Bioorthogonal CRISPR/Cas9-Drug Conjugate: A Combinatorial Nanomedicine Platform

Abstract

Bioconjugation of proteins can substantially expand the opportunities in biopharmaceutical development, however, applications are limited for the gene editing machinery despite its tremendous therapeutic potential. Here, a self-delivered nanomedicine platform based on bioorthogonal CRISPR/Cas9 conjugates, which can be armed with a chemotherapeutic drug for combinatorial therapy is introduced. It is demonstrated that multi-functionalized Cas9 with a drug and polymer can form self-condensed nanocomplexes, and induce significant gene editing upon delivery while avoiding the use of a conventional carrier formulation. It is shown that the nanomedicine platform can be applied for combinatorial therapy by incorporating the anti-cancer drug olaparib and targeting the RAD52 gene, leading to significant anti-tumor effects in BRCA-mutant cancer. The current development provides a versatile nanomedicine platform for combination treatment of human diseases such as cancer.

Keywords: CRISPR/Cas9; bioorthogonal; cancer therapy; chemotherapeutic drugs; combinatorial delivery; gene editing; nanomedicines; unnatural amino acids.

Figure 1

Schematic for the combinatorial nanomedicine platform based on bioorthogonal CRISPR/Cas9 conjugates. a) Development of ComBiNE based on the conjugation of Cas9‐AzF with drug molecules and carrier polymer (CP), and subsequent complexation with sgRNA, forming nano‐assembled RNP complexes. b) Production and selection process of Cas9‐AzF variants. Selected candidate Tyr and Phe positions for AzF incorporation are shown in colored boxes according to the Cas9 domain.

Figure 2

Generation of ComBiNE based on self‐condensation of CRISPR/Cas9 conjugates. a) Schematic for bioconjugation of Cas9‐AzF with either olaparib (Cas9‐Olap), CP (Cas9‐CP), or both olaparib and CP (Cas9‐Olap‐CP), and subsequent complexation with sgRNA, resulting in Cas9 conjugate RNPs. Particularly, Cas9‐Olap‐CP RNPs spontaneously undergo nano‐assembly to form ComBiNE. b) Characterization of Cas9‐AzF variants for different AzF positions by SDS‐PAGE (top), and by reaction with DBCO‐BODIPY to confirm AzF incorporation (bottom). c) Summary showing purification yields, AzF incorporation efficiencies, and cleavage activities of the Cas9‐AzF variants. d–g) Physicochemical characterization of ComBiNE and Cas9 conjugates prepared with the Y373 variant. d) DLS and e) zeta potential measurements of Cas9 conjugates in presence and absence of sgRNA (molar ratio of Cas9:sgRNA = 1:1). f) AFM and g) TEM analyses of ComBiNE and control formulations.

Figure 3

Delivery of ComBiNE for gene editing in vitro. ComBiNE and control formulations were formed using conjugates of the Y373 variant, and complexation with sgRNA targeting RAD52 (molar ratio of Cas9:sgRNA = 1:1). a) Confocal microscopy of BRCA1‐mutant HCC1937 breast cancer cells treated with ComBiNE or control formulations for 4 h at 500 nM Cas9 (blue: DAPI, green: F‐actin, red: Cas9 labeled with AF647; scale bar: 50 µm). b) Quantification of uptake efficiencies by image analysis of (a). c) sgRNA design for the RAD52 gene (blue: sgRNA sequence, black: target sequence of RAD52 gene, green: PAM sequence, red arrowhead: cleavage site). d) Cleavage activities examined by treating Cas9 conjugate RNPs to RAD52 target DNA, and analysis of cleaved DNA fragments. e) Gene editing efficiencies, and f) representative sequence data (green: PAM, ‐: deletion, red: insertion, WT: wild‐type) of HCC1937 cells treated with ComBiNE and control formulations for 48 h at 500 nM Cas9, and targeted deep sequencing analysis. For (e), indel types with frequency values of > 0.5% for at least one formulation are shown in different colors in the stacked bars.

Figure 4

Functional validation of CRISPR/Cas9‐drug conjugates for combination treatment in cancer cells in vitro. a) Schematic showing the principle of synthetic lethality induced by Cas9‐Olap conjugate RNPs, based on the function of olaparib and RAD52 gene editing in BRCA‐mutant cancer. b) Characterization of Cas9‐Olap conjugates prepared with the various Cas9‐AzF variants: olaparib conjugation efficiencies, cleavage activities, and indel frequencies. c) Representative sequence data by targeting deep sequencing of HCC1937 cells treated with Cas9‐Olap RNPs complexed with CMAX for 48 h at 500 nM Cas9. d–f) Combinatorial effect of treating Cas9‐Olap RNPs prepared with the Y373 variant, on anti‐proliferation of cells using conventional carriers for 72 h (500 nM Cas9; n = 3, One‐way ANOVA, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001). d) Effect of Cas9‐Olap RNPs prepared by conjugation of olaparib via azido (Cas9‐Olap1), sulfhydryl (Cas9‐Olap2), and both azido and sulfhydryl (Cas9‐Olap3) groups, complexed with CMAX and treated to HCC1937 cells. e,f) Comparison of the anti‐proliferation effect in HCC1937 and MDA‐MB‐231 cells using e) CMAX, and f) TAT peptide (molar ratio of Cas9:TAT = 1:10) as the carrier. Concentration of free olaparib was adjusted to the conjugated olaparib concentration for the corresponding Cas9‐Olap conjugate in each group. Cas9‐Olap conjugates were complexed with RAD52 sgRNA at a molar ratio of 1:1.

Figure 5

Evaluation of ComBiNE as a self‐delivered nanomedicine platform in cancer cells in vitro. a) Schematic showing the different forms of the ComBiNE platform and delivery to cells. ComBiNE can be prepared from conjugation of Cas9‐AzF with olaparib and CP via the azido and sulfhydryl groups, respectively (Cas9‐Olap1‐CP2), or vice versa (Cas9‐Olap2‐CP1). b,c) Anti‐proliferation effect upon treatment of ComBiNE and control formulations including RAD52 or control sgRNA (NT: non‐target) to HCC1937 cells for 72 h (n = 3, One‐way ANOVA, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001). b) Demonstration of the synergistic effect of ComBiNE compared to Cas9‐CP and Cas9‐Olap conjugate RNPs. c) Comparison of ComBiNE prepared from Cas9‐Olap1‐CP2 and Cas9‐Olap2‐CP1 conjugates. d–f) Functional evaluation of ComBiNE (Cas9‐Olap1‐CP2). d) Western blot analysis showing the expression of RAD52, e) apoptosis levels measured by Annexin V assay, and f) DNA damage measured by the alkaline comet assay, in HCC1937 cells treated with ComBiNE for 48 h (One‐way ANOVA, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001). ComBiNE formulations were prepared with the Y373 variant, and RAD52 sgRNA (molar ratio of Cas9:sgRNA = 1:1), followed by treatment at 500 nM Cas9.

Figure 6

In vivo delivery and efficacy of ComBiNE in a BRCA‐mutant breast cancer model. a) Schematic of the timeline for tumor implantation and treatment of ComBiNE. b) Tumor growth curve based on daily measurements of tumor volumes (n = 6–8, ^**** p < 0.0001), and c) average body weights of mice, from day 0 to day 24. d) Tumor images and (e) tumor weights (n = 6, ^**** p < 0.0001) measured after sacrificing mice on day 24. f) Gene editing efficiencies (n = 2, ^** p < 0.01, ^*** p < 0.001) and g) representative sequence data (green: PAM, ‐: deletion, red: insertion) by analyses of tumor tissues after sacrificing mice on day 5. Examination of h) RAD52 expression by western blot analysis, i) apoptosis levels by the Annexin V assay, and j) cellular DNA damage by the comet assay of excised tumor tissues on day 24 (^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001). ComBiNE (Cas9‐Olap1‐CP2) formulations were prepared with conjugates of the Y373 variant and either RAD52 sgRNA or control (NT) sgRNA (molar ratio of Cas9:sgRNA = 1:1), and locally administered at 25 µg of Cas9 per injection. One‐way ANOVA was performed to obtain P values and determine statistical significance.