diff --git a/src/contents/results.tsx b/src/contents/results.tsx
index 33b422132c3382706f75cd6d1f3b05603c9f0989..44098c7a40570d4b44ed6da21c0120ee11bdc0e5 100644
--- a/src/contents/results.tsx
+++ b/src/contents/results.tsx
@@ -23,7 +23,7 @@ export function Results() {
<p>As proof of concept, we transfected these constructs in <strong>HEK293 and CFB41o- cells</strong> and observed significant prime editing of our reporter via fluorescence microscopy. We identified the PE6c editor and our pegRNA variant 4 as optimal. This resulted in our <strong>New Basic Part</strong>, <strong>PEAR_CFTR</strong>. Furthermore, we extended our approach to primary human nasal epithelial cells generated from our own nasal epithelial cells through nasal swabs. By cultivating them in Air Liquid Culture (ALI) and Apical-Out Airway Organoids (AOAO), we successfully tested our technologies <i>in vitro</i>, mimicking the <i>in vivo</i> situation.</p>
<p>Furthermore, we successfully designed and cloned novel nickases of <strong>Fanzor</strong>, which is special because of its smaller size and eukaryotic origin. This serves as valuable tool for future genome editing applications. </p>
<p>For delivery, lipid nanoparticles (LNPs) are a highly effective and versatile delivery system, valued for their larger cargo capacity, biocompatibility, and ability to protect RNA from degradation. To deliver our Prime Editing construct as pegRNA and mRNA, we optimized a <strong>Selective ORgan Targeting (SORT) LNP</strong> for targeted delivery to the lungs by using the cationic helper lipid DOTAP and encapsulating a stable <strong>chitosan-RNA complex</strong>, achieving significant breakthroughs in transfection of <i>in vitro</i> lung epithelial cells. </p>
-<p>We began by testing three different LNP formulations, starting with the <strong>Cayman LipidLaunc LNP-102 Exploration Kit</strong>. We confirmed by fluorescence microscopy, where Minicircle DNA effectively transfected HEK293 cells. Further experiments with the <strong>Corden LNP Stater Kit #2</strong> failed to achieve successful transfection, likely due to increased cytotoxicity from a more cytotoxic PEG component. Our successful formulation was a <strong>lung-specific SORT LNP</strong>, which demonstrated excellent stability, as confirmed by Zeta potential measurements. Dynamic light scattering (DLS) analysis revealed an optimal particle size of closely smaller than 200 nm, aligning with literature and supporting the ability of the LNPs to penetrate deep lung regions via inhalation. Flow cytometry analysis showed that the SORT LNP had 14 times higher transfection efficiency compared to traditional transfection methods. Moreover, a MTT assay revealed that the SORT LNP, along with Cayman LNPs, exhibited the lowest cytotoxicity, thanks to the use of low-molecular-weight PEG components. </p>
+<p>We began by testing three different LNP formulations, starting with the <strong>Cayman LipidLaunch LNP-102 Exploration Kit</strong>. We confirmed by fluorescence microscopy, where Minicircle DNA effectively transfected HEK293 cells. Further experiments with the <strong>Corden LNP Stater Kit #2</strong> failed to achieve successful transfection, likely due to increased cytotoxicity from a more cytotoxic PEG component. Our successful formulation was a <strong>lung-specific SORT LNP</strong>, which demonstrated excellent stability, as confirmed by Zeta potential measurements. Dynamic light scattering (DLS) analysis revealed an optimal particle size of closely smaller than 200 nm, aligning with literature and supporting the ability of the LNPs to penetrate deep lung regions via inhalation. Flow cytometry analysis showed that the SORT LNP had 14 times higher transfection efficiency compared to traditional transfection methods. Moreover, a MTT assay revealed that the SORT LNP, along with Cayman LNPs, exhibited the lowest cytotoxicity, thanks to the use of low-molecular-weight PEG components. </p>
<p>To further enhance the stability of the LNPs for inhalation, we incorporated <strong>chitosan-RNA complexes</strong>, which provide thermal stability and protect RNA from degradation by RNases. Integration of these complexes into the SORT LNP resulted in a lung-specific delivery platform with superior stability. Using this system, we achieved highly efficient transfection of a bronchial cell line from a Cystic Fibrosis patient (CFBE41o- with F508del mutation), demonstrating the potential of this approach for targeted gene delivery to lung epithelial cells. These results highlight the remarkable efficiency, stability and specificity of our optimized SORT LNP formulation, positioning it as a promising platform for lung-specific genetic therapies. </p>
</Section>
<Section title="Experimental Design" id="Experimental Design">
@@ -221,8 +221,8 @@ export function Results() {
<OneFigure
num={16}
pic1="https://static.igem.wiki/teams/5247/delivery/results/rna-gel-final.png"
- description="Gel of Denaturing RNA Gel Electrophoresis for mRNA synthesized from pcDNA 3.1 eYFP indicating successful RNA synthesis. Lane 1: Low Range Ribo Ruler, Lane 2: FLuc Control Template, Lane 3: Negative Control, Lane 4-9 mRNA from pcDNA 3.1 eYFP"
- alt1="Gel of Denaturing RNA Gel Electrophoresis for mRNA synthesized from pcDNA 3.1 eYFP indicating successful RNA synthesis. Lane 1: Low Range Ribo Ruler, Lane 2: FLuc Control Template, Lane 3: Negative Control, Lane 4-9 mRNA from pcDNA 3.1 eYFP"
+ description="Gel of Denaturing RNA Gel Electrophoresis for mRNA synthesized from pcDNA 3.1 eYFP indicating successful RNA synthesis. Lane 1: Low Range Ribo Ruler, Lane 2: FLuc Control Template, Lane 3: Negative Control, Lane 4-9 mRNA from pcDNA 3.1 eYFP."
+ alt1="Gel of Denaturing RNA Gel Electrophoresis for mRNA synthesized from pcDNA 3.1 eYFP indicating successful RNA synthesis. Lane 1: Low Range Ribo Ruler, Lane 2: FLuc Control Template, Lane 3: Negative Control, Lane 4-9 mRNA from pcDNA 3.1 eYFP."
/>
</div>
<div className="col">
@@ -236,10 +236,10 @@ export function Results() {
</div>
<div className="col">
<OneFigure
- description="Cayman LNP Formation indicated by blue color and turbidity. Mini DNA = Minicircle DNA from PlasmidFactory"
+ description="Cayman LNP Formation indicated by blue color and turbidity. Mini DNA = Minicircle DNA from PlasmidFactory."
num={17}
pic1="https://static.igem.wiki/teams/5247/delivery/results/caymanlnpblue.webp"
- alt1="Cayman LNP Formation indicated by blue color and turbidity. Mini DNA = Minicircle DNA from PlasmidFactory"
+ alt1="Cayman LNP Formation indicated by blue color and turbidity. Mini DNA = Minicircle DNA from PlasmidFactory."
/>
</div>
@@ -252,8 +252,8 @@ export function Results() {
pic1="https://static.igem.wiki/teams/5247/delivery/results/precyse/cayman.png"
num={18}
bg="white"
- description="Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells at 20x magnification 72h post-transfection with different Cayman LNP formulations recorded with Leica DMI6000 B. Lipo= lipofectamine, Mini DNA = Minicircle DNA"
- alt1="Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells at 20x magnification 72h post-transfection with different Cayman LNP formulations recorded with Leica DMI6000 B. Lipo= lipofectamine, Mini DNA = Minicircle DNA"
+ description="Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells at 20x magnification 72h post-transfection with different Cayman LNP formulations recorded with Leica DMI6000 B. Lipo= lipofectamine, Mini DNA = Minicircle DNA."
+ alt1="Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells at 20x magnification 72h post-transfection with different Cayman LNP formulations recorded with Leica DMI6000 B. Lipo= lipofectamine, Mini DNA = Minicircle DNA."
/>
<H5 text="SEM"/>
@@ -265,7 +265,7 @@ export function Results() {
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/results/screenshot-2024-10-01-200629.png"
alt1="CayREM"
- description="SEM image of Cayman LNPs (10,000x magnification) with Topography mode"
+ description="SEM image of Cayman LNPs (10,000x magnification) with Topography mode."
num={19}/>
</div>
</div>
@@ -276,8 +276,8 @@ export function Results() {
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/results/whatsapp-image-2024-09-24-at-12-57-59.jpeg"
num={20}
- description="Turbidity after components of the Corden LNP have been pipetted together indicates particle formation"
- alt1="Turbidity after components of the Corden LNP have been pipetted together indicates particle formation"
+ description="Turbidity after components of the Corden LNP have been pipetted together indicates particle formation."
+ alt1="Turbidity after components of the Corden LNP have been pipetted together indicates particle formation."
/>
</div>
<div className="col">
@@ -292,7 +292,7 @@ export function Results() {
num={21}
bg="white"
description="Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells at 20x magnification 72h post-transfection with different Corden LNP formulations recorded with Leica DMI6000 B. For lipofectamine (lipo) + Minicircle DNA (Mini DNA) only the fluorescence image is shown."
- alt1="Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells"
+ alt1="Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells."
/>
<H5 text="Cryo-EM"/>
<p>Cryo-EM as a form of transmission electron microscopy (TEM) was performed by us using a JEOL JEM-2200FS electron microscope (JEOL, Freising, Germany) operating at 200kV, equipped with a cold field emission electron gun. The sample preparation and imaging were carried out at cryogenic temperatures, which allowed for the visualization of LNPs in their native hydrated state.</p>
@@ -302,7 +302,7 @@ export function Results() {
pic1="https://static.igem.wiki/teams/5247/delivery/results/corden-lnp.jpg"
num={22}
description="Cryo-EM image of Corden LNPs."
- alt1="Cryo-EM image of Corden LNPs"
+ alt1="Cryo-EM image of Corden LNPs."
/>
</div>
<div className="col">
@@ -318,7 +318,7 @@ export function Results() {
num={23}
bg="white"
description="Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells at 20x magnification 48h post-transfection with different SORT LNP formulations recorded with Leica DMI6000 B. Lipo = lipofectamine, Mini DNA = Minicircle DNA."
- alt1="Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells"
+ alt1="Overlay of phase contrast and fluorescence microscopic images of transfected HEK293 cells."
/>
<p>Notably, the SORT LNP formulation with Minicircle DNA showed significant transfection efficiency within 48 hours, making it the most effective delivery method tested among all three LNPs. This early and robust expression demonstrates the clear advantage of this approach for HEK293 cells.</p>
<H5 text="Flow Cytometry"/>
@@ -330,7 +330,7 @@ export function Results() {
num={24}
bg="white"
description="Percentage of fluorescent cells (FITC-A+) performed 72h post-transfection of SORT LNP in HEK293. Mean +/- SEM for n=3. For statistics one-way ANOVA was performed."
- alt1="Percentage of fluorescent cells post-transfection of SORT LNP in HEK293"
+ alt1="Percentage of fluorescent cells post-transfection of SORT LNP in HEK293."
/>
</div>
<div className="col">
@@ -348,7 +348,7 @@ export function Results() {
num={25}
bg="white"
description="Zeta potential of SORT LNP with different cargos measured with Nanotrack Wave II indicating varying degrees of stability but most important good stability for the SORT LNP loaded with pcDNA 3.1 eYFP (LNP DNA). Mean +/- SEM for n=5. For statistics one-way ANOVA was performed."
- alt1="Zeta potential of SORT LNP with different cargos"
+ alt1="Zeta potential of SORT LNP with different cargos."
/>
</div>
</div>
@@ -359,7 +359,7 @@ export function Results() {
num={26}
bg="white"
description="Size distribution for the SORT LNP with different cargos weighted by scattering intensity measured with Nanotrack Wave II."
- alt1="Size distribution for the SORT LNP with different cargos"
+ alt1="Size distribution for the SORT LNP with different cargos."
/>
</div>
<div className="col">
@@ -376,7 +376,7 @@ export function Results() {
pic1="https://static.igem.wiki/teams/5247/delivery/results/sortcryoem.png"
num={27}
description="Cryo-EM image of SORT LNPs. The different colored outlines indicate different size populations of LNPs."
- alt1="Cryo-EM image of SORT LNPs with different size populations"
+ alt1="Cryo-EM image of SORT LNPs with different size populations."
/>
</div>
</div>
@@ -391,7 +391,7 @@ export function Results() {
num={28}
bg="white"
description="Results for hydrodynamic radius determination by DLS Measurements for our SORT LNP, indicating a radius of approximately 100 nm."
- alt1="Hydrodynamic radius of SORT LNP measured by DLS"
+ alt1="Hydrodynamic radius of SORT LNP measured by DLS."
/>
</div>
@@ -407,7 +407,7 @@ export function Results() {
num={29}
bg="white"
description="MTT Assay of LNPs from all iterations performed on HEK293 including Triton as negative control and untreated cells as positive control. Mean +/- SEM for n=6. For statistics one-way ANOVA was performed."
- alt1="MTT Assay results of LNPs on HEK293 cells"
+ alt1="MTT Assay results of LNPs on HEK293 cells."
/>
</div>
<div className="col">
@@ -416,7 +416,7 @@ export function Results() {
</div>
</div>
<H4 text="AirBuddy"/>
- <p>Now we present our results including the chitosan-complexation of the therapeutic RNA cargo, which withdraws AirBuddy from basic lung-specific SORT LNPs</p>
+ <p>Now we present our results including the chitosan-complexation of the therapeutic RNA cargo, which withdraws AirBuddy from basic lung-specific SORT LNPs.</p>
<p>We successfully transfected a well-established lung epithelial cell line CFBE41o- with our chitosan-RNA complexes using our synthesized pcDNA 3.1 eYFP mRNA, which were subsequently incubated with SORT LNPs for packaging. The goal was to encapsulate the chitosan-RNA complexes within the LNPs to enhance mRNA delivery to cells. After formulation, the LNP chitosan-RNA complexes were transfected into CFBE41o- cells to assess the efficiency of mRNA delivery and expression in a CF patient bronchial cell line being closer to the clinically relevant model.</p>
<p>We tested two different chitosan and mRNA concentrations (always used both in the same concentrations), 50 ng/µl and 500 ng/µl, in combination with our LNP to assess their impact on transfection efficiency. Interestingly, both concentrations resulted in similarly high levels of transfection, as evidenced by the fluorescence microscopy images, further validation is necessary to confirm these results.</p>
<p>Fluorescence microscopy was performed 24 hours post-transfection to evaluate the expression of the YFP reporter gene encoded by the pcDNA 3.1 eYFP mRNA. This reporter gene serves as an indicator of successful transfection and translation of the mRNA into protein. The results of the fluorescence microscopy were highly positive and aligned with our expectations, indicating efficient delivery, uptake, and expression of the mRNA in the CFBE41o- cells.</p>
@@ -428,7 +428,7 @@ export function Results() {
pic1="https://static.igem.wiki/teams/5247/delivery/results/precyse/lnp-chito-rna500-t24.webp"
num={30}
description="Overlay of phase contrast and fluorescence microscopic images of with SORT + chitosan 500 transfected CFBE41o- cells at 20x magnification 24h post-transfection recorded with Leica DMI6000 B."
- alt1="Images of SORT + chitosan 500 transfected CFBE41o- cells"
+ alt1="Images of SORT + chitosan 500 transfected CFBE41o- cells."
/>
</div>
<div className="col">
@@ -444,7 +444,7 @@ export function Results() {
pic1="https://static.igem.wiki/teams/5247/delivery/results/precyse/lnpchito-rna50-t24.webp"
num={31}
description="Overlay of phase contrast and fluorescence microscopic images of with SORT + chitosan 50 transfected CFBE41o- cells at 20x magnification 24h post-transfection recorded with Leica DMI6000 B."
- alt1="Images of SORT + chitosan 50 transfected CFBE41o- cells"
+ alt1="Images of SORT + chitosan 50 transfected CFBE41o- cells."
/>
</div>
</div>
@@ -455,7 +455,7 @@ export function Results() {
pic1="https://static.igem.wiki/teams/5247/delivery/results/precyse/chito-t24-1.jpeg"
num={32}
description="Overlay of phase contrast and fluorescence microscopic images of with SORT transfected CFBE41o- cells at 20x magnification 24h post-transfection recorded with Leica DMI6000 B."
- alt1="Images of SORT transfected CFBE41o- cells"
+ alt1="Images of SORT transfected CFBE41o- cells."
/>
</div>
<div className="col">
@@ -463,7 +463,7 @@ export function Results() {
pic1="https://static.igem.wiki/teams/5247/delivery/results/precyse/lnp-leer-t24.webp"
num={33}
description="Overlay of phase contrast and fluorescence microscopic images of with chitosan transfected CFBE41o- cells at 20x magnification 24h post-transfection recorded with Leica DMI6000 B."
- alt1="Images of chitosan transfected CFBE41o- cells"
+ alt1="Images of chitosan transfected CFBE41o- cells."
/>
</div>
</div>
@@ -474,7 +474,7 @@ export function Results() {
pic1="https://static.igem.wiki/teams/5247/delivery/results/precyse/ntc-t24.webp"
num={34}
description="Overlay of phase contrast and fluorescence microscopic images of untransfected CFBE41o- cells at 20x magnification 24h post-transfection recorded with Leica DMI6000 B."
- alt1="Images of untransfected CFBE41o- cells"
+ alt1="Images of untransfected CFBE41o- cells."
/>
</div>
<div className="col">
@@ -502,7 +502,7 @@ export function Results() {
<OneFigure
pic1="https://static.igem.wiki/teams/5247/photos/results/img-2217.webp"
- num={34}
+ num={35}
description="Schematic overview of the isolation and culture of primary human nasal epithelial cells (hNECs). First, nasopharyngeal swabs are taken to obtain the cells. The cells are then cultured in two different culture systems: Apical-Out-Airway-Organoids (AOAO) as a 3D culture (left) and Air-Liquid-Interface (ALI) as a 2D culture (right). In the 3D culture, the cells form a spherical organoid with an outward-facing apical surface, whereas in the 2D culture, the cells form a differentially structured epithelial barrier that mimics different cell types such as ciliated cells, goblet cells and basal cells."
alt1="Overview"
/>
@@ -516,13 +516,13 @@ export function Results() {
<OneFigure
pic1="https://static.igem.wiki/teams/5247/photos/results/a.webp"
- num={35}
- description="Histochemical staining of air liquid interface culture. A) HE staining with goblet cell visible. B) HE staining with goblet cell visible. C) Overview of differnetiated epithelium after PAS staining"
+ num={36}
+ description="Histochemical staining of air liquid interface culture. A) HE staining with goblet cell visible. B) HE staining with goblet cell visible. C) Overview of differnetiated epithelium after PAS staining."
alt1="HE and PAS staining"
/>
</div>
<div className="col">
- <p>The <strong>successful cultivation of primary human nasal epithelial cells (hNECs)</strong> was clearly demonstrated in both two-dimensional and three-dimensional cultures. In the ALI culture, a well-differentiated ciliated epithelium was observed microscopically, exhibiting the characteristic ciliary dynamics. Histological staining, in particular HE and PAS staining, provided confirmation of the differentiation of the cells. The multilayered epithelial structure and the presence of mucus-producing goblet cells were both clearly demonstrated (Figure 38 A, B). </p>
+ <p>The <strong>successful cultivation of primary human nasal epithelial cells (hNECs)</strong> was clearly demonstrated in both two-dimensional and three-dimensional cultures. In the ALI culture, a well-differentiated ciliated epithelium was observed microscopically, exhibiting the characteristic ciliary dynamics. Histological staining, in particular HE and PAS staining, provided confirmation of the differentiation of the cells. The multilayered epithelial structure and the presence of mucus-producing goblet cells were both clearly demonstrated (Figure 36 A, B). </p>
</div>
</div>
<p>The Apical-Out Airway Organoid culture also demonstrated successful differentiation of the cells, which formed an epithelial cell network. The cells exhibited a spherical morphology and were observed to float in the culture, thereby mimicking the natural physiological conditions of the respiratory epithelium. The various 2D and 3D models thus provide a promising foundation for further research and the application of gene therapy approaches. </p>
@@ -535,35 +535,28 @@ export function Results() {
<div className='col'>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/photos/results/patchclamp/pc.webp"
- num={36}
+ num={37}
bg="white"
description="Current density of HEK293, HEK293T CFTR WT and HEK293T CFTR F508del showing significant differences of both HEK293T cell lines compared to HEK293 but no significant differences between them. For statistics one-way ANOVA was performed."
alt1="Current density differences between HEK293 and HEK293T CFTR cell lines"
/>
</div>
<div className='col'>
- <p>In our first set of experiments, we measured current density in <a onClick={() => goToPageAndScroll ('Cell Culture', '/materials-methods')}>HEK293T CFTR wild type (WT) and HEK293T F508del</a> cell lines, comparing them with regular HEK293. The results demonstrated significant differences in chloride ion conductance, with the CFTR-expressing cell lines showing enhanced conductivity compared to HEK293 (Figure 35). However, a drawback was that we did not observe any significant differences between the HEK293T CFTR WT and F508del cell line. This was unexpected, as the F508del mutation typically leads to a knockdown of the CFTR protein[2], impairing chloride ion transport through the CFTR channel.</p>
+ <p>In our first set of experiments, we measured current density in <a onClick={() => goToPageAndScroll ('Cell Culture', '/materials-methods')}>HEK293T CFTR wild type (WT) and HEK293T F508del</a> cell lines, comparing them with regular HEK293. The results demonstrated significant differences in chloride ion conductance, with the CFTR-expressing cell lines showing enhanced conductivity compared to HEK293 (Figure 37). However, a drawback was that we did not observe any significant differences between the HEK293T CFTR WT and F508del cell line. This was unexpected, as the F508del mutation typically leads to a knockdown of the CFTR protein[2], impairing chloride ion transport through the CFTR channel.</p>
</div>
</div>
<H4 text="Further Validation and Challenges"/>
<div className='row align-items-center'>
<div className='col'>
- <p>In light of these results, we improved our experimental setup and performed additional validation experiments. Unfortunately, the repeated measurements yielded similar outcomes, confirming the absence of a significant difference between the two CFTR-expressing cell lines (Figure 36). This finding led us to consult with the research group at <a onClick={() => goToPagesAndOpenTab('mattijsvisit', '/human-practices')}>KU Leuven</a>, who established these cells lines. Although they had not conducted similar Patch Camp measurements, they suggested an alternative approach using Ussing Chamber measurements. This technique, unlike Patch Camp, does not rely on single-cell measurements but rather examines the ion currents across the entire cell monolayer, which may provide a more comprehensive view of CFTR functionality.</p>
+ <p>In light of these results, we improved our experimental setup and performed additional validation experiments. Unfortunately, the repeated measurements yielded similar outcomes, confirming the absence of a significant difference between the two CFTR-expressing cell lines (Figure 38). This finding led us to consult with the research group at <a onClick={() => goToPagesAndOpenTab('mattijsvisit', '/human-practices')}>KU Leuven</a>, who established these cells lines. Although they had not conducted similar Patch Camp measurements, they suggested an alternative approach using Ussing Chamber measurements. This technique, unlike Patch Camp, does not rely on single-cell measurements but rather examines the ion currents across the entire cell monolayer, which may provide a more comprehensive view of CFTR functionality.</p>
</div>
<div className='col'>
<OneFigure
alt1=""
pic1="https://static.igem.wiki/teams/5247/photos/results/patchclamp/pc2.webp"
- num={37}
- bg="white"
- description="
- Repeated validation of current density measurements in HEK293T CFTR WT and HEK293T CFTR-F508del, showing consistent results with the initial experiment. Mean +/- SEM for n=5. For statistics one-way ANOYA was performed"
- />
- <OneFigure
- pic1=""
- alt1="PC1"
num={38}
- description=""
+ bg="white"
+ description="Repeated validation of current density measurements in HEK293T CFTR WT and HEK293T CFTR-F508del, showing consistent results with the initial experiment. Mean +/- SEM for n=5. For statistics one-way ANOYA was performed."
/>
</div>
</div>