Targeted siRNA nanocarrier: a platform technology for cancer treatment

Bäumer, Nicole; Tiemann, Jessica; Scheller, Annika; Meyer, T.; Wittmann, Lisa; Suburu, Matias Ezequiel Gutierrez; Greune, Lilo; Peipp, Matthias; Kellmann, Neele; Gumnior, Annika; Brand, Caroline; Hartmann, Wolfgang; Müller-Tidow, Carsten; Neri, Dario; Strassert, Cristian A.; Rüter, Christian; Dersch, Petra; Lenz, Georg; Koeffler, H. Phillip; Berdel, Wolfgang E.; Bäumer, Sebastian

doi:10.1038/s41388-022-02241-w

Cited by 24 publications

(20 citation statements)

References 22 publications

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“…To this end, we have previously obtained a stable complex comprised of the internalizing anti-EGFR-antibody cetuximab bound to cationic protamine and the anionic siRNA against KRAS and PIK3CA that specifically enters colorectal cancer cells [3][4][5]. Parallel to this study, we observed that these complexes form suprastructures that resemble micelles and act as nanocarriers [10]. As a modular part of our nanocarrier system, siRNA can be designed and synthetized against any oncogenic factor and we here propose a targeted siRNAtransport system composed of an anti-CD33-antibody, protamine and siRNA that efficiently targets the leukemia-related oncogenes DNMT3A and FLT3.…”

Section: Introductionmentioning

confidence: 70%

“…1B). As depicted here, we found out that antibodies coupled with SMCC-protamine form vesicular structures in the presence of free SMCC-protamine when they were complexed with anionic cargo molecules such as siRNA (see below in this manuscript and [10]) or anionic ibrutinib-Cy3.5 [6]. Therefore, we abbreviate the empty carrier components as αCD33-mAB-P/P.…”

Section: Development Of An Aml-specific Sirna Carrier Systemmentioning

confidence: 93%

“…Human AML cell lines KG1 (ACC 14; KRAS-G12D), OCI-AML2 (ACC 99; homozygous DNMT3A-R635W), MV4-11 (FLT3-ITD/MLL-AF4) and OCI-AML3 (ACC 582; DNMT3A-R882C, NPM1c-mut, homozygous NRAS-Q71L) and the DLBCL cell line OCI-Ly19 (NRAS-Q61K; all DSMZ, Braunschweig, Germany) were maintained in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin and penicillin at cell densities of 0.2 -1 × 10 6 cells/ml and incubated at 37 °C with 5% CO 2 and high humidity. The EGFR-positive NSCLC cell line A549 was treated with antibody-siRNA-complexes as described previously [10] and cultivated in DMEM supplemented with 10% FBS and 1% penicillin and streptomycin.…”

Section: Cell Culturementioning

confidence: 99%

“…03231, Stemcell Technologies, Vancouver) in 96-wells (OCI-AML2, OCI-Ly19 and KG1) or 48-wells (OCI-AML3 and MV4-11) for 5-7 days. Colony formation in soft agar for A549 cells was performed as described previously [10].…”

Section: Colony Forming Assaysmentioning

confidence: 99%

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Electrostatic anti-CD33-antibody–protamine nanocarriers as platform for a targeted treatment of acute myeloid leukemia

et al. 2022

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Background Acute myeloid leukemia (AML) is a fatal clonal hematopoietic malignancy, which results from the accumulation of several genetic aberrations in myeloid progenitor cells, with a worldwide 5-year survival prognosis of about 30%. Therefore, the development of more effective therapeutics with novel mode of action is urgently demanded. One common mutated gene in the AML is the DNA-methyltransferase DNMT3A whose function in the development and maintenance of AML is still unclear. To specifically target “undruggable” oncogenes, we initially invented an RNAi-based targeted therapy option that uses the internalization capacity of a colorectal cancer specific anti-EGFR-antibody bound to cationic protamine and the anionic siRNA. Here, we present a new experimental platform technology of molecular oncogene targeting in AML. Methods Our AML-targeting system consists of an internalizing anti-CD33-antibody–protamine conjugate, which together with anionic molecules such as siRNA or ibrutinib-Cy3.5 and cationic free protamine spontaneously assembles into vesicular nanocarriers in aqueous solution. These nanocarriers were analyzed concerning their physical properties and relevant characteristics in vitro in cell lines and in vivo in xenograft tumor models and patient-derived xenograft leukemia models with the aim to prepare them for translation into clinical application. Results The nanocarriers formed depend on a balanced electrostatic combination of the positively charged cationic protamine-conjugated anti-CD33 antibody, unbound cationic protamine and the anionic cargo. This nanocarrier transports its cargo safely into the AML target cells and has therapeutic activity against AML in vitro and in vivo. siRNAs directed specifically against two common mutated genes in the AML, the DNA-methyltransferase DNMT3A and FLT3-ITD lead to a reduction of clonal growth in vitro in AML cell lines and inhibit tumor growth in vivo in xenotransplanted cell lines. Moreover, oncogene knockdown of DNMT3A leads to increased survival of mice carrying leukemia patient-derived xenografts. Furthermore, an anionic derivative of the approved Bruton’s kinase (BTK) inhibitor ibrutinib, ibrutinib-Cy3.5, is also transported by this nanocarrier into AML cells and decreases colony formation. Conclusions We report important results toward innovative personalized, targeted treatment options via electrostatic nanocarrier therapy in AML.

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Section: Introductionmentioning

confidence: 70%

Section: Development Of An Aml-specific Sirna Carrier Systemmentioning

confidence: 93%

Section: Cell Culturementioning

confidence: 99%

Section: Colony Forming Assaysmentioning

confidence: 99%

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Electrostatic anti-CD33-antibody–protamine nanocarriers as platform for a targeted treatment of acute myeloid leukemia

et al. 2022

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“…Due to their numerous advantages, nanocarriers have gained significant interest for drug delivery in the past decade . They have the ability to protect siRNA from enzymatic degradation and improve cellular penetration, and they can be synthesized or assembled to have uniform sizes and shapes to improve cellular delivery. , Many nanocarriers have been reported to carry siRNA in different types of cancers and effectively knock down the gene of interest. − In this study, graphene oxide (GO), lipid nanoparticles (LNPs), and boron nitride nanotubes (BNNTs) as nanocarriers are compared for the delivery of CD47_siRNA in cancer cells and their knockdown efficiency values are monitored.…”

Section: Introductionmentioning

confidence: 99%

Elimination of Cancer Cells in Co-Culture: Role of Different Nanocarriers in Regulation of CD47 and Calreticulin-Induced Phagocytosis

Hassan

McWhirter

Walker

et al. 2023

ACS Appl. Mater. Interfaces

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Under healthy conditions, pro-and anti-phagocytic signals are balanced. Cluster of Differentiation 47 (CD47) is believed to act as an anti-phagocytic marker that is highly expressed on multiple types of human cancer cells including acute myeloid leukemia (AML) and lung and liver carcinomas, allowing them to escape phagocytosis by macrophages. Downregulating CD47 on cancer cells discloses calreticulin (CRT) to macrophages and recovers their phagocytic activity. Herein, we postulate that using a modified graphene oxide (GO) carrier to deliver small interfering RNA (siRNA) CD47 (CD47_siRNA) in AML, A549 lung, and HepG2 liver cancer cells in co-culture in vitro will silence CD47 and flag cancer cells for CRT-mediated phagocytosis. Results showed a high knockdown efficiency of CD47 and a significant increase in CRT levels simultaneously by using GO formulation as carriers in all used cancer cell lines. The presence of CRT on cancer cells was significantly higher than levels before knockdown of CD47 and was required to achieve phagocytosis in co-culture with human macrophages. Lipid nanoparticles (LNPs) and modified boron nitride nanotubes (BNPs) were used to carry CD47_siRNA, and the knockdown efficiency values of CD47 were compared in three cancer cells in co-culture, with an achieved knockdown efficiency of >95% using LNPs as carriers. Interestingly, the high efficiency of CD47 knockdown was obtained by using the LNPs and BNP carriers; however, an increase in CRT levels on cancer cells was not required for phagocytosis to happen in co-culture with human macrophages, indicating other pathways' involvement in the phagocytosis process. These findings highlight the roles of 2D (graphene oxide), 1D (boron nitride nanotube), and "0D" (lipid nanoparticle) carriers for the delivery of siRNA to eliminate cancer cells in co-culture, likely through different phagocytosis pathways in multiple types of human cancer cells. Moreover, these results provide an explanation of immune therapies that target CD47 and the potential use of these carriers in screening drugs for such therapies in vitro.

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Towards targeted Cas9 (CRISPR‐Cas) delivery: Preparation of IgG antibody‐Cas9 conjugates using a split intein

Pasch,

Bäumer,

Bäumer

et al. 2024

Journal of Peptide Science

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The CRISPR‐Cas9 system has revolutionized the field of genetic engineering, but targeted cellular delivery remains a central problem. The delivery of the preformed ribonuclease‐protein (RNP) complex has the advantages of fewer side effects and avoidance of potential permanent effects. We reasoned that an internalizing IgG antibody as a targeting device could address the delivery of Cas9‐RNP. We opted for protein trans‐splicing mediated by a split intein to facilitate posttranslational conjugation of the two large protein entities. We recently described the cysteine‐less CL split intein that efficiently performs under oxidizing conditions and does not interfere with disulfide bonds or thiol bioconjugation chemistries. Using the CL split intein, we report for the first time the ligation of monoclonal IgG antibody precursors, expressed in mammalian cells, and a Cas9 precursor, obtained from bacterial expression. A purified IgG‐Cas9 conjugate was loaded with sgRNA to form the active RNP complex and introduced a double‐strand break in its target DNA in vitro. Furthermore, a synthetic peptide variant of the short N‐terminal split intein precursor proved useful for chemical modification of Cas9. The split intein ligation procedure reported here for IgG‐Cas9 provides the first step towards a novel CRISPR‐Cas9 targeting approach involving the preformed RNP complex.

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Targeted siRNA nanocarrier: a platform technology for cancer treatment

Cited by 24 publications

References 22 publications

Electrostatic anti-CD33-antibody–protamine nanocarriers as platform for a targeted treatment of acute myeloid leukemia

Electrostatic anti-CD33-antibody–protamine nanocarriers as platform for a targeted treatment of acute myeloid leukemia

Elimination of Cancer Cells in Co-Culture: Role of Different Nanocarriers in Regulation of CD47 and Calreticulin-Induced Phagocytosis

Towards targeted Cas9 (CRISPR‐Cas) delivery: Preparation of IgG antibody‐Cas9 conjugates using a split intein

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