Simple In Vivo Gene Editing

Subscriber access provided by UNIV OF DURHAM

Article

Simple In Vivo Gene Editing via Direct Self-Assembly of Cas9 Ribonucleoprotein Complexes for Cancer Treatment Seung Min Kim, Sang Chul Shin, Eunice EunKyeong Kim, SangHeon Kim, Kwideok Park, Seung Ja Oh, and Mihue Jang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b01670 • Publication Date (Web): 20 Jul 2018 Downloaded from http://pubs.acs.org on July 20, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

A manuscript for ACS NANO as an article Simple In Vivo Gene Editing via Direct Self-Assembly of Cas9 Ribonucleoprotein Complexes for Cancer Treatment

Seung Min Kima, Sang Chul Shina, Eunice EunKyeong Kima, Sang-Heon Kimb, Kwideok Parkb, Seung Ja Ohb*and Mihue Janga*

a

Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and

Technology, Seongbuk-Gu, Seoul 136-791, South Korea b

Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science and

Technology, Seongbuk-Gu, Seoul 136-791, South Korea

* The author to whom correspondence: Mihue Jang, Ph.D. Korea Institute of Science and Technology 5, Hwarang-ro 14-gil, Seongbuk-gu, Seoul 02792, Republic of Korea Tel: +82-2-958-6618; fax: +82-2-958-5909 e-mail: [email protected]

* The author to whom correspondence: Seung Ja Oh, Ph.D. Korea Institute of Science and Technology 5, Hwarang-ro 14-gil, Seongbuk-gu, Seoul 02792, Republic of Korea Tel: +82-2-958-5353; fax: +82-2-958-5398 e-mail: [email protected]

ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Cas9 ribonucleoprotein (RNP)-mediated delivery has emerged as an ideal approach for in vivo applications. However, the delivery of Cas9 RNPs requires electroporation or lipid- or cationic-reagent-mediated transfection. Here, we developed a carrier-free Cas9 RNP delivery system for robust gene editing in vivo. For simultaneous delivery of Cas9 and a guide RNA into target cells without the aid of any transfection reagents, we established a multifunctional Cas9 fusion protein (Cas9-LMWP) that forms a ternary complex with synthetic crRNA:tracrRNA hybrids in a simple procedure. Cas9-LMWP carrying both an NLS and a low molecular-weight protamine (LMWP) enables the direct self-assembly of a Cas9:crRNA:tracrRNA ternary complex (a ternary Cas9 RNPs) and allows for the delivery of the ternary Cas9 RNPs into the recipient cells, owing to its intrinsic cellular and nuclear translocation ability with low immunogenicity. To demonstrate the potential of this system, we showed extensive synergistic anti-KRAS therapy (CI value: 0.34) via in vitro and in vivo editing of the KRAS gene by the direct delivery of multifunctional Cas9 RNPs in lung cancer. Thus, our carrier-free Cas9 RNP delivery system could be an innovative platform that might serve as an alternative to conventional transfection reagents for simple gene editing and highthroughput genetic screening.

Key words: CRISPR/Cas9, genome editing, low molecular-weight protamine (LMWP), cancer therapy, in vivo gene editing.

ACS Paragon Plus Environment

Page 2 of 26

Page 3 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

RNA-programmed Cas9 ribonucleoproteins (Cas9 RNPs) have been simplified and adopted for mammalian gene editing with two small RNAs. A CRISPR RNA (crRNA) targets a desired genomic sequence, and a small transactivating crRNA (tracrRNA) binds to the crRNA and Cas9.1-3 For genome editing, a chimeric single-guide RNA (sgRNA) combines the crRNA and tracrRNA.4 The use of the CRISPR-Cas9 system with purified Cas9 RNPs provides an innovative platform for highly efficient genome editing with fewer off-target cleavages than occur with plasmid- or viral-mediated delivery of Cas9 and sgRNAs,5 because the Cas9 RNP system directly introduces RNA or protein into the cells without requiring additional steps, such as transcription and translation. Typically, Cas9 RNP-mediated delivery to target cells is carried out via lipid-mediated transfection or electroporation.3, 6-10 According to a previous report, a cationic lipid-mediated delivery of Cas9 RNPs with sgRNA achieved up to a 20% genome modification in the mouse inner ear in vivo when it was complexed in 50% RNAiMAX or Lipofectamine 2000.1 Recently, the local delivery of an engineered Cas9 with multiple SV40 nuclear localization sequences has been demonstrated for gene editing in the mouse brain in vivo.9 Nevertheless, there are still challenges in Cas9 RNP-mediated in vivo editing.11 Notably, Cas9 RNPs have no intracellular delivery activity, and thus, their direct complexation and cellular internalization in vivo are necessary through conjugation with polycationic polymers or lipid carriers, for which there remain several limitations with regard to the release of payloads into the cytoplasm, nuclear localization, and safety concerns.12 As the most common cause of cancer-related death, non-small cell lung cancer (NSCLC) accounts for 80% of all lung cancers.13 NSCLC is a heterogeneous disease that can be further classified into three major subtypes, including adenocarcinoma, squamous cell carcinoma (SCC) and large cell carcinoma, on the basis of their histologies, which show distinct pathological characteristics.14 A number of genetic, epigenetic and signaling alterations underlie the development of lung cancers. Recently, the third generation EGFR inhibitor osimertinib has been approved for patients with metastasis resulting from a T790M mutation in EGFR.15 The development of the second generation ALK-targeting drugs is ongoing. Although KRAS is considered one of the most promising targets in NSCLC, there are still no FDA approved drugs targeting KRAS.16 Thus, the development of an effective anti-KRAS therapy in NSCLC is necessary.

ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

RESULTS AND DISCUSSION To demonstrate the potential of in vivo genome editing in KRAS mutant cancer as an effective-KRAS therapy in NSCLC, we developed a one-step Cas9 RNP system, denoted a ternary Cas9 RNPs, that uses a multifunctional Cas9 fusion protein (Cas9-LMWP) with synthetic mature crRNA:tracrRNA hybrids (dual RNAs) (Figure. 1). Furthermore, to prove the potential of this platform as an innovative delivery carrier for gene editing, we also developed a synergistic anti-cancer strategy by combining therapeutic genome editing directed against KRAS with a MEK inhibitor to inhibit multiple signaling transductions involving the PI3K/AKT and RAF/MEK/ERK pathways (Figure. 1).

Figure 1. Scheme of a ternary Cas9 RNP-mediated gene editing for cancer therapeutics. The strategy of the synergistic anti-KRAS therapy combined with the ternary Cas9 RNP-induced KRAS disruption and the MEK inhibitor AZD6244. The engineered Cas9-LMWP fusion protein is an RNA-programmable nuclease with multiple complexation reagent abilities and cell penetrating and nuclear translocation properties. The ternary Cas9 RNP enables the selfassembly of a ternary complex of a multifunctional Cas9-LMWP fusion protein and synthetic mature crRNA:tracrRNA hybrids (dual RNAs) by an electrostatically driven interaction and directly targets the recipient cells. The highly cationic LMWP in the Cas9-LMWP functions as a cell penetrating peptide. Consequently, the ternary Cas9 RNP delivers the Cas9 nuclease and dual RNAs into the nucleus, owing to the existence of the NLS in the Cas9-LMWP protein.

A low molecular weight protamine (LMWP), the non-toxic membrane translocon

ACS Paragon Plus Environment

Page 4 of 26

Page 5 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

peptide,is a nature-sourced cell penetrating peptide that might be a powerful tool for in vivo macromolecular delivery.17 We hypothesized that LMWP might provide its intrinsic benefits to Cas9 by conjugating LMWP to Cas9. Therefore, we generated a Cas9 fusion protein, Cas9-LMWP, expressing both the nuclear localization sequence (NLS) and LMWP. The NLS mediates its nuclear localization for functional editing,18 and LMWP, with high arginine amino acids, enables the self-assembly of a ternary complex (Cas9-LMWP/crRNA/tracrRNA) via electrostatically driven interactions and cellular internalization (Figure. 2a-2c and Supporting Information, Figure. S1a).19 Cas9 has a bi-lobed architecture composed of a recognition (REC) lobe and a nuclease (NUC) lobe, with a central channel formed between the two lobes.20-21 Therefore, the positively charged LMWP attaches to the C-terminus of the Cas9 and is accessible as seen in Figure. 2b and 2c. Thus, a Cas9 expressing both an NLS and LMWP has multifunctional abilities with regard to its functions as a complexing agent and as the delivery carrier itself. To prove the electrostatically driven self-assembly of the ternary Cas9 RNPs, a gel mobility shift assay was performed by using incremental amounts of heparin as a cationic competitor (Figure. 2d). The high dose of heparin (over 2 mg/mL) abolished the interaction of Cas9-LMWP with the dual RNAs. Next, we measured the zeta potential and size distribution of the ternary Cas9 RNPs by using dynamic light scattering (DLS) and a Zetasizer, respectively (Figure. 2e and Figure. S2a). After the self-assembly of the ternary Cas9 RNPs, their surface net charge increased from -30 mV to -4 mV and exceeded the 1:1 ratio of the dual RNAs and Cas9-LMWP. Thus, highly enriched cationic LMWP on Cas9LMWP self-assembled with anionic dual RNAs regardless of the addition of any cationic polymers. Interestingly, when the dual RNAs were increased, while the Cas9-LMWP concentration remained constant, the size distribution of the complexation showed a relative decrease. The complex of the dual RNAs and Cas9-LMWP at a 1:1 ratio showed a larger size distribution than that at a 1:5 ratio. We utilized this observation to precisely control the size of the ternary complex at defined ratios of Cas9-LMWP and dual RNAs for the following experiments. Next, the size and morphology of the ternary Cas9 RNPs at given two ratios were evaluated by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) (Figure. 2f-2h and Figure. S2b). SEM, TEM and AFM images revealed the homogenous self-assembly of the ternary Cas9 RNPs. At a ratio of 1:1, the complex was approximately 159 nm, whereas at a 1:5 ratio, it was approximately 89 nm, based on the SEM analysis. The self-assembled complexation at a 1:5 ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ratio was more densely packed than at the 1:1 ratio. Thus, we confirmed that the size of the complexation with the Cas9-LMWP and the dual RNAs could be manipulated by using precisely defined ratios. In addition, the Cas9-LMWP was more efficiently associated with the dual RNAs at 37°C (Figure. S3). To test the ternary Cas9 RNP-induced immunogenicity in vitro, we measured the release of TNF-α or IFN-α, which induces Toll-like receptor (TLR) immune responses at 6 h post treatment from the cells with various treatments by ELISA.22 Compared with the treatments with a conventional transfection reagent, LF2000, the treatment with Cas9-LMWP alone or the ternary Cas9 RNPs produced markedly lower amounts of TNF-α or IFN-α (Figure. 2i). Lipopolysaccharide (LPS) or CpG-oligonucleotide (ODN) served as a positive control for TNF-α or IFN-α induction, respectively. Additionally, we investigated the immunogenicity of the Cas9-LMWP in vivo by intravenous injection of the Cas9-LMWPinto immunocompetent mice (Figure. S4). Intravenous injection of Cas9 or Cas9-LMWP barely induced INF-α even at two different doses, whereas the injection of CpG ODN as a positive control triggered high amounts of INF-α. Thus, the engineered Cas9LMWP fusion protein with high arginine amino acids can be considered as a safe carrier.

ACS Paragon Plus Environment

Page 6 of 26

Page 7 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 2. Characterization of the self-assembled ternary Cas9 RNPs.(a)The generation of the multifunctional Cas9-LMWP fusion protein. The NLS and LMWP are located in the C terminus. (b) A model of the chimeric Cas9-LMWP complexed with the dual RNAs. LMWP is shown in red and is fused to the C-terminus of Cas9, which is shown in a grey molecular surface presentation. The crystal structure of Cas9 from Streptococcus pyogenes complexed with dual RNAs and a partially duplexed target DNA (PDB code: 4UN3) was used.20 (c) The electrostatic surface potential of Cas9 with the dual RNAs and target DNA. (d) The electrostatically driven complex formation of a Cas9-LMWP fusion protein and dual RNAs was validated by a gel mobility shift assay. Six incremental doses of heparin (0.5 – 5 mg/mL), as a cationic competitor, were added. Heparin, at over 2 mg/mL, abolished the interaction of the Cas9-LMWP and the dual RNAs. (e) The zeta potentials (red lines) and the size distributions (blue lines) of the resulting complexes, shown at the indicated ratios, were determined with a Zetasizer and DLS, respectively. (f-h) The morphologies, size distributions

ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

and size homogeneity of the ternary Cas9 RNPs at the two given ratios were investigated by SEM (f), TEM (g) and AFM (h) analyses. (f) The lower panels indicate magnified images from the upper panels. Notably, the sizes of complexes of Cas9-LMWP and dual RNAs can be controlled at precisely defined ratios. The black and white bars represent 5 µm and 100 nm, respectively. (i) The immunogenicity was determined by the detection of the release of TNFα or IFN-α at 6 h post-treatment with the various formulations. The Cas9-LMWP or the ternary Cas9 RNPs caused less immunogenicity, and the Cas9 RNPs complexed with LF2000 increased the induction of large amounts of cytokines. *P< 0.05, **P