Cancer is caused by a series of alterations in genome and epigenome and exists in multiple complex forms, making it difficult to be prevented and/or treated. Telomerase, an enzyme responsible for the maintenance of telomere, is silent in most normal somatic cells but activated in 90% of cancer cells, making it an excellent target for cancer therapy. Therefore, various telomerase activity inhibitors have been developed to treat cancer but all failed due to side effects. Here we acted oppositely to develop a cancer gene therapy named telomerase-activating gene expression (Tage) system by utilizing the telomerase activity in cancer cells. The Tage system consisted of an effector gene expression vector that carried a 3ʹ telomerase-recognizable stick end and an artificial transcription factor expression vector that could express dCas9-VP64 and an sgRNA targeting telomere repeat sequences. By using Cas9 as an effector gene, the Tage system effectively killed various cancer cells, including HepG2, HeLa, PANC-1, MDA-MB-453, A549, HT-29, SKOV-3, Hepa1-6, and RAW264.7, without affecting normal cells MRC-5, HL7702, and bone marrow mesenchymal stem cell (BMSC). More importantly, a four-base 3ʹ stick end produced by the homothallic switching endonuclease in cells could be recognized by telomerase, allowing the Tage system to effectively kill cancer cells in vivo. The Tage system could effectively and safely realize its in vivo application by using adeno-associated virus (AAV) as gene vector. The virus-loaded Tage system could significantly and specifically kill cancer cells in mice by intravenous drug administration without side effects or toxicity.
The human telomeres consist of many repetitive nucleotide sequence (5′-TTAGGG-3′), protecting, positioning and replicating chromosomes [1, 2]. The human telomere has a length of many kilo-base pairs . Eukaryotic telomeres terminate with a 3′ single-stranded overhang that is bound by multiple protein complex, known as shelterin , which is essential for telomere maintenance and capping , and protects the linear chromosome ends from being recognized as double-strand breaks . The inability of eukaryotic DNA replication machinery to replicate the extreme end of chromosomes allows cells to gradually shorten telomeres . Along with ongoing cell division, almost all normal human cells undergo progressive telomere shortening. Once telomeres reach a critically shortened length, it will induce a DNA damage signal that is often referred to as replicative senescence or cell aging . This mechanism appears to prevent genomic instability and cancer development in human aged cells by limiting the number of cell division. Too short telomeres have the potential to unfold from their presumed closed structure, lead to genomic instability, chromosome loss, and might increase cancer susceptibility .
In order to repair the damaged telomere in some special cells, a specialized protein named as telomerase has been developed. Telomerase is a natural RNA-containing enzyme that can synthesize the repetitive telomere sequences , which helps to maintain the integrity of genome in some cells such as embryonic stem cells . Telomerase is responsible for elongating telomeres through the addition of telomere repeats to the ends of chromosomes . Only about 5%−10% of human cancers employ alternative lengthening of telomeres (ALT) pathway . Rarely, cells emerge from crisis immortalized through telomere lengthening by either activated telomerase or ALT [13, 14]. During human development, telomerase activity is terminated by silencing of telomerase reverse transcriptase . Except in a small number of normal cell types such as dividing male germ-line spermatocytes, some types of stem cells and certain white blood cells, telomerase activity is silenced in most normal tissue cells. However, telomerase activity is reactivated in most human cancer cells [16, 17], which provides a potential target to cancer therapy [18, 19]. Many telomerase inhibitors have been therefore developed to treat cancers; however, none of them becomes drugs due to side effects [19,20,21].
With the finding of Clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR therapeutics provided a potential cure for cancers and other genetic diseases . CRISPR is an RNA-guided immune defense system of bacteria and archaea to protect themselves from exogenous viruses or plasmids . On the presence of a protospacer-adjacent motif (PAM) on the opposite strand, single guide RNA (sgRNA) can guide CRISPR-associated protein 9 (Cas9) to a target site for cleavage , resulting in DNA double-strand breaks [25, 26]. Due to advantages such as versatility, simplicity, specificity, and efficiency, the CRISPR/Cas9 system has been widely applied for genome editing and holds tremendous promise for biomedical research [27,28,29].
In this study, by combining the biological functions of telomerase and CRISPR/Cas9, we developed a new cancer gene therapy named telomerase-activating gene expression (Tage). By utilizing telomerase activity, an effector gene could be specifically expressed in cancer cells by Tage. By using Cas9 as an effector gene, cancer cells could be induced to death by Tage. By using adeno-associated virus (AAV) as vector, in vivo tumors could be treated by Tage.
Principle of cancer therapy with Tage
Tage is a new telomerase- and CRISPR/Cas9-based cancer therapy (Fig. 1). In this therapy, an effector gene expression vector (hereinafter referred as to effector) carrying a telomerase-recognizable 3ʹ single-stranded sequence (3ʹ-TT GGGATT GGGATT-5ʹ) is transfected into cancer cells so that it can be elongated by telomerase, which will produce a synthesized double-stranded telomere repeat sequence at the end of effector. Simultaneously, a vector expressing nuclease-dead Cas9 (dCas9)-VP64 and a telomeric DNA-targeting sgRNA (TsgRNA) is co-transfected into cancer cells, which will produce dCas9-VP64-TsgRNA complex, an artificial transcription factor (TF), that can bind with the newly synthesized telomeric DNA at the end of effector. This binding can thus activate the expression of effector gene, Cas9, on effector. The expressed Cas9 protein can associate with TsgRNA to form Cas9-TsgRNA complex. This complex can then damage chromosomes of cancer cells by cutting their telomeres, which will induce the death of cancer cells. However, in normal tissue cells lack of telomerase activity, Tage cannot be activated and thus exerts no effect on normal cells.
Verification of Tage using ZsGreen as effector gene
To verify the effect of Tage system, we first transfected cells with a Tage system that used ZsGreen as effector gene (sTMEP) (Fig. 2a). The results indicated that the ZsGreen was successfully expressed in telomerase-positive cells 293T and HepG2 (Fig. 2b), but not in telomerase-negative cells MRC-5 and HL7702 (Fig. 2b). Moreover, in all cells transfected with various DNAs as controls, the ZsGreen protein was not expressed (Fig. S1). Especially, even though the 293T and HepG2 cells were transfected with a blunt-ended effector (bTMEP) together with TsgRNA and dCas9-VP64 expression vectors, the ZsGreen protein still could not be expressed (Fig. S1). These results revealed that the Tage system could only be activated by telomerase in cancer cells. Without the single-stranded telomerase-recognizable telomere sequence at the end of effector (bTMEP), the telomeric DNA could not be synthesized. These results also suggested that the newly synthesized telomeric DNA at the end of effector was double-stranded as previously testified ; otherwise, the effector gene also could not be expressed in cancer cells because dCas9-VP64-TsgRNA complex only binds with double-stranded DNA.
DNA synthesis at the end of effector
In order to verify that telomeric DNA was synthesized at the end of effector, we extracted the genomic DNA (gDNA) from cells transfected by the Tage system and detected by polymerase chain reaction (PCR) using the TS and CX primers of telomere repeat amplification protocol (TRAP) . The results revealed that the gDNA of 293T and HepG2 transfected with DNAs containing sTMEP could be amplified, and the products appeared as smear in agarose gel electrophoresis (Fig. 2c, lanes 1 and 2). However, the gDNA of MRC-5 and HL7702 transfected with the same vectors could not be amplified (Fig. 2c, lanes 1 and 2). Additionally, the gDNAs of cells transfected by a blunt-ended effector (bTMEP) also could not be amplified (Fig. 2c, lane 3). These results indicated that the single-stranded telomeric sequence at the end of effector can only be elongated by telomerase in cancer cells.
Verification of Tage using Cas9 as effector gene
We then used Cas9 as effector gene of Tage system to kill cancer cells by cutting their telomeres (Fig. 3a). It was conceived that the TsgRNA can guide Cas9 protein to the telomeres of chromosomes, where Cas9 can cut telomeric DNA and induce cell death. Cells were transfected with an effector expressing Cas9 (sTMCP) together with vectors expressing TsgRNA and dCas9-VP64. The results revealed that the Tage system induced the significant death of all cancer cells (Fig. 3b, c). However, the Tage system exerted no effects on normal cells MRC-5 and HL7702 (Fig. 3b, c). Importantly, even though cancer cells were transfected with an effector with 3ʹ blunt-end (bTMCP), their viability was not affected (Fig. S2). In addition, all control transfections also did not induce the death of both cancer and normal cells (Fig. S2).
To further confirm the effect of Tage system to kill cancer cells, we performed more transfections and continuously detected the cell proliferation and viability at 24 h, 48 h and 72 h post transfection. The results indicated that the proliferation and viability of all cancer cells gradually decreased with the increase of cultivation times (Fig. 4 and Fig. S3–S14), showing the most significant cancer cell killing effect of Tage system at 72 h post transfection. Similarly, all control transfections did not affect the proliferation and viability of all transfected cells at any time points (Fig. 4 and Fig. S3–S14). Importantly, both Tage and control transfections did not affect the proliferation and viability of a stem cell, BMSC (Fig. 4 and Fig. S15).
We next quantitatively measured the relative telomere length of all transfected cells at 24 h post transfection with qPCR. The results indicated that the cotransfection of sTMCP & TsgRNA & dCas9-VP64 resulted in the significant shortening of telomeres of all cancer cells in comparison with cells transfected with lipofectin (p < 0.05) (Fig. 5); however, this transfection did not affect the length of telomeres of stem cell BMSC and normal cells MRC-5 and HL7702 (p > 0.05) (Fig. 5). Additionally, all control transfections did not affect the length of telomeres of all transfected cells (Fig. 5). These results suggested that the Tage system killed cancer cells by cutting their telomeric DNA.
Realization of Tage system with HO
Although the linear effector with a telomerase-recognizable stick end can be easily transfected into cells in vitro using lipofectin, it cannot be transfected into cells in vivo using virus vectors. We therefore focused on addressing this issue. It was reported that a four-base overhang, 3ʹ-TTGT-5ʹ, produced by the homothallic switching endonuclease (HO) cutting its target DNA sequence could also be recognized and elongated in Saccharomyces cerevisiae cells by telomerase . To evaluate whether this four-base overhang on 3ʹ end can also be utilized in human cells by the Tage system, we constructed an effector with a stick end of 3ʹ-TTGT-5ʹ that could express reporter gene ZsGreen (HOsite-sTMEP). Cells were then cotransfected by HOsite-sTMEP and vectors expressing TsgRNA and dCas9-VP64. The results indicated that ZsGreen was successfully expressed in 293T and HepG2 cells (Fig. S16), but not expressed in MRC-5 and HL7702 cells (Fig. S16). Similarly, all control transfections did not activate the ZsGreen expression (Fig. S17). These results indicated that the four-base overhang on 3ʹ end produced by HO could also be utilized by the Tage system.
We thus constructed a vector expressing HO enzyme under the control of CMV promoter (C1-HO). It was expected that the expressed HO enzyme could cut a blunt-ended effector containing HO site (HOsite-bTMEP) to produce a stick end of 3ʹ-TTGT-5ʹ, so that it could be elongated by telomerase in cancer cells (Fig. 6a). Cells were then cotransfected by HOsite-bTMEP and vectors expressing TsgRNA, dCas9-VP64, and HO (Fig. 6b). The results indicated that ZsGreen was successfully expressed in 293T and HepG2 cells, but not expressed in MRC-5 and HL7702 cells (Fig. 6b). All control transfections did not activate the ZsGreen expression in all transfected cells (Fig. S17). These results revealed that a four-base telomerase-recognizable stick end could be produced by the intracellularly expressed HO enzyme and used by the Tage system.
In order to further verify the synthesis of telomere repetitive sequence at the stick end of effector cut by HO enzyme, gDNA was extracted from cells at 24 h post transfection and detected with PCR by using TRAP primers. The results revealed that the gDNA of 293T and HepG2 cells transfected with HOsite-bTMEP & TsgRNA & dCas9-VP64 & C1-HO, HOsite-sTMEP & TsgRNA & dCas9-VP64, and HOsite-sTMEP could be amplified, and the products appeared as smear in agarose gel electrophoresis (Fig. S18, lanes 1–3). However, the gDNA of MRC-5 and HL7702 cells transfected with the same vectors could not be amplified (Fig. S18, lanes 1–3). Additionally, the gDNAs of cells transfected with effector containing a blunt HO site (HOsite-bTMEP) could not be amplified as well (Fig. S18, lane 4). These results revealed that the HO enzyme expressed by the C1-HO vector in cells successfully cut the HO site, producing a four-base telomerase-recognizable stick end that could be elongated by telomerase in cancer cells. These data also suggested that the HO-based Tage system could be used in vivo.
Simplification of Tage system
To simplify the Tage system of above four vectors, we next constructed a plasmid TsgRNA-dCas9-VP64 that coexpressed TsgRNA and dCas9-VP64. The plasmid was testified by cotransfecting cells with sTMCP. The results indicated that this two-vector Tage system induced significant death of cancer cell HepG2 (Fig. S19), but not induced death of normal cells MRC-5 and HL7702 (Fig. S19). In addition, all control transfections also induced no cell death in all transfected cells.
With plasmid TsgRNA-dCas9-VP64, we next transfected cells with a Tage system consisting of three plasmid vectors including C1-HO, T-HOsite-TMCP, and TsgRNA-dCas9-VP64, expecting to induce cancer cell death with this Tage system (Fig. 7a). The results revealed that the cancer cell HepG2 was significantly killed by the Tage system (Fig. 7b); however, the growth of normal cells MRC-5 and HL7702 was not affected (Fig. 7b). As controls, the growth of cancer cell HepG2 was not affected by all other transfections (Fig. 7b). By verifying these three vectors, it was possible to apply the Tage system in vivo by using the virus vector.
Cancer therapy with Tage system
Finally, we explored the in vivo application of the Tage system. We firstly packaged C1-HO, T-HOsite-TMCP, and TsgRNA-dCas9-VP64 into the AAV. Then we testified the packaged rAAVs by co-transfecting cells with rAAV-TSD (an equivalent mixture of three viruses including rAAV-HO, rAAV-TsgRNA-dCas9-VP64, and rAAV-HOsite-TMCP). The results revealed that rAAV-TSD induced significant death of cancer cells HepG2 and Hepa1-6, but exerted no effects on normal cells MRC-5 and HL7702 (Fig. S20). All other control transfections did not affect the growth of all transfected cells (Fig. S20).
With the testified rAAV-TSD, we performed animal experiments. In the first animal experiment, mice were transplanted with cancer cell Hepa1-6 mixed with rAAVs. After 2 weeks, the tumors with hyperemia grew on both sides of mice transplanted with the Hepa1-6 cells mixed with rAAV-MCS (Fig. 8a and Fig. S21a), and the mean tumor size of left and right was 83 mm3 and 85 mm3, respectively (Fig. 8b). However, the tumors on mice treated with rAAV-TSD were significantly inhibited (Fig. 8a and Fig. S21b), and the mean tumor size of left and right was only about 14 mm3 and 13 mm3, respectively (Fig. 8b). Most tumors were eradicated in the mice treated by rAAV-TSD.
In the second animal experiment, the tumor-bearing mice were treated by intravenously injected rAAVs. As a result, the mice treated by rAAV-MCS grew large tumors on both sides (Fig. 8c and Fig. S22a), and the mean tumor size of left and right was 237 mm3 and 293 mm3, respectively (Fig. 8d). However, the mice treated by rAAV-TSD grew much smaller tumors on both sides (Fig. 8c and Fig. S22b), and the mean tumor size of left and right was only about 12 mm3 and 20 mm3, respectively (Fig. 8d). In mice treated by rAAV-TSD, most tumors were eradicated. Importantly, no mice died in the two animal experiments after rAAVs were administered, indicating the safety of rAAVs.
Finally, to further explore the cancer cell-specific expression of effector gene of the Tage system, we detected virus DNA and Cas9 mRNA in various tissues of the mice in the second animal experiment. The results indicated that the virus presented in all detected tissues with variant abundance (Fig. 8e); however, the Cas9 mRNA only expressed in tumors of mice intravenously injected with rAAV-TSD (Fig. 8f). These results indicated that the Tage system could only be activated in cancer cells, which led to cancer cell-specific expression of effector gene.
With in vitro cell tests and in vivo animal tests, we developed a novel gene therapy of cancers based on a well-known cancer target, telomerase. Contrary to the current strategy of inhibiting the telomerase activity, we utilized telomerase activity in cancer cells. In the past decades, several of the therapies introduced to treat cancer did not significantly improve mortality and the quality of life due to side effects and toxicities . With the advent of the new gene editing tool CRISPR/Cas9, it provides new therapeutic application potential. Compared to the conventional ZFN and TALEN , CRISPR/Cas9 provides a more simple gene editing and regulation tool. With its merits, CRISPR/Cas9 has been used to recapitulate cancer-associated genomic alterations both in vitro and in vivo [34, 35]. The Tage technique fully developed the functions of CRISPR/Cas9, including the intracellular double-stranded cleavage of target DNA [36,37,38], and gene expression regulation [39, 40].
In recent years, chimeric antigen receptor T-cell (CAR-T) immunotherapy has been developed. Despite CRISPR/Cas9 was reported to be used ex vivo to treat cancers by creating CAR-T cells, to our knowledge, it is still not directly used in vivo to treat cancers. We firstly applied CRISPR-based Tage system to in vivo cancer therapy. Moreover, we fully exploited the two functions of CRISPR/Cas9 in Tage system. The telomere-targeting sgRNA-guided dCas9-VP64 was used as an artificial TF to activate effector gene expression, meanwhile Cas9 was used as effector gene to cut telomere DNA. Therefore, the Tage system provides the first in vivo application of CRISPR technique in cancer gene therapy without visible side effects or toxicity in mice. This promotes the oncotological application of gene therapy when it comes of age .
The HO-based Tage system can be used to treat cancers in vivo by using AAV as gene vector. Several gene therapies had already been permitted in clinics, such as hemophilia  and spinal muscular atrophy . These gene therapies all use AAV as gene vector due to its safety . We treated the tumor-bearing mice with a single-dose intravenous administration of rAAV. Despite some unique advantages, such as no host cell genomic integration, stable transgene expression, long-term expression, low immunogenicity, AAV vector has a major shortcoming of limited packaging capacity (~5 kb). Therefore, we had to package the Tage system into three rAAVs, which may limit the applicable virus dosage and cancer cell killing effect. However, nanoparticle carriers can be used to slim the Tage system.
The Tage system had high cancer cell specificity. In the in vitro experiments, only cancer cells transfected by the Tage system showed effector gene expression. In the in vivo experiments, only cancer tissue infected by AAV showed effector gene expression. It is interesting to find that telomerase-positive 293T cells appeared to undergo no obvious cell death when treated with the Tage system. HEK 293 cell was derived from normal human embryonic kidney tissue cultured in vitro, which underwent a series of adenovirus transfection to obtain the continuously cultured 293 cell lines. In this process, telomerase was activated. However, 293T was not regarded as cancer cells. The results of 293T cell transfection revealed that the Tage system possessed high specificity to cancer cells. Further, there were some noncancer cells naturally with telomerase activity, such as stem cells and splitting epithelial cells. This study revealed that the BMSC did not show death when treated with the Tage system. This phenomenon also indicated that the Tage system had no significant effect on these noncancer cells.
Clearly, the Tage system is a versatile technical platform that is adaptable. In our view, the Tage vector is a powerful missile, whereas the effector gene is its loaded warhead. The warhead is changeable, including “nuclear warhead” like Cas9 used in this study and other “conventional warhead” like variant functional proteins or microRNAs that can lead to cancer cell apoptosis, suicide, reprogramming, and differentiation. For example, using the Tage system to express some membrane or secretary proteins intrigues immunotherapy to cancers . Besides effector gene, the artificial TF in the Tage system can also be changed into other proteins with similar function. For example, using the effector sTMEP, we demonstrated that the artificial TF dCas9-VP64 and TsgRNA used in the Tage system could be replaced by another artificial TF, transcription activator-like effector (TALE), that targeted the telomeric DNA (Fig. S23).
Materials and methods
The detailed information of vector construction was provided in supplementary methods. The linear vectors including TsgRNA, double-stranded blunt-ended TMEP (bTMEP), stick-ended TMEP (sTMEP), double-stranded blunt-ended TMCP (bTMCP), stick-ended TMCP (sTMCP), HOsite-sTMEP, HOsite-bTMEP, and HOsite-sTMCP were constructed. The plasmid vectors including T-Msps, T-TMEP, T-TMP, T-TMCP, TsgRNA-Cas9, TsgRNA-dCas9-VP64, T-HOsite-TMEP, T-HOsite-TMCP, C1-HO, pAAV-MCS, pAAV-TsgRNA-dCas9-VP64, pAAV-HOsite-TMCP, pAAV-HO, and TALE were constructed. The oligonucleotides used in vector construction and PCR amplification were shown in Tables S1–S6.
Cell culture and transfection
RAW264.7, Hepa1-6, HEK-293T, HepG2, HeLa, PANC-1, MDA-MB-453, and BMSC cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (HyClone). A549, HT-29, SKOV-3, MRC-5, and HL7702 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium (HyClone). All cell culture media were supplemented with 10% fetal bovine serum (FBS) (HyClone), 100 U/mL penicillin and 100 µg/mL streptomycin. All cell lines were obtained from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and maintained in a humidified incubator set at 37 °C and 5% CO2. Cells were seeded into 24-well plates at a density of 0.5 × 105 cells/well and cultivated overnight, then transfected with various vectors (see supplementary methods) using Lipofectamine 2000 (ThermoFisher Scientific) according to the manufacturer’s instructions. After transfection, cells were further incubated for 24 h. All transfection experiments were performed in triplicates and repeated a minimum of three times.
Telomerase-synthesized telomeric DNA detection
The gDNA was extracted from cells at 24 h post transfection using the TIANamp Genomic DNA Kit (TIANGEN) according to the manufacturer’s instructions. The gDNA was amplified with PCR using primers TS and CX. PCR detections were performed using 2× Premix Taq (TaKaRa) according to the manufacturer’s instructions with 50 ng of gDNA as template. The PCR products were analyzed in 1.5% agarose gel electrophoresis.
Cells were transfected with 100 ng of various combinations of vectors including sTMCP, bTMCP, TsgRNA, and dCas9-VP64 in 96-well plates, using Lipofectamine 2000 according to the manufacturer’s instructions. After incubation for 5 h, the medium of each well was replaced with 100 μL of fresh DMEM or RPMI 1640 medium containing 10% FBS. At 0 h, 24 h, 48 h, and 72 h post transfection, cells were then added with 10 μL of Cell Counting Kit-8 (CCK-8) (Biosharp) per well. After being cultivated for 4 h, the absorbance of each well was measured at 450 nm using BioTek plate reader (BioTek). Without transfection wells were regarded as blank wells, and the percentage of cell viability (%) was calculated by (A450 of treated wells – A450 of blank wells)/(A450 of control-treated wells – A450 of blank wells) × 100%. All experiments were carried out in triplicates and repeated a minimum of three times.
Relative telomere length detection
The gDNA was extracted from cells at 24 h post transfection using the TIANamp Genomic DNA Kit (TIANGEN) according to the manufacturer’s instructions. The relative telomere length was measured by qPCR as previously reported . The qPCR detection was performed using 2× SYBR Green Real-time PCR Master Mix (Roche) according to the manufacturer’s instructions with 20 ng of gDNA as template. A melting curve was conducted to monitor the PCR amplification. The qPCR programs were run on a StepOne Plus real-time PCR machine (Applied Biosystems). The Ct values of telomere (T) were normalized against that of single-copy internal gene β-globin (S). The relative telomere length was calculated as T/S ratio according to the equation: RQ = 2ΔCt. All experiments were carried out in triplicates and repeated a minimum of three times.
HEK-293T cells were cotransfected with 4 μg of plasmid pAAV-RC (Stratagene), 4 μg of plasmid pHelper (Stratagene), and 4 μg of one of following plasmids, including pAAV-HOsite-TMCP, pAAV-TsgRNA-dCas9-VP64, pAAV-HO, and pAAV-MCS (Stratagene) in 75-cm2 flask, by using Lipofectamine 2000 according to the manufacturer’s instructions. After 72 h, cells were scraped off and subjected to three rounds of freeze–thaw–vortex cycles to release rAAV. Virus was purified with the PEG 8000 (Sigma) method and named as rAAV-HOsite-TMCP, rAAV-TsgRNA-dCas9-VP64, and rAAV-HO, rAAV-MCS, respectively.
The titer of rAAVs was analyzed by quantitative PCR (qPCR) using primers listed in Table S6. The qPCR detection was performed using 2× SYBR Green Real-time PCR Master Mix (Roche) according to the manufacturer’s instructions. All qPCR programs were run on the ABI StepOne Plus real-time PCR system (Applied Biosystems). Melting curve analysis revealed a single PCR product. A series of dilutions of known copies of DNA fragment were simultaneously detected to construct a standard curve. All experiments were carried out in triplicates and repeated a minimum of three times.
Cells were transfected with viruses including rAAV-HOsite-TMCP, rAAV-TsgRNA-dCas9-VP64, rAAV-HO, and rAAV-MCS at the dose of 1×104 vg per cell in 24-well plates for 24 h. Cells were then stained with acridine orange (Solarbio) and photographed under a fluorescence microscope (Olympus) at a constant magnification of ×200. All experiments were carried out in triplicates and repeated a minimum of three times.
Four-week-old female nude mice (BALB/c-Foxn1nu) (weighed 18−22 g) were purchased from the Model Animal Research Center of Nanjing (Nanjing, China). All animal experiments in this study followed the guidelines and ethics of the Animal Care and Use Committee of Southeast University (Nanjing, China). In the first animal experiment, the nude mice were subcutaneously transplanted with the mixture of 1 × 107 of Hepa1-6 cells mixed with 1 × 109 vg of rAAV-MCS or rAAV-TSD on both sides of abdomen. After 2 weeks, all mice were sacrificed and photographed. In the second animal experiment, the nude mice were subcutaneously transplanted with 1 × 107 of Hepa1-6 cells on both sides of abdomen to produce the tumor-bearing mice. After 1 week, the tumor-bearing mice were randomly assigned to each treatment group and intravenously injected with 1 × 109 vg of purified rAAV-TSD and rAAV-MCS, respectively. After 1 week, all mice were sacrificed and photographed. The tumor size of all mice was measured with a precision caliper. Tumor volume was calculated with the formula V = (Dd2)/2, in which D was the major tumor axis and d was the minor tumor axis. Investigators were not blinded in the animal experiments.
Virus DNA and gene expression detection
The gDNA was extracted from various tissues of mice in the second animal experiment using TIANamp Genomic DNA Kit (TIANGEN) according to the manufacturer’s instructions. Total RNA was extracted from above tissues with Trizol® reagent (Invitrogen). The cDNA was reversely transcribed from total RNA using PrimeScriptTM RT Master Mix (TaKaRa) according to the manufacturer’s instructions. Then, AAV DNA abundance and Cas9 mRNA expression level were analyzed by qPCR using 2× SYBR Green Real-time PCR Master Mix (Roche) according to the manufacturer’s instructions. All qPCR programs were run on the ABI StepOne Plus real-time PCR system (Applied Biosystems). Melting curve analysis revealed a single PCR product. The threshold value Ct for each individual PCR product was calculated by the Applied Biosystems StepOne software v2.3, and Ct values were normalized by subtracting the Ct values obtained for GAPDH. The primers used in qPCR were provided in Table S6. The resulting ΔCt values were then used to calculate relative abundance of viral DNA or changes of Cas9 mRNA expression as relative quantity (RQ) according to the equation: RQ = 2−ΔΔCt. All experiments were carried out in triplicates and repeated a minimum of three times.
Data were expressed as mean ± standard deviation (SD) of at least three independent biological or experimental replicates, and analyzed by a two-tailed unpaired Student’s t test, using GraphPad Prism 5.0 Software. Differences at p < 0.05 were considered statistically significant.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Blackburn EH. Telomeres: do the ends justify the means? Cell. 1984;37:7–8.
Blackburn EH. Structure and function of telomeres. Nature. 1991;350:569–73.
Shampay J, Szostak JW, Blackburn EH. DNA sequences of telomeres maintained in yeast. Nature. 1984;310:154–7.
Moyzis RK, Buckingham JM, Cram LS, Dani M, Deaven LL, Jones MD, et al. A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes. Proc Natl Acad Sci USA. 1988;85:6622–6.
Williams TL, Levy DL, Maki-Yonekura S, Yonekura K, Blackburn EH. Characterization of the yeast telomere nucleoprotein core: Rap1 binds independently to each recognition site. J Biol Chem. 2010;285:35814–24.
Martinez P, Blasco MA. Role of shelterin in cancer and aging. Aging Cell. 2010;9:653–66.
Watson JD. Origin of concatemeric T7 DNA. Nat New Biol. 1972;239:197–201.
Wright WE, Shay JW. Historical claims and current interpretations of replicative aging. Nat Biotechnol. 2002;20:682–8.
Stewart SA, Bertuch AA. The role of telomeres and telomerase in cancer research. Cancer Res. 2010;70:7365–71.
Aschacher T, Wolf B, Enzmann F, Kienzl P, Messner B, Sampl S, et al. LINE-1 induces hTERT and ensures telomere maintenance in tumour cell lines. Oncogene. 2016;35:94–104.
Deng Y, Chang S. Role of telomeres and telomerase in genomic instability, senescence and cancer. Lab Invest. 2007;87:1071–6.
Henson JD, Neumann AA, Yeager TR, Reddel RR. Alternative lengthening of telomeres in mammalian cells. Oncogene. 2002;21:598–610.
Colgin LM, Baran K, Baumann P, Cech TR, Reddel RR. Human POT1 facilitates telomere elongation by telomerase. Curr Biol. 2003;13:942–6.
Colgin LM, Reddel RR. Telomere maintenance mechanisms and cellular immortalization. Curr Opin Genet Dev. 1999;9:97–103.
Maciejowski J, de Lange T. Telomeres in cancer: tumour suppression and genome instability. Nat Rev Mol Cell Biol. 2017;18:175–86.
Wright WE, Piatyszek MA, Rainey WE, Byrd W, Shay JW. Telomerase activity in human germline and embryonic tissues and cells. Dev Genet. 1996;18:173–9.
Shay JW, Wright WE. Role of telomeres and telomerase in cancer. Semin Cancer Biol. 2011;21:349–53.
Huang FW, Hodis E, Xu MJ, Kryukov GV, Chin L, Garraway LA. Highly recurrent TERT promoter mutations in human melanoma. Science. 2013;339:957–9.
Jafri MA, Ansari SA, Alqahtani MH, Shay JW. Roles of telomeres and telomerase in cancer, and advances in telomerase-targeted therapies. Genome Med. 2016;8:69.
Mosoyan G, Kraus T, Ye F, Eng K, Crispino JD, Hoffman R, et al. Imetelstat, a telomerase inhibitor, differentially affects normal and malignant megakaryopoiesis. Leukemia. 2017;31:2458–67.
Tefferi A, Lasho TL, Begna KH, Patnaik MM, Zblewski DL, Finke CM, et al. A pilot study of the telomerase inhibitor imetelstat for myelofibrosis. N Engl J Med. 2015;373:908–19.
Khan FA, Pandupuspitasari NS, Chun-Jie H, Ao Z, Jamal M, Zohaib A, et al. CRISPR/Cas9 therapeutics: a cure for cancer and other genetic diseases. Oncotarget. 2016;7:52541–52.
Horvath P, Barrangou R. CRISPR/Cas, the immune system of bacteria and archaea. Science. 2010;327:167–70.
Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 2011;471:602–7.
Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014;346:1258096.
Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339:823–6.
Bikard D, Euler CW, Jiang W, Nussenzweig PM, Goldberg GW, Duportet X, et al. Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. Nat Biotechnol. 2014;32:1146–50.
Samanta MK, Dey A, Gayen S. CRISPR/Cas9: an advanced tool for editing plant genomes. Transgenic Res. 2016;25:561–73.
Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L, et al. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell. 2014;156:836–43.
Diede SJ, Gottschling DE. Telomerase-mediated telomere addition in vivo requires DNA primase and DNA polymerases alpha and delta. Cell. 1999;99:723–33.
Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, et al. Specific association of human telomerase activity with immortal cells and cancer. Science. 1994;266:2011–5.
Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC, et al. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol. 2009;10:459–66.
Sachdeva M, Sachdeva N, Pal M, Gupta N, Khan IA, Majumdar M, et al. CRISPR/Cas9: molecular tool for gene therapy to target genome and epigenome in the treatment of lung cancer. Cancer Gene Ther. 2015;22:509–17.
Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell. 2014;159:440–55.
Xue W, Chen S, Yin H, Tammela T, Papagiannakopoulos T, Joshi NS, et al. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature. 2014;514:380–4.
Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci USA. 2012;109:E2579–86.
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819–23.
Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J. RNA-programmed genome editing in human cells. eLife. 2013;2:e00471.
Chavez A, Tuttle M, Pruitt BW, Ewen-Campen B, Chari R, Ter-Ovanesyan D, et al. Comparison of Cas9 activators in multiple species. Nat Methods. 2016;13:563–7.
Banerjee B, Sherwood RI. A CRISPR view of gene regulation. Curr Opin Syst Biol. 2017;1:1–8.
Dunbar CE, High KA, Joung JK, Kohn DB, Ozawa K, Sadelain M. Gene therapy comes of age. Science. 2018;359:1–10.
George LA. Hemophilia gene therapy comes of age. Blood Adv. 2017;1:2591–9.
Mendell JR, Al-Zaidy S, Shell R, Arnold WD, Rodino-Klapac LR, Prior TW, et al. Single-dose gene-replacement therapy for spinal muscular atrophy. N Engl J Med. 2017;377:1713–22.
Valdmanis PN, Kay MA. Future of rAAV gene therapy: platform for RNAi, gene editing, and beyond. Hum Gene Ther. 2017;28:361–72.
Nissim L, Wu MR, Pery E, Binder-Nissim A, Suzuki HI, Stupp D, et al. Synthetic RNA-based immunomodulatory gene circuits for cancer immunotherapy. Cell. 2017;171:1138–50 e15.
Cawthon RM. Telomere measurement by quantitative PCR. Nucleic Acids Res. 2002;30:e47.
This work was supported by the National Natural Science Foundation of China (61571119) and the National Key Research and Development Program of China (2017YFA0205502).
About this article