Intracranial Delivery of Synthetic mRNA Using Common Transfection Reagent

Background: Owing to messenger RNA’s unique biological advantages, it has received increasing attention to be used as a therapeutic gene carrier, known as mRNA-based gene therapy. It is critical to precisely deliver specic mRNA into targeted part of the body to achieve effective treatment. Methods: In the present study, a mouse model of intracranial delivery of synthetic mRNA was established using commonly used transfection reagent. Synthetic luciferase mRNAs were wrapped with two different transfection reagents and microinjected into the brain at the xed point. The expression status of delivered mRNA was monitored by a small animal imaging system. The possible reagent-induced biological toxicity was evaluated by behavioral and blood biochemical measurements. Synthetic modied TRAIL mRNA was also used as an example of therapeutic application. Results: This model demonstrated that synthetic mRNA could be successfully delivered into the brain with commonly used transfection reagents without measurable toxicity. The expression of exogenous mRNA persisted in a reasonable period following intracranial injection. Conclusions: This mouse model of intracranial delivery of synthetic mRNA can be applied in preclinical studies of mRNA-based gene therapy.

Longjun Dai Taihe Hospital therapies, the existence of blood-brain barrier (BBB) and brain-tumor cell barrier (BTB) in the brain further increases the intractability of GBM [5]. Therefore, under certain circumstances, intracranial injection of synthetic mRNA may be an ideal choice for the treatment of gliomas. In the present study, in vitro synthetized luciferase-mRNA (Luc-mRNA) was used as the target gene that was wrapped with commonly used transfection regents. Synthetic Luc-mRNA was microinjected into mouse brain at the xed point. The expression status of delivered mRNA was monitored and the possible reagent-induced biological toxicity was evaluated. Meanwhile, synthetic TRAIL-mRNA was used as an example of therapeutic application in a mouse xenografted tumor model. This mouse model of intracranial delivery of synthetic mRNA could provide additional option for mRNA-based gene therapy.

Methods
In vitro synthesis of Luc-mRNA and TRAIL-mRNA Luc-mRNA and TRAIL-mRNAs were synthesized in vitro as previously described [6]. Brie y, the human 5'UTR with Kozak sequence and 3'UTR sequence were commercially synthesized by Integrated DNA Technologies (Coralville, Iowa) and sub-cloned into pcDNA3. 3. The DNA templates of human TRAIL and luciferase were obtained from our previously constructed expression vectors through restriction enzyme digestion. MEGAscript T7 kit (Ambion) was used to synthesize mRNAs, whereas m7GpppG was replaced with ARCA cap analog (New England Biolabs) and cytidine and uridine were replaced with 5methylcytidine triphosphate and pseudouridine triphosphate (TriLink Biotechnologies) respectively. Intracranial injection of synthetic mRNA in mice The C57BL/6J mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. A sagittal incision (1.0 to 1.5 cm) was made on the scalp, and the calvarium was exposed by blunt dissection. A tiny parietal hole was created on the sagittal suture of skull. The microinjector was positioned at the right caudate nucleus (2 mm forward and 6 mm right side the anterior fontanelle) and vertically punctured 3 mm. The Luc-mRNA or TRAIL-mRNA solution (20 µl) was injected at a rate of 2 µl/min through the microinjector. The TransIT-mRNA kit-mediated mRNA solution was composed of 1 µl of synthetic mRNA (1 or 2 µg/µl Luc-mRNA or 1 µg/µl TRAIL-mRNA), 15 µl of Opti-MEM, 2 µl of Boost reagent and 2 µl of TransIT-mRNA. The in vivo-jetPEI kit-mediated mRNA solution was composed of 1 µl of synthetic mRNA (1 µg/µl Luc-mRNA), 0.12 µl or 0.16 µl of in vivo-jetPEI reagent (N/P ratio 6 or 8), 2 µl of 10% glucose solution, 0.88 or 0.84 1µl DEPC water. After injection, the microinjector was kept in place for 5 minutes.

Establishment of xenografted tumor model
Since IVIS Spectrum System is preferentially sensitive to bioluminescence, luciferase gene-transfected DBTRG (DBTRG-Luc) cells were used for the xenograft test. A total of 3 × 10 5 DBTRG-Luc cells were implanted into the right frontal lobe of nude mice. This xenografted tumor model was used to test the therapeutic effect of synthetic TRAIL-mRNA through intracranial injection. Seven days after in situ implantation of DBTRG-Luc cells, 20 µl of cock tail solution (containing 1 µg TRAIL-mRNA) was intracranially injected to each mouse. The bioluminescence was determined at day 0, 14, 28 and 60 using IVIS spectrum system. The body weight was recorded at day 0, 14, 28 and 60. At last, statistical analysis of survival rate of mice was performed at day 75.

Determination of tumor size by MRI
In order to detect the tumor formation of DBTRG-Luc cells, 75 days after in situ implantation of DBTRG-Luc cells, the mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital, and the tumors size were detected by MRI (General Electric, GE).

Immunohistochemistry
The tumors were isolated from the brain tissues. One section per sample was depara nized, rehydrated, and stained with hematoxylin and eosin (H&E). Additional sections were immunostained using the automated immunohistochemistry (IHC) and in situ hybridization staining system Bond RX (Leica Biosystems). Sections were stained for proliferating cells using rabbit anti-Ki67 antibody (1:50, Abcam).

Determination of blood biochemical indexes in exogenous mRNA-injected mice
To assess the toxicity of luciferase mRNA and Mirus reagents, routine blood examinations were performed for both control and Luc-mRNA injected mice. Blood was collected by cardiac puncture in EDTA-K2 blood collection tubes at the day of sacri ce. The white blood cells (WBC), hemoglobin (HGB), aspartate transaminase (AST), alanine transaminase (ALT), creatinine (Cr) and C-reactive protein (CRP) were determined using Mindray BC-6900 Vet animal automatic hematology analyzer. The blood cells of the mice were analyzed by using Mindray BC-5300 Vet animal automatic hematology analyzer (Mindray, China).

Behavioral evaluation of mice injected with synthetic mRNA
According to previous literature reports, behavioral test has been used for the evaluation of drug-induced potential biological toxicity in animals [7,8]. In the present study, open eld test and Y maze test were performed to determine the possible transfection reagent-induced neurotoxicity. These two behavioral tests were conducted in C57BL/6J mice on day 14 post-inoculation of Luc-mRNA wrapped with Mirus TransIT-mRNA or Mirus TransIT-mRNA alone. For the open eld test, each mouse was placed in the center of a darkened white box (50 × 50 × 38 cm) and monitored using an infrared video tracking system (Ethovision XT 9.0, Noldus Information Technology) for 10 min. A 30-by-30-cm square in the center of the box was de ned as the "zone," and the peripheral arena was de ned as the "residual". The distance traveled and time spent in the zone and residual were recorded for further analysis.
For the Y maze test, the Y-maze was fabricated from gray plastic and consisted of three arms (21 cm long, 15.5 cm high, 7 cm wide at the bottom, and 10 cm wide at the top) with an angle of 120°. Visual cues were placed outside each arm, and the apparatus was illuminated at 10 lux. Each mouse was placed at the end of one arm and allowed to freely explore the maze for 10 minutes. An arm entry was de ned as all four paws of the mouse being in the arm, and the sequence of arm entries was monitored with a video camera and counted manually. An alternation was de ned as successive entries into the three arms on overlapping triplet sets. Viability assay of DBTRG-Luc glioma cells The cell viability was detected by real-time assessment by the xCELLigence cell analyzer (RTCA, Roche, USA) as previously described [6]. A volume of 100 μl of DBTRG-Luc cell suspension (5 × 10 3 cells) was seeded in E-Plate 16. All cells were allowed to settle at the bottom of the wells at room temperature (RT) for 15 min, and then incubated at 37°C and 5% CO 2 . The impedance signals were recorded every 5 min for the rst 6 h. After 6 h of base line measurement, 0.1 µg synthetic TRAIL-mRNA mixture was added into each well. The impedance signals were recorded using the same time intervals until the end of the experiment (up to 72 h). Cell index (CI) value was de ned as relative change in measured impedance to background impedance and represented cell status, which is directly proportional to the quantity, size, and attachment forces of the cells.

Immunoblotting analysis of TRAIL-induced apoptosis-related proteins expression
Immunoblotting analysis was used to detect the cellular expression of TRAIL induced apoptosis-related proteins (caspase-3, Bcl-2 and Bax) in DBTRG-Luc cells. Brie y, DBTRG-Luc ells were washed with PBS three times and collected with the cell lysis RIPA buffer (Cowin Bio, China). Cell lysates were incubated on ice for 30 min. Protein concentration was determined using BCA protein assay reagents (Beyotime) according to the manufacturer's protocol. Equal amounts of protein (50 μg/each sample) were loaded on each lane and separated by electrophoresis in 12% SDS ployacrylamide gel and electrotransferred to nitrocellulose membranes. The membrane was put in blocking buffer for 1h at RT followed by overnight incubation at 4°C with appropriate primary TRAIL antibody (1:1000, CST, USA), caspase-3 antibody

Results
Expression veri cation of synthesized Luc-mRNA and TRAIL-mRNA The expression of Luc-mRNA and TRAIL-mRNA was veri ed in 293T cells. After transfection of 293T cells with Luc-mRNA, the bioluminescence was detected at 12 h, 24 h and 48 h using IVIS Spectrum System.
The luciferase expression maintained at least 48 h (Fig.1A). To verify the expression of TRAIL-mRNA, TRAIL-mRNA was transfected into 293T cells and detected by western blot. As shown in Fig.1B, the expression of TRAIL was highly upregulated by TRAIL-mRNA transfection.
Intracranial expression of synthetic mRNA delivered with common transfection reagents Two commercially available transfection reagents, in vivo-jetPEI from Polypus Transfection and TransIT-mRNA from Mirus, were used for the intracranial mRNA delivery. As shown in Fig. 2, the intracranial injected synthetic Luc-mRNA was successfully expressed either with jetPEI or with TransIT-mRNA. The peak bioluminescence signals of Luc-mRNA injected with these two reagents appeared in 12 h after intracranial injection ( Fig. 2A-B). However, the bioluminescence intensity produced by Luc-mRNA delivered with TransIT-mRNA was signi cantly higher than that produced by Luc-mRNA delivered with jetPEI at every time points. As shown in Fig. 2C, the duration of luciferase expression was much longer in Luc-mRNA injection with TransIT-mRNA (60 hours) than that in Luc-mRNA injection with jetPEI (36 hours).
The evaluation of biochemical toxicity of intracranially injected Luc-mRNA with TransIT-mRNA After injecting 1 µg mRNA mixture, all mice remained healthy without any toxic symptoms or behavioral abnormalities. There were no signi cant differences between TransIT-mRNA injection and controls on all determined blood biochemical parameters, including HGB, WBC, ALT, AST, Cr and CRP (Fig. 3A-3F). No signi cant transfection reagent-related neurovirulence was detected by both Y maze (Fig. 4A-E) tests and open eld (Fig. 4F-J). The results of blood test and behavioral test indicated that this transfection reagent is safe to be used as a wrapping material of synthetic mRNA for intracranial injection.

The effects of synthetic TRAIL-mRNA on DBTRG-Luc glioma cells
The effect of synthetic TRAIL-mRNA on the viability of DBTRG-Luc glioma cells was determined using RTCA, and TRAIL-induced apoptosis was detected by ow cytometric analysis. As shown in Fig. 5A, the mean apoptotic population of normal DBTRG-Luc cells was 4.38% + 4.7%, however, the apoptotic population of DBTRG-Luc cells transfected with 1 μg TRAIL-mRNA was 11.01% + 17.96%. RTCA results indicated that synthetic TRAIL-mRNA signi cantly inhibited the viability of DBTRG-luc cells (Fig. 5B). Figure. 5C showed the results of immunoblotting analysis of apoptosis-related proteins in DBTRG-Luc cells after transfection with synthetic TRAIL-mRNA for 24 h. DBTRG-Luc cells expressed similar amount of total caspase-3. However, the cleaved form of caspase-3 and Bax were obviously upregulated by the treatment of synthetic TRAIL-mRNA transfection. Bcl-2 was down regulated by synthetic TRAIL-mRNA transfection.
Therapeutic application of synthetic TRAIL-mRNA through intracranial injection in orthotopic glioma mouse model A DBTRG-Luc cell-derived xenografted glioma mouse model was used to test the therapeutic application of synthetic TRAIL-mRNA through intracranial injection. As shown in Fig. 6A, the intensity of luminescence in DBTRG-Luc cells pretreated with synthetic TRAIL-mRNA for 6 h was not different from DBTRG-Luc cells without synthetic TRAIL-mRNA pretreatment. DBTRG-Luc cell-derived and TRAIL-mRNApretreated DBTRG-Luc cell-derived xenografted glioma mouse models were used to further evaluate the effect of intracranial injection of synthetic TRAIL-mRNA. The tumor growth was signi cantly inhibited by both TRAIL-mRNA pretreatment and TRAIL-mRNA intracranial injection ( Fig. 6B-C). Fig. 6D and 6E showed the changes of body weight and animal survival rate under different conditions. At the end point, tumor size was detected with brain MRI scan and the measurement of isolated tumors (Fig. 6F). The inhibitory effect of injected TRAIL-mRNA on tumor cell proliferation was also con rmed by immunohistochemical staining of tumor tissues (Fig. 6G).

Discussion
Because mRNA possesses high positive charge and high hydrophilicity, it is usually delivered into cells as a complex with transfection reagent. TransIT-mRNA and jetPEI are commonly used for mRNA transfection in most in vitro studies. It is essential to verify the safety of these reagents in vivo application and their effects on the expression of associated genes after intracranial injection. In the present study, luciferase mRNA was synthesized in vitro and its expression was veri ed in 293T cells. In order to study the safety of TransIT-mRNA and jetPEI in vivo application and their effects on the expression of Luc-mRNA after intracranial injection, we injected Luc-mRNA wrapped with TransIT-mRNA or jetPEI into the brain of C57BL/6J mice. Considering the volume safety of intracranial injection, we compared the transfection e ciency of 20 µl system containing 1 µg Luc-mRNA and 30 µl system containing 2 µg Luc-mRNA. After intracranial injection of Luc-mRNA, the longest time, in which Luc-mRNA-produced bioluminescence can be detected by IVIS, was 72 h for 20 µl system and 48 h for 30 µl system respectively. The poor performance of 30 µl system might be due to the large volume leading to an incomplete injection into the brain. So, 20 µl system containing 1 µg Luc-mRNA was used for right caudate nucleus injection in the related in vivo experiments. In addition, according to in vivo jetPEI manufacturer's instruction, a 5 µl system containing 1 µg Luc-mRNA at N/P ratio 6 was injected into the right caudate nucleus. The longest detectable time of Luc-mRNA-produced bioluminescence was 36 hours. When the N/P ratio was increased to 8, the bioluminescence intensity was signi cantly higher than that at N/P ratio 6, but the longest detectable time retained 36 hours.
The results of comparative experiment clearly indicate that TransIT-mRNA is much better than jetPEI in terms of assisting intracranial Luc-mRNA delivery. TransIT-mRNA is preferred to be used for mRNA delivery through intracranial injection. However, long-term safety is an important prerequisite for it to be routinely used in mRNA-based gene therapy studies. The test results of blood biochemical parameters showed relatively stable internal environment after intracranial injection of TransIT-mRNA, indicating that TransIT-mRNA solution was harmless to the metabolism of tested subjects. Two behavioral methods were used to test the potential neurotoxicity of TransIT-mRNA. Fourteen days after the intracranial injection, the results of open eld test and Y-maze test demonstrated that the behaviors of tested mice were not signi cantly affected by intracranial injection of TransIT-mRNA reagent. Taken together, TransIT-mRNA reagent-assisted synthetic mRNA delivery is safe for in vivo intracranial administration.
The major reasons that limit the effectiveness of conventional therapies for GBM include tumors' exceptional anatomy location and the existence of BBB. Intracranial injection of synthetic mRNA is able to directly deliver speci c anticancer genes to the tumor. TRAIL (TNF-related apoptosis-inducing ligand) is an anticancer gene. Its protein product can speci cally kill cancer cells without harming normal cells [9][10][11][12][13]. Owing to its tumor cell-speci c killing effect, TRAIL has been widely used in preclinical and clinical studies [12,[14][15][16][17][18][19][20][21][22][23]]. In the current study, we used synthetic TRAIL-mRNA as an example to verify whether this method of intracranial injection can be applied for the treatment of GBM. First,the tumor cell killing effect of synthetic TRAIL-mRNA was veri ed in vitro with DBTRG-Luc cells. Then, the in vivo antitumor effect of intracranially injected TRAIL-mRNA was investigated with DBTRG-Luc cell-derived xenografted glioma mouse model. The antitumor effect of intracranially injected TRAIL-mRNA was comparable to that of pretreatment of DBTRG-Luc cells with TRAIL-mRNA, and the combination of pretreatment and intracranial injection of TRAIL-mRNA showed the best antitumor effect. This result shows that the method of synthetic mRNA intracranial delivery using common transfection reagent is suitable for the experimental studies of intracranial tumors.
In summary, the in vitro synthetic luciferase mRNAs were highly expressed in the mouse brain through intracranial injection, in which the injected mRNAs were wrapped with commonly used transfection reagents. Mouse behavioral and blood biochemical measurements veri ed the biological safety of related reagent's intracranial application for mRNA transfection. Using synthetic TRAIL-mRNA as an experimental therapeutic example, its intracranial delivery with common transfection reagents signi cantly inhibited the tumor growth in DBTRG cell-derived xenografted glioma mouse model. This study provides an intracranial synthetic mRNA delivery model for preclinical studies of mRNA-based gene therapy.