P-03.01 Generation and characterization of recombinant Vpx coupled to cell penetrating peptides
Year: 2025
Type: conference-paper
Abstract: Background The triphosphorylase SAM domain and HD domain containing protein 1 (SAMHD1) has been identified as a biomarker for Ara-Cytarabine sensitivity in patients with acute myeloid leukemia (AML).1 It recognizes Ara-Cytarabine in its active form and contributes to chemotherapy resistance.2 Lentiviral accessory Vpx proteins degrade SAMHD1, posing a potential asset against chemotherapy resistance.3 This project focuses on cell-penetrating peptides (CPPs) as a non-cytotoxic, non-invasive transient delivery method of Vpx into AML cells.4 Materials and Methods Based on our previous experience,5 we recombinantly expressed Vpx proteins coupled to CPPs from HEK293T cells stably expressing the respective constructs. Proteins were purified using StrepTactin®XT 4Flow beads and digested with Tobacco-Etch-Virus protease. Protein yield was quantified using HiBiT assays, Western Blots and Coomassie staining. As a proof of concept, the recombinant Vpx proteins were directly nucleofected into resting primary CD4+ T cells to test for CPP-Vpx functionality. Results We successfully optimized purification conditions for recombinant CPP-Vpx proteins with high yield and purity and tested their SAMHD1 degradation potency after nucleofection into resting CD4+ T cells, which naturally express high levels of SAMHD1. Further characterization in AML cell lines is currently ongoing and will be presented. Conclusions We have succeeded in purifying recombinant CPP-Vpx proteins and are currently testing their potency and specificity in AML cell lines. As a future possibility, transient delivery of recombinant Vpx proteins conjugated to CPPs may provide an opportunity to widen the therapeutic window for patients with high endogenous SAMHD1 levels who do not respond well to Ara-C induced chemotherapy. References Schneider C, Oellerich T, Baldauf HM, et al. SAMHD1 is a biomarker for cytarabine response and a therapeutic target in acute myeloid leukemia [published correction appears in Nat Med. 2017 Jun 6;23(6):788.] Nat Med. 2017;23(2):250–255. Knecht KM, Buzovetsky O, Schneider C, et al. The structural basis for cancer drug interactions with the catalytic and allosteric sites of SAMHD1. Proc Natl Acad Sci U S A. 2018;115(43):E10022-E10031. Laguette N, Sobhian B, Casartelli N, et al. SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx. Nature. 2011;474(7353):654–657. Zhang D, Wang J, Xu D, et al. Cell-penetrating peptides as noninvasive transmembrane vectors for the development of novel multifunctional drug-delivery systems: J Control Release 2016;229:130–139. Nair R, Pignot Y, Salinas-Illarena A, et al. Purified recombinant lentiviral Vpx proteins maintain their SAMHD1 degradation efficiency in resting CD4+ T cells. Anal Biochem 2023;670(1):115153. S. Aidin: None. A. Salinas-Illarena: None. R. Nair: None. H.M. Baldauf: None.
Source: Poster presentations
Authors: S Aidin, A Salinas-Illarena, R Nair, HM Baldauf
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W4408828432