNA which leading CRISPR a technology can effectively repair, correct or regulate genetic mutations associated with genetic diseases, providing patients with new treatment options. However, despite the remarkable progress, CRISPR technology still faces many challenges in its clinical application, including ethics and safety. This article reviews the principle and development of CRISPR technology, as well as its application in the treatment of cystic fibrosis, and summarizes the challenges of CRISPR in clinical applications. More importantly, this article focuses on some current improvements to CRIPSR to make CRISPR gene editing technology more valuable and meaningful in the treatment of genetic diseases in the future.

Down-Regulation of Rice Glutelin by CRISPR-Cas9 Gene Editing Decreases Carbohydrate Content and Grain Weight and Modulates Synthesis of Seed Storage Proteins during Seed Maturation

The glutelins are a family of abundant plant proteins comprised of four glutelin subfamilies (GluA, GluB, GluC, and GluD) encoded by 15 genes. In this study, expression of subsets of rice glutelins were suppressed using CRISPR-Cas9 gene-editing technology to generate three transgenic rice variant lines, GluA1, GluB2, and GluC1. Suppression of the targeted glutelin genes was confirmed by SDS-PAGE, Western blot, and q-RT-PCR. Transgenic rice variants GluA1, GluB2, and GluC1 showed reduced amylose and starch content, increased prolamine content, reduced grain weight, and irregularly shaped protein aggregates/protein bodies in mature seeds. Targeted transcriptional profiling of immature seeds was performed with a focus on genes associated with grain quality, starch content, and grain weight, and the results were analyzed using the Pearson correlation test (requiring correlation coefficient absolute value ≥ 0.7 for significance). Significantly up- or down-regulated genes were associated with gene ontology (GO) and KEGG pathway functional annotations related to RNA processing (spliceosomal RNAs, group II catalytic introns, small nucleolar RNAs, microRNAs), as well as protein translation (transfer RNA, ribosomal RNA and other ribosome and translation factors). These results suggest that rice glutelin genes may interact during seed development with genes that regulate synthesis of starch and seed storage proteins and modulate their expression via post-transcriptional and translational mechanisms.

Targeted genome engineering based on CRISPR/Cas9 system to enhance FVIII expression in vitro

BackgroundHemophilia A is caused by a deficiency of coagulation factor VIII in the body due to a defect in the F8 gene. The emergence of CRISPR/Cas9 gene editing technology will make it possible to alter the expression of the F8 gene in hemophiliacs, while achieving a potential cure for the disease. MethodsInitially, we identified high-activity variants of FVIII and constructed donor plasmids using enzymatic digestion and ligation techniques. Subsequently, the donor plasmids were co-transfected with sgRNA-Cas9 protein into mouse Neuro-2a cells, followed by flow cytometry-based cell sorting and puromycin selection. Finally, BDD-hF8 targeted to knock-in the mROSA26 genomic locus was identified and validated for FVIII expression. ResultsWe identified the p18T-BDD-F8-V3 variant with high FVIII activity and detected the strongest pX458-mROSA26-int1-sgRNA1 targeted cleavage ability and no cleavage events were found at potential off-target sites. Targeted knock-in of BDD-hF8 cDNA at the mROSA26 locus was achieved based on both HDR/NHEJ gene repair approaches, and high level and stable FVIII expression was obtained, successfully realizing gene editing in vitro. ConclusionsKnock-in of exogenous genes based on the CRISPR/Cas9 system targeting genomic loci is promising for the research and treatment of a variety of single-gene diseases.

Unraveling Pathophysiology and Hematopoiesis of Vexas Syndrome By Multi-Omics Analyses and Targeted Gene Editing

Background and Rationale VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is a newly described, treatment-refractory, severe, highly prevalent disease (~1:4,000 males aged >50 years) caused by somatic mutations in the UBA1 (ubiquitin-activating enzyme 1) gene of hematopoietic stem/progenitor cells (HSPCs). VEXAS is hallmarked by vacuoles in myeloid/erythroid precursors and presents with systemic inflammation. About 50% patients develop hematologic malignancies, including myelodysplasia, contributing to the poor prognosis. The pathophysiology and impact of VEXAS syndrome on human hematopoiesis remain elusive. Moreover, the lack of suitable disease models hampers investigation of disease development and preclinical drug testing. To fill these gaps, here we: i) integrated multiparametric flow cytometry and multi-omics analyses on VEXAS patients to achieve comprehensive molecular and phenotypic characterization of hematopoiesis; and ii) successfully developed in vitro and in vivo models of VEXAS syndrome by cutting-edge targeted base editing strategies. Methods Multiparametric immunophenotypic analyses and single-cell transcriptomics were performed on peripheral blood and bone marrow (BM) cells from six VEXAS patients (p.Met41>Thr; p.Met41>Val; p.Met41>Leu; c.118-1 G>C) recruited by our Unit. Circulating monocytes were analyzed by whole RNA-sequencing (RNA-seq) and metabolomics. UBA1 mutations were introduced by gene editing technologies in myeloid cell lines, primary T cells and healthy human HSPCs. Engineered HSPCs were used to reconstitute the hematopoietic compartment of immunodeficient mice to generate a humanized VEXAS model. Variant allele frequency of UBA1 mutant cells was quantified by targeted sequencing in isolated hematopoietic lineages and HSPCs. Results i) Multiparametric immunophenotypic analyses of BM cells showed unbalanced composition of the HSPC compartment in VEXAS patients compared to age-matched healthy individuals, with lower abundance of stem cells, multipotent and lymphoid progenitors and expanded myeloid progenitors. This was paralleled by a significant increase of circulating myeloid-biased HSPCs and immature myeloid cells. Transcriptomic analyses of patient-derived peripheral monocytes showed sustained VEXAS-associated inflammatory signatures and heightened glycolysis, which were confirmed by metabolomic profiling. Single-cell transcriptomics on the BM hematopoietic compartment of VEXAS patients harboring different UBA1 mutations displayed genotype-dependent gene expression patterns. Single-cell RNA-seq of peripheral blood mononuclear cells from VEXAS patients is ongoing. ii) In vitro and in vivo models of VEXAS, generated by UBA1 gene editing, faithfully recapitulated patient hematopoiesis and pathophysiology . Editing efficiency approached 90% in wild-type myeloid cell lines, primary human T cells and HSPCs. While UBA1-mutant T cells were counterselected over time in culture, myeloid ones were enriched. Colony forming unit assays of UBA1-edited HSPCs revealed an exclusive myeloid output in vitro and a significant loss of the erythroid HSPC differentiation potential compared to mock-edited controls. Transplantation of edited HSPCs in immunodeficient mice resulted in a 100-fold reduction in circulating B cells, while NK and myeloid compartments were preserved. Human BM HSPCs were 5-fold lower than in control mice, largely myeloid-biased, and displayed abnormal vacuolar morphology. Concordantly, while myeloid cells and HSPCs were mainly (>80%) UBA1 mutated, the lymphoid compartment showed predominance of wild-type alleles. Strikingly, the distribution of UBA1-mutant cells across lineages in humanized mice mirrored the composition of our VEXAS patient cohort, where UBA1-mutant cells accounted for 90% of total myeloid cells and HSPCs and for less than 5% of lymphoid lineages. Conclusions Our findingsprovide evidence thatmutations in UBA1 drive accumulation of myeloid-biased HSPCs and immature myeloid precursors. Mutant lymphoid cells are negatively selected and their peripheral myeloid counterpart displays inflammatory activation. Gene editing-based in vitro and in vivo models phenocopy most hematopoietic features of the disease and hold promise to enable preclinical testing and validation of novel therapeutics to treat VEXAS syndrome.

Intracellular Retention of Tcrαβ/CD3 to Generate Novel Allogeneic CAR-T Cells (ThisCART19A) with Enhanced Antitumor Potency for Treating B-ALL

Background: Autologous CAR-T therapy has been complicated by long production time, high-cost and risks of manufacturing failure. Allogeneic CAR-T cells can overcome these hurdles, but would subsequently require specific strategies to inhibit allogeneic TCR responses and GvHD. Gene-editing technologies can efficiently deplete endogenous TCR, but also leads to off-target edits and chromosomal abnormality. Furthermore, genetic depletion of TCR disrupts the intracellular T cell activation signal and may compromise CAR-T cytotoxicity. It is thus necessary to develop non-gene-editing allogeneic CAR-T platforms and enhance the potency of allogeneic CAR-T cells. Methods: We developed a novel non-gene-editing platform named ThisCART ( TCRαβ/CD3 and/or HLA-I intracellular sequestered) to manufacture allogeneic CAR-T cells. The platform was based on the intracellular retention of TCRαβ/CD3 complex, allowing for allogeneic CAR-T production with a single lentiviral vector without genetic depletion of TCR. Allogeneic CD19 CAR-T cells (ThisCART19A) was a prototypic product for the platform. The construct contains a CD19-targeted CAR and a KDEL-tagged anti-CD3 single chain antibody (scFv) which prevents TCRαβ/CD3 from being secreted from the endoplasmic reticulum (ER) (Figure A). The efficacy and safety of ThisCART19A were tested in xenograft models. Finally, a phase I study was conducted to assess the safety, efficacy and pharmacokinetics in patients with relapsed or refractory (R/R) B-ALL (NCT05350787). All patients received intravenous fludarabine (30mg/m 2/d), cyclophosphamide (300mg/m 2/d) and etoposide (100mg/d) for 5 days followed by a single infusion of thisCART19A. Results: The manufacturing platform of ThisCART19A was able to achieve over 150-folds of ex vivo CAR-T expansion in all batches, with the purity of products (CAR-positive/TCRαβ-negative) above 99%. In preclinical models, ThisCART19A did not induce GvHD, and exhibited superior antitumor function compared to conventional CD19 CAR-T cells. In the Phase I study (Figure B), 10 patients were enrolled and 8 received thisCART19A at doses of 3 (n =5) and 5 (n = 3)×10 6/kg. All patients were diagnosed as relapse/refractory acute B cell leukemia (R/R B-ALL). Three patients previously received CD19-or CD22-targeted therapies (autologous CAR-T, BiTE or ADC). Grade 3-4 treatment-related adverse events were reported in 8/8 (100%) patients, the most frequent being neutropenia (100%) and thrombocytopenia (87.5%), which most likely related to lymphodepletion. Grade 3-4 CRS was reported in 2/8(25%) patients, and ICANS was reported in 3/8 (37.5%) patients which were all reversible with steroid treatment. 7 patients were evaluable for efficacy analyses (one died from CRS and infection at 5 days post infusion), and MRD-negative CR/CRi was achieved in 100% of these patients. With a median follow-up of 146 days (range, 56 to 407), 4/7 patients remained MRD-negative. Two patients were bridged to allo-HSCT. The mean peak of CAR-T number by FCM was 5908.8(0.51-17457.9) cells/µL , which occurred on day 9 (7-9). Conclusions: We report for the first time that intracellular retention of TCRαβ/CD3 complex can successfully manufacture allogeneic CAR-T cells, with superior activation potential. In patients with R/R B-ALL, ThisCART19A demonstrated acceptable safety, robust expansion and encouraging clinical response profiles. With streamlined single-vector-based production and enhanced CAR signaling, ThisCART platform represents an attractive alternative to gene-editing-based allogeneic CAR-T platforms.

Targeted, Safe, and Efficient Gene Delivery to Human Hematopoietic Stem and Progenitor Cells In Vivo Using Engineered Avid Adenovirus Vector Platform

Precise editing of human genome using CRISPR-Cas nucleases, base editors, prime editors, or other technologies represents a promising approach for ameliorating or correcting many human genetic diseases. Although numerous pre-clinical and clinical proof-of-concept studies demonstrated feasibility of gene correction in the desired regions of the human genome ex vivo, targeted delivery to and cell-type-specific expression of gene editing proteins in various cell types in vivo represent major challenges for all viral and non-viral delivery platforms developed to date. Here, we describe the development and analysis of AVIDs, a novel adenovirus-based gene delivery platform, that allows for a highly targeted, safe, and efficient gene delivery to human hematopoietic stem and progenitor cells (HSPCs) in vivo after intravenous vector administration. The AVID vectors attach to human HSPCs via engineered fibers that bind to either CD46 or DSG2 receptors, which are highly expressed on human HSPCs. The efficient entry of AVIDs into these cells is mediated via a mutated penton that was engineered to interact with CD49f, α6 integrin class, whose expression is restricted to cells with long-term repopulating and stem cell capacity. To improve virus safety after intravenous administration, as well as to increase the efficacy of virus production for a cost-effective high-yield vector manufacturing, AVIDs further comprise mutations in hypervariable loops of the major capsid protein hexon. While in vitro infection with AVIDs leads to about 30% transduction of a pool of human CD34 + cells, the efficacy of transduction of CD34 +CD38 -CD45RA -CD90 + subsets, highly enriched for hematopoietic stem cells (HSCs), ranged from 80% to 100% for individual donors. Single intravenous administration of AVIDs to humanized mice, grafted with human CD34 + cells, after mobilization led to up to 20% transduction of CD34 +CD38 -CD45RA- HSPC subsets in the bone marrow. Importantly, targeted in vivo transduction of CD34 +CD38 -CD45RA -CD90 -CD49f + subsets, highly enriched for human HSCs, reached up to 19%, which represented a 1900-fold selectivity in gene delivery to HSC-enriched over lineage committed CD34-negative cell populations in the bone marrow. Because the AVID platform allows for regulated, cell type-specific expression of gene editing technologies as well as for expression of immunomodulatory proteins to ensure persistence of corrected HSCs in vivo, the HSC-targeted AVID platform may enable development of novel curative therapies through safe and effective in vivo correction of disease-causing mutations in human HSCs after a single intravenous administration.

Knockout of eight hydroxyproline-O-galactosyltransferases cause multiple vegetative and reproductive growth defects

Arabinogalactan-proteins (AGPs) are a family of hyperglycosylated hydroxyproline-rich cell wall proteins found throughout the plant kingdom. To date, eight Hydroxyproline-galactosyltransferases (Hyp-GALTs), named GALT2-GALT9, are known to catalyze the addition of the first galactose sugar to Hyp residues in AGP protein cores. The generation and characterization of galt23456789 octuple mutants using CRISPR-Cas9 gene editing technology, provided strong reverse genetic evidence that AG glycans are essential for normal vegetative and reproductive growth, as these mutants demonstrated stunted growth, greatly delayed flowering and significant defects in floral organ development and morphogenesis. Compared to the lower seed set of galt25789 quintuple mutants being more so contributed by female gametophytic defects, dramatically low seed-set of octuple mutants was largely due to impaired male reproductive function, specifically due to shorter filaments, delayed anther dehiscence, and large decreases in pollen quantity and viability. Octuple mutant pollen had severely distorted reticulate exine, tectum patterning and intine thickness. Reduced amounts of galactose and arabinose in overall lower amounts of β-Yariv precipitated AGPs illustrated how biological functions of AGPs are affected by abnormal glycosylation.

Targeted mutagenesis of Medicago truncatula Nodule-specific Cysteine-Rich (NCR) genes using the Agrobacterium rhizogenes-mediated CRISPR/Cas9 system

The host-produced nodule specific cysteine-rich (NCR) peptides control the terminal differentiation of endosymbiotic rhizobia in the nodules of IRLC legumes. Although the Medicago truncatula genome encodes about 700 NCR peptides, only few of them have been proven to be crucial for nitrogen-fixing symbiosis. In this study, we applied the CRISPR/Cas9 gene editing technology to generate knockout mutants of NCR genes for which no genetic or functional data were previously available. We have developed a workflow to analyse the mutation and the symbiotic phenotype of individual nodules formed on Agrobacterium rhizogenes-mediated transgenic hairy roots. The selected NCR genes were successfully edited by the CRISPR/Cas9 system and nodules formed on knockout hairy roots showed wild type phenotype indicating that peptides NCR068, NCR089, NCR128 and NCR161 are not essential for symbiosis between M. truncatula Jemalong and Sinorhizobium medicae WSM419. We regenerated stable mutants edited for the NCR068 from hairy roots obtained by A. rhizogenes-mediated transformation. The analysis of the symbiotic phenotype of stable ncr068 mutants showed that peptide NCR068 is not required for symbiosis with S. meliloti strains 2011 and FSM-MA either. Our study reports that gene editing can help to elicit the role of certain NCRs in symbiotic nitrogen fixation.