Study Of Protamine-conjugated Human PSMA Antibody To Deliver TRIM24 SiRNA In The Treatment Of CRPC

Objective:Previously,our collaborator OriMAbs company obtained a PSMA specific single-chain antibody fragment(scFv)(named gy1)from a large yeast-display naive human scFv library which specifically recognizes the extracellular domain of PSMA.Then,we reconstructed this scFv into a fully human antibody(named PSMAb)and demonstrated that PSMAb could specifically bind with and internalize into PSMA~+prostate cancer cells.TRIM24,whose expression was upregulated in both primary prostate cancer(PCa)and CRPC,could promote the proliferation of CRPC cells under low androgen condition through activating AR signaling,indicating that TRIM24 could be an ideal therapeutic target in CRPC.In this study,we examined the efficacy of the PSMAb-based platform for the targeted delivery of TRIM24 siRNA and its therapeutic effect in CRPC both in vitro and in vivo.Methods:1.Perparation of PSP and PSPS.2.Gel shift and fluorometric assay were performed to detect the siRNA payload and release efficacy of PSMAb-sulfo-SMCC-protamine(PSP)complexes.3.FCM and IF analyses were performed to detect the binding and internalized ability of PSP and PSP-FAM siRNA(PSPS)complex in CRPC cells in vitro.4.FLI was performed to detect the distribution and siRNA target delivery capability of PSP and PSPS in vivo.5.qRT-PCR,WB,and IF assays were performed to detect the silence effects of PSP-TRIM24 siRNA complex on TRIM24 expression in vitro and in vivo.6.CCK-8,colony formation assay,FCM,wound healing and Transwell assays were performed to detect the effects of PSP-TRIM24 siRNA complex on cell proliferation,colony formation,apoptosis,cell cycle,migration and invasion of CRPC cells in vitro.7.BLI,x-ray,micro CT assays were performed to detect the effects of PSP-TRIM24siRNA complex on tumor growth and bone loss in vivo.TUNEL and Ki-67 staining was performed to detect the effects of PSP-TRIM24 siRNA complex on cell apoptosis and cell proliferation in vivo.8.Body weight analyses,liver and kidney function analyses,and HE staining were performed to examine the bio-safety of PSP-TRIM24 siRNA complexes after repeated injection.Results:1.Gel shift assay showed that PSP encapsulated siRNA duplexes and released them in the cytoplasm.Fluorometric assay showed that each PSP molecule can bind with approximately 5 FAM-labeled siRNAs.Gel shift assay and fluorometric assay indicated that PSP could protect siRNA from enzymatic digestion.2.Flow cytometry and immunofluorescence staining analyses showed that PSP can specifically bind with and internalized into PSMA~+prostate cancer cells,but not PSMA~-cells.In addition,PSPS can specifically deliver siRNA to PSMA~+cells.3.ICG labeled PSP and PSPS diffused rapidly throughout the whole body at 6 h after intravenous injection,then they were gradually cleared from the body,but still specifically retained in PSMA~+tumor tissues even at 96 h after injection.Fluorescence imaging study confirmed that ICG-labeled PSP and PSPS specifically distributed in PC-3-PSMA~+tumor tissues but not PC-3 tumor tissues.In addition,with ICG-labeled PSP and PSPS could also be observed in metabolic organs,such as liver and kidneys,and organs with rich blood supply,such as lung and spleen.4.Both TRIM24 and PSMA expression were upregulated and their expressions were positively correlated in prostate cancer.5.TRIM24 expression was significantly downregulated in C4-2 cells incubated with PSP-TRIM24 siRNA complex,compared with the control group.Cell proliferation and colony formation of C4-2 cells incubated with PSP-TRIM24 siRNA complex was significantly inhibited,while cell apoptosis increased and more cells were arrested at G1phase.In addition,cell migration and invasion were significantly inhibited in C4-2 cells incubated with PSP-TRIM24 siRNA complex.6.In PC-3-PSMA~+xenograft model,compared with control group,TRIM24expression was significantly downregulated in PSP-TRIM24 siRNA complex treated mice.And slower tumor growth,smaller tumor volume,lighter tumor weight,more TUNEL fluorescent signal,and less tense Ki-67 fluorescent signal was observed in mice receiving PSP-NC and PSP-TRIM24 siRNA complex than the mice receiving IgG-P-TRIM24siRNA or PBS.PSP-TRIM24 siRNA complex treated group showed better therapeutic effect than PSP-NC group.H&E staining showed that morphological abnormalities can be observed in PC-3-PSMA~+xenograft mice receiving PSP-TRIM24 siRNA complex.7.In CRPC bone metastasis model,cancer-induced bone loss was significantly alleviated in mice receiving PSP-NC and PSP-TRIM24 siRNA complex,compared with IgG-P-TRIM24 siRNA or PBS treated groups.And PSP-TRIM24 siRNA complex treated group showed better alleviation than PSP-NC group.8.After receiving intravenous injection of PSP-TRIM24 siRNA for 5 times,mice body weight,liver and kidney function,and morphology of major organs did not show significant changes.Conclusion:We demonstrated that PSPS could protect siRNA from enzymatic digestion,and specifically bind with and internalized into PSMA~+prostate cancer cells both in vitro and in vivo.Furthermore,we found that inhibition of TRIM24 expression by PSP-TRIM24siRNA complex inhibited cell proliferation,migration and invasion,and induced apoptosis and cell cycle arrest in PSMA~+CRPC cells in vitro.We also found that PSP-TRIM24siRNA complex inhibited tumor growth of PSMA~+CRPC xenografts through apoptosis induction and cell proliferation inhibition.More significantly,PSP-TRIM24 siRNA complex inhibited bone loss in the bone metastasis model of PSMA~+CRPC.