Floxed exon (Flexon): A flexibly positioned stop cassette for recombinase-mediated conditional gene expression

Conditional gene expression is a powerful tool for genetic analysis of biological phenomena. In the widely used “lox-stop-lox” approach, insertion of a stop cassette consisting of a series of stop codons and polyadenylation signals flanked by lox sites into the 5′ untranslated region (UTR) of a gene prevents expression until the cassette is excised by tissue-specific expression of Cre recombinase. Although lox-stop-lox and similar approaches using other site-specific recombinases have been successfully used in many experimental systems, this design has certain limitations. Here, we describe the Floxed exon (Flexon) approach, which uses a stop cassette composed of an artificial exon flanked by artificial introns, designed to cause premature termination of translation and nonsense-mediated decay of the mRNA and allowing for flexible placement into a gene. We demonstrate its efficacy in Caenorhabditis elegans by showing that, when promoters that cause weak and/or transient cell-specific expression are used to drive Cre in combination with a gfp(flexon) transgene, strong and sustained expression of green fluorescent protein (GFP) is obtained in specific lineages. We also demonstrate its efficacy in an endogenous gene context: we inserted a flexon into the Argonaute gene rde-1 to abrogate RNA interference (RNAi), and restored RNAi tissue specifically by expression of Cre. Finally, we describe several potential additional applications of the Flexon approach, including more precise control of gene expression using intersectional methods, tissue-specific protein degradation, and generation of genetic mosaics. The Flexon approach should be feasible in any system where a site-specific recombination-based method may be applied.

A Transgenic Murine Model Expressing Hyperactive STAT3 Recapitulates the Features of MDS/AML

Myelodysplastic syndromes (MDS) are clonal, myeloid malignancies that emerge and progress due to the expansion of disease-initiating aberrant hematopoietic stem cells that can evolve into Acute Myeloid Leukemia (AML). FDA approved therapies such as the recently approved Bcl-2 inhibitor venetoclax, FLT3 inhibitors, among others, have moved the field forward in newly diagnosed MDS/AML. However, relapsed/refractory (R/R) disease, as well as leukemic transformation post-MDS continues to have a poor prognosis. A pool of hematopoietic stem and progenitor cells (HSPCs) escape chemotherapy, proliferate during disease remission, and causes relapse partly in effect due to signaling effector mutations. It is imperative, for future therapeutic agents, to target these HSPCs populations to achieve a durable remission for aggressive myeloid malignancies. There is an urgent need to develop mouse models that recapitulate human disease for the study of pathogenesis and drug development in these disorders. Signal transducer and activator of transcription 3 (STAT3) belongs to the STAT family of transcription factors that are inappropriately activated in several malignancies. Our preliminary data indicates that STAT3 is overexpressed in MDS and AML stem cells and is associated with an adverse prognosis in a large cohort of patients. (Shastri et al, JCI 2018). We have successfully demonstrated that a selective antisense oligonucleotide inhibitor of STAT3, Danvatirsen, is rapidly incorporated into MDS/AML HSPCs and induces selective apoptosis and downregulation of STAT3 in these cells in comparison with healthy control HSPCs. To determine the role of STAT3 in the initiation of myeloid malignancies, a murine model was generated by crossing R26STAT3C stopfl/fl mice with vavCre transgenic mice. In this model, a hyperactive version of STAT3, STAT3C, is knocked into the Rosa26 locus with an upstream floxed stop cassette (R26STAT3C stopfl). Excision of the stop cassette by Cre recombinase leads to expression of a flag-tagged STAT3C protein and concomitant expression of EGFP in hematopoietic cells. GFP expression allows tracking of cells in which the floxed stop/Neo cassette is deleted and STAT3C is expressed. STAT3C-vavCre double transgenic mice were validated by GFP expression in HSPCs and differentiated hematopoietic cells. The STAT3C-vavCre mice developed ruffled fur, a hunched phenotype and weight-loss by five months of age. CBC analysis of STAT3C-vavCre mice shows a proliferative phenotype reminiscent of high-risk MDS/AML with higher WBC & platelet counts and lower hemoglobin (Figure 1A). Review of the peripheral smear showed an increase in granulocytic precursors that are likely leukemic blasts (Fig 1E). In addition, STAT3C-vavCre mice developed massive splenomegaly (Figure 1B). HSC lineage analysis by FACS showed the presence of GFP positive cells (Figure 1C) with increased expansion of the MPP and HSC compartment compared to controls, suggesting a stem and progenitor phenotype (Figure 1D). Murine myeloid colony assays showed larger colonies in the STAT3C-vavCre mice compared to controls. At this time, single cell RNA sequencing, and bulk RNA sequencing are being performed and will be used to further characterize the phenotype of the STAT3C-vavCre transgenic mice in addition to bone marrow and splenic aspirates & biopsies. Through the generation of a STAT3C-vavCre mouse model, that recapitulates the features of MDS/AML, we aim to further our understanding of the molecular mechanisms and pathways that play an important role in MDS to AML transformation and will help us identify downstream mediators of this event that can be therapeutically targeted. We would also like to use this murine model as an ideal substrate for preclinical studies of STAT3 targeting therapies in hematologic malignancies such as previously reported antisense inhibitors of STAT3 and STAT3 degraders. Figure 1 Figure 1. Disclosures Frank: Roche Genentech: Research Funding; Kymera: Consultancy, Research Funding; Revitope: Consultancy; Vigeo: Consultancy. Verma: Throws Exception: Current equity holder in publicly-traded company; BMS: Research Funding; GSK: Research Funding; Acceleron: Consultancy; Incyte: Research Funding; Stelexis: Current equity holder in publicly-traded company; Medpacto: Research Funding; Curis: Research Funding; Eli Lilly: Research Funding; Celgene: Consultancy; Stelexis: Consultancy, Current equity holder in publicly-traded company; Novartis: Consultancy. Shastri: Kymera Therapeutics: Research Funding; GLC: Consultancy; Guidepoint: Consultancy; Onclive: Honoraria.

Mouse Models of Achromatopsia in Addressing Temporal “Point of No Return” in Gene-Therapy

Achromatopsia is characterized by amblyopia, photophobia, nystagmus, and color blindness. Previous animal models of achromatopsia have shown promising results using gene augmentation to restore cone function. However, the optimal therapeutic window to elicit recovery remains unknown. Here, we attempted two rounds of gene augmentation to generate recoverable mouse models of achromatopsia including a Cnga3 model with a knock-in stop cassette in intron 5 using Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR) and targeted embryonic stem (ES) cells. This model demonstrated that only 20% of CNGA3 levels in homozygotes derived from target ES cells remained, as compared to normal CNGA3 levels. Despite the low percentage of remaining protein, the knock-in mouse model continued to generate normal cone phototransduction. Our results showed that a small amount of normal CNGA3 protein is sufficient to form “functional” CNG channels and achieve physiological demand for proper cone phototransduction. Thus, it can be concluded that mutating the Cnga3 locus to disrupt the functional tetrameric CNG channels may ultimately require more potent STOP cassettes to generate a reversible achromatopsia mouse model. Our data also possess implications for future CNGA3-associated achromatopsia clinical trials, whereby restoration of only 20% functional CNGA3 protein may be sufficient to form functional CNG channels and thus rescue cone response.

A Floxed exon (Flexon) approach to Cre-mediated conditional gene expression

Conditional gene expression allows for genes to be manipulated and lineages to be marked during development. In the established "lox-stop-lox" approach, Cre-mediated tissue-specific gene expression is achieved by excising the stop cassette, a lox-flanked translational stop that is inserted into the 5' untranslated region of a gene to halt its expression. Although lox-stop-lox has been successfully used in many experimental systems, the design of traditional stop cassettes also has common issues and limitations. Here, we describe the Floxed exon (Flexon), a stop cassette within an artificial exon that can be inserted flexibly into the coding region of any gene to cause premature termination of translation and nonsense-mediated decay of the mRNA. We demonstrate its efficacy in C. elegans by showing that, when promoters that cause weak and/or transient cell-specific expression are used to drive Cre in combination with a gfp(flexon) transgene, strong and sustained expression is obtained in specific lineages. We also describe several potential additional applications for using Flexon for developmental studies, including more precise control of gene expression using intersectional methods, tissue-specific protein degradation or RNAi, and generation of genetic mosaics. The Flexon approach should be feasible in any system where any site-specific recombination-based method may be applied.

Novel transgenic mice with Cre-dependent co-expression of GFP and human ACE2: a safe tool for study of COVID-19 pathogenesis

Current coronavirus disease (COVID-19) pandemia still belongs to the most serious problems of public health. A growing body of data shows that infection caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a very complex and multifaceted disease requiring its detailed study to fight with. Nevertheless, there is a row of stumbling blocks in the way of experimental research of COVID-19 determined by the deficiency of appropriate animal models. Herein, we report novel humanized mice with Cre-dependent expression of hACE2, the main entry receptor of SARS-CoV-2. These mice carry hACE2 and GFP transgenes floxed by the STOP-cassette, allowing them to be used as breeders for the creation of animals with the tissue-specific co-expression of hACE2 and GFP. Moreover, inducible expression of hACE2 makes this line biosafe, whereas co-expression with GFP simplifies the detection of transgene-expressing cells. In our study, we tested our line by crossing with Ubi-Cre mice, characterized by tamoxifen-dependent ubiquitous activation of Cre-recombinase. After tamoxifen administration, copy number of the STOP-cassette was decreased and the offspring expressed hACE2 and GFP, confirming the efficiency of our system. We believe that our model can be a useful tool for studying COVID-19 pathogenesis because the selective expression of hACE2 can shed light on the role of different tissues in SARS-CoV-2 associated complications. Obviously, it can also be used for preclinical trials of antiviral drugs and new vaccines.

Towards Improved Models of NPM-ALK Induced T-Cell Lymphoma

Time- and tissue-specific expression of transgenes is essential for the accurate representation of human disease in in vivo models. To improve flexibility but also fidelity of ALK+ ALCL models, we developed new approaches enabling lineage specific expression of NPM-ALK cDNAs, as well as a novel system for CRISPR/Cas induced Npm-Alk recombination. First, we have designed a new system for lineage-restricted expression of transgenes based on a retroviral vector incorporating a translational stop-cassette flanked by loxP recombination sites. Conditional transgene expression in chimeric mice is rapidly achieved through retroviral infection and subsequent transplantation of hematopoietic stem cells (HSC) derived from transgenic mice expressing Cre-recombinase from a lineage specific promoter. To validate the model, we directed expression of NPM-ALK, the fusion oncogene present in anaplastic large cell lymphoma (ALCL), to T-cells by infecting hematopoietic stem cells from Lck-Cre transgenic mice with a retroviral construct containing the Npm-Alk cDNA preceded by a translational stop cassette. The approach efficiently induced T-cell lymphomas within 12-16 weeks closely resembling the human disease including the expression of the ALCL hallmark antigen CD30. Since NPM-ALK overexpressed from a cDNA acts as a very strong oncogene transforming a range of cell types, we were interested to develop a more physiologic model based on chromosomal recombination, enabling NPM-ALK expression from the endogenous Npm promoter. To achieve this, we have designed guide RNAs (gRNAs) directed to either the intron between exon 4 and 5 for Npm1 on mouse chr. 11, or the intron between exons 19 and 20 for Alk on mouse chr. 17., enabling targeted translocation between the two chromosomes. For further analysis, the IL-3 dependent murine pro-B cell line Ba/F3 stably expressing the Cas9 recombinase was transduced with the respective gRNAs and subsequently grown in the absence of IL-3 to allow positive selection of cells transformed by productive t(11;17) NA recombination. A PCR reaction on genomic DNA using primers covering the translocation breakpoint resulted in a product and Sanger sequencing of the amplicon confirmed the intended recombination at the targeted genomic positions. The translocation was also detectable by fluorescence in-situ hybridization (FISH), and Western blot analysis demonstrated expression of a highly phosphorylated Npm-Alk fusion protein. To further probe for oncogene dependency, we treated Npm-Alk translocated cells and control cells with a specific Alk inhibitor, resulting in rapid cell depletion of the Npm-Alk translocated cells, but not controls. The described Cre/loxP-based system represents a versatile tool for the rapid functional analysis of gene function in a defined lineage or in a developmental stage in vivo, and faithfully recapitulates many features of ALCL. Furthermore, using Crispr/Cas to induce targeted double-strand breaks, we have been able to generate specific Npm-Alk translocations in murine cells, paving the way for novel models which may help to further define the initial pathogenetic event underlying lymphomagenesis in ALCL. Disclosures No relevant conflicts of interest to declare.

Deficiency of the Endocytic Protein Hip1 Leads to DecreasedGdpd3Expression, Low Phosphocholine, and Kypholordosis

ABSTRACTDeficiency of huntingtin-interacting protein 1 (Hip1) results in degenerative phenotypes. Here we generated aHip1deficiency allele where a floxed transcriptional stop cassette and a humanHIP1cDNA were knocked into intron 1 of the mouseHip1locus.CMV-Cre-mediated germ line excision of the stop cassette resulted in expression of HIP1 and rescue of theHip1knockout phenotype.Mx1-Cre-mediated excision led to HIP1 expression in spleen, kidney and liver, and also rescued the phenotype. In contrast,hGFAP-Cre-mediated, brain-specific HIP1 expression did not rescue the phenotype. Metabolomics and microarrays of severalHip1knockout tissues identified low phosphocholine (PC) levels and low glycerophosphodiester phosphodiesterase domain containing 3 (Gdpd3) gene expression. Since Gdpd3 has lysophospholipase D activity that results in the formation of choline, a precursor of PC,Gdpd3downregulation could lead to the low PC levels. To test whetherGdpd3contributes to theHip1deficiency phenotype, we generatedGdpd3knockout mice. Double knockout ofGdpd3andHip1worsened the Hip1 phenotype. This suggests that Gdpd3 compensates for Hip1 loss. More-detailed knowledge of howHip1deficiency leads to low PC will improve our understanding of HIP1 in choline metabolism in normal and disease states.

Deficiency of the Endocytic ProteinHip1Leads to DecreasedGdpd3expression, Low Phosphocholine, and Kypholordosis

ABSTRACTDeficiency of huntingtin interacting protein 1 (Hip1) results in degenerative phenotypes. Here we generated aHip1deficiency allele where a floxed transcriptional stop-cassette and a humanHIP1cDNA were knocked-in to intron 1 of mouseHip1locus.CMV-Cre-mediated germline excision of the stop-cassette resulted in expression of HIP1 and rescue of theHip1knockout phenotype.Mxl-Cre--mediated excision led to HIP1 expression in spleen, kidney and liver, and also rescued the phenotype. In contrast,GFAP-Cre-mediatedHIP1expression in brain did not rescue the phenotype. Metabolomics and microarrays of severalHip1knockout tissues identified low phosphocholine (PC) levels and lowGlycerophosphodiester Phosphodiesterase Domain Containing 3 (Gdpd3) expression. Since Gdpd3 has lysophospholipase D activity that results in the formation of choline, a precursor of PC,Gdpd3downregulation could lead to the low PC levels. To test ifGdpd3contributes to the Hip1 deficiency phenotype, we generatedGdpd3knockout mice. Double knockout ofGdpd3andHip1worsened the Hip1 phenotype. This suggests that Gdpd3 compensates for Hip1 loss. More detailed knowledge of how Hip1 deficiency leads to low PC will improve our understanding of HIP1 in choline metabolism in normal and disease states.