In vivo genome editing represents a powerful strategy for both understanding basic biology and treating inherited diseases. However, it remains a challenge to develop universal and efficient in vivo genome-editing tools for tissues that comprise diverse cell types in either a dividing or non-dividing state. Here, we describe a versatile in vivo gene knock-in methodology that enables the targeting of a broad range of mutations and cell types through the insertion of a minigene at an intron of the target gene locus using an intracellularly linearized single homology arm donor. As a proof-of-concept, we focused on a mouse model of prematureaging caused by a dominant point mutation, which is difficult to repair using existing in vivo genome-editing tools. Systemic treatment using our new method ameliorated aging-associated phenotypes and extended animal lifespan, thus highlighting the potential of this methodology for a broad range of in vivo genome-editing applications.

Retrospective lineage tracing harnesses naturally occurring mutations in cells to elucidate single cell development. Common single-cell phylogenetic fate mapping methods have utilized highly mutable microsatellite loci found within the human genome. Such methods were limited by the introduction of in vitro noise through polymerase slippage inherent in DNA amplification, which we characterized to be approximately 10–100× higher than the in vivo replication mutation rate. Here, we present RETrace, a method for simultaneously capturing both microsatellites and methylation-informative cytosines to characterize both lineage and cell type, respectively, from the same single cell. An important unique feature of RETrace was the introduction of linear amplification of microsatellites in order to reduce in vitro amplification noise. We further coupled microsatellite capture with single-cell reduced representation bisulfite sequencing (scRRBS), to measure the CpG methylation status on the same cell for cell type inference. When compared to existing retrospective lineage tracing methods, RETrace achieved higher accuracy (88% triplet accuracy from an ex vivo HCT116 tree) at a higher cell division resolution (lowering the required number of cell division difference between single cells by approximately 100 divisions). Simultaneously, RETrace demonstrated the ability to capture on average 150,000 unique CpGs per single cell in order to accurately determine cell type. We further formulated additional developments that would allow high-resolution mapping on microsatellite-stable cells or tissues with RETrace. Overall, we present RETrace as a foundation for multi-omics lineage mapping and cell typing of single cells.

Single chain fragment variable (scFv) phage display libraries of randomly paired VH-VL antibody domains are a powerful and widely adopted tool for the discovery of antibodies of a desired specificity. Characterization of full length VH-VL constructs using synthetic long read assemblies of short read next-generation sequencing data has emerged as a powerful approach to identify antibody candidates with greater speed and sensitivity than classical screening methods. Here we introduce a new version of the synthetic long read approach, which we denote the Extended Range Targeted Sequencing method. We apply the method to demonstrate accurate and high throughput analysis of full-length VH-VL constructs from a commercial scFv combinatorial display library.

T-cell acute lymphoblastic leukemia (T-ALL) is a common hematological malignancy associated with poor prognosis and significant risk of relapse. Loss of PTEN function due to mutations or deletions is common in primary T-ALL samples and is associated with resistance to therapeutic agents. We previously developed a VEC-Cre+;Ptenloxp/loxp (Pten null) T-ALL model to investigate the molecular and cellular mechanisms underlying T-ALL pathogenesis and resistance to therapy. Pten-null T-ALL develops after acquisition of a T cell receptor α/δ-c-myc translocation that causes c-Myc over-expression, and the leukemia-initiating cells, or leukemia stem cells (LSCs), are enriched in a c-kitmidCD3+ subpopulation. The goal of our current study is to develop improved therapeutic approaches to target LSCs and eliminate T-ALL. We hypothesize that small molecule inhibitors that target molecular lesions and pathways required for the development and survival of Pten null LSCs and T-ALL may be potent in eliminating LSCs and abolishing T-ALL. Rapamycin, an inhibitor of the PI3K downstream kinase mTOR, can suppress leukemia development in Pten null pre-leukemic mice, but is insufficient to eliminate LSCs and T-ALL. Therefore, we are investigating the therapeutic utility of combinational therapies that co-target multiple deregulated pathways that are critical for LSC formation and T-ALL survival. We found that combination treatment of Pten null T-ALL mice with rapamycin and VX-680, an Aurora kinase inhibitor shown to have synthetic lethality with Myc over-expression, causes robust elimination of leukemia blasts and significantly diminishes the LSC population. Functional studies for leukemia-initiating-cell activity using transplantation of treated bone marrow into NOD-SCID-IL2rγ-/-(NSG) recipients showed that dual treatment reduced leukemia-initiating-cell activity in bone marrow by at least 100-fold in comparison to single agent alone. Importantly, a large portion of Pten null T-ALL LSCs are actively cycling (~50% in S-phase), which may contribute to the mechanism underlying the efficacy of VX-680. We have developed a T-ALL in vitro culture system derived from primary Pten null T-ALL mice to conduct more detailed evaluation of the cellular and biochemical effects of rapamycin and VX-680, perform mechanistic analysis, and screen additional cell cycle-directed and Myc-targeted agents. We found that Pten null T-ALL is highly sensitive to killing by the CDK1 inhibitor purvalanol and JQ1, a bromodomain inhibitor reported to down-regulate c-myc expression that is suitable for use in vivo. Rapamycin and JQ1 combination treatment of Pten null T-ALL mice causes a significant reduction in both the leukemia blast and LSC populations, similar to the effects of rapamycin and VX-680 combination therapy. Our results highlight the importance of understanding the mechanisms underlying T-ALL development and survival and show potent synthetic lethality of PI3K and Myc pathway co-inhibition in PTEN null T-ALL LSCs. Importantly, such targeted combination therapy may offer an improved therapeutic approach to treat human T-ALL. Citation Format: Suzanne Schubbert, Anjelica Cardenas, Christopher Wei, Lingda Ma, Consuelo Garcia, Wei Guo, Hong Wu. Co-inhibition of PI3K and Myc pathways is synthetic lethal to PTEN null T-ALL leukemia-initiating cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B12.