Homology-directed Repair Research Articles

Progress in gene editing tools, implications and success in plants: a review.

Genetic modifications are made through diverse mutagenesis techniques for crop improvement programs. Among these mutagenesis tools, the traditional methods involve chemical and radiation-induced mutagenesis, resulting in off-target and unintended mutations in the genome. However, recent advances have introduced site-directed nucleases (SDNs) for gene editing, significantly reducing off-target changes in the genome compared to induced mutagenesis and naturally occurring mutations in breeding populations. SDNs have revolutionized genetic engineering, enabling precise gene editing in recent decades. One widely used method, homology-directed repair (HDR), has been effective for accurate base substitution and gene alterations in some plant species. However, its application has been limited due to the inefficiency of HDR in plant cells and the prevalence of the error-prone repair pathway known as non-homologous end joining (NHEJ). The discovery of CRISPR-Cas has been a game-changer in this field. This system induces mutations by creating double-strand breaks (DSBs) in the genome and repairing them through associated repair pathways like NHEJ. As a result, the CRISPR-Cas system has been extensively used to transform plants for gene function analysis and to enhance desirable traits. Researchers have made significant progress in genetic engineering in recent years, particularly in understanding the CRISPR-Cas mechanism. This has led to various CRISPR-Cas variants, including CRISPR-Cas13, CRISPR interference, CRISPR activation, base editors, primes editors, and CRASPASE, a new CRISPR-Cas system for genetic engineering that cleaves proteins. Moreover, gene editing technologies like the prime editor and base editor approaches offer excellent opportunities for plant genome engineering. These cutting-edge tools have opened up new avenues for rapidly manipulating plant genomes. This review article provides a comprehensive overview of the current state of plant genetic engineering, focusing on recently developed tools for gene alteration and their potential applications in plant research.

ATM/ATR Phosphorylation of CtIP on Its Conserved Sae2-like Domain Is Required for Genotoxin-Induced DNA Resection but Dispensable for Animal Development.

Homology-directed repair (HDR) of double-strand DNA breaks (DSBs) is dependent on enzymatic resection of DNA ends by the Mre11/Rad50/Nbs1 complex. DNA resection is triggered by the CtIP/Sae2 protein, which allosterically promotes Mre11-mediated endonuclease DNA cleavage at a position internal to the DSB. Although the mechanics of resection, including the initial endonucleolytic step, are largely conserved in eucaryotes, CtIP and its functional counterpart in Saccharomyces cerevisiae (Sae2) share only a modest stretch of amino acid homology. Nonetheless, this stretch contains two highly conserved phosphorylation sites for cyclin-dependent kinases (T843 in mouse) and the damage-induced ATM/ATR kinases (T855 in mouse), both of which are required for DNA resection. To explore the function of ATM/ATR phosphorylation at Ctip-T855, we generated and analyzed mice expressing the Ctip-T855A mutant. Surprisingly, unlike Ctip-null mice and Ctip-T843A-expressing mice, both of which undergo embryonic lethality, homozygous CtipT855A/T855A mice develop normally. Nonetheless, they are hypersensitive to ionizing radiation, and CtipT855A/T855A mouse embryo fibroblasts from these mice display marked defects in DNA resection, chromosomal stability, and HDR-mediated repair of DSBs. Thus, although ATM/ATR phosphorylation of CtIP-T855 is not required for normal animal development, it enhances CtIP-mediated DNA resection in response to acute stress, such as genotoxin exposure.

Genomic Engineering of Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Recent advances in patient-derived induced Pluripotent Stem Cell (iPSC) generation, improvement of cardiomyocyte-directed differentiation protocols, and the availability of new genome editing techniques have opened up new avenues for disease modeling of cardiomyopathies. Patients with cardiomyopathies often harbor a single-base substitution believed to be linked to the disease phenotype. Somatic cells derived from patients can be efficiently reprogrammed into iPSCs and subsequently engineered. The targeting of a precise mutation can be achieved by the introduction of double stranded breaks with CRISPR-Cas9 and by homology-directed repair when using a DNA donor template. This allows for the correction of a mutation in a patient iPSC line to generate an isogenic control. In addition, key mutations associated with cardiomyopathies can be introduced in an iPSC line derived from a healthy individual using the same techniques. In this chapter, we describe in detail how to engineer pluripotent stem cells to model cardiomyopathy in a dish using CRISPR-Cas9 technology.

MutSβ protects common fragile sites by facilitating homology-directed repair at DNA double-strand breaks with secondary structures.

Common fragile sites (CFSs) are regions prone to chromosomal rearrangements, thereby contributing to tumorigenesis. Under replication stress (RS), CFSs often harbor under-replicated DNA regions at the onset of mitosis, triggering homology-directed repair known as mitotic DNA synthesis (MiDAS) to complete DNA replication. In this study, we identified an important role of DNA mismatch repair protein MutSβ (MSH2/MSH3) in facilitating MiDAS and maintaining CFS stability. Specifically, we demonstrated that MutSβ is required for the increased mitotic recombination induced by RS or FANCM loss at CFS-derived AT-rich and structure-prone sequences (CFS-ATs). We also found that MSH3 exhibits synthetic lethality with FANCM. Mechanistically, MutSβ is required for homologous recombination (HR) especially when DNA double-strand break (DSB) ends contain secondary structures. We also showed that upon RS, MutSβ is recruited to Flex1, a specific CFS-AT, in a PCNA-dependent but MUS81-independent manner. Furthermore, MutSβ interacts with RAD52 and promotes RAD52 recruitment to Flex1 following MUS81-dependent fork cleavage. RAD52, in turn, recruits XPF/ERCC1 to remove DNA secondary structures at DSB ends, enabling HR/break-induced replication (BIR) at CFS-ATs. We propose that the specific requirement of MutSβ in processing DNA secondary structures at CFS-ATs underlies its crucial role in promoting MiDAS and maintaining CFS integrity.

Highly Efficient Ex Vivo Correction of COL7A1 through Ribonucleoprotein-Based CRISPR/Cas9 and Homology-Directed Repair to Treat Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare and severe genetic skin disease responsible for blistering of the skin and mucosa after minor trauma. RDEB is caused by a wide variety of variants in COL7A1 encoding type VII Collagen, the major component of anchoring fibrils that form key attachment structures for dermal-epidermal adherence. In this study, we achieved highly efficient COL7A1 editing in primary RDEB keratinocytes and fibroblasts from 2 patients homozygous for the c.6508C>T (p.Gln2170∗) variant through CRISPR/Cas9-mediated homology-directed repair. Three guide RNAs targeting the c.6508C>T variant or harboring sequences were delivered together with high-fidelity Cas9 as a ribonucleoprotein complex. Among them, one achieved 73% cleavage activity in primary RDEB keratinocytes and RDEB fibroblasts. Then, we treated RDEB keratinocytes and RDEB fibroblasts with this specific ribonucleoprotein complex and the corresponding donor template delivered as single-stranded oligodeoxynucleotide and achieved up to 58% of genetic correction as well as type VII Collagen rescue. Finally, grafting of corrected 3-dimensional skin onto nude mice induced re-expression and normal localization of type VII Collagen as well as anchoring fibril formation at the dermal-epidermal junction 5 and 10 weeks after grafting. With this promising nonviral approach, we achieved therapeutically relevant specific gene editing that could be applicable to all variants in exon 80 of COL7A1 in primary RDEB cells.

Efficient selection of knocked-in pluripotent stem cells using a dual cassette cellular elimination system

Although recent advances in genome editing technology with homology-directed repair have enabled the insertion of various reporter genes into the genome of mammalian cells, the efficiency is still low due to the random insertion of donor vectors into the host genome. To efficiently select knocked-in cells without random insertion, we developed the "double-tk donor vector system," in which the expression units of the thymidine kinase of herpes simplex virus (HSV-tk) are placed on both outer sides of homology arms. This system is superior in enriching knocked-in human induced pluripotent stem cells (hiPSCs) than conventional donor vector systems with a single or no HSV-tk cassette. Using this system, we efficiently generated fluorescent reporter knockin hiPSCs targeting POU5F1 (OCT3/4), EEF1A1, H2BC21 (H2B clustered histone 21), ISL1, and MYH7 genes. These results indicate that the double-tk donor vector system enables efficient selection of knocked-in hiPSCs carrying reporter proteins.

Abstract C002: Identification of biomarkers predictive of sensitivity to the CHK1/2 inhibitor ACR-368 using high-resolution phosphoproteomics and development of an ACR-368-tailored patient responder identification 3-marker test, ACR-368 OncoSignature

Abstract Introduction ACR-368 (prexasertib) is a potent and selective CHK1/2 inhibitor with demonstrated durable, single-agent activity in patients with advanced solid tumors. Genomic biomarkers have been unsuccessful in predicting response to ACR-368 due in part to the complex genetic changes in cancer that translate into dysregulated protein signaling pathways. To address this challenge, we sought to identify protein-based, predictive biomarkers that measure the ACR-368-sensitive dysregulated signaling driving tumorigenesis using our proprietary approach, AP3 (Acrivon Predictive Precision Proteomics). Methods ACR-368 anticancer activity was measured in a panel of ovarian cancer cell lines using CellTiter-Glo. Quantitative phosphoproteomics was performed on ACR-368 sensitive and resistant ovarian cancer cell lines using data-independent acquisition mass spectrometry (DIA-MS). Kinase activity inference analysis and signaling pathway analyses were conducted to uncover ACR-368-regulated pathways associated with tumorigenesis. A quantitative, multiplexed immunofluorescent (IF) assay was developed based on three biomarkers, termed ACR-368 OncoSignature. Ovarian PDX studies were conducted in female athymic nude or CB-17 Scid mice. Results ACR-368 demonstrated diverse anti-proliferative activity in ovarian cancer cell lines. Phosphoproteome-profiling analysis from ACR-368 sensitive and resistant cells exposed to ACR-368 yielded >17,000 confidently localized phospho-sites (localization probability > 0.75), with 8272 being significantly regulated by ACR-368 (FC > 1.5, Q-value < 0.05, Limma t-test). Unsupervised hierarchical clustering analysis revealed overrepresentation of ATM/ATR and CDK1/2-associated substrate sites in the upregulated group, while the downregulated group showed overrepresentation of CHK1-associated sites. Pathway enrichment analysis highlighted the differential regulation of the homology-directed repair (HDR) pathway between sensitive and non-sensitive ovarian cancer cells. Through analysis of HDR-related signaling networks, three functionally orthogonal predictive biomarkers were assembled into a quantitative multiplex in situ assay for FFPE tissue that provides a direct readout of a tumor’s dependency on the signaling axis inhibited by ACR-368. Quantitation of these biomarkers in cancer cell lines using the IF-based OncoSignature demonstrated efficient classification based on ACR-368 sensitivity. The assay accurately predicted sensitivity to ACR-368 across ovarian cancer PDX models with an AUC of 0.9 (95% confidence interval: 0.71 to 1; p-value = 0.025). Conclusions Employing our AP3 platform, which combines MS-based phosphoproteomics and quantitative multiplexed-IF staining of drug-tailored biomarkers, we developed a response-predictive test for ACR-368 that enables identification of responders to ACR-368 treatment. A clinical trial (NCT05548296) evaluating the efficacy of ACR-368 based on the OncoSignature test status is currently recruiting in patients with platinum-resistant ovarian, endometrial, and urothelial cancer. Citation Format: Caroline Wigerup, Michail Shipitsin, Ayesha Murshid, Lei Shi, Magnus E. Jakobsson, Dorte Bekker-Jensen, Sibgat Choudry, James Dunyak, David Proia, Jesper V. Olsen, Kristina Masson, Peter Blume-Jensen. Identification of biomarkers predictive of sensitivity to the CHK1/2 inhibitor ACR-368 using high-resolution phosphoproteomics and development of an ACR-368-tailored patient responder identification 3-marker test, ACR-368 OncoSignature [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C002.

Life, the genome and everything.

In this issue of the Journal of Bacteriology, N. J. Bonde, E. A. Wood, K. S. Myers, M. Place, J. L. Keck, and M. M. Cox (J Bacteriol 205:e00184-23, 2023, https//doi.org/10.1128/jb.00184-23) used an unbiased transposon-sequencing (Tn-seq) screen to identify proteins required for life when cells lose the RecG branched-DNA helicase (synthetic lethality). The proteins' identities indicate pathways that prevent endogenous DNA damage, pathways that prevent its homology-directed repair (HDR) "strand-exchange" intermediates between sister chromosomes, and pathways that resolve those intermediates. All avoid intermediate pile-up, which blocks chromosome segregation, causing "death-by-recombination." DNA damage is managed to regulate crucial but potentially lethal HDR.

The strategy of knock-in with homology-directed genome editing in the model ornamental plant Petunia using CRISPR/Cas9 ribonucleoprotein complex

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system 9 (Cas9) ribonucleoprotein (RNP) complex offers numerous advantages, including increased genome editing efficiency, reduced off-target effects, minimized risk of genomic integration, expanded applicability, and the ability to address specific ethical concerns. These benefits establish the CRISPR/Cas9 RNP complex as a valuable technique for precise genome editing in plant research and applications. Nevertheless, achieving precise knock-in using CRISPR/Cas9-mediated precision knock-in of genes by homology-directed repair (HDR) remains challenging due to the infrequent occurrence of HDR and the limited availability of DNA repair templates near double-strand breaks (DSBs). In this study, we presented the efficient utilization strategy of HDR-mediated gene knock-in through co-infection of the CRISPR/Cas9 RNP complex and donor plasmid in Petunia protoplast. We accomplish this by designing a donor plasmid targeting the chalcone synthase (CHS) in Petunia, consisting of a CRISPR RNA (crRNA)-PAM sequences flanked by a donor DNA cassette with homology arms. These findings offer insights into the application of these techniques in ornamental crops to enhance CRISPR/Cas9 RNA complex-mediated gene knock-in strategies.

Sensitivity to Targeted UBA1 Inhibition in a Myeloid Cell Line Model of Vexas Syndrome

Somatic UBA1 mutations in hematopoietic cells are a hallmark of VEXAS (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) syndrome, which is a late-onset, male-predominant myeloid autoinflammatory disease associated with bone marrow failure and high mortality. UBA1 resides on the X chromosome and encodes the E1 enzyme isoforms UBA1a and UBA1b, which are essential for nuclear and cytosolic protein ubiquitination, respectively. The majority of UBA1 mutations in VEXAS syndrome comprise hemizygous missense mutations affecting methionine-41 (M41), leading to loss of UBA1b, gain of a truncated protein isoform (UBA1c),and globally reduced polyubiquitination. The relationship between impaired polyubiquitination, autoinflammation, and dyspoiesis in VEXAS syndrome is poorly understood, highlighting the need for experimental models that reproduce the genetic, biochemical, morphological, and inflammatory features associated with somatic UBA1 M41 missense mutations. To investigate the effects of VEXAS syndrome-associated UBA1 mutations, we used CRISPR-Cas9-based homology directed repair (HDR) in the male myeloid cell line 32D to generate isogenic single cell clones expressing Uba1 WT or Uba1 M41L from the endogenous mouse Uba1 locus. Sanger sequencing analysis of genomic and complementary DNA indicated the presence of a single Uba1 allele in Uba1 WT and Uba1 M41L single cell clones. Consistent with prior analyses of VEXAS syndrome patient samples, UBA1 and ubiquitin immunoblots showed that hemizygous Uba1 M41L expression was associated with loss of the UBA1b protein isoform, gain of the UBA1c protein isoform and reduced polyubiquitination. Uba1 M41L cells also contained abnormal cytoplasmic vacuoles, which were largely absent from Uba1 WT cells. Electron microscopy revealed that vacuoles in Uba1 M41L cells contained a variety of endolysosomal membranes, including small vesicles, multivesicular bodies, and multilamellar lysosomes. Similar to cytokine profiles in VEXAS syndrome patient samples, concentrations of IL1β and CXCL10 were significantly increased in supernatants from Uba1 M41L cells compared to Uba1 WT cells; Uba1 M41L cell supernatants also contained higher concentrations of CCL5, CCL17, CXCL9, and CXCL12. Thus, hemizygous Uba1 M41L expression in a myeloid cell line recapitulates key biochemical, morphological, and inflammatory features of VEXAS syndrome. The UBA1c protein isoform generated via UBA1 M41 missense mutations has reduced capacity to form the UBA1-ubiquitin thioester bond necessary for ubiquitin transfer to E2 enzymes. We hypothesized that decreased UBA1 activity would render Uba1 M41L cells vulnerable to the UBA1 inhibitor TAK243. In cell proliferation assays, Uba1 M41L cells were more sensitive to TAK243 compared to Uba1 WT cells (IC 50 mean±s.d., 1.4±0.6nM vs. 63±28nM, respectively). TAK243 promoted apoptosis in Uba1 M41L cells, as indicated by increased annexin-V/propidium iodide staining, H2AX, H2AX pS139, and PARP1 cleavage. TAK243 also led to preferential loss of Uba1 M41L cells in competition assays with Uba1 WT cells. To determine if on-target UBA1 inhibition was responsible for the increased TAK243 sensitivity of Uba1 M41L cells, we used CRISPR-HDR to generate a TAK243 drug-binding mutant, Uba1 A580S. Knock-in of Uba1 A580S rendered Uba1 M41L cells resistant to TAK243; the TAK243 IC 50 was significantly higher in Uba1 M41L;A580S cells compared to Uba1 M41L cells (mean±s.d., 73±16nM vs. 3.8±1.2nM, respectively). Together, these data demonstrate that hemizygous expression of Uba1 M41L in 32D cells confers TAK243 sensitivity via on-target inhibition of UBA1. Notably, increased TAK243 sensitivity and reduced polyubiquitination in Uba1 M41L cells were rescued by overexpression of UBA1b, but not UBA1b/C632A (catalytically inactive) or luciferase, indicating that loss of UBA1b enzymatic function contributes to the phenotype conferred by Uba1 M41L. In summary, we present a myeloid cell line with hemizygous expression of Uba1 M41L that exhibits the biochemical, morphological, and inflammatory features of VEXAS syndrome. We find that loss of UBA1b is a cause of reduced polyubiquitination and renders Uba1 M41L cells vulnerable to targeted UBA1 inhibition by TAK243. Our Uba1 M41L knock-in cell line is a faithful model of VEXAS syndrome that will aid in the study of disease pathogenesis and the development of effective therapies.

Development of an Ex Vivo Precision Gene Engineered B Cell Medicine That Produces Active and Sustained Levels of FIX for the Treatment of Hemophilia B

Hemophilia B is an X-linked recessive bleeding disorder that affects approximately 1:20,000 males. It is caused by mutations in the F9 gene that encodes for the factor IX (FIX) protein, an essential enzyme in the coagulation cascade. The current recommended therapy for hemophilia B patients is prophylactic administration of exogenous FIX derived from recombinant protein. The short biological half-life of FIX requires frequent infusions to maintain therapeutic level. More recently, an adeno-associated virus (AAV) vector-based gene therapy, etranacogene dezaparvovec, has been approved for some adults as a potential new option. Despite these advances, there remains a significant unmet medical need in hemophilia B. Intravenous infusions of weekly (or more frequently, 3 times per week) factor replacements are an enormous burden to patients and their caregivers. Gene therapy, while a promising option for some, carries potential for significant risk. Importantly, the limitations associated with increasing the dose of FIX needed to treat pediatric patients as they age make immunogenic AAV-based gene therapy inappropriate for this patient population. Terminally differentiated human plasma cells derived from genetically engineered B cells (termed B Cell Medicines, BeCMs), potentially offer natural longevity (persisting for decades), capacity for high levels of protein secretion (thousands of Ig molecules/cell/sec), the ability to engraft without host preconditioning, and the ability to re-dose, making them an attractive platform for the sustained supply of biologics where continuous dosing is required to achieve therapeutic benefit. We have developed an ex vivo precision engineered BeCM platform with modularity and broad therapeutic utility. In this study, primary human B cells were isolated, activated, and engineered by CRISPR/Cas9 genome editing followed by AAV-mediated homology directed repair (HDR) insertion of human F9 gene into the C-C chemokine receptor type 5 (CCR5) safe harbor locus. The cells were then further expanded and differentiated towards the plasma cell lineage, resulting in FIX-producing BeCMs. We achieved approximately 40% targeted integration as measured by droplet digital PCR (ddPCR). Engineered BeCMs secreted up to 60 ng/1e6 cells/hour of FIX protein, approaching 40% of IgG secretion rate as measured by ELISA. BeCM-produced FIX was analyzed by LC-MS which demonstrated gamma carobxylation of FIX protein Gal-domain. Vitamin K-dependent activated partial thromboplastin time (aPTT), using the one stage clotting assay, was employed to verify biological activity of BeCM-produced FIX. Similarly, FIX-expressing BeCMs exhibited vitamin K-dependent activity in vitro in the chromogenic assay. FIX-expressing BeCMs were transferred into immunodeficient NOG-hIL6 mice, with FIX production demonstrated at least 20 weeks in vivo. The safety of FIX-expressing BeCMs has been characterized based on 28-day and 5-month in vivo studies in NOG-hIL6 mice. Neither abnormal clinical observations nor mortality were observed in those studies. Biodistribution of the FIX expressing BeCM product was assessed using a qPCR assay. The qPCR data confirms the expected biodistribution of FIX-expressing BeCMs, which engraft and persist in bone marrow tissue over time. In summary, we have developed an ex vivo precision gene engineered B cell medicine that produces active and sustained levels of FIX for the treatment of hemophilia B. The potential therapeutic application of this unique biologic delivery system could afford a novel treatment modality for hemophilia B.

Using Inducible Signaling Receptors to Increase Erythropoietic Output from Genome-Edited Hematopoietic Stem Cells

Introduction: Oxygen is delivered to all tissues in the body through the hemoglobin molecule uniquely present in red blood cells (RBCs). Proper production and function of RBCs is therefore critical to human health. However, genetic defects of the hematologic system such as anemias and hemoglobin disorders (broadly termed “hemoglobinopathies”) are among the most common genetic disorders in the world. Therefore, developing strategies to regulate RBC development may allow researchers and clinicians to increase therapeutic potential of hematologic cell and gene therapies. Erythropoietin (EPO) signaling is indispensable to RBC development. In its native form, two EPO receptor (EPOR) monomers dimerize in the presence of EPO cytokine to initiate a downstream signaling cascade. Prior work has shown that conformational requirements of EPOR dimerization are rather “loose” and signaling may also be initiated by agonistic diabodies or in the context of chimeric receptors. While these chimeric receptors have limited clinical applicability, inducible dimerization domains such as engineered FKBPs are currently being used in clinical trials in the form of inducible safety switches. Due to the modular nature of cytokine receptors, we hypothesized that inducible dimerization domains (such as the FKBP domain) could be paired with endogenous receptors and signaling pathways to generate chimeric receptors that place RBC development under the control of a bioavailable small molecule - and therefore in the control researchers and clinicians. This study presents a set of chimeric, synthetic receptors that support HSPC output towards the erythroid lineage via a non-immunogenic, non-toxic small molecule (AP30187, hereafter termed “BB”). Methods: In-vitro editing was performed in primary human CD34+ HSPCs by electroporating Cas9 protein complexed with sgRNA along with an AAV6 DNA repair template to facilitate gene insertion via homology-directed repair (HDR). Bulk edited CD34+ HSPCs were then differentiated into RBCs using established differentiation protocols in the presence and absence of both erythropoietin and BB. Differentiation was determined by staining for RBC surface markers (CD71 and GPA) and subsequent flow cytometry analysis. Erythropoietic output was determined by cell density counts. Allele frequency of the edited cell population was determined through droplet digital PCR. Results: We generated seven FKBP-EPOR chimeric receptors - with the basic structure of SFFV-[FKBP-EPOR]-2A-YFP-bGH - that placed the FKBP binding domain at different regions of the endogenous receptor (Figure 1) and analyzed their ability to support RBC differentiation in the absence of EPO but with BB. Out of the seven constructs we found a set of receptors - 1.4 and 1.5 - that demonstrated erythropoietin-independent, BB-mediated erythroid differentiation at 108% and 94% mock RBC differentiation respectively. Construct 1.5 - dubbed inducible EPOR (iEPOR) - was chosen over 1.4 for further optimization because it was also no longer EPO responsive. We further optimized the design of iEPOR by 1) introducing a secretory signal peptide to improve trafficking of the receptor to the cell surface; 2) inclusion of a naturally occurring EPOR truncation that results in supraphysiological RBC differentiation; and 3) modification of the genomic insertion site and promoter of the iEPOR cassette to generate a toolbox of expression profiles. Our results suggest that iEPOR mimics the native EPOR signaling cascade with high fidelity and sensitivity according to bulk RNA-Seq data. We also observe a significant increase in erythropoietic output for iEPOR version 1.4 (6.7-fold by day 11 of RBC differentiation in +BB over -BB conditions, P=0.003), which was further enhanced by the above modifications for vector iEPOR version 3.5.2 (12.9-fold by day 11 of RBC differentiation in +BB over -BB conditions, P<0.0001). Additionally, iEPOR is demonstrated to be erythropoietin-independent, dose-responsive, and tunable via expression from different loci and promoters. Conclusion:iEPORs represent a powerful new tool to alter the cellular dynamics of red blood cell development. The work demonstrated in this study has the potential to dramatically improve the efficacies of existing gene therapies, offer a route to EPO-free ex-vivo RBC production, as well as serve as a new tool by which to better understand stem cell biology and differentiation.

Liver Sinusoidal Endothelial Cells but Not Hepatocytes Express a Fluorescent Reporter Under the Endogenous F8 Promoter in a New Hemophilia a Mouse Model

Hemophilia A (HA) is an inherited bleeding disorder caused by mutations in the F8 gene leading to a deficiency in coagulation factor VIII (FVIII). Treatment of patients with HA has centered around replacement of FVIII protein. Gene therapy provides the possibility of permanently altering the HA phenotype. Gene therapy for HA induces FVIII expression in hepatocytes, despite the well supported belief that FVIII is not made within hepatocytes but rather in liver sinusoidal endothelial cells (LSECs). Ectopic expression of FVIII in hepatocytes has been implicated in mild toxicities and FVIII decline observed in adeno-associated viral (AAV) vector-based gene therapy, the causes of which remain unknown. Extrahepatic production of FVIII, e.g. in lymphoid tissues, has been controversial. A better understanding of where FVIII is produced is needed to improve gene therapy for HA and inform development of other therapeutic interventions. We developed in collaboration with Jackson Laboratory a new strain of hemophilic mice on the C57BL/6J background expressing the fluorescent protein mScarlet under the endogenous F8 promoter. Using the CRISPR/Cas9 gene editing system, we inserted a transgene encoding mScarlet into exon 1 of the mouse F8 gene through homology-directed repair. We selected mScarlet for its relative brightness and stability, to maximize the chances of detecting its expression despite weak F8 promoter activity. The donor DNA consisted of two homology arms flanking the mScarlet-encoding transgene followed by the bovine growth hormone polyadenylation [poly(A)] signal. Insertion of the transgene at the first F8 codon in exon 1 and inclusion of poly(A) ensured knock-out of F8. Male animals showed a bleeding phenotype consistent with other HA mouse strains, such as C57BL/6J HA and B6/129 HA. We performed retro-orbital bleeding and tested mouse plasma samples using the activated partial thromboplastin time (aPTT) assay. Animals expressing mScarlet (n=4) and C57BL/6J HA mice (n=3) had similarly prolonged aPTT (mean 57 seconds) compared to non-hemophilic C57BL/6J mice (n=5, 41 seconds). Production of mScarlet was first evaluated in the hepatocytes and LSECs of mScarlet mice (n=3) using flow cytometry. The livers were perfused in situ via the portal vein with a perfusion and collagenase medium. The livers were then processed into single cell suspensions, and hepatocytes and LSECs were separated by centrifugation. An anti-CD31 antibody was used to identify LSECs. Hepatocytes were identified by exclusion of CD45.2 + cells. CD31 +cells from mScarlet mice but not from control C57BL/6J mice expressed mScarlet. Neither mouse strain had detectable mScarlet in hepatocytes. Flow cytometry was then used to evaluate mScarlet in the splenic endothelial cells (ECs) and leukocytes. Using in situtranscardiac perfusion, mScarlet or control C57BL/6J mice received a perfusion and collagenase medium. Following processing into single cell suspensions, ECs were isolated from other splenic cells by magnetic separation using CD31 microbeads. Anti-CD31 and anti-CD45.2 antibodies were used to identify ECs and splenic leukocytes, respectively. Neither cell type from mScarlet or control mice had detectable mScarlet. We next evaluated mScarlet expression via fluorescent microscopy of liver and spleen sections. Before sectioning, tissues were fixed using in situ transcardiac perfusion fixation. Since mScarlet fluorescence was not detectable in tissue sections, we employed an anti-mScarlet antibody. Anti-mScarlet binding was detectable in liver sections from mScarlet but not control C57BL/6J mice. The staining pattern was consistent with the sinusoidal arrangement of hepatic capillaries running between rows of hepatocytes toward central venules. We found the same staining pattern using an anti-CD31 antibody, supporting that the mScarlet-expressing cells were LSECs but not hepatocytes. No anti-mScarlet binding was detected in the spleens from either mScarlet or C57BL/6J mice. We propose that FVIII is produced in LSECs but not in hepatocytes, splenic endothelial cells, splenic or hepatic leukocytes. Our novel mouse model expressing a well-defined and readily detectable reporter protein overcomes the limitations of immunohistochemistry using anti-FVIII antibodies, which complicated previous studies on the cellular origins of FVIII. Further investigation of extrahepatic FVIII production is ongoing.

A Novel Strategy to Produce CAR-Γδ T Cells Via Targeted Knockout By CRISPR/Cas9 and Targeted Knockin By AAV

Chimeric antigen receptor (CAR)-γδ T cells are promising in immunotherapy owing to the dual recognition system of γδ T cells, which is potential to exhibit stronger anti-tumor effect. Up to date, established CAR-γδ T cells exhibit poor anti-tumor efficiency and are commonly generated using lentiviral (LV) or gamma-retroviral (γRV) vectors, which may increase the risk of insertional mutagenesis. Nowadays, clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) has been widely used to cleave the genomic sequence precisely, as well as Adeno-associated Virus (AAV) being used to deliver DNA templates for target sequence knock-in (KI) mediated by homology-directed repair, which could contribute to achieve the accurate insertion of CAR at a specific site. Furthermore, the endogenous promoter of TCR has been used to express CAR molecules with stable expression and better antitumor function in the production of CAR-αβ T cells. In the present study, we established and optimized the strategy to generate CAR-γδ T cells, combining the site-specific knock-out (KO) targeting the TCR delta chain constant region (TRDC) site of γδ T cells using CRISPR/Cas9 and site-specific CAR knock-in using AAV. Firstly, we screened out a functional guide RNA (gRNA) targeting the γδ TCR TRDC locus by prediction tools and validation in cell line model experiments, and achieved an INDEL frequency of 31.8%, which showed a high activity of gRNA. Then, we optimized the option to increase KO efficiency including exploring appropriate Cas9 amount, ratio of gRNA to Cas9 and cell number titration. Besides, we designed AAV vector carrying CAR sequence targeting the gRNA aiming site selected above (Figure1A). After that, we used CRISPR/Cas9 and AAV to knock out TCR and knock in CAR sequence in γδ T cells. We tried different electroporation procedures (X001, T007, U014, T023) and cell culture conditions (cultured in medium containing zoledronic acid (ZOL)/ IL2 for 5 days or for 10 days) to select out the optimal procedure to acquire high KO/KI efficiency and high cell proliferation rate. When we used PBMC being cultured in medium containing zoledronic acid (ZOL)/ IL2 for 5 days (Day 5) to generate CAR-γδ T cells, cells electroporated using X001 procedure showed the best TCR KO efficiency of ~80%（p<0.05）and CAR expression of ~20% (p< 0.01), as well as keeping the highest cell proliferation rate than other procedures (p<0.0001) (Figure 1B and 1C). If we used PBMC being cultured in medium containing zoledronic acid (ZOL)/ IL2 for 10 days (Day 10) to generate CAR-γδ T cells, U014 program also exhibited a relative high CAR expression and the significant quicker cell growth than others in the later period (p<0.01) (Figure 1B and 1C). However, the later proliferation rate of CAR-γδ T cells generated at Day 5 was superior to that generated at Day10. Thus, we determined that a 5 days' cell culture and the use of X001 program were the optimal condition to generate CAR-γδ T cells. Besides, we found that KI efficiency is positively correlative with Multiplicity of infection (MOI). While MOI comes to 1.2×10 5, the KI efficiency couldn't be increased (Figure 1D). Finally, we evaluated the anti-tumor function of CAR-γδ T cells . Antigen stimulated CAR-γδ T cells significantly released TNFα and INFγ, but failed to produce IL2 (Figure 1E). Besides, CAR-γδ T cells could significantly lysis different tumor cells even at a low effector/target ratio (E/T) (Figure 1G). Interestingly, the proportion of memory phenotype (CD62L + ) in CAR-γδ T cells after being stimulated was comparable to that of CAR-γδ T cells without stimulation, indicating the potential of the great anti-tumor activity of CAR-γδ T cells after being stimulated in tumor environment (Figure 1F). Our new method successfully generated CAR-γδ T cells with a CAR expression of 20% and in a high proliferation rate, which was determined to exert effective anti-tumor effects.

Towards Automated Engineering of Donor-Derived T Lymphocytes into CRISPR/Cas9-Mediated CAR T Cells in a Closed-System

Chimeric antigen receptor (CAR)-expressing T cells have revolutionized immunotherapy of cancer. However, some patients derive no benefit from approved CARTcell therapies due to limitations such as toxicity and use of autologous cells which are sometimes unfit and risk manufacturing failures. Additional or more effective CARTcell therapies are needed. Harnessing the endogenous homology-directed DNA repair pathway via precision genome editing based on Cas9RNP/rAAV6, we and others have demonstrated the feasibility of targeting transgene(s) integration into specific gene site(s) including their incorporation under endogenous regulatory elements. Using this strategy, we aimed to engineer healthy donor-derived CAR T cells from αβ+ T lymphocytes. For enhanced safety, we metabolically reprogrammed the CAR T cells by inactivating the UMPS gene needed for uridine biosynthesis. The UMPS-/-CAR T cells become dependent on exogenous uridine thereby serving as a cell control mechanism. We hypothesized that donor-derived αβ+ T lymphocytes can be engineered using the Cas9RNP-rAAV6 strategy in an automated closed-system into controllable CAR T cells. We inactivated TRAC and UMPS gene loci in αβ+T lymphocytes using Lonza4D nucleofector. Electroporated cells were simultaneously transduced with CD19CAR-containing rAAV6 DNA vector. We obtained TRAC gene knockout up to 97% with knock-in up to 89%. Disruption of the UMPS gene would transform the cells to being auxotrophic and depend on uridine supply for viability and growth. To evaluate the CART cells' antitumor potency, we cocultured the CD19CART cells with GFP-expressing Nalm6 leukemia cell lines. The CD19CART cells eradicated the leukemia cells within 72 hours. To mimic relapse, we re-challenged cocultured CART with Nalm6 cells and the tumor cells were again depleted within 72 hours. Expectedly, IL-2 production was elevated in CD19CART cells challenged with Nalm6. Next, we tested the ability to control the cytotoxicity of the CD19CART cells by coculturing them with Nalm6 cells in separate groups with/without exogenous uridine. Interestingly, the CD19CART cells cocultured without uridine eradicated the target cells within 72 hours but lost their potency upon subsequent challenge (Figure 1). In contrast, CD19CAR T cells supplied with uridine remained potent and repeatedly eradicated target cells indicating the ability to turn off the potency of CART cells with uridine supply/withdrawal. To establish an automated closed-system for CART cells, we adopted the Miltenyi CliniMACS Prodigy with in-line electroporator (Elpo) for process engineering. First, we determined optimized parameters for electroporating αβ+ T lymphocytes on Elpo(Table1). Cells electroporated with these settings were (>80% viability) comparable to untreated cells. We first engineered CART cells with Elpo/Miltenyi reagents in an open system(i.e.cultivated Elpo-electroporated cells in culture vessels/incubators). We obtained 95% of TRAC and UMPS gene knockouts and up to 90% CD19CAR transgene knock-in to the TRAC locus - an efficiency slightly higher than using Lonza 4D nucleofector. To scale up the Cas9-based T cell engineering into a single automated process on the CliniMACS Prodigy, the Cas9 and guide RNA reagents would be scaled up 1000fold. To make the process cost-effective, we tested AZD7468, a small molecule DNAPKcs inhibitor. We optimized/reduced the amounts of reagents upto 90% less of Cas9RNP used without any significant reduction in gene editing outcomes. AZD7648 reduced the MOI needed for viral transduction by 50% while maintaining 80% or higher levels of transgene knock-in efficiency. Next, we commenced pilot studies to automate the CAR T cell engineering process in closed-system. Currently, we have achieved an automated TRAC gene knockout of over 90% and CAR transgene knock-in of slightly >10%. Further optimization studies are underway to improve automated gene knock-in efficiency. We have engineered donor-derived αβ+ T lymphocytes into potent/controllable CD19CART cells. The UMPS-deficient CD19CART cells lose antileukemia activity without uridine. We show that metabolic reprogramming can be combined with precision genome editing to generate controllable CART cells. Finally, our data demonstrate the feasibility of automating CRISPR/Cas9-mediated engineering of CAR T cells within closed-system.

Platinum Talen-Mediated Knock-in of Single-Stranded DNA Templates Facilitates Manufacturing of Clinical-Scale Non-Virally Gene-Edited T Cells from Peripheral Blood

Background: During the past decade, technologies that manufacture gene-modified immune cells, such as chimeric antigen receptor T cells, have been rapidly evolved and introduced in human clinics. T cells reprogrammed to express antigen-specific T-cell receptors (TCR-T) are an up-and-coming tool to treat intractable infection or malignant neoplasm in a personalized manner. However, several caveats still exist in the production of TCR-T for clinical use, because competitive surface expression of endogenous and transduced TCRs might hamper desired expression of transduced TCR. Moreover, mispairing of TCR subunits could mediate unexpected harmful immune complications. Therefore, in this study, we aimed to develop a method to introduce any desired TCRs in human primary T cells independent of interference with endogenous TCR by way of targeted knockout of TRA and TRB gene loci using highly-efficient transcription activator-like effector nuclease (TALEN), named Platinum TALEN (Sakuma T, Sci Rep 2013). Methods: We designed and synthesized two pairs of 5'-Cap-1 Platinum TALEN mRNA targeting the gene sequences that encode constant regions of human TRA ( TRAC) and TRB ( TRBC). TCR knockout efficiencies by these TALEN pairs were assessed by flow cytometric analysis using anti-CD3/TCR antibodies. Next, we constructed single-stranded DNA (ssDNA) homology-directed repair templates for the knock-in of desired TCR transgene into the TALEN target sequence of the TRAC locus. As a model TCR, we selected a 1G4 clone that reacts with NY-ESO-1-derived peptide in an HLA-A*02-restricted manner. We delivered these TALEN mRNA pairs and 1G4-encoding ssDNA by electroporation into primary human T cells collected from healthy volunteer adult donors after 72 hours of anti-CD3/CD28 beads activation. Genome-edited T cells were subsequently cultured in Therapeak TM X-VIVO15 (Lonza) supplemented with 10% human AB serum in the presence of 2-mercaptoethanol and interleukin-2 followed by second anti-CD3/CD28 beads activation. Density of T cells were maintained approximately at 1.0-3.0 x10 6/mL and transferred to larger flasks with additional fresh media as appropriate according to cell counts throughout the culture.1G4-transduced T cells were detected and counted by HLA-A*02:01 NY-ESO-1 tetramers at 17-22 days after electroporation. Additionally, we performed optical genome mapping analysis (Bionano Genomics) of Platinum TALEN-treated cells to screen genome-editing-associated large chromosomal structural variations (SVs) including unintended translocation. Results: Platinum TALEN mRNA pairs targeting TRAC and TRBC yielded a high proportion (>70%) of CD3/TCR negative T cells without the cost of compromised cell viability. By co-electroporation of 1G4 ssDNA and these Platinum TALEN mRNA pairs, we successfully obtained an average of 10.5 (range 2.2-19.0) million (n=5)NY-ESO-1-tetramer-positive cells with high viability from 3 millionprimary T cells until 3 weeks after electroporation. By flow cytometric analysis, a proportion of 1G4-tetramer positive cells that express Vβ chains other than Vβ13 (Vβ of 1G4) was approximately 10%. The PD-1-positive fraction in the expanded 1G4-expressing T cells was less than 10%, while levels of TIM-3 expression were 40-60%. These tetramer-positive cells showed specific cytotoxicities against HLA-A*02-positive cells pulsed with cognate NY-ESO-1 peptides. In an optical genome mapping analysis of genomic DNA obtained from TALEN-treated T cells, we found unique SVs with relatively small sizes and low coverage but no large unique SVs with high coverage when compared with control genomic DNA. Additionally, no interchromosomal translocation was observed. Conclusions: We have successfully designed Platinum TALEN mRNA pairs targeting TRAC and TRBC which facilitate the production of genome-edited human primary T cells. We also developed an electroporation and expansion culture protocol that enables us to produce viable genome-edited 1G4-transduced T cells at a large cell dose equivalent to a clinical scale. Collectively, these results suggest that targeted orthotopic knock-in of antigen-specific TCR-encoding ssDNA into the TCR locus by the use of Platinum TALEN is a promising strategy that can be applied for the clinical manufacturing of therapeutic TCR-T cells.

Genome editing approaches with CRISPR/Cas9: the association of NOX4 expression in breast cancer patients and effectiveness evaluation of different strategies of CRISPR/Cas9 to knockout Nox4 in cancer cells

BackgroundThe increasing prevalence of cancer detection necessitated practical strategies to deliver highly accurate, beneficial, and dependable processed information together with experimental results. We deleted the cancer biomarker NOX4 using three novel genetic knockout (KO) methods. Homology-directed repair (HDR), Dual allele HITI (Du-HITI) and CRISPR-excision were utilized in this study.MethodsThe predictive value of the NOX4 expression profile was assessed using a combined hazard ratio (HR) with a 95% confidence interval (CI). With a 95% confidence interval, a pooled odd ratio (OR) was used to calculate the relationship between NOX4 expression patterns and cancer metastasis. There were 1060 tumor patients in all sixteen research that made up this meta-analysis. To stop the NOX4 from being transcribed, we employed three different CRISPR/Cas9-mediated knockdown methods. The expression of RNA was assessed using RT-PCR. We employed the CCK-8 assay, colony formation assays, and the invasion transwell test for our experiments measuring cell proliferation and invasion. Using a sphere-formation test, the stemness was determined. Luciferase reporter tests were carried out to verify molecular adhesion. Utilizing RT-qPCR, MTT, and a colony formation assay, the functional effects of NOX4 genetic mutation in CRISPR-excision, CRISPR-HDR, and CRISPR du-HITI knockdown cell lines of breast cancer were verified.ResultsThere were 1060 malignant tumors in the 16 studies that made up this meta-analysis. In the meta-analysis, higher NOX4 expression was linked to both a shorter overall survival rate (HR = 1.93, 95% CI 1.49–2.49, P < 0.001) and a higher percentage of lymph node metastases (OR = 3.22, 95% CI 2.18–4.29, P < 0.001). In breast carcinoma cells, it was discovered that NOX4 was overexpressed, and this increase was linked to a poor prognosis. The gain and loss-of-function assays showed enhanced NOX4 breast carcinoma cell proliferation, sphere-forming capacity, and tumor development. To activate transcription, the transcriptional factor E2F1 also attaches to the promoter region of the Nanog gene. The treatment group (NOX4 ablation) had substantially more significant levels of proapoptotic gene expression than the control group (P < 0.01). Additionally, compared to control cells, mutant cells expressed fewer antiapoptotic genes (P < 0.001). The du-HITI technique incorporated a reporter and a transcription termination marker into the two target alleles. Both donor vector preparation and cell selection were substantially simpler using this approach than with “CRISPR HDR” or “CRISPR excision.“ Furthermore, single-cell knockouts for both genotypes were created when this method was applied in the initial transfection experiment.ConclusionsThe NOX4 Knockout cell lines generated in this research may be used for additional analytical studies to reveal the entire spectrum of NOX4 activities. The du-HITI method described in this study was easy to employ and could produce homozygous individuals who were knockout for a specific protein of interest.

Modulation of cell cycle increases CRISPR-mediated homology-directed DNA repair

BackgroundGene knock‐in (KI) in animal cells via homology‐directed repair (HDR) is an inefficient process, requiring a laborious work for screening from few modified cells. HDR tends to occur in the S and G2/M phases of cell cycle; therefore, strategies that enhance the proportion of cells in these specific phases could improve HDR efficiency.ResultsWe used various types of cell cycle inhibitors to synchronize the cell cycle in S and G2/M phases in order to investigate their effect on regulating CRISPR/Cas9-mediated HDR. Our results indicated that the four small molecules—docetaxel, irinotecan, nocodazole and mitomycin C—promoted CRISPR/Cas9-mediated KI with different homologous donor types in various animal cells. Moreover, the small molecule inhibitors enhanced KI in animal embryos. Molecular analysis identified common signal pathways activated during crosstalk between cell cycle and DNA repair. Synchronization of the cell cycle in the S and G2/M phases results in CDK1/CCNB1 protein accumulation, which can initiate the HDR process by activating HDR factors to facilitate effective end resection of CRISPR-cleaved double-strand breaks. We have demonstrated that augmenting protein levels of factors associated with the cell cycle via overexpression can facilitate KI in animal cells, consistent with the effect of small molecules.ConclusionSmall molecules that induce cell cycle synchronization in S and G2/M phases promote CRISPR/Cas9-mediated HDR efficiency in animal cells and embryos. Our research reveals the common molecular mechanisms that bridge cell cycle progression and HDR activity, which will inform further work to use HDR as an effective tool for preparing genetically modified animals or for gene therapy.

Biallelic MAD2L1BP (p31comet) mutation is associated with mosaic aneuploidy and juvenile granulosa cell tumors.

MAD2L1BP-encoded p31comet mediates Trip13-dependent disassembly of Mad2- and Rev7-containing complexes and, through this antagonism, promotes timely spindle assembly checkpoint (SAC) silencing, faithful chromosome segregation, insulin signaling, and homology-directed repair (HDR) of DNA double-strand breaks. We identified a homozygous MAD2L1BP nonsense variant, R253*, in 2 siblings with microcephaly, epileptic encephalopathy, and juvenile granulosa cell tumors of ovary and testis. Patient-derived cells exhibited high-grade mosaic variegated aneuploidy, slowed-down proliferation, and instability of truncated p31comet mRNA and protein. Corresponding recombinant p31comet was defective in Trip13, Mad2, and Rev7 binding and unable to support SAC silencing or HDR. Furthermore, C-terminal truncation abrogated an identified interaction of p31comet with tp53. Another homozygous truncation, R227*, detected in an early-deceased patient with low-level aneuploidy, severe epileptic encephalopathy, and frequent blood glucose elevations, likely corresponds to complete loss of function, as in Mad2l1bp-/- mice. Thus, human mutations of p31comet are linked to aneuploidy and tumor predisposition.

Generation of iPSC lines with SLC16A2:G401R or SLC16A2 knock out

The X-linked Allan-Herndon-Dudley syndrome (AHDS) is characterized by severely impaired psychomotor development and is caused by mutations in the SLC16A2 gene encoding the thyroid hormone transporter MCT8 (monocarboxylate transporter 8). By targeting exon 3 of SLC16A2 using CRISPR/Cas9 with single-stranded oligodeoxynucleotides as homology-directed repair templates, we introduced the AHDS patient missense variant G401R and a novel knock-out deletion variant (F400Sfs*17) into the male healthy donor hiPSC line BIHi001-B. We successfully generated cerebral organoids from these genome-edited lines, demonstrating the utility of the novel lines for modelling the effects of MCT8-deficency on human neurodevelopment.