A Cell-Permeant Nanobody-Based Degrader That Induces Fetal Hemoglobin

Abstract

Proximity-based strategies to degrade proteins have enormous therapeutic potential in medicine, but the technologies are limited to proteins for which small molecule ligands exist. The identification of such ligands for therapeutically relevant but "undruggable" proteins remains challenging. Herein, we employed yeast surface display of synthetic nanobodies to identify a protein ligand selective for BCL11A, a critical repressor of fetal globin gene transcription. Fusion of the nanobody to a cell-permeant miniature protein and an E3 adaptor creates a degrader that depletes cellular BCL11A in differentiated primary erythroid precursor cells, thereby inducing the expression of fetal hemoglobin, a modifier of clinical severity of sickle cell disease and β-thalassemia. Our strategy provides a means of fetal hemoglobin induction through reversible, temporal modulation of BCL11A. Additionally, it establishes a new paradigm for the targeted degradation of previously intractable proteins.

Dose-Dependent Nuclear Delivery and Transcriptional Repression with a Cell-Penetrant MeCP2

“…We showed previously that the mini-protein ZF5.3 ,− is taken up by the endosomal pathway and released efficiently into the cytosol and nucleus of live cells, alone and when fused to certain protein cargos. Proteins successfully delivered using ZF5.3 include the self-labeling protein SNAP-tag, the metabolic enzyme argininosuccinate synthetase, the proximity labeling tool APEX2, and a nanobody-based degrader . These proteins differ in molecular weight, stoichiometry, isoelectric point, and the presence of bound cofactors.…”

Emerging Strategies in Proteolysis-Targeting Chimeras (PROTACs): Highlights from 2022

“…BCL11A subjugates HbF expression, thereby switching from fetal to adult hemoglobin expression during erythropoiesis [ 56 ]. Dassama et al (2022) developed a cell-permeant nanobody fused to a miniature protein and an E3 adaptor to form a degrader 21 ( Figure 18 ) [ 57 ]. This nanobody degrader 21 efficiently depleted cellular BCL11A in differentiated primary erythroid precursor cells.…”

Structures and Mechanisms of a Novel Bacterial Transport System for Fatty Acids

“…SEC-MALS was performed using an in-line Superdex 200 Increase 3.2/300 GL SEC column (Cytiva), as previously reported. [9,25] Proteins were separated at a flow rate of 0.15 mL/min at room temperature, and molecular masses were determined by the Astra6.1 software (Wyatt Technology).…”

“…His-tagged BstC, Tp0956, Es_TatT and Es_BstC were fluorescently labelled, as reported. [9,25] 4,4-dimethylsterol was extracted from M. capsulatus as previously described [9] while lanosterol (79630, Sigma-Aldrich), cholesterol (AAA1147018, Fisher Scientific), palmitic acid (P0500-10G, Sigma-Aldrich) and linoleic acid (L1376-5G, Sigma-Aldrich) were purchased. 50 nM of each labeled protein (diluted in assay buffer) was incubated with ligands (dissolved in 5 % DMSO) and loaded into Monolith NT.115 Capillaries (NanoTemper Technologies); MST measurements were performed and the data analyzed as reported previously.…”

“…50 nM of each labeled protein (diluted in assay buffer) was incubated with ligands (dissolved in 5 % DMSO) and loaded into Monolith NT.115 Capillaries (NanoTemper Technologies); MST measurements were performed and the data analyzed as reported previously. [9,25]…”