Adenine base editing reduces misfolded protein accumulation and toxicity in alpha-1 antitrypsin deficient patient iPSC-hepatocytes.

Alpha-1 antitrypsin deficiency (AATD) is most commonly caused by the Z mutation, a single-base substitution that leads to AAT protein misfolding and associated liver and lung disease. In this study, we apply adenine base editors to correct the Z mutation in patient induced pluripotent stem cells (iPSCs) and iPSC-derived hepatocytes (iHeps). We demonstrate that correction of the Z mutation in patient iPSCs reduces aberrant AAT accumulation and increases its secretion. Adenine base editing (ABE) of differentiated iHeps decreases ER stress in edited cells, as demonstrated by single-cell RNA sequencing. We find ABE to be highly efficient in iPSCs and do not identify off-target genomic mutations by whole-genome sequencing. These results reveal the feasibility and utility of base editing to correct the Z mutation in AATD patient cells.

Directed evolution of adenine base editors with increased activity and therapeutic application.

The foundational adenine base editors (for example, ABE7.10) enable programmable A•T to G•C point mutations but editing efficiencies can be low at challenging loci in primary human cells. Here we further evolve ABE7.10 using a library of adenosine deaminase variants to create ABE8s. At NGG protospacer adjacent motif (PAM) sites, ABE8s result in ~1.5× higher editing at protospacer positions A5-A7 and ~3.2× higher editing at positions A3-A4 and A8-A10 compared with ABE7.10. Non-NGG PAM variants have a ~4.2-fold overall higher on-target editing efficiency than ABE7.10. In human CD34+ cells, ABE8 can recreate a natural allele at the promoter of the γ-globin genes HBG1 and HBG2 with up to 60% efficiency, causing persistence of fetal hemoglobin. In primary human T cells, ABE8s achieve 98-99% target modification, which is maintained when multiplexed across three loci. Delivered as messenger RNA, ABE8s induce no significant levels of single guide RNA (sgRNA)-independent off-target adenine deamination in genomic DNA and very low levels of adenine deamination in cellular mRNA.

Individualized Molecular Analyses Guide Efforts (IMAGE): A Prospective Study of Molecular Profiling of Tissue and Blood in Metastatic Triple-Negative Breast Cancer.

PURPOSE: The clinical utility of next-generation sequencing (NGS) in breast cancer has not been demonstrated. We hypothesized that we could perform NGS of a new biopsy from patients with metastatic triple-negative breast cancer (TNBC) in a clinically actionable timeframe. EXPERIMENTAL DESIGN: We planned to enroll 40 patients onto a prospective study, Individualized Molecular Analyses Guide Efforts (IMAGE), to evaluate the feasibility of obtaining a new biopsy of a metastatic site, perform NGS (FoundationOne), and convene a molecular tumor board to formulate treatment recommendations within 28 days. We collected blood at baseline and at time of restaging to assess cell-free circulating plasma tumor DNA (ptDNA). RESULTS: We enrolled 26 women with metastatic TNBC who had received ≥1 line of prior chemotherapy, and 20 (77%) underwent NGS of a metastatic site biopsy. Twelve (60%) evaluable patients received treatment recommendations within 28 days of consent. The study closed after 20 patients underwent NGS, based on protocol-specified interim futility analysis. Three patients went on to receive genomically directed therapies. Twenty-four of 26 patients had genetic alterations successfully detected in ptDNA. Among 5 patients, 4 mutations found in tumor tissues were not identified in blood, and 4 mutations found in blood were not found in corresponding tumors. In 9 patients, NGS of follow-up blood samples showed 100% concordance with baseline blood samples. CONCLUSIONS: This study demonstrates challenges of performing NGS on prospective tissue biopsies in patients with metastatic TNBC within 28 days, while also highlighting the potential use of blood as a more time-efficient and less invasive method of mutational assessment. Clin Cancer Res; 23(2); 379-86. ©2016 AACR.

Integrated genomic DNA/RNA profiling of hematologic malignancies in the clinical setting.

The spectrum of somatic alterations in hematologic malignancies includes substitutions, insertions/deletions (indels), copy number alterations (CNAs), and a wide range of gene fusions; no current clinically available single assay captures the different types of alterations. We developed a novel next-generation sequencing-based assay to identify all classes of genomic alterations using archived formalin-fixed paraffin-embedded blood and bone marrow samples with high accuracy in a clinically relevant time frame, which is performed in our Clinical Laboratory Improvement Amendments-certified College of American Pathologists-accredited laboratory. Targeted capture of DNA/RNA and next-generation sequencing reliably identifies substitutions, indels, CNAs, and gene fusions, with similar accuracy to lower-throughput assays that focus on specific genes and types of genomic alterations. Profiling of 3696 samples identified recurrent somatic alterations that impact diagnosis, prognosis, and therapy selection. This comprehensive genomic profiling approach has proved effective in detecting all types of genomic alterations, including fusion transcripts, which increases the ability to identify clinically relevant genomic alterations with therapeutic relevance.

Hyaluronan-tethered opioid depots: synthetic strategies and release kinetics in vitro and in vivo.

We proposed the use of opioid drug bound covalently to hyaluronan (HA) via ester linkages as a method to prolong drug delivery and to possibly increase the quality of perioperative pain management. The in vitro release profile of morphine conjugated to HA (1.3 million MW) was studied. The influence of parameters such as conjugation site and steric protection of the labile ester bonds was investigated in phosphate buffered saline (PBS) medium. HA--codeine and HA--naloxone conjugates were used as structural controls. Codeine and morphine conjugated via the allylic hydroxyl group had a release half-life of 14.0 days in PBS. Naloxone conjugated via the phenolic hydroxyl group showed a half-life of 0.3 days, and all drugs admixed in HA showed half-lives of 0.1 days. Methyl, ethyl, or n-propyl introduced in vicinal position to the ester bond prolonged release of naloxone with half-lives of 0.5, 4.0, and 4.0 days in PBS, respectively. Incorporation of a methyl group prolonged codeine release with a half-life of 55.0 days in PBS. Drugs were released chemically unaltered from the conjugates as confirmed by LC-MS/MS. Further, morphine was conjugated to divinylsulfone cross-linked HA (Hylan B) particles and the release profiles in rat plasma were studied in vitro and in vivo. Release in rat plasma was faster than in PBS with a half-life of 2.5 days, but the release was similar (ca. 12 days) when a cocktail of protease inhibitors was added to the plasma. Sustained release of morphine was observed in a rat surgical model over 30 h. Morphine was released chemically unaltered from the conjugate and morphine intermediates were not detected in significant amounts as confirmed by LC-MS/MS. These results suggest that the morphine release profile from the HA conjugates depends on the alkyl groups vicinal to the ester and the nature of the leaving group. In rat plasma, hydrolysis seems to be controlled by esterase activity.