Lentiviral Engineered cells for production of miR34a loaded exosomes

Lentiviral Engineered cells for production of miR34a loaded exosomes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Lentiviral Engineered cells for production of miR34a loaded exosomes Sahar Abdi Sarkami, Sajjad Molavipordanjani, Saeed Abedian Kenari, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2825672/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Sep, 2023 Read the published version in Molecular Biology Reports → Version 1 posted 5 You are reading this latest preprint version Abstract Background: RNA antisense emerging as novel candidates for therapeutic purposes. As an RNA, miR-34a involves in P53 function, and triggers cancer cells apoptosis. The clinical applications of miRNAs face some limitation which can be potentially resolved using exosome as a transporting vehicle. Aims: The aim of this study is to create a cell factory to generate miR34a-enriched exosomes. Methods: First exosome specific sequences were inserted into miR34a. The resulting miR34a gene were transduced HEK293T cells genome with a lentiviral system. In the structure of miR34a gene 6 nucleotides were substituted to increase its packaging rate into exosomes. To maintain the secondary structure, stability and expression of the miRNA gene, changes to the miR34a gene were made using PCR Extension. Results: The results confirmed the created changes in the miR34a gene do not affect its secondary structure. The energy level of the manipulated miR34a gene was not changed in comparison with the original one. Conclusion: Our results suggested that the induced mutation (replacing 6 nucleic acids at the 3' end of miR34a) increases loading of miR34a into the exosome by 3-fold. exosome miR34a HEK293T lentiviral vector Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction RNA antisense has captured a lot of attention as a new candidate for developing therapeutic drug. These biomolecules occur naturally in all cells and regulate various genes through different signaling pathways [1]. To that end, microRNA (miRNA) family suppress cancer related genes and has different members including miR-34a. As a critical mediator of p53 function, miR-34a targets cancer signaling pathway such as cell proliferation downregulation, apoptosis induction, cell-cycle arrest, and etc. miR-34 contributes to cell-cycle arrest by downregulating CDK4/6, Cyclin E2, MET, and Bcl-2 mRNAs to enrich seed-matching sequences in their 30-UTRs directly [2]. Moreover, it can also inhibit several pro-apoptotic proteins by translational repression of SIRT1, a NAD-dependent deacetylase [3]. To that end, restoration of miR-34a activity is helpful to prevent chemotherapy resistance [4, 5]. Biomolecules such as miR-34 as a medicine suffer from structural instability and susceptibility to nucleases; hence, using different delivery systems could be helpful. Although several nanoparticle packaging systems were designed and applied for RNA interference (RNAi) based cancer therapy, the toxicity and immunogenicity of these approaches are still troublesome [6-8]. Using nanocarriers such as exosome with feature similar to that of cell could resolve the problem. Exosome is a naturally occurring nano-carrier for cell to cell communication in all biological systems. Exosome as a nanocarriers is cup likes structure with 30-100 nm diameter which possesses bold features such as, biocompatibility, no toxicity, and no immunogenicity. Having that said, exosome seems to be a proper candidate for RNAi delivery and targeted therapy [9, 10]. It worth to note, that the application of these bionanoparticles also struggles with efficient cargo loading, efficient isolation methods, purity, and microbial infection. Various studies tried to remove these obstacles [11]. Exosome as a communication devise of the cells carry RNA, more specifically miRNA, which sparked the idea of applying exosome as a proper device for RNA delivery [12]. The exosome's contents lack ribosomal RNA (rRNA) and have a high amount of small RNAs, messenger RNAs (mRNAs) and miRNA, which are named exosomal shuttle RNAs (esRNAs) [13]. Exosomes extracted from body fluid such as plasma, amniotic fluid, and saliva can also carry their specific esRNAs which have a function in recipient cells [14]. The cell transporting machinery distinguishes esRNAs from its cytosolic form due to the difference in their sequence with a not well-understood mechanism [15]. Recognition of esRNA specific sequences can be applied in creating a specific cell factory that produces targeted exosomes containing a definitely identified exosomal miRNA. This study aims to design a specific miR34a expression system and create a stable cell line that releases exosomes with elevated miR34a cargo. 2. Material and Methods 2.1. Software and primer designing The sequence of the miR34a gene was taken from the NCBI database (GenBank: EF609116.1) and was checked using CLC main version 5 software. The secondary structures of the wild-type and mutant miR34a gene were also analyzed by RNA software. Gene Runner and OLIGO7 software were applied to design six primers to amplify the miR34a gene, site-directed mutagenesis, and cloning. The primers have restriction site sequences compatible with pCDH vector and spacer sequences. 2.2. miR34a gene amplification and site directed mutation Total DNA was extracted from whole blood cells by QIAamp DNA extraction kit (Qiagen, Germany). Two primer pairs were separately used to amplify the 250bp DNA encoding miR34a by PCR. The F-34a (5-TGGGGAGAGGCAGGACAGG-3) and R-34a primers (5-TCCGAAGTCCTGGCGTCTCC-3) were used for amplification of the miR34a gene from DNA . PCR reactions were performed using Takara puf kit for 30 cycles (reaction conditions were as follows: 30s at 95 ºC, 30s at 60 ºC, and 30s at 72 ºC). The first pair, 34a FWD (5'- TGGGGAGAGGCAGGACAGG – 3' and 5'- TCCGAAGTCCTGGCGTCTCC-3') and S2 (5' TTA CTA TTG CTC ACA A CA TCC TC C TAA GAC ACT GC 3′) were used for amplification of the first DNA fragment with the first set of 6 nucleotides deserted point mutation. In comparison, the second pair S1 (5′CTCGAGTACAACTATGCGGCCGCAGCAGAATG-GGAGATGAATTTCA3′) and 34a REV (5′ACGACGCGTCG TTAGTGTTACAGAGTCT-GATATCC3′) were used for amplification of the second fragment of DNA with a second set of 6 nucleated deserted point mutation encoding miR34a pre-miRNA. PCR reactions were performed using Takara puf kit for 30 cycles (reaction conditions were as follow: 30s at 95 ºC, 30s at 60 ºC, 30s at 72 ºC for the first reaction and 30s at 95 ºC, 30s at 60 ºC, 30s at 72 ºC for the second reaction). Both S1 and S2 primers were designed to include an overlapping sequence. In the next step, these overlapping fragments were used to assemble two amplified sequences into a full-length DNA through an overlap extension of PCR (30s at 95 ºC, 30s at 60 ºC, and 120s at 72 ºC). Finally, a primer pair composed of 34a FWD and 34a REV was used to amplify the assembled fragment following reaction conditions as the 30s at 95ºC, 30s at 60ºC, and 90s at 72º C). 2.3. miR34a gene cloning pCDH lentiviral vector and the amplified miR34aW and miR34aM gene fragments were digested separately with BamH1 and not1 restriction enzymes (Roche, Germany) and ligated by T4 DNA ligase (Fermentas, Germany). The ligation mixture was transformed to Stbl4 competent bacteria by CaCl 2 method. The transformed bacteria were cultured on LB-agar medium containing ampicillin (100µg/ml). Colony PCR was performed to screen the positive colon by universal primers of pCDH vector (pCDH-FWD 5′-CACCAAAATCAACGGGACTT-3′ and pCDH-REV 5′-ATATAGACAAACGCACACCGGCCT-3′). Positive bacterial colonies were harvested and cultured overnight in LB medium for plasmid extraction. DNA sequencing with universal primers was done to confirm the accuracy of the cloned product. 2.4. Lentiviral vector packaging, titration, and cell transduction Lentiviral particles were produced in HEK293T cells as described by Kutner et al. with some modifications [18]. Briefly, 24 hours before transfection, a 5 million concentration of HEK 293T cells were plated in a 10cm dish in DMEM for confluency of 80% to 90% in the next day. Cell culture medium was replaced with fresh complete DMEM two hours before transfection. Then, cells were transfected with Lenti ORF pcdh-mir34a (wild and mutant separately), pMD2G, and pSPAX by calcium phosphate method. Culture medium was replaced with fresh medium 14 hours post-transfection. For virus isolation, the Culture medium was collected three times at 12 hours intervals. To increase virus particle concentration, cell debris was removed following centrifugation of culture medium at 300g for 5 minutes, and the supernatant was subsequently filtrated through Millex-HV 0.45μm PVDF filter (Millipore). Lentiviral particles were precipitated via ultracentrifugation at 45000g for 1.5 hours, and the virus particles in the pellet were re-suspended in 1ml of PBS. Concentrated virus particles were used to transduce HEK 293T cells. Three days post-transduction, flow cytometric analysis was performed for lentivirus titration [15]. Briefly, serial dilution (0.5, 1, 5, 25, and 50) of concentrated virus particles were added to 100000 HEK 293T cells which were cultured in 500 µl of DMED medium. Three days later, cells were harvested and washed twice with PBS, and GFP positive cells population were counted by flow cytometry. Viral transduction performed by the concentrated virus, briefly HEK293T cells (6.0×10 4 ) were cultured in 6 well plates. After 24 hours, 10ulit of concentrated virus dissolved to 2 ml DMEM free FBS medium, and the final solution was added to each well. After 16 hours, the medium was replaced with a fresh DMED complete medium. The GFP fluorescent microscopy was applied for transduction rate detection after 72 hours. 2.5. Puromycin kill curve and stable cell line selection First, HEK293T cells (6.0×10 4 , not infected with the virus) were cultured in 5 wells of a 24-well plate containing 1 ml of complete DMEM medium. On the next day, when the cells stick to the bottom of the plate, we replace the culture medium of the cells with 1 ml of the culture medium containing zero, 0.5, 1, 1.5 and 2 micrograms of antibiotic puromycin, respectively. On the first day after the treatment, the culture medium of the cells was replaced with the selected medium containing the antibiotic puromycin, and daily the number of living cells in each well was counted by vital staining. The best result is obtained 1 to 4 days after antibiotic treatment. During this period, the number of living cells for each concentration was shown as a curve. The least amount of antibiotic that destroys the cells at this time was selected for the selection of cells after transduction. 2.6. Isolation of exosomes To produce miR34a enriched exosomes, HEK293T stable cells were cultured in DMEM supplied with 10% exosome-free FBS. The culture media were periodically collected at 48 hours intervals, and then exosomes were isolated via sequential centrifugation and re-suspended in PBS. Aliquots of exosomes were stored at -80 ºC. 2.7. Characterization of exosome Transmission electron microscopy (TEM), zeta sizer analysis, and western blot analysis were carried out to characterize the obtained exosome. For electron microscopy imaging, the ultra-concentrated exosomes were fixed with 4% paraformaldehyde in PBS. The fixed samples were air-dried on Formvar-carbon coated TEM grids and stained with 0.5% uranyl acetate in 30% ethanol for 10 min, followed by staining with lead citrate 10 min. The sample was sequentially rinsed in 0.05 M NaOH and distilled water for removing lead precipitates on the grid sections. Then, the grids were dried at room temperature, and the stained sections were scanned under the transmission electron microscope (Philips) operating at 150 kV. According to the manufacturer's instructions, the purified exosomes' dimensions were measured using a zeta sizer (Malvern Corp.). The transduced cells and exosomes were lysed with RIPA buffer containing protease inhibitor cocktail (Roche, Germany), and the obtained total protein was subjected to Western Blot analysis. Briefly, the cells were washed twice with ice-cold PBS and centrifuged at 4000 rpm for 5min. The cells (and exosomes) were treated with lysis buffer and incubated on ice for 20min with shaking. Cell debris was precipitated by centrifugation at 13000 rpm for 20 min, and the upper phase was collected as a source of the total protein. The number of total proteins was calculated using the Bradford assay. The extracted proteins (10 μg) were boiled for 5 min in 10X SDS buffer (1 μg) and resolved on 10 % polyacrylamide gel for 2 hours. Protein fragments were transferred to nitrocellulose membrane under the constant current of 300 mA for 2 hours, and the membrane was blocked with 5% nonfat dry skim milk in PBS for 2 hours. The membrane was washed with PBS and treated with HRP-conjugated anti-His tag antibody for 16 hours to detect LAMP-DARPin G3 chimeric protein. After three times of washing with PBST, the related band was detected using ECL solution. In addition, the expression of CD63 as an exosome marker was investigated by flow cytometry. 2.8. Revers Transcription and quantitative PCR Total RNA was extracted using Qiagen RNeasy mini kit from purified exosomes three days post-transduction of HEK293T cells with miR-34a virus. Revert-Aid™ First Strand cDNA Synthesis Kit (Fermentas, Germany) was applied for cDNA synthesis according to the manufacturer's instructions. The relative qPCR experiments were performed on the ABI thermal cycler in the total volume of 20 µl containing 10 µl of Qiagen qPCR Master Mix (Qiagen), 10 pmol of each primer, and 2 µl of each cDNA. 16 s RNA was used as a reference gene. 3. Results 3.1. miR34a gene manipulation The sequence of wild type miR34a was taken from NCBI data-base (GenBank: EF609116.1), and its sequence was manipulated for more expression as illustrated in figure 1a. The bioinformatics analysis using RNA software indicated that the changes of miR34a did not change its secondary structure compared to wild-type miR34a (Fig. 1b). MiR34a gene amplification and site directed mutagenesis were performed by six primers (Fig. 2). At first, the 300 bp DNA fragment of human genome, including miR34a gene, was amplified, sequenced fallow blast, and confirmed miR34a gene. This segment was re-amplified by two specific primers, which have restriction site in 5' end for gene cloning into PCDH lentiviral vector, after digestion of miR34a and PCDH, gene and vector ligated and pcdh-mir34w was constructed and cloned into stbl4 (Fig. 2b). We used overlap extention PCR method to change 6 nucleotide in 3p and 5p coding sequence of miR34a. Therefore, two segments of miR34a were amplified and manipulated then ligated by PCR, one PCR amplified half of 5p segment of mir3a and stem-lope and change the 5p miR34a by R Soeing reverse primer and the second PCR performed to amplified 3p segment of miR34a and stem lope and introduce diversity in 3-p miR34a gene (Fig. 2a), the stem lope is the segment has overlap function in the final PCR which soied to segment of mutated miR34a (Fig. 2b). This fragment digested and insert to pCDH and pCDH-mir34M was constructed and cloned into stbl4 bacteria. As pCDH vector has a reporter gene to evaluate transfection efficiency, the GFP expression was used for transfection of HEK293T cells. We found that up to 90% of the cells express the GFP reporter gene; In addition, HEK293T virus producer cells were nucleated due to VSV-G protein expression during virus production (Fig. 2c). The virus particles were concentrated by ultra-centrifugation and the titer of lentiviral stock was determined by flow cytometry. Transduction of 8×10 4 HEK cells by 5µl of the virus suspension, induced GFP expression in the 8×10 3 cells [16]. The lentiviral titer was estimated by flow cytometry as 1.6 × 10 9 TdU/ml. The electron microscopy result of the HEK293 T cells showed that 100% of the cells were transduced with pCDH-miR34a Vector (Fig. 2d). 3.3. HEK293T cells transduction and stable cell line production The HEK293T cells were transduced with virus particles bearing mir34w and mir34M gene (MOI=5). Puromycin kill curve for HEK293T (Fig. 3) showed that 1.5 µg/ml of Puromycin is the minimum concentration of antibiotic that kills non-transduced HEK293T cells. To select a stable cell line containing mir34M and mir34W within its genome, Puromycin was added to kill non-transduced cells in the cell culture medium for 2 weeks. Exosome characterization result In the current study, we hypothesized that modified HEK293T cells with pCDH-miR34a vector secrete functional miR-34a into exosomes. To confirm this hypothesis, we initially performed isolation and characterization of transduced HEK293T cell derives exosomes. According to the results, exosome fractions showed around 75 nm sizes in diameter (Fig. 4b). Exosomes demonstrated a spherical shape with a uniform particle size. The particle size distribution obtained by DLS was in accordance with the images obtained by TEM (Fig. 4a). Western blot analysis of the exosomes extracted from medium of transduced cells indicated expression of CD63 but did not show calnexin expression (Fig. 5 a and b). As illustrated in Fig. 5c immunophenotyping analysis showed that the exosomes were positive for CD81 (54.4 ± 0.5%) (Fig. 5c). Expression of wild type and manipulated miR34a: According to the results of relative qPCR, miR34a expression extracted from exosomes of three sources; non-transduced HEK293T, HEK293T transduced with mir34W lentivirus and HEK293T cells transduced by mir34M lentivirus vectors. The results indicated that intact cells have no delectable miR34a expression while its expression on HEK293T cells which were transduced by mutate type of mir34 increased by approximately 2.9-fold in comparison to hek293 cells transduced by wild type miR-34a (p < 0.01) (Fig. 6). Discussion miRNAs, non-coding RNAs, have an effect on various biological processes, they play a key role in the growth and development of organisms[17]. miRNAs have been introduced as novel drug candidates. Assessing their safety and effectiveness is the most important step in their entry into the clinical field[18]. On the other hand, the low stability of miRNAs, difficult delivery to the target cell, rapid degradation in the blood by ribonucleases, clearance by the reticuloendothelial system, being trapped in the endosome and then degradation in the lysosome limit their clinical applications. Considering the development of nanoscience and the progress of nanoparticles in the field of drug delivery, it can be said that the delivery of miRNAs by nanoparticles can be an effective method in the transfer of miRNAs[19-21]. Due to nanoparticles, the entry of miRNA into the target cells is multiplied [22]. On the other side, unlike nanoparticles that are synthesized manually, exosomes as nanoparticle of endogenous origin have unique features such as better targeting capacity, stability in body fluids, biocompatibility, safety, and nontoxicity [23]. The aim of this study is to create a cell factory to generate miR34a-enriched exosomes. Mir-34a is a member of the mir34 family involved in P53 regulation, cell signaling, and cell survival pathways. MiR34a stops the cell cycle at the G1 step and induces apoptosis or cell death. MiR34a is a tumor suppressor molecule, and the degradation of miR34a was observed in several types of cancers [24-26]. The expression of miR-34a is suppressed in malignancies and some molecular processes such as cell differentiation and proliferation[26]. MiR34a affects osteosarcoma's metastasis, and decreases in its expression could lead to increased cell growth and metastasis[27]. Applying mimetic miR34a as a candidate for cancer therapy has been progressed to phase I of the clinical trial and production and packaging of miR34a into liposome has been successfully done[28]. There are several methods for loading miRNAs into exosomes. The easiest procedure to generate miRNA-enriched EVs is to incubate them with miRNA-secreting cells. Using a concentration gradient for this method can be a natural process that does not create a risk factor for cells. However, this approach has the disadvantage that it cannot predict the concentration of loaded miRNAs in exosomes and has low efficiency due to the instability of miRNA[29]. Another method is electroporation. For the first time, L Alvarez-Erviti et al.[30] used exosomes to transport siRNA to the brain cells. They insert the desired siRNA into the exosomes using electroporation approaches. JH Wang et al.[31] used electroporation to load EVs with HChrR6 mRNA. But the results of this method seemed inadequate. Using electroporation for transfection of nucleic acids inside exosomes is a simple method, but it is not efficient. This method requires the purification and isolation of extracellular vesicles before and after the transfection process, which repeated purification can lead to the loss of exosomes[32]. In another study, TN Lamichhane et al.[33] investigated the transport of siRNAs loaded by sonication in extracellular vesicles. Contrary to the high cellular uptake of extracellular vesicles (80%), the overall delivery of siRNA to cells was very low (2.96%). Sonication also has limitations such as changing the shape of the extracellular vesicle membrane, generating heat, destroying surface proteins and not being suitable for the delivery of hydrophobic drugs[34]. Cell-engineering is another approach to producing miRNA-enriched exosomes. Overexpression of the desired miRNA in exosome-producing cells by cellular machinery and mimicking the spontaneous miRNA loading and packaging system are two approaches used to generate miRNA-loaded exosomes[35]. In the present research, we used both systems. First, we inserted exosome specific sequences into miR34a then we transduced the manipulated miR34a gene via a lentiviral system into HEK293T genome, which applied CMV promoter for high and permanent expression of miR34a gene. Various studies suggest that there is a sorting sequence at the 3' end of miRNA sequences. It is predicted that the transfer of miRNA into the exosome is carried out by this sequence [36]. This sequence can be used to transfer miRNA into the exosome. MF Bolukbasi et al.[37] investigated a 25-nt sequence containing "CTGCC" on a stem-loop structure and a miR-1289 binding site in the 3UTRs of many mRNAs enriched in MVs derived from GBM cells. Their findings indicate the important role of the presence of the CTGCC sequence in the loop structure and the binding site of miR-1289 in the 3'UTR, in increasing the integration of mRNAs in MVs. By RNA sequencing (RNA-seq) from a panel of human B cells and their secreted exosomes, D Koppers-Lalic et al.[38] demonstrated that miRNAs that are 3' adenylated were relatively enriched in cells, while 3' uridylated isoforms were overexpressed in exosomes. limits are shown. Therefore, it can be said that these post-transcriptional modifications help to guide the sorting of ncRNA to EVs to some extent. Villarroya-Beltri et al.[15] described sequence motifs present in miRNAs that control their localization into exosomes (EXOmiRNA). These specific sequences (GGAG) are present at the end of miRNA and lead the miRNA to the endosomes and finally into exosomes. Sumoylated hnRNPA2B1 binds to EXOmiRNA and controls its loading into exosomes. In this study we substituted 6 nucleotides of miR34a gene to increase its packaging rate into exosomes. Since substitution of nucleotides at 5' and 3' ends of the miR34a gene could affect its expression, stability and secondary structure, a 250 bp fragment capturing and containing miR34a sequences was selected for cloning in the present research. Moreover, any changes in one part of the gene may require other changes in other parts. For manipulating the 3' ends of mir5p, the manipulation of a complementary sequence at mir3p is also required. The required changes to the miR34a gene were created using the overlap extension PCR method by four specific primers at two gene regions. One pair for insertion of GGAGAG sequence at 3 end of mir34-5p, and another pair for insertion of 6 nucleotides CTCTCC that is complementary to the first manipulated change for stabilization of the secondary structure. The results confirmed the created changes in the miR34a gene, and bioinformatics analysis has also indicated that these changes do not affect the secondary structure of the molecule. The energy level of the manipulated molecule was not changed in comparison to the original one. High expression of the desired RNA in exosome-producing cells via lentiviral vectors can lead to enrichment of that RNA and its encoded protein in the produced exosomes[39]. Lentiviral vectors can transduce both dividing and non-dividing cells, while retroviral vectors only transduce non-dividing cells [40]. Therefore, in the present research, a lentiviral vector was chosen for overexpression of miR34a and creation of a stable cell line that produces miR34a loaded exosomes and releases them to medium. Conclusion In conclusion, our results showed that normal HEK293T didn't express miR34a while lentiviral transduced miR34a gene induced loading of miR34a into exosome, in comparison, induced mutation by replacing of 6 nucleic acids in 3' end of miR34a increases 3-fold loading of miR34a to exosome. Declarations Compliance with Ethical Standards: Disclosure of potential conflicts of interest The authors declare no competing interest. Research involving human participants and/or animals This research dose not involve human tissue or animals. Informed consent All the authors consent to publish this article findings. Data Availability Statement All the data are reported as they obtained. And the original data will be presented upon a reasonable request. The RNA folding (Fig. 1) was obtained from http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi website. 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Cite Share Download PDF Status: Published Journal Publication published 02 Sep, 2023 Read the published version in Molecular Biology Reports → Version 1 posted Editorial decision: Minor Revisions Needed 03 Jul, 2023 Reviewers agreed at journal 23 May, 2023 Reviewers invited by journal 02 May, 2023 Editor assigned by journal 28 Apr, 2023 First submitted to journal 24 Apr, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2825672","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":196854215,"identity":"6c75a3fb-417b-4645-a87f-6681c8a6075f","order_by":0,"name":"Sahar Abdi Sarkami","email":"","orcid":"","institution":"Mazandaran University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sahar","middleName":"Abdi","lastName":"Sarkami","suffix":""},{"id":196854216,"identity":"e06cdbb4-fb79-49d3-8e33-f604daa4b481","order_by":1,"name":"Sajjad Molavipordanjani","email":"","orcid":"","institution":"Mazandaran University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sajjad","middleName":"","lastName":"Molavipordanjani","suffix":""},{"id":196854217,"identity":"fc7cefdc-e406-486e-83c9-b4ab2298d57f","order_by":2,"name":"Saeed Abedian Kenari","email":"","orcid":"","institution":"Mazandaran University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Saeed","middleName":"Abedian","lastName":"Kenari","suffix":""},{"id":196854218,"identity":"b7a3596e-4b9c-4894-a0c1-396d3b9d43b7","order_by":3,"name":"Javad Akhtari","email":"","orcid":"","institution":"Mazandaran University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Javad","middleName":"","lastName":"Akhtari","suffix":""},{"id":196854219,"identity":"6a095473-9366-420f-adfe-14c090cb67ea","order_by":4,"name":"Pooria Gill","email":"","orcid":"","institution":"Mazandaran University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Pooria","middleName":"","lastName":"Gill","suffix":""},{"id":196854220,"identity":"7103336c-a74b-4aba-abd2-8edac17d755a","order_by":5,"name":"Hossein Ghalehnoei","email":"","orcid":"","institution":"Mazandaran University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hossein","middleName":"","lastName":"Ghalehnoei","suffix":""},{"id":196854221,"identity":"f756a983-4460-4046-a577-992ef4f5bb22","order_by":6,"name":"Shabanali Khodashenas","email":"data:image/png;base64,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","orcid":"","institution":"Mazandaran University of Medical Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Shabanali","middleName":"","lastName":"Khodashenas","suffix":""}],"badges":[],"createdAt":"2023-04-17 07:02:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2825672/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2825672/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11033-023-08754-1","type":"published","date":"2023-09-02T15:10:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":36790126,"identity":"597f6da1-7689-4501-8aa3-cc1bf5da3961","added_by":"auto","created_at":"2023-05-10 21:28:59","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":228999,"visible":true,"origin":"","legend":"\u003cp\u003e(a) The sequence of wild type and manipulated miR34a. The wild type sequences was manipulated for more expression. (b)The bioinformatics analysis Infelunce of the changes of miR34a in its secondary structure compared to wild-type miR34a\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2825672/v1/a5fb5acf96aa75a2cfca174e.jpg"},{"id":36790127,"identity":"5b3ae166-349b-42d0-bea6-d2d67a229889","added_by":"auto","created_at":"2023-05-10 21:28:59","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":164177,"visible":true,"origin":"","legend":"\u003cp\u003e(a) amplification of 200bp miR34a gen fragment contains overlapping fragment and the mutated fragment, Fs2 amplified 5p segment of mir3a and stem-lope and s1R primer amplified the 3p segment of miR34a (b) miR34a gen amplification including the miR34a mutant (soeing from two fragments of miR34a amplified from first PCR rection (s1 and s2 in the Fig2b)), wild, the miR34a product (which is a wild type of miR34a amplified from human DNA), and the ladder (c) transfection of HEK293T for miR34a-lentivirus production (d) transduction of HEK293T with miR34a-lentivirus\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2825672/v1/098a10c19e624045af8a3c02.jpg"},{"id":36790566,"identity":"f3b03a97-7d41-43ed-9a74-8998eeaa4621","added_by":"auto","created_at":"2023-05-10 21:36:59","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":31330,"visible":true,"origin":"","legend":"\u003cp\u003ePuromycin kill curve for HEK293T\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2825672/v1/6d4e955fb38a92e1fe8232d3.jpg"},{"id":36790124,"identity":"a3c4bf5d-a970-4280-aa03-86e8e032e927","added_by":"auto","created_at":"2023-05-10 21:28:59","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":109945,"visible":true,"origin":"","legend":"\u003cp\u003eTEM and DLS analysis of the produced exsome\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2825672/v1/788b1ffdb87702502d9550ba.jpg"},{"id":36790125,"identity":"3280f087-b346-4554-b4e9-e6b4956677a6","added_by":"auto","created_at":"2023-05-10 21:28:59","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":143909,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Western Blot The results showed the positive expression of exosomal markers and negative expression of calnexin. Immunophenotyping results According to the results of flow cytometry, the expression of exosomal marker CD63 was positive\u003c/p\u003e","description":"","filename":"5j.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2825672/v1/cac7a314d95b1d5d4655ce36.jpg"},{"id":36790123,"identity":"6d28a59e-5652-4187-97c1-68ba4952c173","added_by":"auto","created_at":"2023-05-10 21:28:59","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2938,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of miR34a 3-folds more loading of miR34a into the exosome than when miR34a was only overexpressed in the cell.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2825672/v1/80f723ccc78fd633ba0fe794.png"},{"id":42782819,"identity":"2479d051-dbaa-417c-9fe0-1e002270b248","added_by":"auto","created_at":"2023-09-07 15:17:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":789609,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2825672/v1/a628ae8b-083f-42e0-ba09-79e6f64452b8.pdf"}],"financialInterests":"","formattedTitle":"Lentiviral Engineered cells for production of miR34a loaded exosomes","fulltext":[{"header":"1.\tIntroduction","content":"\u003cp\u003eRNA antisense has captured a lot of attention as a new candidate for developing therapeutic drug. These biomolecules occur naturally in all cells and regulate various genes through different signaling pathways [1]. To that end, microRNA (miRNA) family suppress cancer related genes and has different members including miR-34a. As a critical mediator of p53 function, miR-34a targets cancer signaling pathway such as cell proliferation downregulation, apoptosis induction, cell-cycle arrest, and etc. miR-34 contributes to cell-cycle arrest by downregulating CDK4/6, Cyclin E2, MET, and Bcl-2 mRNAs to enrich seed-matching sequences in their 30-UTRs directly [2]. Moreover, it can also inhibit several pro-apoptotic proteins by translational repression of SIRT1, a NAD-dependent deacetylase [3]. To that end, restoration of miR-34a activity is helpful to prevent chemotherapy resistance [4, 5]. \u003c/p\u003e\n\u003cp\u003eBiomolecules such as miR-34 as a medicine suffer from structural instability and susceptibility to nucleases; hence, using different delivery systems could be helpful. Although several nanoparticle packaging systems were designed and applied for RNA interference (RNAi) based cancer therapy, the toxicity and immunogenicity of these approaches are still troublesome [6-8]. Using nanocarriers such as exosome with feature similar to that of cell could resolve the problem. \u003c/p\u003e\n\u003cp\u003eExosome is a naturally occurring nano-carrier for cell to cell communication in all biological systems. Exosome as a nanocarriers is cup likes structure with 30-100 nm diameter which possesses bold features such as, biocompatibility, no toxicity, and no immunogenicity. Having that said, exosome seems to be a proper candidate for RNAi delivery and targeted therapy [9, 10]. It worth to note, that the application of these bionanoparticles also struggles with efficient cargo loading, efficient isolation methods, purity, and microbial infection. Various studies tried to remove these obstacles [11]. Exosome as a communication devise of the cells carry RNA, more specifically miRNA, which sparked the idea of applying exosome as a proper device for RNA delivery [12]. The exosome\u0026apos;s contents lack ribosomal RNA (rRNA) and have a high amount of small RNAs, messenger RNAs (mRNAs) and miRNA, which are named exosomal shuttle RNAs (esRNAs) [13]. Exosomes extracted from body fluid such as plasma, amniotic fluid, and saliva can also carry their specific esRNAs which have a function in recipient cells [14]. The cell transporting machinery distinguishes esRNAs from its cytosolic form due to the difference in their sequence with a not well-understood mechanism [15]. Recognition of esRNA specific sequences can be applied in creating a specific cell factory that produces targeted exosomes containing a definitely identified exosomal miRNA. This study aims to design a specific miR34a expression system and create a stable cell line that releases exosomes with elevated miR34a cargo.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1. Software and primer designing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequence of the miR34a gene was taken from the NCBI database (GenBank: EF609116.1) and was checked using CLC main version 5 software. The secondary structures of the wild-type and mutant miR34a gene were also analyzed by RNA software. Gene Runner and OLIGO7 software were applied to design six primers to amplify the miR34a gene, site-directed mutagenesis, and cloning. The primers have restriction site sequences compatible with pCDH vector and spacer sequences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. miR34a gene amplification and site directed mutation \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal DNA was extracted from whole blood cells by QIAamp DNA extraction kit (Qiagen, Germany). Two primer pairs were separately used to amplify the 250bp DNA encoding miR34a by PCR. The F-34a (5-TGGGGAGAGGCAGGACAGG-3) and R-34a primers (5-TCCGAAGTCCTGGCGTCTCC-3) were used for amplification of the miR34a gene from DNA\u003cspan dir=\"RTL\"\u003e.\u003c/span\u003e PCR reactions were performed using Takara puf kit for 30 cycles (reaction conditions were as follows: 30s at 95 \u0026ordm;C, 30s at 60 \u0026ordm;C, and 30s at 72 \u0026ordm;C). The first pair, 34a FWD (5\u0026apos;- TGGGGAGAGGCAGGACAGG \u0026ndash; 3\u0026apos; and 5\u0026apos;- TCCGAAGTCCTGGCGTCTCC-3\u0026apos;) and S2 (5\u0026apos; TTA CTA TTG CTC ACA A CA TCC TC C TAA GAC ACT GC 3\u0026prime;) were used for amplification of the first DNA fragment with the first set of 6 nucleotides deserted point mutation.\u003c/p\u003e\n\u003cp\u003eIn comparison, the second pair S1 (5\u0026prime;CTCGAGTACAACTATGCGGCCGCAGCAGAATG-GGAGATGAATTTCA3\u0026prime;) and 34a REV (5\u0026prime;ACGACGCGTCG TTAGTGTTACAGAGTCT-GATATCC3\u0026prime;) were used for amplification of the second fragment of DNA with a second set of 6 nucleated deserted point mutation encoding miR34a pre-miRNA. PCR reactions were performed using Takara puf kit for 30 cycles (reaction conditions were as follow: 30s at 95 \u0026ordm;C, 30s at 60 \u0026ordm;C, 30s at 72 \u0026ordm;C for the first reaction and 30s at 95 \u0026ordm;C, 30s at 60 \u0026ordm;C, 30s at 72 \u0026ordm;C for the second reaction). Both S1 and S2 primers were designed to include an overlapping sequence. In the next step, these overlapping fragments were used to assemble two amplified sequences into a full-length DNA through an overlap extension of PCR (30s at 95 \u0026ordm;C, 30s at 60 \u0026ordm;C, and 120s at 72 \u0026ordm;C). Finally, a primer pair composed of 34a FWD and 34a REV was used to amplify the assembled fragment following reaction conditions as the 30s at 95\u0026ordm;C, 30s at 60\u0026ordm;C, and 90s at 72\u0026ordm; C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. miR34a gene cloning \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003epCDH lentiviral vector and the amplified miR34aW and miR34aM gene fragments were digested separately with BamH1 and not1 restriction enzymes (Roche, Germany) and ligated by T4 DNA ligase (Fermentas, Germany). The ligation mixture was transformed to Stbl4 competent bacteria by CaCl\u003csub\u003e2\u003c/sub\u003e method. The transformed bacteria were cultured on LB-agar medium containing ampicillin (100\u0026micro;g/ml). Colony PCR was performed to screen the positive colon by universal primers of pCDH vector (pCDH-FWD 5\u0026prime;-CACCAAAATCAACGGGACTT-3\u0026prime; and pCDH-REV 5\u0026prime;-ATATAGACAAACGCACACCGGCCT-3\u0026prime;). Positive bacterial colonies were harvested and cultured overnight in LB medium for plasmid extraction. DNA sequencing with universal primers was done to confirm the accuracy of the cloned product.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. Lentiviral vector packaging, titration, and cell transduction \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLentiviral particles were produced in HEK293T cells as described by Kutner et al. with some modifications [18]. Briefly, 24 hours before transfection, a 5 million concentration of HEK 293T cells were plated in a 10cm dish in DMEM for confluency of 80% to 90% in the next day. Cell culture medium was replaced with fresh complete DMEM two hours before transfection. Then, cells were transfected with Lenti ORF pcdh-mir34a (wild and mutant separately), pMD2G, and pSPAX by calcium phosphate method. Culture medium was replaced with fresh medium 14 hours post-transfection. For virus isolation, the Culture medium was collected three times at 12 hours intervals. \u003c/p\u003e\n\u003cp\u003eTo increase virus particle concentration, cell debris was removed following centrifugation of culture medium at 300g for 5 minutes, and the supernatant was subsequently filtrated through Millex-HV 0.45\u0026mu;m PVDF filter (Millipore). Lentiviral particles were precipitated via ultracentrifugation at 45000g for 1.5 hours, and the virus particles in the pellet were re-suspended in 1ml of PBS. Concentrated virus particles were used to transduce HEK 293T cells.\u003c/p\u003e\n\u003cp\u003eThree days post-transduction, flow cytometric analysis was performed for lentivirus titration [15]. Briefly, serial dilution (0.5, 1, 5, 25, and 50) of concentrated virus particles were added to 100000 HEK 293T cells which were cultured in 500 \u0026micro;l of DMED medium. Three days later, cells were harvested and washed twice with PBS, and GFP positive cells population were counted by flow cytometry.\u003c/p\u003e\n\u003cp\u003eViral transduction performed by the concentrated virus, briefly HEK293T cells (6.0\u0026times;10\u003csup\u003e4\u003c/sup\u003e) were cultured in 6 well plates. After 24 hours, 10ulit of concentrated virus dissolved to 2 ml DMEM free FBS medium, and the final solution was added to each well. After 16 hours, the medium was replaced with a fresh DMED complete medium. The GFP fluorescent microscopy was applied for transduction rate detection after 72 hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5. Puromycin kill curve and stable cell line selection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirst, HEK293T cells (6.0\u0026times;10\u003csup\u003e4\u003c/sup\u003e, not infected with the virus) were cultured in 5 wells of a 24-well plate containing 1 ml of complete DMEM medium. On the next day, when the cells stick to the bottom of the plate, we replace the culture medium of the cells with 1 ml of the culture medium containing zero, 0.5, 1, 1.5 and 2 micrograms of antibiotic puromycin, respectively. On the first day after the treatment, the culture medium of the cells was replaced with the selected medium containing the antibiotic puromycin, and daily the number of living cells in each well was counted by vital staining.\u003c/p\u003e\n\u003cp\u003eThe best result is obtained 1 to 4 days after antibiotic treatment. During this period, the number of living cells for each concentration was shown as a curve. The least amount of antibiotic that destroys the cells at this time was selected for the selection of cells after transduction.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6. Isolation of exosomes \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo produce miR34a enriched exosomes, HEK293T stable cells were cultured in DMEM supplied with 10% exosome-free FBS. The culture media were periodically collected at 48 hours intervals, and then exosomes were isolated via sequential centrifugation and re-suspended in PBS. Aliquots of exosomes were stored at -80 \u0026ordm;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7. Characterization of exosome\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransmission electron microscopy (TEM), zeta sizer analysis, and western blot analysis were carried out to characterize the obtained exosome. For electron microscopy imaging, the ultra-concentrated exosomes were fixed with 4% paraformaldehyde in PBS. The fixed samples were air-dried on Formvar-carbon coated TEM grids and stained with 0.5% uranyl acetate in 30% ethanol for 10 min, followed by staining with lead citrate 10 min. The sample was sequentially rinsed in 0.05 M NaOH and distilled water for removing lead precipitates on the grid sections. Then, the grids were dried at room temperature, and the stained sections were scanned under the transmission electron microscope (Philips) operating at 150 kV. According to the manufacturer\u0026apos;s instructions, the purified exosomes\u0026apos; dimensions were measured using a zeta sizer (Malvern Corp.). \u003c/p\u003e\n\u003cp\u003eThe transduced cells and exosomes were lysed with RIPA buffer containing protease inhibitor cocktail (Roche, Germany), and the obtained total protein was subjected to Western Blot analysis. Briefly, the cells were washed twice with ice-cold PBS and centrifuged at 4000 rpm for 5min. The cells (and exosomes) were treated with lysis buffer and incubated on ice for 20min with shaking. Cell debris was precipitated by centrifugation at 13000 rpm for 20 min, and the upper phase was collected as a source of the total protein. The number of total proteins was calculated using the Bradford assay. The extracted proteins (10 \u0026mu;g) were boiled for 5 min in 10X SDS buffer (1 \u0026mu;g) and resolved on 10 % polyacrylamide gel for 2 hours. Protein fragments were transferred to nitrocellulose membrane under the constant current of 300 mA for 2 hours, and the membrane was blocked with 5% nonfat dry skim milk in PBS for 2 hours. The membrane was washed with PBS and treated with HRP-conjugated anti-His tag antibody for 16 hours to detect LAMP-DARPin G3 chimeric protein. After three times of washing with PBST, the related band was detected using ECL solution. In addition, the expression of CD63 as an exosome marker was investigated by flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8. Revers Transcription and quantitative PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted using Qiagen RNeasy mini kit from purified exosomes three days post-transduction of HEK293T cells with miR-34a virus. Revert-Aid\u0026trade; First Strand cDNA Synthesis Kit (Fermentas, Germany) was applied for cDNA synthesis according to the manufacturer\u0026apos;s instructions.\u003c/p\u003e\n\u003cp\u003eThe relative qPCR experiments were performed on the ABI thermal cycler in the total volume of 20 \u0026micro;l containing 10 \u0026micro;l of Qiagen qPCR Master Mix (Qiagen), 10 pmol of each primer, and 2 \u0026micro;l of each cDNA. 16 s RNA was used as a reference gene. \u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1. miR34a gene manipulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequence of wild type miR34a was taken from NCBI data-base (GenBank: EF609116.1), and its sequence was manipulated for more expression as illustrated in figure 1a. The bioinformatics analysis using RNA software indicated that the changes of miR34a did not change its secondary structure compared to wild-type miR34a (Fig. 1b).\u003c/p\u003e\n\u003cp\u003eMiR34a gene amplification and site directed mutagenesis were performed by six primers (Fig. 2). At first, the 300 bp DNA fragment of human genome, including miR34a gene, was amplified, sequenced fallow blast, and confirmed miR34a gene. This segment was re-amplified by two specific primers, which have restriction site in 5\u0026apos; end for gene cloning into PCDH lentiviral vector, after digestion of miR34a and PCDH, gene and vector ligated and pcdh-mir34w was constructed and cloned into stbl4 (Fig. 2b).\u003c/p\u003e\n\u003cp\u003eWe used overlap extention PCR method to change 6 nucleotide in 3p and 5p coding sequence of miR34a. Therefore, two segments of miR34a were amplified and manipulated then ligated by PCR, one PCR amplified half of 5p segment of mir3a and stem-lope and change the 5p miR34a by R Soeing reverse primer and the second PCR performed to amplified 3p segment of miR34a and stem lope and introduce diversity in 3-p miR34a gene (Fig. 2a), the stem lope is the segment has overlap function in the final PCR which soied to segment of mutated miR34a (Fig. 2b). This fragment digested and insert to pCDH and pCDH-mir34M was constructed and cloned into stbl4 bacteria. \u003c/p\u003e\n\u003cp\u003eAs pCDH vector has a reporter gene to evaluate transfection efficiency, the GFP expression was used for transfection of HEK293T cells. We found that up to 90% of the cells express the GFP reporter gene; In addition, HEK293T virus producer cells were nucleated due to VSV-G protein expression during virus production (Fig. 2c). \u003c/p\u003e\n\u003cp\u003eThe virus particles were concentrated by ultra-centrifugation and the titer of lentiviral stock was determined by flow cytometry. Transduction of 8\u0026times;10\u003csup\u003e4\u003c/sup\u003e HEK cells by 5\u0026micro;l of the virus suspension, induced GFP expression in the 8\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells [16]. The lentiviral titer was estimated by flow cytometry as 1.6 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e TdU/ml. The electron microscopy result of the HEK293 T cells showed that 100% of the cells were transduced with pCDH-miR34a Vector (Fig. 2d).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. HEK293T cells transduction and stable cell line production\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe HEK293T cells were transduced with virus particles bearing mir34w and mir34M gene (MOI=5). Puromycin kill curve for HEK293T (Fig. 3) showed that 1.5 \u0026micro;g/ml of Puromycin is the minimum concentration of antibiotic that kills non-transduced HEK293T cells. To select a stable cell line containing mir34M and mir34W within its genome, Puromycin was added to kill non-transduced cells in the cell culture medium for 2 weeks. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExosome characterization result\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the current study, we hypothesized that modified HEK293T cells with pCDH-miR34a vector secrete functional miR-34a into exosomes. To confirm this hypothesis, we initially performed isolation and characterization of transduced HEK293T cell derives exosomes. According to the results, exosome fractions showed around 75 nm sizes in diameter (Fig. 4b). Exosomes demonstrated a spherical shape with a uniform particle size. The particle size distribution obtained by DLS was in accordance with the images obtained by TEM (Fig. 4a).\u003c/p\u003e\n\u003cp\u003eWestern blot analysis of the exosomes extracted from medium of transduced cells indicated expression of CD63 but did not show calnexin expression (Fig. 5 a and b). As illustrated in Fig. 5c immunophenotyping analysis showed that the exosomes were positive for CD81 (54.4 \u0026plusmn; 0.5%) (Fig. 5c).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression of wild type and manipulated miR34a:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to the results of relative qPCR, miR34a expression extracted from exosomes of three sources; non-transduced HEK293T, HEK293T transduced with mir34W lentivirus and HEK293T cells transduced by mir34M lentivirus vectors. The results indicated that intact cells have no delectable miR34a expression while its expression on HEK293T cells which were transduced by mutate type of mir34 increased by approximately 2.9-fold in comparison to hek293 cells transduced by wild type miR-34a (p \u0026lt; 0.01) (Fig. 6).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003emiRNAs, non-coding RNAs, have an effect on various biological processes, they play a key role in the growth and development of organisms[17]. miRNAs have been introduced as novel drug candidates. Assessing their safety and effectiveness is the most important step in their entry into the clinical field[18]. On the other hand, the low stability of miRNAs, difficult delivery to the target cell, rapid degradation in the blood by ribonucleases, clearance by the reticuloendothelial system, being trapped in the endosome and then degradation in the lysosome limit their clinical applications. Considering the development of nanoscience and the progress of nanoparticles in the field of drug delivery, it can be said that the delivery of miRNAs by nanoparticles can be an effective method in the transfer of miRNAs[19-21]. Due to nanoparticles, the entry of miRNA into the target cells is multiplied [22].\u003c/p\u003e\n\u003cp\u003eOn the other side, unlike nanoparticles that are synthesized manually, exosomes as nanoparticle of endogenous origin have unique features such as better targeting capacity, stability in body fluids, biocompatibility, safety, and nontoxicity [23].\u003c/p\u003e\n\u003cp\u003eThe aim of this study is to create a cell factory to generate miR34a-enriched exosomes. \u003c/p\u003e\n\u003cp\u003eMir-34a is a member of the mir34 family involved in P53 regulation, cell signaling, and cell survival pathways. MiR34a stops the cell cycle at the G1 step and induces apoptosis or cell death. MiR34a is a tumor suppressor molecule, and the degradation of miR34a was observed in several types of cancers [24-26].\u003c/p\u003e\n\u003cp\u003eThe expression of miR-34a is suppressed in malignancies and some molecular processes such as cell differentiation and proliferation[26]. MiR34a affects osteosarcoma\u0026apos;s metastasis, and decreases in its expression could lead to increased cell growth and metastasis[27]. Applying mimetic miR34a as a candidate for cancer therapy has been progressed to phase I of the clinical trial and production and packaging of miR34a into liposome has been successfully done[28].\u003c/p\u003e\n\u003cp\u003eThere are several methods for loading miRNAs into exosomes. The easiest procedure to generate miRNA-enriched EVs is to incubate them with miRNA-secreting cells. Using a concentration gradient for this method can be a natural process that does not create a risk factor for cells. However, this approach has the disadvantage that it cannot predict the concentration of loaded miRNAs in exosomes and has low efficiency due to the instability of miRNA[29]. Another method is electroporation. For the first time, L Alvarez-Erviti et al.[30] used exosomes to transport siRNA to the brain cells. They insert the desired siRNA into the exosomes using electroporation approaches. JH Wang et al.[31] used electroporation to load EVs with HChrR6 mRNA. But the results of this method seemed inadequate. Using electroporation for transfection of nucleic acids inside exosomes is a simple method, but it is not efficient. This method requires the purification and isolation of extracellular vesicles before and after the transfection process, which repeated purification can lead to the loss of exosomes[32]. In another study, TN Lamichhane et al.[33] investigated the transport of siRNAs loaded by sonication in extracellular vesicles. Contrary to the high cellular uptake of extracellular vesicles (80%), the overall delivery of siRNA to cells was very low (2.96%). Sonication also has limitations such as changing the shape of the extracellular vesicle membrane, generating heat, destroying surface proteins and not being suitable for the delivery of hydrophobic drugs[34].\u003c/p\u003e\n\u003cp\u003eCell-engineering is another approach to producing miRNA-enriched exosomes. Overexpression of the desired miRNA in exosome-producing cells by cellular machinery and mimicking the spontaneous miRNA loading and packaging system are two approaches used to generate miRNA-loaded exosomes[35]. In the present research, we used both systems. First, we inserted exosome specific sequences into miR34a then we transduced the manipulated miR34a gene via a lentiviral system into HEK293T genome, which applied CMV promoter for high and permanent expression of miR34a gene.\u003c/p\u003e\n\u003cp\u003eVarious studies suggest that there is a sorting sequence at the 3\u0026apos; end of miRNA sequences. It is predicted that the transfer of miRNA into the exosome is carried out by this sequence [36]. This sequence can be used to transfer miRNA into the exosome. MF Bolukbasi et al.[37] investigated a 25-nt sequence containing \u0026quot;CTGCC\u0026quot; on a stem-loop structure and a miR-1289 binding site in the 3UTRs of many mRNAs enriched in MVs derived from GBM cells. Their findings indicate the important role of the presence of the CTGCC sequence in the loop structure and the binding site of miR-1289 in the 3\u0026apos;UTR, in increasing the integration of mRNAs in MVs. By RNA sequencing (RNA-seq) from a panel of human B cells and their secreted exosomes, D Koppers-Lalic et al.[38] demonstrated that miRNAs that are 3\u0026apos; adenylated were relatively enriched in cells, while 3\u0026apos; uridylated isoforms were overexpressed in exosomes. limits are shown. Therefore, it can be said that these post-transcriptional modifications help to guide the sorting of ncRNA to EVs to some extent. Villarroya-Beltri et al.[15] described sequence motifs present in miRNAs that control their localization into exosomes (EXOmiRNA). These specific sequences (GGAG) are present at the end of miRNA and lead the miRNA to the endosomes and finally into exosomes. Sumoylated hnRNPA2B1 binds to EXOmiRNA and controls its loading into exosomes. In this study we substituted 6 nucleotides of miR34a gene to increase its packaging rate into exosomes. Since substitution of nucleotides at 5\u0026apos; and 3\u0026apos; ends of the miR34a gene could affect its expression, stability and secondary structure, a 250 bp fragment capturing and containing miR34a sequences was selected for cloning in the present research. Moreover, any changes in one part of the gene may require other changes in other parts. For manipulating the 3\u0026apos; ends of mir5p, the manipulation of a complementary sequence at mir3p is also required. The required changes to the miR34a gene were created using the overlap extension PCR method by four specific primers at two gene regions. One pair for insertion of GGAGAG sequence at 3 end of mir34-5p, and another pair for insertion of 6 nucleotides CTCTCC that is complementary to the first manipulated change for stabilization of the secondary structure. The results confirmed the created changes in the miR34a gene, and bioinformatics analysis has also indicated that these changes do not affect the secondary structure of the molecule. The energy level of the manipulated molecule was not changed in comparison to the original one. \u003c/p\u003e\n\u003cp\u003eHigh expression of the desired RNA in exosome-producing cells via lentiviral vectors can lead to enrichment of that RNA and its encoded protein in the produced exosomes[39]. Lentiviral vectors can transduce both dividing and non-dividing cells, while retroviral vectors only transduce non-dividing cells [40]. Therefore, in the present research, a lentiviral vector was chosen for overexpression of miR34a and creation of a stable cell line that produces miR34a loaded exosomes and releases them to medium. \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our results showed that normal HEK293T didn\u0026apos;t express miR34a while lentiviral transduced miR34a gene induced loading of miR34a into exosome, in comparison, induced mutation by replacing of 6 nucleic acids in 3\u0026apos; end of miR34a increases 3-fold loading of miR34a to exosome.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure of potential conflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResearch involving human participants and/or animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research dose not involve human tissue or animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors consent to publish this article findings.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data are reported as they obtained. And the original data will be presented upon a reasonable request. The RNA folding (Fig. 1) was obtained from http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi website.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported financially by Grant No. #2837 from Mazandaran University of Medical Sciences, Sari, Iran.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHuppi, K., S.E. Martin, and N.J. Caplen, \u003cem\u003eDefining and assaying RNAi in mammalian cells.\u003c/em\u003e Molecular cell, 2005. \u003cstrong\u003e17\u003c/strong\u003e(1): p. 1-10.\u003c/li\u003e\n\u003cli\u003eChen, F. and S.J. Hu, \u003cem\u003eEffect of microRNA‐34a in cell cycle, differentiation, and apoptosis: a review.\u003c/em\u003e Journal of biochemical and molecular toxicology, 2012. \u003cstrong\u003e26\u003c/strong\u003e(2): p. 79-86.\u003c/li\u003e\n\u003cli\u003eYamakuchi, M., M. 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Cell Rep 8: 1649\u0026ndash;1658\u003c/em\u003e. 2014.\u003c/li\u003e\n\u003cli\u003eChen, L., et al., \u003cem\u003eExosomes derived from GDNF-modified human adipose mesenchymal stem cells ameliorate peritubular capillary loss in tubulointerstitial fibrosis by activating the SIRT1/eNOS signaling pathway.\u003c/em\u003e Theranostics, 2020. \u003cstrong\u003e10\u003c/strong\u003e(20): p. 9425.\u003c/li\u003e\n\u003cli\u003eDufait, I., et al., \u003cem\u003eRetroviral and lentiviral vectors for the induction of immunological tolerance.\u003c/em\u003e Scientifica, 2012. \u003cstrong\u003e2012\u003c/strong\u003e.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"exosome, miR34a, HEK293T, lentiviral vector ","lastPublishedDoi":"10.21203/rs.3.rs-2825672/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2825672/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e RNA antisense emerging as novel candidates for therapeutic purposes. As an RNA, miR-34a involves in P53 function, and triggers cancer cells apoptosis. The clinical applications of miRNAs face some limitation which can be potentially resolved using exosome as a transporting vehicle.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAims:\u003c/strong\u003e The aim of this study is to create a cell factory to generate miR34a-enriched exosomes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e First exosome specific sequences were inserted into miR34a. The resulting miR34a gene were transduced HEK293T cells genome with a lentiviral system. In the structure of miR34a gene 6 nucleotides were substituted to increase its packaging rate into exosomes. To maintain the secondary structure, stability and expression of the miRNA gene, changes to the miR34a gene were made using PCR Extension.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The results confirmed the created changes in the miR34a gene do not affect its secondary structure. The energy level of the manipulated miR34a gene was not changed in comparison with the original one.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Our results suggested that the induced mutation (replacing 6 nucleic acids at the 3' end of miR34a) increases loading of miR34a into the exosome by 3-fold.\u003c/p\u003e","manuscriptTitle":"Lentiviral Engineered cells for production of miR34a loaded exosomes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-05-10 21:28:54","doi":"10.21203/rs.3.rs-2825672/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor Revisions Needed","date":"2023-07-03T08:01:57+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2023-05-23T07:54:40+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-05-02T12:16:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-04-28T18:09:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2023-04-25T02:17:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"cf6e80d3-11cf-4c4f-98c4-25bebad48343","owner":[],"postedDate":"May 10th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-09-07T15:17:03+00:00","versionOfRecord":{"articleIdentity":"rs-2825672","link":"https://doi.org/10.1007/s11033-023-08754-1","journal":{"identity":"molecular-biology-reports","isVorOnly":false,"title":"Molecular Biology Reports"},"publishedOn":"2023-09-02 15:10:16","publishedOnDateReadable":"September 2nd, 2023"},"versionCreatedAt":"2023-05-10 21:28:54","video":"","vorDoi":"10.1007/s11033-023-08754-1","vorDoiUrl":"https://doi.org/10.1007/s11033-023-08754-1","workflowStages":[]},"version":"v1","identity":"rs-2825672","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2825672","identity":"rs-2825672","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}