Letters Cite This: ACS Chem. Biol. XXXX, XXX, XXX−XXX
Generation of Optogenetically Modified Adenovirus Vector for Spatiotemporally Controllable Gene Therapy Kazuo Takayama*,†,‡,§ and Hiroyuki Mizuguchi*,†,§,∥ †
Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan ‡ PRESTO, Japan Science and Technology Agency, Saitama 332-0012, Japan § Laboratory of Hepatocyte Regulation, National Institute of Biomedical Innovation, Health and Nutrition, Osaka 567-0085, Japan ∥ Global Center for Medical Engineering and Informatics, Osaka University, Osaka 565-0871, Japan S Supporting Information *
ABSTRACT: Gene therapy is expected to be utilized for the treatment of various diseases. However, the spatiotemporal resolution of current gene therapy technology is not high enough. In this study, we generated a new technology for spatiotemporally controllable gene therapy. We introduced optogenetic and CRISPR/Cas9 techniques into a recombinant adenovirus (Ad) vector, which is widely used in clinical trials and exhibits high gene transfer efficiency, to generate an illumination-dependent spatiotemporally controllable gene regulation system (designated the Opt/Cas-Ad system). We generated an Opt/Cas-Ad system that could regulate a potential tumor suppressor gene, and we examined the effectiveness of this system in cancer treatment using a xenograft tumor model. With the Opt/Cas-Ad system, highly selective tumor treatment could be performed by illuminating the tumor. In addition, Opt/Cas-Ad system-mediated tumor treatment could be stopped simply by turning off the light. We believe that our Opt/Cas-Ad system can enhance both the safety and effectiveness of gene therapy.
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engineering modalities.9−14 Gersbach and his colleagues developed a light-activated CRISPR-Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of blue light.14 This is accomplished by fusing the light-inducible heterodimerizing proteins Cryptochrome-2 (CRY2) and calcium and integrin-binding protein 1 (CIB1) to a transactivation domain and the catalytically inactive dCas9, respectively.14 Sato and his colleagues developed an engineered photoactivatable Cas9 (paCas9) that enables optogenetic control of CRISPR/Cas9 genome editing.12 To apply these attractive technologies to the clinical treatment of disease, it will be necessary to design an optogenetically controlled CRISPR/ Cas9 system that can be carried in a vector suitable for use in clinical investigation. Human Ad vectors are widely used in gene therapy for cancer and genetic disorders.1−3 Compared with episomal, lentivirus, and retrovirus vectors, Ad vectors can perform more highly efficient gene transfer into a wider spectrum of dividing and nondividing cells both in vitro and in vivo. In addition,
ecently, the effectiveness of adenovirus (Ad)-based gene therapy in cancer and genetic disorder treatments has been proven in various clinical trials.1−3 However, the spatiotemporal resolution of current Ad-based gene therapy technology is not high enough. Even when Ad vectors are injected intratumorally, it is not easy to avoid leaky expression into the nontarget cells and organ,4,5 which, in turn, can lead to unexpected adverse reactions, such as hepatotoxicity. Although it is known that Ad vector system does not integrate the intended transgene into cellular genome, it is difficult to stop the transgene expression at the intended timing if when unexpected adverse reaction occurs by the transgene expression. Taken together, there is need of a new technology to spatiotemporally control gene therapy so that it is safer and more effective. Optogenetics is a technology that allows targeted and fast control of precisely defined events in biological systems.6 Genome engineering technologies, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated 9 (Cas9) system,7,8 have enabled not only doublestrand break-mediated genome editing but also regulation of endogenous gene expression and epigenome status at a desired position.9 However, the spatiotemporal resolution of genome engineering technologies is not high enough. Recently, several groups have introduced optogenetic technologies into genome © XXXX American Chemical Society
Special Issue: Chemical Biology of CRISPR Received: December 11, 2017 Accepted: January 9, 2018
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DOI: 10.1021/acschembio.7b01058 ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
ACS Chemical Biology
Figure 1. Generation of the Opt/Cas-Ad system. (A) Schematic diagram of the Opt/Cas-Ad system. The Opt/Cas-Ad system consists of AdK7CIBN-dCas9-CIBN and AdK7-CMV-CRY2-VP64-U6-gRNAs. CIBN: N-terminal region of CIB1, dCas9: catalytically inactive Cas9, AdK7: fibermodified adenovirus, CRY2: Cryptochrome 2, VP64: VP64 transactivation domain, hU6: human U6 promoter, gRNA: guide RNA. Note that AdK7CMV-CRY2-VP64-U6-gRNAs contain four gRNA-expressing cassettes targeting the same genomic loci. (B) Schematic diagram of the Opt/Cas-Ad vector-mediated gene expression activation system. (C−G) The PC-3 cells were transduced with 3000 VP/cell of AdK7-CIBN-dCas9-CIBN and 3000 VP/cell of AdK7-CMV-CRY2-VP64-U6-gRNAs, and cultured for 24 h. The transduced PC-3 cells were cultured in the dark or under blue light illumination for 48 h. In panel (C), the gene expression levels of Dkk-3 were examined by real-time RT-PCR. The dCas9-VP64-expressing Ad vector was used as a positive control. The gene expression level in the dark group was taken as 1.0. Statistical significance was evaluated by one-way ANOVA followed by Tukey’s posthoc tests to compare all groups. Groups that do not share the same letter are significantly different from each other (p < 0.05). In panel (D), the temporal gene expression profiles of Dkk-3 were examined by real-time RT-PCR under blue light illumination. In panel (E), after the blackout, the temporal gene expression profiles of Dkk-3 were examined by real-time RT-PCR. The gene expression level at “0 h” was taken as 1.0. In panel (F), the temporal Dkk-3 protein expression profiles were examined by Western blotting under blue light illumination. In panel (G), the temporal Dkk-3 protein expression profiles were examined by ELISA under blue light illumination. In panels (C), (D), (E), and (G), the results are shown as the mean ± SE (n = 3). In panel (F), the images represent typical results from three independent measurements.
vectors containing the RGD or a stretch of lysine residue (K7) peptides on the fiber knob (AdRGD or AdK7, respectively), which were able to perform efficient gene transfer, irrespective of the CAR expression levels.16−18 In this study, we generated a fiber-modified Ad vector carrying a light-activated CRISPR-Cas9 effector (LACE) system to generate an illumination-dependent spatiotemporally controllable gene regulation system, which we designated the Opt/Cas-Ad system. The Opt/Cas-Ad system will be available
conventional Ad vectors based on human Ad type 5 can package ∼8.1−8.2 kbp of foreign DNA, versus 4.5 kbp for adeno-associated virus (AAV) vectors. Thus, Ad vectors can carry large expression cassettes such as SpCas9 (4.2 kbp without a promoter and pA signal). However, one of the limitations in the use of Ad vectors for gene transfer is the inefficient gene transfer into cells that lack one of the adenovirus receptors: the coxsackievirus and adenovirus receptor (CAR).15 We previously generated improved Ad B
DOI: 10.1021/acschembio.7b01058 ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
ACS Chemical Biology
Figure 2. Spatiotemporally controllable gene therapy using the Opt/Cas-Ad system. (A) PC-3 cells were transduced with 3000 VP/cell of AdK7CIBN-dCas9-CIBN and 3000 VP/cell of AdK7-CMV-CRY2-VP64-U6-gRNAs, and cultured for 24 h. The transduced PC-3 cells were cultured in the dark or under blue light illumination for 48 h. The percentage of TUNEL-positive cells was examined by FACS. The results are shown as the mean ± SE (n = 3). Statistical significance was evaluated by unpaired two-tailed Student’s t-tests (** indicate p < 0.01). (B−E) PC-3 xenograft tumors in nu/nu mice were intratumorally administered 2.5 × 109 VP/mouse of AdK7-CIBN-dCas9-CIBN and 2.5 × 109 VP/mouse of AdK7CMV-CRY2-VP64-U6-gRNAs at day −1. The tumor was illuminated by blue light for 6 h/day (from day 0 to 7). In panel (B), at day 2 after the illumination, the protein expression levels of Dkk-3 and TUNEL in the tumor were examined by immunostaining. The images represent typical results from three independent measurements. In panel (C), the mean volumes of tumors were estimated from the diameters in five nude mice in each group. The results are shown as the mean ± SE (n = 5). Statistical significance was evaluated by two-way repeated ANOVA followed by Tukey’s posthoc tests. At 2, 3, and 4 weeks after the illumination, the mean tumor volumes of “light” were higher than those of other groups (* denotes p < 0.05, ** denotes p < 0.01). The experimental design of panels (E) and (F) is summarized in panel (D). In panel (E), at day 2 after the illumination, human and mouse Dkk-3 mRNA expression levels in the tumor and liver, respectively, were examined by real-time RT-PCR. The ratio of Dkk-3 mRNA to the Ad genome copy number is shown in panel (F). In panels (E) and (F), the results are shown as the mean ± SE (n = 5). Statistical significance was evaluated by one-way ANOVA, followed by Bonferroni’s posthoc tests (** denotes p < 0.01: compared with “Opt/Cas-Ad+dark” or “Ad-LacZ”). (G) After Ad transduction into the tumor, the tumor was illuminated with blue light for 2 days, and then the mice were kept under dark conditions. The temporal gene expression levels of Dkk-3 mRNA in the tumor were examined by real-time RT-PCR (“day −1”, before Ad vectors transduction; “day 0”, before blue light illumination; “day 2”, after blue light illumination; “day 4”, dark condition). The results are shown as the mean ± SE (n = 5). Statistical significance was evaluated by one-way ANOVA followed by Tukey’s posthoc tests. Groups that do not share the same letter are significantly different from each other (p < 0.01). In panel (H), the global gene expression profiles were compared between illuminated and nonilluminated Opt/Cas-Ad-transduced PC3 cells. In panel (I), the off-target effect of the Opt/Cas-Ad system was examined. The gene expression levels of the potential off-target sites, which were predicted by sequence similarity of up to four mismatches with the on-target site, were examined. C
DOI: 10.1021/acschembio.7b01058 ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
ACS Chemical Biology
recombinant Ad vector. The CIBN-dCas9-CIBN- and Cry2VP64-U6-gRNAs-expressing cassettes were mounted into AdK7 (Figure 1A). We previously developed a fiber-modified Ad vector, AdK7, that contained a stretch of lysine residue (K7) peptides which target heparan sulfates on the cellular surface.18 The efficient transduction of various cancer cells and stem cells could be performed by using AdK7 vectors.18−20 Generally, high-grade cancer cells express only low levels of CAR.15 In this study, we confirmed that the expression level of CAR in the high-grade prostate cancer cell line PC-3 was significantly lower than that in the low-grade prostate cancer cell line LNCaP (Figure S1A in the Supporting Information). The AdK7 vector showed higher gene transfer efficiency compared with the conventional nonfiber modified ad vector, even in the highgrade cancer cell line PC-3 (Figure S1B). By transducing AdK7CIBN-dCas9-CIBN and AdK7-CMV-CRY2-VP64-U6-gRNAs, the gRNAs would direct the binding of dCas9 to the intended promoter region under both light and dark conditions. However, gene expression is upregulated only in the presence of blue light, because VP64 co-localizes with dCas9 via CRY2CIBN interactions (Figure 1B). Next, we checked the operation of the Opt/Cas-Ad system. It is known that Ad vector-mediated Dkk-3 gene transfer inhibits the growth and metastasis of prostate cancer.23,24 In this study, we generated an Opt/Cas-Ad system that can spatiotemporally regulate Dkk-3 expression. The Dkk-3 mRNA expression level was increased by blue light illumination (Figure 1C) as well as positive control (dCas9-VP64). The Dkk-3 mRNA expression level continued to increase, in response to blue light illumination for 48 h (Figure 1D), and then reached a plateau. The Dkk-3 mRNA expression level was dramatically decreased by turning off the light (Figure 1E). In agreement with this finding, the Dkk-3 protein expression level was increased by turning on the blue light (Figure 1F). In addition, the amount of Dkk-3 protein secreted into the culture medium was also increased by blue light illumination (Figure 1G). We also confirmed that multiplex control of four different target genes could be performed by using the Opt/Cas-Ad system (Figure S2 in the Supporting Information). In addition, the target gene expression level was enhanced by using two gRNAs targeting different genomic loci of Dkk-3 than using only single gRNA targeting a genomic locus of Dkk-3 (Figure S3 in the Supporting Information). These results suggest that the gene expression could be spatiotemporally controlled by using the Opt/Cas-Ad system. We next examined whether spatiotemporally controlled gene therapy could be achieved by using an Opt/Cas-Ad system, since this system was found to spatiotemporally regulate Dkk-3 expression. The percentage of TUNEL-positive cells among the Opt/Cas-Ad-transduced PC-3 cells was significantly increased by blue light illumination (Figure 2A). Next, we verified the usefulness of the Opt/Cas-Ad system by using a PC-3 xenograft tumor model. After intratumoral injection of the Opt/Cas-Ad system, the signal intensities of Dkk-3 and TUNEL were increased by blue light illumination (Figure 2B). These results suggest that the Dkk-3 expression induction-mediated apoptosis of PC-3 cells was successfully induced by blue light illumination. When the tumor was grown to ∼5−6 mm in diameter, the mice were intratumorally injected with Opt/CasAd vectors at a dose of 5 × 109 VP/mouse. The growth of subcutaneous PC-3 tumors in the mice was efficiently suppressed by blue light illumination (Figure 2C). These
as a new technology for spatiotemporally controllable gene therapy.
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MATERIALS AND METHODS
Ad Vector. Ad vectors were prepared as follows. PacI-digested pAdK7-CIBN-dCas9-CIBN (CIBN: N-terminal fragment of calcium and integrin-binding protein 1, dCas9: catalytically inactive Cas9) and pAdK7-CMV-CRY2-VP64-U6-gRNAs (CMV: CMV promoter, Cry2: Cryptochrome-2, VP64: VP64 activation domain, U6: human U6 promoter) were transfected into HEK293 cells by using Lipofectamine 2000 (Thermo Fisher Scientific), resulting in AdK7-CIBN-dCas9CIBN and AdK7-CMV-CRY2-VP64-U6-gRNAs, respectively. These Ad vectors were amplified and purified by two rounds of cesium chloride gradient ultracentrifugation, dialyzed, and stored at −80 °C. AdK7 contains a stretch of lysine residue (K7) peptides in the Cterminal region of the fiber knob for efficient transduction of various cancer cells and stem cells.18−21 The vector particle (VP) titer was determined by using a spectrophotometric method.22 gRNA Design. The gRNA targeting sequence for both human and mouse Dkk-3 was 5′-GTTCCGGGACACACAGGCGG-3′ (used in Figures 1 and 2). The gRNA targeting sequences for human p53, IL12A, IL12B, and Dkk-3 were 5′-GCAGGTAGCTGCTGGGCTCC3′, 5′-GAAAGCGCCGCAAGCCCCGC-3′, 5′-GGGCAGGACGGAGAGTCCAA-3′, and 5′-GATCGCGTTCCGGGACACAC-3′, respectively (used in Figure S2). The gRNA × 2 targeting sequences for human Dkk-3 were 5′-GATCGGGGCTCGGGCTGGAG-3′ and 5′GTTCCGGGACACACAGGCGG-3′ (used in Figure S3). The sequence of the chimeric guide RNA expression cassette was obtained from the plasmid #42230 (Addgene). Tandem expression cassettes of the gRNAs were synthesized by using a Standard GENE SYNTHESIS service (GENEWIZ). To predict the off-target candidate sequences, the potential off-target sites, which were predicted by sequence similarity up to four mismatches with the on-target site, were extracted by using Cas-OFFinder (v2.2). Illumination. Cells were illuminated by using an ultrahigh-power light-emitting diode (LED) (Prizmatix). Blue light was measured by using a Photodiode Power Sensors (PD300) held directly above the LEDs (∼15 mW/cm2). At 24 h after the transduction, the cells were illuminated from the bottom of the dish. Under dark conditions, cellcontaining plates were wrapped in aluminum foil. Opt/Cas-Ad Vector Treatment of Xenograft Tumors. PC-3 cells (5 × 106 cells in 50 μL PBS) were mixed with 50 μL Matrigel (BD Biosciences) and were injected subcutaneously into the flanks of 8-week-old female BALB/c nu/nu mice (Japan SLC, Inc.). When the tumors were grown to ∼5−6 mm in diameter, the mice were intratumorally injected with AdK7-CIBN-dCas9-CIBN and AdK7CMV-CRY2-VP64-U6-gRNAs at a dose of 5 × 109 VP/mouse. At 24 h after the transduction, illumination of the tumors was initiated. Because blue light does not readily penetrate tissue, only tumors, but not other organs, are illuminated by blue light. The tumors were measured every week, and the tumor volume was calculated by using the following formula:
tumor volume (mm 3) = A × B2 × 3.14 × 6−1 where A is the longest diameter and B is the shortest. The PC-3 xenograft tumors were resected three days after administration (two days after illumination), followed by homogenization. Total DNA, including the viral genome, was extracted from the whole-tumor homogenates by using a DNeasy Blood and Tissue Kit (Qiagen). After the isolation, the Ad genome copy number was quantified as described above. These experiments were approved by the Animal Experiment Committee of the National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN).
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RESULTS AND DISCUSSION In this study, we attempted to generate a new technology for spatiotemporally controllable gene therapy. We introduced the light-activated CRISPR-Cas9 effector (LACE) system14 into a D
DOI: 10.1021/acschembio.7b01058 ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
ACS Chemical Biology
Unlike the blue light, near-infrared (NIR) light (650−1450 nm) penetrates deep into the tissues. In this study, we showed that our Opt/Cas-Ad system has the potential to be utilized in cancer gene therapy. Currently, oncolytic viruses are being used more frequently than replication-incompetent viral vectors for cancer gene therapy. A safer and more effective cancer gene therapy could be achieved by equipping oncolytic viruses with an illuminationdependent spatiotemporally controllable gene expressioninducing system. Although many issues remain to be addressed before practical application of our Opt/Cas-Ad system, the system has the potential to dramatically increase the safety and effectiveness of gene therapy.
results indicated that the Opt/Cas-Ad system could be utilized for spatiotemporally controlled gene therapy. Next, we examined whether the Opt/Cas-Ad system can regulate gene expression at a high spatiotemporal resolution. It is known that Ad vectors have a tendency to accumulate in the liver.25 In the Opt/Cas-Ad-transduced PC-3 xenograft mice, higher levels of Ad genome were found in the liver than in the other organs, such as the spleen, lung, and heart (Figure S4 in the Supporting Information). To examine whether the Opt/ Cas9-Ad system can induce illuminated tissue-specific Dkk-3 expression, Dkk-3 expression levels and Ad genome copy numbers in the tumor, liver, and isolated hepatocytes of the Opt/Cas-Ad-transduced PC-3 xenograft mice were measured in the presence or absence of blue light illumination (Figure 2D). The Dkk-3 expression levels (Figure 2E) and ratio of Dkk-3 mRNA to Ad genome (Figure 2F) in the liver were significantly lower than those in the tumor and isolated hepatocytes, which were directly illuminated in vitro. These results suggest that the Dkk-3 expression level was not enhanced, although the Ad vector was accumulated in the liver. This might be because only tumors, but not other organs, including the liver, were illuminated by the blue light, which does not readily penetrate tissue. In addition, we also confirmed that the Dkk-3 expression levels were rapidly decreased by turning off the blue light (Figure 2G). Taken together, these results showed that the Opt/Cas-Ad system can regulate gene expression at a high spatiotemporal resolution. Finally, we examined the off-target effect of the Opt/Cas-Ad system. The global gene expression analysis indicated that there were no significant gene expression alterations except for Dkk-3 and its target genes such as cancerassociated genes (Figure 2H). The gene expression levels of the potential off-target sites, which were predicted by sequence similarity of up to four mismatches with the on-target site, were examined by real-time RT-PCR (Figure 2I). There was no unexpected gene expression induction of the potential off-target sites. We have developed an illumination-dependent spatiotemporally controllable gene expression inducing system: the Opt/ Cas-Ad system. Our system is capable of carrying multiple types of gRNA sequences to concurrently control the expression of multiple genes. It is expected that illuminationdependent repression of gene expression or histone acetylation would be performed by using a Krüppel-associated box (KRAB)26 or histone acetyltransferases (such as p300),27 respectively, rather than VP64. Moreover, optogenetic genome editing could be performed by using split Cas9 fragments12 in place of dCas9. Although genome editing technologies are expected to be utilized for the treatment of genetic disorders, the constitutive expression of nucleases, such as Cas9, runs a risk of unexpected genome editing at a nontarget site. Therefore, optogenetic genome editing technologies might be a useful technique for safer genome editing in humans. Blue light does not readily penetrate tissue. Although, in the present study, blue light was successfully transmitted into the subcutaneous space, it might be impossible to transmit blue light into the deep tissue regions. Recently, a tiny blue LED capable of generating blue light of ∼250 μm in diameter was developed.28,29 Such a small blue LED could be attached to a catheter, allowing the illumination of deep tissues, such as the prostate and liver. Moreover, the targeted deep tissues could also be illuminated by using up-conversion particles,30 which convert different invisible infrared (IR) light into blue light.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.7b01058. Efficient gene transfer into high-grade prostate cancer using AdK7 vectors (Figure S1); regulation of multiple gene expressions by the Opt/Cas-Ad system (Figure S2); Dkk-3 mRNA expression regulation using an Opt/CasAd system carrying multiple gRNA expression cassettes (Figure S3); distribution of the Ad genome after intratumoral injection of the Opt/Cas-Ad system (Figure S4); supporting information and methods; supplemental references (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*Tel.: +81-6-6879-8187. Fax: +81-6-6879-8186. E-mail:
[email protected]. *Tel.: +81-6-6879-8185. Fax: +81-6-6879-8186. E-mail:
[email protected]. ORCID
Kazuo Takayama: 0000-0003-1562-3924 Funding
This research was supported by JST’s PRESTO funding program (Grant No. JPMJPR1687). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Y. Hagihara, N. Mimura, and A. Sakamoto for her excellent technical support. We also thank for Y. Yamaguchi (KCONNEX, Kyoto University) and E. Ono (K-CONNEX, Kyoto University) for helpful discussion.
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REFERENCES
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DOI: 10.1021/acschembio.7b01058 ACS Chem. Biol. XXXX, XXX, XXX−XXX
Letters
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DOI: 10.1021/acschembio.7b01058 ACS Chem. Biol. XXXX, XXX, XXX−XXX
