TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells

Abstract CRISPR/Cas9 mediated gene editing of patient-derived hematopoietic stem and progenitor cells (HSPCs) ex vivo followed by autologous transplantation of the edited HSPCs back to the patient can provide a potential cure for monogenic blood disorders such as β-hemoglobinopathies. One challenge for this strategy is efficient delivery of the ribonucleoprotein (RNP) complex, consisting of purified Cas9 protein and guide RNA, into HSPCs. Because β-hemoglobinopathies are most prevalent in developing countries, it is desirable to have a reliable, efficient, easy-to-use and cost effective delivery method. With this goal in mind, we developed TRansmembrane Internalization Assisted by Membrane Filtration (TRIAMF), a new method to quickly and effectively deliver RNPs into HSPCs by passing a RNP and cell mixture through a filter membrane. We achieved robust gene editing in HSPCs using TRIAMF and demonstrated that the multilineage colony forming capacities and the competence for engraftment in immunocompromised mice of HSPCs were preserved post TRIAMF treatment. TRIAMF is a custom designed system using inexpensive components and has the capacity to process HSPCs at clinical scale

Structure of lipoprotein lipase in complex with GPIHBP1.

Lipoprotein lipase (LPL) plays a central role in triglyceride (TG) metabolism. By catalyzing the hydrolysis of TGs present in TG-rich lipoproteins (TRLs), LPL facilitates TG utilization and regulates circulating TG and TRL concentrations. Until very recently, structural information for LPL was limited to homology models, presumably due to the propensity of LPL to unfold and aggregate. By coexpressing LPL with a soluble variant of its accessory protein glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) and with its chaperone protein lipase maturation factor 1 (LMF1), we obtained a stable and homogenous LPL/GPIHBP1 complex that was suitable for structure determination. We report here X-ray crystal structures of human LPL in complex with human GPIHBP1 at 2.5-3.0 Å resolution, including a structure with a novel inhibitor bound to LPL. Binding of the inhibitor resulted in ordering of the LPL lid and lipid-binding regions and thus enabled determination of the first crystal structure of LPL that includes these important regions of the protein. It was assumed for many years that LPL was only active as a homodimer. The structures and additional biochemical data reported here are consistent with a new report that LPL, in complex with GPIHBP1, can be active as a monomeric 1:1 complex. The crystal structures illuminate the structural basis for LPL-mediated TRL lipolysis as well as LPL stabilization and transport by GPIHBP1

Structure of lipoprotein lipase in complex with GPIHBP1.

Lipoprotein lipase (LPL) plays a central role in triglyceride (TG) metabolism. By catalyzing the hydrolysis of TGs present in TG-rich lipoproteins (TRLs), LPL facilitates TG utilization and regulates circulating TG and TRL concentrations. Until very recently, structural information for LPL was limited to homology models, presumably due to the propensity of LPL to unfold and aggregate. By coexpressing LPL with a soluble variant of its accessory protein glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) and with its chaperone protein lipase maturation factor 1 (LMF1), we obtained a stable and homogenous LPL/GPIHBP1 complex that was suitable for structure determination. We report here X-ray crystal structures of human LPL in complex with human GPIHBP1 at 2.5-3.0 Å resolution, including a structure with a novel inhibitor bound to LPL. Binding of the inhibitor resulted in ordering of the LPL lid and lipid-binding regions and thus enabled determination of the first crystal structure of LPL that includes these important regions of the protein. It was assumed for many years that LPL was only active as a homodimer. The structures and additional biochemical data reported here are consistent with a new report that LPL, in complex with GPIHBP1, can be active as a monomeric 1:1 complex. The crystal structures illuminate the structural basis for LPL-mediated TRL lipolysis as well as LPL stabilization and transport by GPIHBP1

Design, Synthesis, and In Vitro and In Vivo Evaluation of Cereblon Binding Bruton’s Tyrosine Kinase (BTK) Degrader CD79b Targeted Antibody–Drug Conjugates

Antibody–drug conjugates (ADCs) are an established modality that allow for targeted delivery of a potent molecule, or payload, to a desired site of action. ADCs, wherein the payload is a targeted protein degrader, are an emerging area in the field. Herein we describe our efforts of delivering a Bruton’s tyrosine kinase (BTK) bifunctional degrader 1 via a CD79b mAb (monoclonal antibody) where the degrader is linked at the ligase binding portion of the payload via a cleavable linker to the mAb. The resulting CD79b ADCs, 3 and 4, exhibit in vitro degradation and cytotoxicity comparable with that of 1, and ADC 3 can achieve more sustained in vivo degradation than intravenously administered 1 with markedly reduced systemic exposure of the payload