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 <span style="font-size:10pt"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""="">'''<span lang="EN-US" style="font-size:14.0pt"><span style="line-height:107%"><span style="font-family:" arial",sans-serif"="">Necessity of RNA Sequencing in Human BBB Models</span></span></span>'''</span></span></span><br/> <span style="font-size:10pt"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""="">'''<span lang="EN-US" style="font-size:14.0pt"><span style="line-height:107%"><span style="font-family:" arial",sans-serif"="">&>&nbsp;and Drug Delivery Research</span></span></span>'''</span></span></span><br/> <span style="font-size:10pt"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""=""><span lang="EN-US" style="font-family:" arial",sans-serif"="">20191193 HyeongJin Yun</span></span></span></span><br/> <br/> <span style="font-size:10pt14px;"><span style="line-height:107%">'''<span stylelang="fontEN-family:" 맑은="" 고딕""="US">Introduction</span>'''</span></span> <span lang="EN-US" style="font-familysize:14px;" arial">I have always been deeply interested in diseases caused by brain dysfunction,sans-serif"="">Introduction</span>'''</span></span></span><br/> <span style="fontsuch as brain cancer, depression, and dementia. This interest led me to pursue research and development in the field of drug delivery to the brain, particularly through the blood-size:10pt"><span style="linebrain barrier (BBB). I am currently developing a human blood-height:107%"><span style="font-family:" 맑은="" 고딕""=""><span lang="EN-US" style="font-family:" arial",sans-serif"="">I am currently developing a human blood brain barrier (BBB) model brain barrier (BBB) model and conducting extensive research to understand how carriers penetrate the BBB for drug delivery. My work primarily involves building basic cell culture techniques and creating an in vitro BBB model. The carrier samples used in this experiment are provided by our collaborative research partner. We plan and conducting extensive research to understand how execute further experimental procedures by determining the permeability of these carriers penetrate across the BBB for drug delivery. My work is primarily to build basic cell culture techniques and an in vitro BBB model. The carrier samples used in this experiment are provided by our collaborative research partner. We plan and execute further experimental procedures by determining </span> <span style="font-size:14px;">Currently, I have completed the permeability process of these carriers across determining whether lipid nanoparticles (LNPs) can pass through the BBB.</span></span></span></span><br/> <span style="fontNow, I have shifted my focus to exploring the mechanisms by which these nanoparticles pass through the barrier. I became particularly interested in this area because I believe RNA-size:10pt"><span style="linesequencing, which was mentioned in the Genomics course, would help me explore these mechanisms further.&nbsp;RNA-height:107%"><span sequencing can provide detailed insights into the gene expression changes and molecular pathways activated during nanoparticle transport across the BBB, thereby elucidating the specific biological processes and interactions involved.</span> <br/> <br/> <span style="font-familysize:14px;" 맑은="" 고딕""><span style="line-height:107%">'''<span lang="EN-US" style="font>Blood-family:" arialBrain Barrier and Lipid Nanoparticles</span>'''</span><br/> <span style=",sansline-serifheight:107%"=><span lang="EN-US">Currently, I have completed the process of determining whether lipid nanoparticles The human blood-brain barrier (LNPsBBB) can pass through BBB. Now I have shifted my focus is a unique and selective barrier that regulates the transport of substances from Blood to exploring the mechanisms by which these nanoparticles pass through brain. It plays an important role in maintaining the barrierneurons and glia function. I came to find out because I thought RNA-sequencingBBB is composed of Brain microvascular endothermic cells (BMVECs), Astrocytes, which was mentioned in Genomics coursePericytes, would help me explore this mechanism.and ECM (</spanstyle="color:#0070c0">1,2</span></span><). BMVECs have a much lower degree of endocytosis/transcytosis activity than peripheral endothelium contributing to the characteristics of BBB as a barrier (<spanstyle="color:#0070c0">3<br/span> ).<br/span> </span style="font-size:10pt"><br/><span style="line-height:107%"><span stylelang="fontEN-family:" 맑은="" 고딕""="">'''<span lang="EN-US" style="font-family:" arialUS">&nbsp;Lipid nanoparticles (LNPs) are widely used in the field of drug delivery. LNPs are primarily composed of cholesterol and lipids and can encapsulate DNA,sans-serif"RNA, or drugs. Additionally, ligands or antibodies can be attached to the surface to aid in targeting (<span style=""color:#0070c0">Blood-Brain Barrier and Lipid Nanoparticles</span>'''4</span>).</span></span><br/> <span style="fontline-sizeheight:10pt107%"><span stylelang="lineEN-height:107%US"><span style="font-family:" 맑은="" 고딕""=""><span lang="ENMy research is conducting a study on whether LNPs using a new fabrication method called Liposome Under Cryo-US" style="font-family:" arial",sans-serif"="">The Assembly (Luca) can pass through the human blood-brain barrier (BBB) model. The identification of LNP samples that penetrate well is a unique finished and selective barrier that regulates the transport of substances from Blood to mechanism study process is in the brainprocess. It plays an important role in maintaining the neurons and glia function. BBB is composed of Brain microvascular endothermic cells (BMVECs), Astrocytes, Pericytes, and ECM (</span></span><br/> <br/> <span style="colorline-height:#0070c0107%">'''<span lang="EN-US">1,2Overview of RNA sequencing technology</span>). BMVECs have a much lower degree of endocytosis'''</transcytosis activity than peripheral endothelium contributing to the characteristics of BBB as a barrier (span><span stylebr/> <span style="colorline-height:#0070c0107%">3</span>).</span></spanlang="EN-US">Principles of RNA Sequencing</span></span><br/> <span style="font-size:10pt"><span style="line-height:107%"><span stylelang="fontEN-family:" 맑은="" 고딕""="US"><span lang="ENRNA sequencing (RNA-US" style="fontseq) is a high-family:" arial",sans-serif"="">&nbsp;Lipid nanoparticles (LNPs) are widely throughput sequencing technology used in to determine the field nucleotide sequence of drug deliveryRNA molecules and quantify specific RNA species within a population. LNPs are primarily composed The process of cholesterol and lipids and can encapsulate DNA, RNA, or drugs. Additionally, ligands or antibodies can be attached to sequencing includes the surface to aid in targeting following steps (<span style="color:#0070c0">45</span>).:</span></span></span></span><br/>  #<span style="font-size:10pt14px;"><span style="line-height:107%"><span stylelang="fontEN-family:" 맑은="" 고딕""="US">RNA Extraction: Extracting RNA from biological samples such as cells or tissues.</span></span></span>  &nbsp;<ol start="2"><li><span lang="EN-US" style="font-family:" arial",sans-serif"="">My research is conducting a study on whether LNPs using a new fabrication method called Liposome Under Cryo-Assembly (Luca) can pass through the human BBB model. The identification of LNP samples that penetrate well is finished and the mechanism study process is in the process.</span></span></span></span><br/> <br/> <span style="font-size:10pt"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""="">'''<span lang="EN-US" style="font-family:" arial",sans-serif"="">Overview of RNA sequencing technology</span>'''</span></span></span><br/> <span style="font-size:10pt"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""=""><span lang="EN-US" style="font-family:" arial",sans-serif"="">Principles of RNA Sequencing</span></span></span></span><br/> <span style="font-size:10pt"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""=""><span lang="EN-US" style="font-family:" arial",sans-serif"="">RNA sequencing (RNA-seq) is a high-throughput sequencing technology used to determine the nucleotide sequence of RNA molecules and quantify specific RNA species within a population. The process of RNA sequencing includes the following steps (<span style="color:#0070c0">5</span>):</span></span></span></span> #<span style="font-size:10pt"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""=""><span lang="EN-US" style="font-family:" arial",sans-serif"="">RNA Extractionsize: Extracting RNA from biological samples such as cells or tissues.</span></span></span></span> &nbsp14px; <ol start="2"><li><span style="font-size:10pt"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""=""><span lang="EN-US" style="font-family:" arial",sans-serif"="">cDNA Synthesis: Converting the extracted RNA into complementary DNA (cDNA) using reverse transcriptase.</span></span></span></span></li>

<br/> &nbsp;<ol start="3"><li><span style="font-size:10pt14px;"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""=""><span lang="EN-US" style="font-family:" arial",sans-serif"="">Library Preparation: Fragmenting the cDNA, attaching adapters, and amplifying the cDNA fragments through PCR.</span></span></span></span></li>

 &nbsp; <ol start="4"><li><span style="font-size:10pt14px;"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""=""><span lang="EN-US" style="font-family:" arial",sans-serif"="">Sequencing: Sequencing the prepared library using platforms such as Illumina or Nanopore.</span></span></span></span></li>

 &nbsp; <ol start="5"><li><span style="font-size:10pt14px;"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""=""><span lang="EN-US" style="font-family:" arial",sans-serif"="">Data Analysis: Aligning the generated sequences to a reference genome and quantifying gene expression levels​.</span></span></span></span></li>

 <br/> <span style="font-size:10pt14px;"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""="">'''<span lang="EN-US" style="font-family:" arial",sans-serif"="">Methods for Gene Expression Analysis Using RNA Sequencing</span>'''</span></span></span><br/> <span style="font-size:10pt"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""=""><span lang="EN-US" style="font-family:" arial",sans-serif"="">The analysis of gene expression using RNA sequencing involves several steps:</span></span></span></span><br/> &nbsp;  #<span style="font-size:10pt14px;"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""=""><span lang="EN-US" style="font-family:" arial",sans-serif"="">Data Quality Control: Assessing the quality of sequencing data and removing low-quality reads.</span></span></span></span>  &nbsp; <ol start="2"><li><span style="font-size:10pt14px;"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""=""><span lang="EN-US" style="font-family:" arial",sans-serif"="">Data Alignment: Aligning the reads to a reference genome to determine the location of each read.</span></span></span></span></li>

<br/> &nbsp;<ol start="3"><li><span style="font-size:10pt14px;"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""=""><span lang="EN-US" style="font-family:" arial",sans-serif"="">Gene Expression Quantification: Calculating the expression levels of genes, typically represented as RPKM, FPKM, or TPM values.</span></span></span></span></li>

 &nbsp; <ol start="4"><li><span style="font-size:10pt14px;"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""=""><span lang="EN-US" style="font-family:" arial",sans-serif"="">Differential Expression Analysis: Identifying differentially expressed genes between specific conditions to understand changes in gene expression due to factors such as inflammation, stress, or drug treatment​</span></span></span></span></li>

 <br/> <br/> <span style="font-size:10pt14px;"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""="">'''<span lang="EN-US" style="font-family:" arial",sans-serif"="">Necessity of RNA Sequencing in BBB Models</span>'''</span></span></span><br/> <span style="font-size:10pt"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""=""><span lang="EN-US" style="font-family:" arial",sans-serif"="">Through fluorescence image analysis, I found out that LNPs migrate to the transcytosis pathway. In addition, I checked the difference in transmittance by performing several transcytosis inhibitor treatments.</span></span></span></span><br/> <span style="font-size:10pt"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""=""><span lang="EN-US" style="font-family:" arial",sans-serif"="">Through RNA sequencing, I would like to check whether the permeability has changed due to changes in the genetic level of the cells that actually make up BBB.</span></span></span></span><br/> <br/> <span style="font-size:10pt"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""="">'''<span lang="EN-US" style="font-family:" arial",sans-serif"="">Future Plan</span>'''</span></span></span><br/> <span style="font-size:10pt"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""=""><span lang="EN-US" style="font-family:" arial",sans-serif"="">Listening to the Genomics course, I thought that conducting research at various gene levels was essential to conduct research at a high level. Later, the goal is to proceed with the RNA sequencing mentioned above to find out the detailed mechanisms that have not yet been found. After finding out the mechanism, I will treat the Aptamer on the surface to find out the change in the permeability and put the actual drug inside to deliver it.</span></span></span></span><br/> <br/> <br/> <span style="font-size:10pt"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""="">'''<span lang="EN-US" style="font-family:" arial",sans-serif"="">References</span>'''</span></span></span><br/> <span style="font-size:10pt"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""=""><span lang="EN-US" style="line-height:107%"><span style="font-family:" arial",sans-serif"="">(1) Cecchelli, Romeo, et al., Modelling of the blood–brain barrier in drug discovery and development., '''2007''', ''Nature Reviews Drug Discovery''. vol. 6, no. 8, pp. 650–661,.</span></span></span></span></span><br/> <br/> <span style="font-size:10pt"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""=""><span lang="EN-US" style="line-height:107%"><span style="font-family:" arial",sans-serif"="">(2) Park, Tae-Eun, et al., Hypoxia-enhanced blood-brain barrier chip recapitulates human barrier function and shuttling of drugs and antibodies., '''2019''', ''Nature Communications'', vol. 10, no. 1, 13</span></span></span></span></span><br/> <br/> <span style="font-size:10pt"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""=""><span lang="EN-US" style="line-height:107%"><span style="font-family:" arial",sans-serif"="">(3) Abbott, N. Joan, Lars Rönnbäck, and Elisabeth Hansson., Astrocyte–Endothelial Interactions at the Blood–Brain Barrier., '''2006''', ''Nature Reviews Neuroscience 7'', no. 1: 41–53.</span></span></span></span></span><br/> <br/> <span style="font-size:10pt"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""=""><span lang="EN-US" style="line-height:107%"><span style="font-family:" arial",sans-serif"="">(4) Mehta, M., Bui, T. A., Yang, X., Aksoy, Y., Goldys, E. M., & Deng, W., Lipid-based nanoparticles for drug/gene delivery: An overview of the production techniques and difficulties encountered in their industrial development., '''2023''', ''ACS Materials Au'', 3(6), 600–619.</span></span></span></span></span><br/> <br/> <span style="font-size:10pt"><span style="line-height:107%"><span style="font-family:" 맑은="" 고딕""=""><span lang="EN-US" style="font-family:" arial",sans-serif"="">(5)</span> <span lang="EN-US" style="font-family:" arial",sans-serif"="">Deshpande, Dhrithi, et al., RNA-seq data science: From RAW data to effective interpretation.,'''2023''', ''Frontiers in Genetics'', vol. 14,</span></span></span></span><br/> &nbsp;