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. 2018 Feb 23;9:203-210.
doi: 10.1016/j.omtm.2018.02.009. eCollection 2018 Jun 15.

A Non-integrating Lentiviral Approach Overcomes Cas9-Induced Immune Rejection to Establish an Immunocompetent Metastatic Renal Cancer Model

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Free PMC article

A Non-integrating Lentiviral Approach Overcomes Cas9-Induced Immune Rejection to Establish an Immunocompetent Metastatic Renal Cancer Model

Junhui Hu et al. Mol Ther Methods Clin Dev. .
Free PMC article

Abstract

The CRISPR-based technology has revolutionized genome editing in recent years. This technique allows for gene knockout and evaluation of function in cell lines in a manner that is far easier and more accessible than anything previously available. Unfortunately, the ability to extend these studies to in vivo syngeneic murine cell line implantation is limited by an immune response against cells transduced to stably express Cas9. In this study, we demonstrate that a non-integrating lentiviral vector approach can overcome this immune rejection and allow for the growth of transduced cells in an immunocompetent host. This technique enables the establishment of a von Hippel-Lindau (VHL) gene knockout RENCA cell line in BALB/c mice, generating an improved model of immunocompetent, metastatic renal cell carcinoma (RCC).

Keywords: CRISPR/Cas9; RENCA; ccRCC; immunocompetent mouse model; immunotherapy; kidney cancer; non-integrated lentivirus.

Figures

Figure 1
Figure 1
Generation of Cas9-Transduced, Non-integrating Cells (A) Schematic representation of lentiCRISPR with gRNA targeting murine VHL downstream of a U6 promoter. Cells were derived by infection with this lentivirus packaged with either wild-type or mutated integrase. Other elements of the virus include tandem long terminal repeat sequences (LTR), Psi signal, central polypurine tract (cPPT), EFS promoter, Cas9 sequence, P2A element, puromycin resistance gene (Puro), and WPRE. (B) Cas9 gene expression in RC clonal lines were assessed by RT-PCR. The expression of NIL-generated clones RC(NIC1), RC(NIC2), and RC(NIC3) relative to the integrated lentiCRISPR-generated line, RC1, is shown. The Cas9 expression in RC1 is normalized to 1. (C) Western blot for Cas9 in RENCA, RC1, and RC(NIC1) cells. Cas9 protein is shown for positive control in the left lane. (D) Light microscopic images of RC1 and RC(NIC1) cells at 10× magnification. (E) qRT-PCR analysis of genes in RC1 and RC(NIC1) cells.
Figure 2
Figure 2
Using Non-integrating Lentivirus Allows for Growth of lentiCRISPR-Modified Cells in an Immunocompetent BALB/c Host (A) Growth curves for subcutaneous tumors implanted with RENCA, RC(NIC1), and RC1 cells over 20 days in BALB/c mice. n = 4 per group. (B) Images of gross tumors from three representative animals in each group are shown. The RC1 tumor in the fourth animal completely regressed. (C) Representative H&E images of tumors from each of three groups in both 4× and 20× magnification are shown. (D) Representative images of immunohistochemistry staining of CD3 in RC(NIC1) and RC1 tumors (**p < 0.01).
Figure 3
Figure 3
Generation of Clonally Selected, Non-integrating, VHL Knockout RENCA Cells (A) Cas9 gene expression levels in seven clonal, non-integrating RVN lines (RVN(NIC1–7)) were assessed by RT-PCR. The expression level in an RC1 integrated lentiCRISPR generated line is set at 1. (B) The DNA sequences in the mVHL coding region from RC(NIC1) and RVN(NIC5) are shown with the reference sequence. A 10-base-pair deletion within the region targeted by our VHL gRNA was found in RVN(NIC5) cells, but not in RC(NIC1). Please note: from here on, the RC(NIC1) clonal line will be designated as VHL-WT and the RVN(NIC5) clonal line as VHL-KO. (C) Representative images of VHL-WT and VHL-KO cells under light microscopy with 10× fields. (D) Cell proliferation assay and (E) western blot for VHL, HIF-1α, E-cadherin, and β-actin in VHL-WT and VHL-KO cells. Gene expression was assessed by RT-PCR for (F) Glut-1, (G) E-cadherin, and (H) N-cadherin (*p < 0.05, **p < 0.01, ***p < 0.001). (I) Either VHL-WT or VHL-KO cells were implanted into the subcapsular space of the left kidney in NU/J nude female 5-week-old mice. VHL-WT tumors grew well, while VHL-KO tumors grew poorly, as monitored by firefly-luciferase-based in vivo bioluminescence imaging. No lung metastasis was seen in both tumor groups.
Figure 4
Figure 4
A VHL-Knockout, Immunocompetent, Metastatic RCC Model Intrarenal tumors were established in BALB/c mice with 106VHL-WT cells or a 1:4 ratio of VHL-WT and VHL-KO cells. (A) BLI was used to detect firefly-luciferase-expressing tumors in vivo. Three representative tumor-bearing mice in each group at the endpoint 6 weeks after tumor implantation were shown. (B) Gross images and unmagnified H&E stain of harvested organs, including the primary kidney tumor and contralateral normal kidney, as well as lung and heart from both groups, were shown. (C) Higher magnification H&E stains of primary tumors and lungs were shown. (D) Immunohistochemical stain of CD3 in the primary tumor to identify infiltrating T lymphocytes in both groups. (E) Immunohistochemistry staining of CD31 depicting the blood vessels within the primary tumor were performed. Whole-section scanning was performed by Applied Imaging Leica Aperio Versa high-throughput scanning system, and quantification was performed by Definiens’ Tissue Studio at TPCL of UCLA. The angiogenesis was evaluated by CD31 positivity area within tumor region and denoted by % as shown in the right. (*p < 0.05)

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