High-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome

Gene editing in C. elegans using plasmid-based CRISPR reagents requires microinjection of many animals to produce a single edit. Germline silencing of plasmid-borne Cas9 is a major cause of inefficient editing. Here, we present a set of C. elegans strains that constitutively express Cas9 in the germline from an integrated transgene. These strains markedly improve the success rate for plasmid-based CRISPR edits. For simple, short homology arm GFP insertions, 50–100% of injected animals typically produce edited progeny, depending on the target locus. Template-guided editing from an extrachromosomal array is maintained over multiple generations. We have built strains with the Cas9 transgene on multiple chromosomes. Additionally, each Cas9 locus also contains a heatshock-driven Cre recombinase for selectable marker removal and a bright fluorescence marker for easy outcrossing. These integrated Cas9 strains greatly reduce the workload for producing individual genome edits.

Simultaneous Disruption of XPO1 and A20 in Murine B Cells Influences Both B and T Cell Repertoire

Introduction & Objectives: Efforts to characterize the heterogeneity of advanced hematologic malignancies using large-scale genomic studies have identified recurrent monoallelic mutations affecting the E571 residue of the essential nuclear exporter, Exportin-1 (XPO1; E571K in ~80% of cases, E571G in ~15% of cases). E571-XPO1 mutations alter the charge and structural basis of the cargo-binding region, disrupting critical biophysical interactions between XPO1 and its' cargos. Enriched in hematologic malignancies, E571-XPO1 mutations are predominantly reported in chronic lymphocytic leukemia (CLL; 5-10% of cases), classical Hodgkin's lymphoma (~25% of cases), and primary mediastinal B cell lymphoma (PMBCL; 25-30% of cases). The subsequent change in XPO1-cargo localization alters the transcriptional profile and overall phenotype of the leukemic cell, with evidence suggesting hyper-active NF-κB and NFAT signaling pathways as leading leukemogenic mechanisms. Moreover, while overall immune dysfunction in CLL leads to infections as a major cause of morbidity and mortality, CLL patients with E571-XPO1 mutations are more susceptible to death by infection, suggesting these mutations may exacerbate the leukemia-induced immunosuppressive phenotype. Similarly, inactivating mutations/deletions to A20 (TNFAIP3 gene), the master regulator of NF-κB, are recurrently reported in several B cell malignancies but most frequently observed in PMBCL (~30% of cases). E571-XPO1 mutations and TNFAIP3 deletions/mutations have been found as co-occurring genetic abnormalities in PMBCL, and while TNFAIP3 mutations in CLL are rare, functional convergence on NF-κB and immune signaling suggests altered XPO1 and A20 activity may have unreported pathogenic significance in CLL. Thus, we aimed to explore the oncogenic and immunologic consequence of co-occurring XPO1 and A20 abnormalities by evaluating a novel in-vivo model recapitulating this scenario. Methods: To explore concurrent aberrations to XPO1 and A20, we developed a novel mouse model to recapitulate this event (Eµ-XPO1xA20 KO). This model was generated by crossing the Eµ-XPO1 transgenic mouse - which overexpresses wildtype (WT), E571K, or E571G-XPO1 under control of a VH promoter-IgH-Eµ enhancer to target transgene expression to immature and mature B cells - with a B cell-specific A20 inactivation mouse (A20 KO) - which lacks functional A20 as a result of Cre recombinase-mediated excision of TNFAIP3 exon 3 via loxP recombination sites flanking this region and Cre recombinase expressed under CD19 promoter/enhancer elements. Eµ-XPO1 and Eµ-XPO1xA20 KO mice were aged and followed, and their B and T cell repertoire was assessed via flow cytometry. Results & Conclusion: We previously demonstrated Eµ-XPO1 mice develop a CLL-like disease (CD19+/CD5+/B220dim B lymphocytes), but leukemia development is significantly delayed - evident between 20-30 months of age. Preliminary analysis in adolescent animals revealed irregular lymphocyte populations as early as 6 months of age in the blood and spleen of Eµ-XPO1xA20 KO mice when compared to non-transgenic and Eµ-XPO1 mice; highlighted by elevated populations of CD93+/CD23+ transitional B cells and CD3+ T cells, and reduced populations of CD21+/IgM+ marginal zone B cells. Moreover, development of a circulating CLL-like disease accompanied by palpable lymphadenopathy and splenomegaly was observed in Eµ-XPO1xA20 KO mice as early as 17-20 months of age, again presenting a distinct immunophenotype inconsistent with that observed in Eµ-XPO1 mice. Additionally, progressive accumulation of CD3+/CD19- T cell leukemia-like populations were observed in a subset of Eµ-XPO1xA20 KO and A20 KO mice, indicating these aberrations may further disrupt and stimulate uncontrolled proliferation affecting the overall immune repertoire. Significance: We report that simultaneous disruption of essential regulators XPO1 and A20 in murine B cells encourages development of irregular B and T cell populations, and can stimulate a progressive CLL-like or T cell leukemia-like expansion. Continued investigation with these models can further our understanding of the relationship between overall immune function and these critical regulatory molecules, and can provide considerable insight to identifying pathways for selective targeting as a personalized therapy in several high-risk cancer types. Disclosures Byrd: AstraZeneca: Consultancy; Takada: Consultancy; Novartis: Consultancy; Pharmacyclics: Consultancy; Syndex: Consultancy; Trillium: Consultancy; Vincera Pharmaceuticals: Current equity holder in publicly-traded company.

Screening of Chemically Distinct Lipid Nanoparticles In Vivo Using DNA Barcoding Technology Towards Effectively Delivering Messenger RNA to Hematopoietic Stem and Progenitor Cells

Using adenine base editors, we aim to treat sickle cell disease by generating single nucleotide polymorphisms in human CD34+ hematopoietic stem and progenitor cells (HSPCs) at specific target sites by mediating A-T to G-C base conversions. While ex vivo gene editing approaches show great therapeutic promise, access is limited due to the requirement of an autologous hematopoietic stem cell (HSC) transplant to deliver the ex vivo edited cells. To further increase the number of patients eligible for base editing therapy, we are developing an alternative approach to directly deliver base editors to HSCs in vivo through non-viral delivery methods. Lipid Nanoparticles (LNPs) are a clinically validated, non-viral approach that enables the delivery of nucleic acid payloads, which may avoid the challenges associated with ex vivo approaches including the transplantation of edited CD34+ HSPCs. Here we describe the development and characterization of LNPs for the delivery of messenger RNA (mRNA) to HSPCs in vivo in both mice and cynomolgus macaques. By screening >1,000 chemically distinct LNPs in vivo utilizing a DNA barcoding technology, we identified several hit LNPs capable of biodistribution to HSPCs. Upon individual validation of these hit LNPs by delivery of Cre recombinase mRNA in a Cre-reporter mouse model (Ai14), which expresses the fluorescent protein tdTomato under a constitutive CAG promoter following Cre-meditate gene editing, we confirmed that several LNPs efficiently delivered Cre recombinase mRNA to mouse Lin-Sca-1+c-Kit+ (LSK) HSPCs. We next confirmed the most potent hit LNP (LNP-HSC1) identified from the in vivo screen to transfect LSK HSPCs in a dose-dependent manner between 0.1 and 1.0 mg/kg Cre recombinase mRNA, transfecting over 40% of LSK HSPCs in Ai14 mice at 1.0mg/kg. In a transfection durability study using Ai14 mice, we observed maintenance of tdTomato+ LSK HSPCs levels in the bone marrow at 10 weeks post-LNP delivery. As LNP-HSC1 had been identified and validated in mice of a C57BL6/j background, we next confirmed its ability to transfect a reporter mRNA into HSPCs in Balb/c mice and in 5 cynomolgus macaques. LNP-HSC1 efficiently transfected LSK HSPCs in Balb/c mice at doses ranging from 0.3 to 1.0 mg/kg. In 5 cynomolgus macaques (n=5 across two experiments), we observed a dose-dependent increase in reporter mRNA delivery with an average of 19% of bone marrow-derived CD34+ HSPCs (n=3) expressing the reporter protein at the highest dose tested. Taken together, these data demonstrate the value of our in vivo high-throughput LNP screening approach to identify novel LNPs capable of delivering to HSPCs, providing a promising delivery platform for an in vivo HSC gene editing approach for the treatment of hemoglobinopathies. Disclosures Sago: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Campbell: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lutz: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Patwardhan: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Hamilton: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Wong: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lee: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Keating: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Murray: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Singh: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ciaramella: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

STEM-25. ADENO-ASSOCIATED VIRUS INTRAVENTRICULAR INJECTION ALLOWS THE RESTRICTED TRANSDUCTION OF GLIOBLASTOMA CELLS NESTED IN THE PERI- AND SUB-VENTRICULAR ZONE IN A PATIENT-DERIVED ORTHOTOPIC XENOGRAFT MODEL

INTRODUCTION: Glioblastoma (GBM) seems to arise from cells nested in the subventricular zone (SVZ), a neurogenic zone in the adult brain. After striatal engraftment of patient-derived GBM cells in mice, we showed that some tumor cells escape the tumor mass to colonize the SVZ. We aimed to determine if the SVZ-nested GBM cells could be highlighted by the intraventricular injection of a viral vector. MATERIALS AND METHODS: We determined the optimal Adeno-Associated Virus (AAV) serotype to transduce patient-derived GBM cells cultured in 3D (GB1). We studied the spreading of the virus 4 weeks after intraventricular injection in mice. We realized striatal engraftment with GB1 cells, previously transfected with a lentiviral construction in order to express the Red-Fluorescent-Protein (RFP) spontaneously, while they express Green-Fluorescent-Protein (eGFP) only in presence of Cre-recombinase. 10 weeks after engraftment, an AAV expressing Cre-Recombinase was injected in the lateral ventricle and mice were perfused at 14 weeks. RESULTS: AAV serotype DJ (AAVDJ) efficiently transduced GB1 cells. 4 weeks after intraventricular injection, the AAVDJ transduced cells in the SVZ and the medial part of the caudoputamen (mCP). The median of the longest distance between the right ventricle and the transduced cells in the mCP was 293.8µm [245 – 376.5]. In 5 mice, the median of the shortest distance between RFP-positive GBM cells and right ventricle was 580.1µm [535 – 785.1]. SVZ was not colonized and eGFP signal was not found. In the 4 other mice, SVZ was invaded and eGFP signal was detected. The median of the longest distance between the right ventricle and eGFP-positive tumor cells was 240.5µm [195.7 – 372.5]. The median of the eGFP volume was 9 499 404µm3. CONCLUSION: It is possible to transduce GBM cells nested in the peri- and sub-ventricular zone thanks to AAV intraventricular injection.

A New Mouse Model of Diffuse Midline Glioma to Test Targeted Immunotherapies

BACKGROUND: Diffuse midline gliomas remain incurable, with consistently poor outcomes in children despite radiotherapy. Immunotherapeutic approaches hold promise, with the integration of the host's immune system fundamental to their design. Here, we describe a new, genetically engineered immunocompetent model that incorporates interleukin 13 receptor alpha 2 (IL13Rα2), a tumor-associated antigen, which is suitable for further evaluation of the antitumor activity of IL13Rα2-targeted immunotherapeutics in preclinical studies. METHODS: The RCAS-Tv-a delivery system was used to induce gliomagenesis through overexpression of PDGFB and p53 deletion with and without human IL13Rα2 in Nestin-Tva; p53fl/fl mice. Neonatal pups were infected with Cre recombinase and PDGFB+IL13Rα2 or Cre recombinase and PDGFB in forth ventricle or right cortex of the brain to model diffuse midline glioma and pediatric high-grade glioma, respectively. Immunoblotting and flow cytometry was used to confirm target expression. Kaplan-Meier survival curves were established to compare tumor latency in both models. Tumor tissue was analyzed through immunohistochemistry and H&E staining. Cell lines generated from tumor-bearing mice were used for in vitro studies and orthotopic injections. RESULTS: The protein expression of PDGFB and IL13Rα2 was confirmed by flow cytometry and western blot. In both groups, de novo tumors developed without significant difference in median survival between PDGFB and p53 loss (n=25, 40 days) and PDGB, IL13Rα2, and p53 loss (n=33, 38 days, p=0.62). Tumors demonstrated characteristics of high-grade glioma such as infiltration, palisading necrosis, microvascular proliferation, high Ki-67 index, heterogeneous IL13Rα2 expression, and CD11b+ macrophages, along with a low proportion of CD3+ T cells. Orthotopic tumors developed from cell lines retained histopathological characteristics of de novo tumors. Mice orthotopically implanted with cells in the 4th ventricle or right cortex showed a median survival of 42 days and 41 (p=0.56) days, respectively. CONCLUSION: Generation of de novo tumors using the RCAS-Tv-a delivery system was successful, with tumors possessing histopathologic features common to pediatric diffuse gliomas. The development of these models opens the opportunity for preclinical assessment of IL13Rα2-directed immunotherapies with the potential for clinical translation.

Generation of scalable cancer models by combining AAV-intron-trap, CRISPR/Cas9, and inducible Cre-recombinase

Scalable isogenic models of cancer-associated mutations are critical to studying dysregulated gene function. Nonsynonymous mutations of splicing factors, which typically affect one allele, are common in many cancers, but paradoxically confer growth disadvantage to cell lines, making their generation and expansion challenging. Here, we combine AAV-intron trap, CRISPR/Cas9, and inducible Cre-recombinase systems to achieve >90% efficiency to introduce the oncogenic K700E mutation in SF3B1, a splicing factor commonly mutated in multiple cancers. The intron-trap design of AAV vector limits editing to one allele. CRISPR/Cas9-induced double stranded DNA breaks direct homologous recombination to the desired genomic locus. Inducible Cre-recombinase allows for the expansion of cells prior to loxp excision and expression of the mutant allele. Importantly, AAV or CRISPR/Cas9 alone results in much lower editing efficiency and the edited cells do not expand due to toxicity of SF3B1-K700E. Our approach can be readily adapted to generate scalable isogenic systems where mutant oncogenes confer a growth disadvantage.

Rapid conversion of replicating and integrating Saccharomyces cerevisiae plasmid vectors via Cre recombinase

Plasmid shuttle vectors capable of replication in both Saccharomyces cerevisiae and Escherichia coli and optimized for controlled modification in vitro and in vivo are a key resource supporting yeast as a premier system for genetics research and synthetic biology. We have engineered a series of yeast shuttle vectors optimized for efficient insertion, removal and substitution of plasmid yeast replication loci, allowing generation of a complete set of integrating, low copy and high copy plasmids via predictable operations as an alternative to traditional subcloning. We demonstrate the utility of this system through modification of replication loci via Cre recombinase, both in vitro and in vivo, and restriction endonuclease treatments.

The Optimal Condition of Poly(I: C)-Induced Knockout of Cre Recombinase for Mx1-Cre/ Cre-loxP Mice

The full text of this preprint has been withdrawn by the authors due to author disagreement with the posting of the preprint. Therefore, the authors do not wish this work to be cited as a reference. Questions should be directed to the corresponding author.

Tamoxifen administration induces histopathologic changes within the lungs of Cre-recombinase-negative mice: A case report

Tamoxifen is commonly used as a cancer treatment in humans and for inducing genetic alterations using Cre-lox mouse models in the research setting. However, the extent of tamoxifen off-target effects in animal research is underappreciated. Here, we report significant changes in cellular infiltration in Cre-recombinase-negative mice treated with tamoxifen intraperitoneally. These changes were noted in the lungs, which were characterized by the presence of alveolitis, vasculitis, and pleuritis. Despite significant immunological changes in response to tamoxifen treatment, clinical symptoms were not observed. This study provides a cautionary note that tamoxifen treatment alone leads to histologic alterations that may obscure research interpretations and further highlights the need for the development of alternative mouse models for inducible Cre-mediated deletion.