diff --git a/src/components/Link.tsx b/src/components/Link.tsx
index 34daa8f36dcf81eeef858ae08aac0b18072306cd..dc9555d3e268cb0a51a9c41bb0a38ef21ed307bd 100644
--- a/src/components/Link.tsx
+++ b/src/components/Link.tsx
@@ -12,6 +12,7 @@ export function TabScrollLink({tab, scrollId, num}:{tab: string, scrollId: strin
// 2^4 = 16 possible combinations + special cases müssen eventuell ergänzt werden
export function OurLink({path, scrollToId, tabId, subTabId, collapseId, text, tabincolId}:Ourlink){
+
const {goToPlace} = useNavigation();
// 1. [1-1-1-1] go to page and open tab and open subTab and open colapsible in subtab and scroll to something
if(tabId && subTabId && scrollToId && collapseId && !tabincolId) {
diff --git a/src/contents/engineering.tsx b/src/contents/engineering.tsx
index 30d196aa06772815eea0170f676a3747137c6654..ac48bc601a2d0c7b741f94c069872adf545ed57c 100644
--- a/src/contents/engineering.tsx
+++ b/src/contents/engineering.tsx
@@ -558,24 +558,46 @@ export function Engineering() {
<p id="peg1">
<H3 text="Initial pegRNA Design and Silent Edits" id="peg1head"/>
<p>
- The first iteration of our engineering cycle, we designed our first set of pegRNAs targeting the <a onClick={() => goToPageWithTabAndScroll ({scrollToId: 'reporter-header', path: '/engineering', tabId: 'reporter' })}>modified pPEAR_CFTR reporter</a>. We also focused on the incorporation of silent edits.
+ The first iteration of our engineering cycle, we designed our first set of pegRNAs targeting the
+ <a onClick={() => goToPageWithTabAndScroll ({scrollToId: 'reporter-header', path: '/engineering',
+ tabId: 'reporter' })}>modified pPEAR_CFTR reporter</a>. We also focused on the incorporation of
+ silent edits.
</p>
<H4 text="Design" id="design-head"/>
<p>
- Following an interview with <a onClick={() => goToPagesAndOpenTab('JPpegRNA', '/human-practices')}>Jan-Phillipp Gerhard</a>, we came across the concept of silent edits. Silent edits refer to single-base alterations of the nucleotide sequence that do not change the encoded amino acid. Jan-Phillipp pointed out that introducing silent edits in addition to the intended edit offers two major advantages.
+ Following an interview with <a onClick={() => goToPagesAndOpenTab('JPpegRNA', '/human-practices')}>
+ Jan-Phillipp Gerhard</a>, we came across the concept of silent edits. Silent edits refer to single-base
+ alterations of the nucleotide sequence that do not change the encoded amino acid. Jan-Phillipp pointed out
+ that introducing silent edits in addition to the intended edit offers two major advantages.
</p>
<p>
- Firstly, silent edits can increase the likelihood of flap incorporation during the prime editing process, especially in the context of MMR (Mismatch Repair) in the cell. Without silent edits, the cell is more likely to detect the mismatches that only occur at the desired mutation site, leading to a higher chance of the wild-type flap being reinserted. By introducing silent edits, multiple mismatches are present which this increases the probability of the synthesized flap being incorporated.
+ Firstly, silent edits can increase the likelihood of flap incorporation during the prime editing process,
+ especially in the context of MMR (Mismatch Repair) in the cell. Without silent edits, the cell is more likely
+ to detect the mismatches that only occur at the desired mutation site, leading to a higher chance of the
+ wild-type flap being reinserted. By introducing silent edits, multiple mismatches are present which this
+ increases the probability of the synthesized flap being incorporated.
</p>
<p>
- Secondly, silent edits can prevent re-binding of the prime editing complex to the target region after successful editing. This is be achieved by introducing silent edits to the regions making up PAM sequence and/or protospacer. PAM or protospacer disruption make the editing process more secure. This is because it reduces the likelihood of editing the target region repeatedly, which would increase the probability of on-target undesired editing outcomes. He suggested that swapping cytosine or guanine bases for these silent edits can be particularly effective in improving prime editing efficiency.
+ Secondly, silent edits can prevent re-binding of the prime editing complex to the target region after
+ successful editing. This is be achieved by introducing silent edits to the regions making up PAM sequence
+ and/or protospacer. PAM or protospacer disruption make the editing process more secure. This is because it
+ reduces the likelihood of editing the target region repeatedly, which would increase the probability of
+ on-target undesired editing outcomes. He suggested that swapping cytosine or guanine bases for these silent
+ edits can be particularly effective in improving prime editing efficiency.
</p>
<H4 text="Build" id="build-head"/>
<p>
- We designed several pegRNAs, both with and without silent edits. To assist with this, we used the pegFinder software<TabScrollLink tab="tab-pegrna" num="1" scrollId="desc-1"/>, which generated possible variations of pegRNAs based on the sequence of the reporter plasmid. We selected the optimal pegRNA as suggested by the software, and then tested it in two forms: one unmodified and one with silent edits. For the unmodified variant, we included a single silent edit that introduced a PAM disrupt in terms of our biosafety measures. For the modified variant, we introduced three silent edits in total, adding two more to the initial edit.
+ We designed several pegRNAs, both with and without silent edits. To assist with this, we used the pegFinder
+ software<TabScrollLink tab="tab-pegrna" num="18" scrollId="desc-18"/>, which generated possible variations of
+ pegRNAs based on the sequence of the reporter plasmid. We selected the optimal pegRNA as suggested by the
+ software, and then tested it in two forms: one unmodified and one with silent edits. For the unmodified
+ variant, we included a single silent edit that introduced a PAM disrupt in terms of our biosafety measures.
+ For the modified variant, we introduced three silent edits in total, adding two more to the initial edit.
</p>
<p>
- Once we had designed these variants, we ordered them in their individual components and cloned them into a pU6-peg-GG-acceptor backbone using Golden Gate cloning according to the protocol from Anzalone et al. 2019<TabScrollLink tab="tab-pegrna" num="2" scrollId="desc-2"/>. We then screened the assembled pegRNAs to ensure that the individual components had the correct orientation and then cloned them into the pU6-GG-pegRNA-acceptor plasmid so that they were ready to be tested.
+ Once we had designed these variants, we ordered them in their individual components and cloned them into a
+ pU6-peg-GG-acceptor backbone using Golden Gate cloning according to the protocol from Anzalone et al.
+ 2019<TabScrollLink tab="tab-pegrna" num="19" scrollId="desc-19"/>. We then screened the assembled pegRNAs to ensure that the individual components had the correct orientation and then cloned them into the pU6-GG-pegRNA-acceptor plasmid so that they were ready to be tested.
</p>
<H4 text="Test" id="test-head"/>
<p>
@@ -598,7 +620,7 @@ export function Engineering() {
Based on literature reviews and our interview with <a onClick={() => goToPagesAndOpenTab('mattijsinv', '/human-practices')}> Mattijs Bulcaen </a>, we decided to modify our pegRNA by adding a stem loop to enhance its stability. Specifically, Mattijs recommended using the tevopreQ1 stem loop, a small structural motif that increases the pegRNA's resistance to RNases. This stem loop was added to the 3' end of the pegRNA, positioned after the PBS.
</p>
<p>
- Additionally, during a webinar with B. Sc. Jordan Doman<TabScrollLink tab="tab-pegrna" num="3" scrollId="desc-3"/>, we learned that it is important to test various lengths of PBS and RTT, as there is no universally optimal length for all applications. Instead, the ideal lengths are application specific. Following this advice, we designed six different pegRNA variants with combinations of two different PBS lengths (16 and 17 nucleotides) and three different RTT lengths (27, 30, and 33 nucleotides).
+ Additionally, during a webinar with B. Sc. Jordan Doman<TabScrollLink tab="tab-pegrna" num="20" scrollId="desc-20"/>, we learned that it is important to test various lengths of PBS and RTT, as there is no universally optimal length for all applications. Instead, the ideal lengths are application specific. Following this advice, we designed six different pegRNA variants with combinations of two different PBS lengths (16 and 17 nucleotides) and three different RTT lengths (27, 30, and 33 nucleotides).
</p>
<p>
We chose the PBS lengths of 16 and 17 nucleotides based on an earlier recommendation from Jan-Phillipp Gerhard, who emphasised that the annealing temperature of the PBS should match the environmental conditions relevant to the intended application. In our case, since we are exploring a potential therapeutic approach, it is important that the annealing temperature of the PBS is close to the body temperature of 37 °C, which is the case for these lengths. The RTT lengths were selected based on suggestions from the pegFinder software. As with our previous insights, we designed all six variants both with and without silent edits for a wider comparison of the silent edits, making it 12 variants in total.
@@ -657,7 +679,7 @@ export function Engineering() {
</p>
<H4 text="Test" id="test-head"/>
<p>
- The next step is to test the correction of CFTR F508del using these pegRNAs in the CFBE41o- epithelial cells. Additionally, we also want to test the pegRNAs in primary cells derived from friend of the team and Cystic Fibrosis patient <a onClick={() => goToPagesAndOpenTab('maxfirst', '/human-practices')}> Max </a>, testing whether our approaches are applicable not only in model systems, but also work in patient cells. To validate the editing efficiency of our designed pegRNAs were going to co-transfect a plasmid carrying an eYFP variant which is sensitive to chloride and iodide ion concentrations<TabScrollLink tab="tab-pegrna" num="4" scrollId="desc-4"/><TabScrollLink tab="tab-pegrna" num="5" scrollId="desc-5"/>. The intensity of the fluorescence correlates with these ion concentrations, which in turn reflects the functionality of the CFTR channel. This enables us to evaluate the editing efficiency of the different pegRNA variants on a phenotypic level. After 72 hours, we are going to perform a final analysis using flow cytometry to quantify the results and determine the editing efficiency of each pegRNA. Secondly, we wanted to detect the editing on a genomic level by facilitating a qPCR with a primer specific only to the corrected F508del locus.
+ The next step is to test the correction of CFTR F508del using these pegRNAs in the CFBE41o- epithelial cells. Additionally, we also want to test the pegRNAs in primary cells derived from friend of the team and Cystic Fibrosis patient <a onClick={() => goToPagesAndOpenTab('maxfirst', '/human-practices')}> Max </a>, testing whether our approaches are applicable not only in model systems, but also work in patient cells. To validate the editing efficiency of our designed pegRNAs were going to co-transfect a plasmid carrying an eYFP variant which is sensitive to chloride and iodide ion concentrations<TabScrollLink tab="tab-pegrna" num="21" scrollId="desc-21"/><TabScrollLink tab="tab-pegrna" num="22" scrollId="desc-22"/>. The intensity of the fluorescence correlates with these ion concentrations, which in turn reflects the functionality of the CFTR channel. This enables us to evaluate the editing efficiency of the different pegRNA variants on a phenotypic level. After 72 hours, we are going to perform a final analysis using flow cytometry to quantify the results and determine the editing efficiency of each pegRNA. Secondly, we wanted to detect the editing on a genomic level by facilitating a qPCR with a primer specific only to the corrected F508del locus.
</p>
<H4 text="Learn" id="learn-head"/>
<p>
@@ -673,10 +695,10 @@ export function Engineering() {
</p>
<H4 text="Design" id="design-head"/>
<p>
- As we continued to refine our approach, further literature research was conducted, and new design ideas considered. The overarching goal remained to enhance both the stability and editing efficiency of the pegRNAs. One concept we are already exploring involves the incorporation of 3’ and 5’ UTRs (Untranslated Regions)<TabScrollLink tab="tab-pegrna" num="6" scrollId="desc-6"/>. These elements, typically found in mRNA, could be added to the pegRNA to increase its stability.
+ As we continued to refine our approach, further literature research was conducted, and new design ideas considered. The overarching goal remained to enhance both the stability and editing efficiency of the pegRNAs. One concept we are already exploring involves the incorporation of 3’ and 5’ UTRs (Untranslated Regions)<TabScrollLink tab="tab-pegrna" num="23" scrollId="desc-23"/>. These elements, typically found in mRNA, could be added to the pegRNA to increase its stability.
</p>
<p>
- Another promising idea is the use of circular RNA (circRNA)<TabScrollLink tab="tab-pegrna" num="7" scrollId="desc-7"/>, which could provide additional stability by maintaining the closed-loop structure of the pegRNA. This would prevent degradation and increase the longevity of the pegRNA in the cell.
+ Another promising idea is the use of circular RNA (circRNA)<TabScrollLink tab="tab-pegrna" num="24" scrollId="desc-24"/>, which could provide additional stability by maintaining the closed-loop structure of the pegRNA. This would prevent degradation and increase the longevity of the pegRNA in the cell.
</p>
<p>
Additionally, further nucleotide modifications could be explored, such as experimenting with alternative silent edits to see if this leads to improved editing efficiency. We also nucleotide substitutions in the scaffold region to enhance RNA-binding affinity to the protein complex could be of use.
@@ -720,10 +742,10 @@ export function Engineering() {
<H3 text="SpuFz1 Zink Finger Mutation" id="nic1head"/>
<H4 text="Design" id="text"/>
<p>
- In our quest to identify smaller endonucleases suitable for creating nickases, we focused on a newly characterized family of eukaryotic endonucleases known as Fanzor proteins first described in June 2023<TabScrollLink tab="tab-nickase" num="1" scrollId="desc-1"/>, with SpuFz1 (Fig. 1) being a standout candidate due to its smaller size compared to Cas9 (SpuFz1 consists of 638 amino acids<TabScrollLink tab="tab-nickase" num="1" scrollId="desc-1"/>, whereas Cas9 has a size of 1368 amino acids<TabScrollLink tab="tab-nickase" num="2" scrollId="desc-2"/>). We selected SpuFz1 not only because of its smaller size, but also due to structural advantages, such as the reversed positioning of the spacer, which provides better protection from RNase degradation and improves editing precision.
+ In our quest to identify smaller endonucleases suitable for creating nickases, we focused on a newly characterized family of eukaryotic endonucleases known as Fanzor proteins first described in June 2023<TabScrollLink tab="tab-nickase" num="25" scrollId="desc-25"/>, with SpuFz1 (Fig. 1) being a standout candidate due to its smaller size compared to Cas9 (SpuFz1 consists of 638 amino acids<TabScrollLink tab="tab-nickase" num="25" scrollId="desc-25"/>, whereas Cas9 has a size of 1368 amino acids<TabScrollLink tab="tab-nickase" num="26" scrollId="desc-26"/>). We selected SpuFz1 not only because of its smaller size, but also due to structural advantages, such as the reversed positioning of the spacer, which provides better protection from RNase degradation and improves editing precision.
</p>
<p>
- The Cas9 endonuclease contains two active domains, each responsible for cutting one of the two DNA strands. Cas9 uses the RuvC and HNH domains, with each domain making a cut on a different strand of the target DNA<TabScrollLink tab="tab-nickase" num="3" scrollId="desc-3"/>. To create a nickase from Cas9, scientists deactivate one of these active domains, typically the HNH domain, so that the enzyme only cuts one strand instead of both, producing a single-strand break rather than a double-strand break<TabScrollLink tab="tab-nickase" num="4" scrollId="desc-4"/>.
+ The Cas9 endonuclease contains two active domains, each responsible for cutting one of the two DNA strands. Cas9 uses the RuvC and HNH domains, with each domain making a cut on a different strand of the target DNA<TabScrollLink tab="tab-nickase" num="27" scrollId="desc-27"/>. To create a nickase from Cas9, scientists deactivate one of these active domains, typically the HNH domain, so that the enzyme only cuts one strand instead of both, producing a single-strand break rather than a double-strand break<TabScrollLink tab="tab-nickase" num="27" scrollId="desc-27"/>.
</p>
<p>
Based on the function of this prototypical Cas9 nickase, we assumed that SpuFz1 would operate similarly, with two active centers—RuvC and TNB—each cutting one DNA strand. Following this logic, we hypothesized that by deactivating the TNB domain, which contains a zinc finger motif (Fig. 2) crucial for DNA coordination, we could convert SpuFz1 into a nickase. To test this, we aimed to replace the cysteine residues involved in zinc ion coordination within the TNB domain with alanine, thereby impairing its DNA-binding ability and producing a SpuFz1 nickase that cuts only one strand. At that time, we believed both domains in SpuFz1 were directly responsible for DNA cleavage, and our strategy was based on this assumption.
@@ -770,7 +792,7 @@ export function Engineering() {
<H3 text="Fusion Protein from GtFz1 & SpuFz1" id="nic2head"/>
<H4 text="Design" id="design-head"/>
<p>
- In our ongoing exploration of Fanzor proteins, we identified another potential candidate, GtFz1, which had a suitable TAM sequence for our target application of correcting the F508del mutation in Cystic Fibrosis. However, GtFz1 showed low cutting efficiency in the tests reported in the literature<TabScrollLink tab="tab-nickase" num="1" scrollId="desc-1"/>. To address this, we devised a strategy to combine the favorable TAM-binding properties of GtFz1 with the higher cutting efficiency of SpuFz1. Specifically, we planned to engineer a fusion protein by replacing the TAM-binding domain of SpuFz1 with that of GtFz1. This approach aims to create an endonuclease that retains the strong TAM-binding ability of GtFz1 while utilizing the robust cutting efficiency of SpuFz1, optimizing it for our Prime Editing application.
+ In our ongoing exploration of Fanzor proteins, we identified another potential candidate, GtFz1, which had a suitable TAM sequence for our target application of correcting the F508del mutation in Cystic Fibrosis. However, GtFz1 showed low cutting efficiency in the tests reported in the literature<TabScrollLink tab="tab-nickase" num="25" scrollId="desc-25"/>. To address this, we devised a strategy to combine the favorable TAM-binding properties of GtFz1 with the higher cutting efficiency of SpuFz1. Specifically, we planned to engineer a fusion protein by replacing the TAM-binding domain of SpuFz1 with that of GtFz1. This approach aims to create an endonuclease that retains the strong TAM-binding ability of GtFz1 while utilizing the robust cutting efficiency of SpuFz1, optimizing it for our Prime Editing application.
</p>
<p>
Given that we were swapping entire domains rather than just single amino acids, we realized that the fusion protein might not retain the ideal TAM-binding efficiency or cutting efficiency of the original proteins. Our strategy was to create a fusion protein that could bind to the TAM site and perform DNA cutting to a certain extent, albeit weakly. We planned to use directed evolution techniques, such as Phage Assisted Continuous Evolution (PACE), to enhance these functionalities over time. This approach relies on having a starting point with some degree of the desired activity, which can then be incrementally improved through evolution.
@@ -830,10 +852,10 @@ export function Engineering() {
<H3 text="nCas12 analog Mutations" id="nic4head"/>
<H4 text="Design" id="design-head"/>
<p>
- After concluding that the zinc finger mutation approach was not suitable for converting SpuFz1 into a nickase, we revisited our understanding of its cutting mechanism. Initially, we believed that SpuFz1, similar to Cas9, contained two active centers that each cut one of the DNA strands, and that by deactivating one of these centers, we could generate a nickase that only cuts one strand. However, through further research, we discovered that this assumption was incorrect. SpuFz1 actually functions with a different cutting mechanism: the RuvC domain is responsible for cutting the non-target strand, while the TNB domain does not directly cut the DNA. Instead, it assists the process by guiding the target strand into the RuvC domain for sequential cleavage<TabScrollLink tab="tab-nickase" num="5" scrollId="desc-5"/>. This discovery shifted our focus from simply deactivating an active site to better understanding how the sequential cleavage works in order to inform future mutation strategies.
+ After concluding that the zinc finger mutation approach was not suitable for converting SpuFz1 into a nickase, we revisited our understanding of its cutting mechanism. Initially, we believed that SpuFz1, similar to Cas9, contained two active centers that each cut one of the DNA strands, and that by deactivating one of these centers, we could generate a nickase that only cuts one strand. However, through further research, we discovered that this assumption was incorrect. SpuFz1 actually functions with a different cutting mechanism: the RuvC domain is responsible for cutting the non-target strand, while the TNB domain does not directly cut the DNA. Instead, it assists the process by guiding the target strand into the RuvC domain for sequential cleavage<TabScrollLink tab="tab-nickase" num="29" scrollId="desc-29"/>. This discovery shifted our focus from simply deactivating an active site to better understanding how the sequential cleavage works in order to inform future mutation strategies.
</p>
<p>
- In addition to these insights, we noticed a significant phylogenetic relationship between Fanzor endonucleases, like SpuFz1, and Cas12 endonucleases<TabScrollLink tab="tab-nickase" num="1" scrollId="desc-1"/>. This connection was crucial, as Cas12 proteins have a similar cutting mechanism to Fanzor proteins, utilizing a single active site for cleavage while coordinating both DNA strands. More importantly, we identified a precedent in the literature where a Cas12a endonuclease was successfully converted into a nickase by substituting a single amino acid in the TNB domain<TabScrollLink tab="tab-nickase" num="6" scrollId="desc-6"/> (Fig. 5 and 6). This provided us with a clear model strategy to follow, as this targeted mutation allowed the endonuclease to selectively cut only one DNA strand, effectively converting it into a nickase.
+ In addition to these insights, we noticed a significant phylogenetic relationship between Fanzor endonucleases, like SpuFz1, and Cas12 endonucleases<TabScrollLink tab="tab-nickase" num="25" scrollId="desc-25"/>. This connection was crucial, as Cas12 proteins have a similar cutting mechanism to Fanzor proteins, utilizing a single active site for cleavage while coordinating both DNA strands. More importantly, we identified a precedent in the literature where a Cas12a endonuclease was successfully converted into a nickase by substituting a single amino acid in the TNB domain<TabScrollLink tab="tab-nickase" num="30" scrollId="desc-30"/> (Fig. 5 and 6). This provided us with a clear model strategy to follow, as this targeted mutation allowed the endonuclease to selectively cut only one DNA strand, effectively converting it into a nickase.
</p>
<TwoFigureRow
pic1="https://static.igem.wiki/teams/5247/engineering-cycle/cas12-nikase.webp"
@@ -925,7 +947,7 @@ export function Engineering() {
<H3 text="Ongoing: In Vitro Cleavage Assays" id="nic5head"/>
<H4 text="Design" id="design-head"/>
<p>
- In this iteration, our focus shifted to testing whether our mutation candidates had successfully converted the endonucleases into functional nickases. To do this, we adapted an existing assay that had been originally designed to determine whether mutated endonucleases exhibited nickase activity<TabScrollLink tab="tab-nickase" num="1" scrollId="desc-1"/>. We tailored this assay to fit our specific needs, allowing us to accurately assess the properties of our mutated proteins in the lab. The key question was whether the mutations had rendered the proteins dysfunctional, left them as endonucleases, or successfully converted them into nickases.
+ In this iteration, our focus shifted to testing whether our mutation candidates had successfully converted the endonucleases into functional nickases. To do this, we adapted an existing assay that had been originally designed to determine whether mutated endonucleases exhibited nickase activity<TabScrollLink tab="tab-nickase" num="25" scrollId="desc-25"/>. We tailored this assay to fit our specific needs, allowing us to accurately assess the properties of our mutated proteins in the lab. The key question was whether the mutations had rendered the proteins dysfunctional, left them as endonucleases, or successfully converted them into nickases.
</p>
<H4 text="Build" id="build-head"/>
<p>
@@ -1007,14 +1029,14 @@ export function Engineering() {
<div className="box" >
<p id="del1">
<H3 text="Iteration 1 - AVVs vs LNPs" id="del1head" />
- <p>Initially, this project part started with a discussion with <a onClick={() => goToPagesAndOpenTab('kristian', '/human-practices')}> Prof. Dr. Krisitan Müller</a>, PI of our team with expertise in Adeno-associated viruses (AAVs), focusing on whether to pursue LNPs or AAVs for mRNA delivery. The deciding factor leaned towards LNPs, as they offered a significant advantages including less immunogenic potential<TabScrollLink tab="tab-delivery" num="1" scrollId="desc-1"/> and bigger loading capacity<TabScrollLink tab="tab-delivery" num="2" scrollId="desc-2"/>. LNPs loading capacity depends on various factors, but in general they offer a bigger cargo size compared to 4.7 kb for AVVs<TabScrollLink tab="tab-delivery" num="3" scrollId="desc-3"/>. This allows the delivery of bigger mRNA constructs compared to AAVs, which is needed for our Prime Editing construct.</p>
+ <p>Initially, this project part started with a discussion with <a onClick={() => goToPagesAndOpenTab('kristian', '/human-practices')}> Prof. Dr. Krisitan Müller</a>, PI of our team with expertise in Adeno-associated viruses (AAVs), focusing on whether to pursue LNPs or AAVs for mRNA delivery. The deciding factor leaned towards LNPs, as they offered a significant advantages including less immunogenic potential<TabScrollLink tab="tab-delivery" num="31" scrollId="desc-31"/> and bigger loading capacity<TabScrollLink tab="tab-delivery" num="32" scrollId="desc-32"/>. LNPs loading capacity depends on various factors, but in general they offer a bigger cargo size compared to 4.7 kb for AVVs<TabScrollLink tab="tab-delivery" num="33" scrollId="desc-33"/>. This allows the delivery of bigger mRNA constructs compared to AAVs, which is needed for our Prime Editing construct.</p>
<p><a onClick={() => goToPagesAndOpenTab('weber', '/human-practices')}>Prof. Wolf-Michael Weber and Dr. Jörg Große-Onnebrink</a> from the UKM in Münster were our first point of contact for the development of our LNP for CFTR treatment. Moreover, <a onClick={() => goToPagesAndOpenTab('radukic', '/human-practices')}>Dr. Marco Radukic </a>form Bielefeld University provided us with a very useful cargo, namely minicircle DNA carrying the EYFP gene from <a href="https://www.plasmidfactory.com/custom-dna/minicircle-dna/" title="PlasmidFactory" >PlasmidFactory</a> as a positive control for our experiments. He also helped us establish protocols for LNP synthesis and LNP transfection in our lab.</p>
</p>
</div>
<div className="box" >
<p id="del2">
<H3 text="Interation 2 - Cayman LNP" id="del2head" />
- In the first experimental phase, LNPs from <strong>Cayman Chemical LNP Exploration Kit (LNP-102)</strong> consisting of SM-102, 1,2-DSPC, Cholesterol, and DMG-PEG(2000)<TabScrollLink tab="tab-delivery" num="4" scrollId="desc-4"/> were tested with mRNA encoding fluorescent protein to evaluate their transfection efficiency. However, the results indicated good non-lung-specific transfection efficiency, which was a critical factor for the project. This led the team to reconsider their choice of this LNP.
+ In the first experimental phase, LNPs from <strong>Cayman Chemical LNP Exploration Kit (LNP-102)</strong> consisting of SM-102, 1,2-DSPC, Cholesterol, and DMG-PEG(2000)<TabScrollLink tab="tab-delivery" num="34" scrollId="desc-34"/> were tested with mRNA encoding fluorescent protein to evaluate their transfection efficiency. However, the results indicated good non-lung-specific transfection efficiency, which was a critical factor for the project. This led the team to reconsider their choice of this LNP.
</p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/cayman-lnp-freigestellt.webp"
@@ -1027,7 +1049,7 @@ export function Engineering() {
<div className="box" >
<p id="del3">
<H3 text="Interation 3 - Corden LNP" id="del3head" />
- In the next phase, we chose to use a new LNP formulation, namely the <strong>LNP Starter Kit #2</strong><TabScrollLink tab="tab-delivery" num="5" scrollId="desc-5"/> of <a onClick={() => goToPagesAndOpenTab('corden', '/human-practices')}>Corden Pharma</a>, because it offered several advantages over the initial option. The key benefit of this new LNP lies in the use of DOTAP, a cationic lipid that enhances interaction with negatively charged cell membranes in the lungs, improving cellular uptake efficiency. While SM-102 in the Cayman LNP-102 is effective for systemic delivery, it lacks the same specificity for lung tissue. Additionally, Corden Pharma’s plant-based BotaniChol® prevents animal-sourced contamination and helps address the global lipid shortage for vaccine production. mPEG-2000-DSPE provides superior stability and reduces immune system activation over time, making it particularly suitable for pulmonary delivery. This made the new formulation a better choice for safely and effectively targeting lung tissue, especially in delivering therapies for CFTR-related diseases. During this time, the team encountered a paper on capsaicin-chitosan nanoparticles, which explored its use in targeted delivery and higher transfection efficiency. However, after further investigation and consultation of <a onClick={() => goToPagesAndOpenTab('kolonkofirst', '/human-practices')}>Dr. Katharina Kolonko</a>, it was determined that capsaicin was not suitable in our way of pulmonary application.
+ In the next phase, we chose to use a new LNP formulation, namely the <strong>LNP Starter Kit #2</strong><TabScrollLink tab="tab-delivery" num="35" scrollId="desc-35"/> of <a onClick={() => goToPagesAndOpenTab('corden', '/human-practices')}>Corden Pharma</a>, because it offered several advantages over the initial option. The key benefit of this new LNP lies in the use of DOTAP, a cationic lipid that enhances interaction with negatively charged cell membranes in the lungs, improving cellular uptake efficiency. While SM-102 in the Cayman LNP-102 is effective for systemic delivery, it lacks the same specificity for lung tissue. Additionally, Corden Pharma’s plant-based BotaniChol® prevents animal-sourced contamination and helps address the global lipid shortage for vaccine production. mPEG-2000-DSPE provides superior stability and reduces immune system activation over time, making it particularly suitable for pulmonary delivery. This made the new formulation a better choice for safely and effectively targeting lung tissue, especially in delivering therapies for CFTR-related diseases. During this time, the team encountered a paper on capsaicin-chitosan nanoparticles, which explored its use in targeted delivery and higher transfection efficiency. However, after further investigation and consultation of <a onClick={() => goToPagesAndOpenTab('kolonkofirst', '/human-practices')}>Dr. Katharina Kolonko</a>, it was determined that capsaicin was not suitable in our way of pulmonary application.
</p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/corden-lnp-freigestellt.webp"
@@ -1040,7 +1062,7 @@ export function Engineering() {
<div className="box" >
<p id="del4">
<H3 text="Interation 4 - Spray-dried SORT LNP called Airbuddy" id="del4head" />
- The next design iteration incorporated the insights from Wang's LNP work for building upon SORT principles to make the nanoparticles lung-specific<TabScrollLink tab="tab-delivery" num="6" scrollId="desc-6"/>. The main components include DMG-PEG 2000, cholesterol, DOPE and DOTAP as phospholipids and cationic lipids such as 4A3-SC8. In our LNP development, we carefully considered the use of PEG. While PEG can improve stability, it can also reduce cellular uptake and induce immune responses, necessitating a balanced approach to its inclusion<TabScrollLink tab="tab-delivery" num="7" scrollId="desc-7"/>.
+ The next design iteration incorporated the insights from Wang's LNP work for building upon SORT principles to make the nanoparticles lung-specific<TabScrollLink tab="tab-delivery" num="36" scrollId="desc-36"/>. The main components include DMG-PEG 2000, cholesterol, DOPE and DOTAP as phospholipids and cationic lipids such as 4A3-SC8. In our LNP development, we carefully considered the use of PEG. While PEG can improve stability, it can also reduce cellular uptake and induce immune responses, necessitating a balanced approach to its inclusion<TabScrollLink tab="tab-delivery" num="37" scrollId="desc-37"/>.
<Collapsible id="Col1" open={false} title="Ambivalence of PEG and our implementation">
<p>
<H4 text="What is PEG and why is it important for LNPs?" id="text" />
@@ -1088,7 +1110,7 @@ export function Engineering() {
<p>The final LNP solution can be stored at 4 °C for a few days. It is recommended to use the formulated LNPs as soon as possible to maintain consistent results. Storage at RT is not recommended. Storage at freezing temperatures is also not recommended unless optimized cryoprotectants are used.</p>
</Collapsible>
- <p>The final innovation for our LNP to become <strong>AirBuddy</strong> came through consultation with Benjamin Winkeljann from <a onClick={() => goToPagesAndOpenTab('rnhale', '/human-practices')}> RNhale</a>, when the use of spray-drying techniques was discussed. Spray-drying the LNPs, instead of using traditional methods, helps improve stability and eco-friendliness of the product<TabScrollLink tab="tab-delivery" num="8" scrollId="desc-8"/>. Our samples are set for transfer to RNhale for spray-drying, with scheduling aligned to resume promptly after the wiki freeze. Meanwhile, the discussion with <a onClick={() => goToPagesAndOpenTab('moorlach', '/human-practices')}>Benjamin Moorlach</a>, chitosan expert working at FH Bielefeld, provided new ideas for improvement by <strong>complexing the cargo with chitosan</strong> to improve the stability of the cargo during spray drying and nebulization. The positive effect of chitosan-complexing for opimized LNP delivery could be confirmed in our lab. In conclusion, we created a stable LNP for efficient delivery of RNA therapeutics to the lungs since the successful delivery of the prime editing complex via inhalation is key to our project. </p>
+ <p>The final innovation for our LNP to become <strong>AirBuddy</strong> came through consultation with Benjamin Winkeljann from <a onClick={() => goToPagesAndOpenTab('rnhale', '/human-practices')}> RNhale</a>, when the use of spray-drying techniques was discussed. Spray-drying the LNPs, instead of using traditional methods, helps improve stability and eco-friendliness of the product<TabScrollLink tab="tab-delivery" num="38" scrollId="desc-38"/>. Our samples are set for transfer to RNhale for spray-drying, with scheduling aligned to resume promptly after the wiki freeze. Meanwhile, the discussion with <a onClick={() => goToPagesAndOpenTab('moorlach', '/human-practices')}>Benjamin Moorlach</a>, chitosan expert working at FH Bielefeld, provided new ideas for improvement by <strong>complexing the cargo with chitosan</strong> to improve the stability of the cargo during spray drying and nebulization. The positive effect of chitosan-complexing for opimized LNP delivery could be confirmed in our lab. In conclusion, we created a stable LNP for efficient delivery of RNA therapeutics to the lungs since the successful delivery of the prime editing complex via inhalation is key to our project. </p>
<OneFigure
pic1="https://static.igem.wiki/teams/5247/delivery/big-plan-inhalation-teil-del.webp"
@@ -1101,7 +1123,7 @@ export function Engineering() {
<div className="box" >
<p id="del5">
<H3 text="Outlook" id="del5head" />
- Ultimately, through continuous cycles of experimentation, feedback, and optimization, our LNP formulation called AirBuddy was designed using SORT LNPs incorporating chitosan-complexation of the cargo and processing via spray-drying, achieving lung-specificity and improved stability suited for inhalation strategies. We also want to state that for our LNP is further room for improvement. Intensive research led us to the realization that, among other modifications, <strong>antibody conjugation</strong> as a surface modification of our LNP for cell type-specific administration, more specifically club cells<TabScrollLink tab="tab-delivery" num="9" scrollId="desc-9"/> and ionocytes<TabScrollLink tab="tab-delivery" num="10" scrollId="desc-10"/> as most CFTR-expressing lung epithelial cells, would round off our most important aspect of precision.
+ Ultimately, through continuous cycles of experimentation, feedback, and optimization, our LNP formulation called AirBuddy was designed using SORT LNPs incorporating chitosan-complexation of the cargo and processing via spray-drying, achieving lung-specificity and improved stability suited for inhalation strategies. We also want to state that for our LNP is further room for improvement. Intensive research led us to the realization that, among other modifications, <strong>antibody conjugation</strong> as a surface modification of our LNP for cell type-specific administration, more specifically club cells<TabScrollLink tab="tab-delivery" num="39" scrollId="desc-39"/> and ionocytes<TabScrollLink tab="tab-delivery" num="40" scrollId="desc-40"/> as most CFTR-expressing lung epithelial cells, would round off our most important aspect of precision.
</p>
</div>
<Section title="References" id="references">
diff --git a/src/sources/eng-delivery-sources.tsx b/src/sources/eng-delivery-sources.tsx
index c70fa0b6a3aef5d3051fda54acebd208560f1f33..ce76d8136fec9b99625b327692169178af475909 100644
--- a/src/sources/eng-delivery-sources.tsx
+++ b/src/sources/eng-delivery-sources.tsx
@@ -3,7 +3,7 @@ import BibtexParser from "../components/makeSources";
export default function EngDelsources(){
return (
<div>
- <BibtexParser bibtexSources={bibtexSources} />
+ <BibtexParser bibtexSources={bibtexSources} start={30}/>
</div>
);
}
diff --git a/src/sources/eng-nickases-sources.tsx b/src/sources/eng-nickases-sources.tsx
index d3e9dbb6f0dd81568a5c9179928c79ea8991f0a1..8e6948fe11d5070ac3fa982470da9d7410078c66 100644
--- a/src/sources/eng-nickases-sources.tsx
+++ b/src/sources/eng-nickases-sources.tsx
@@ -3,7 +3,7 @@ import BibtexParser from "../components/makeSources";
export default function EngNicksources(){
return (
<div>
- <BibtexParser bibtexSources={bibtexSources} />
+ <BibtexParser bibtexSources={bibtexSources} start={24}/>
</div>
);
}
diff --git a/src/sources/eng-peg-sources.tsx b/src/sources/eng-peg-sources.tsx
index f4ed5481f43b096937233a1f1c5e98eda0c9a324..0f4f521486b64339e2843d36db31179c96bfd12d 100644
--- a/src/sources/eng-peg-sources.tsx
+++ b/src/sources/eng-peg-sources.tsx
@@ -3,7 +3,7 @@ import BibtexParser from "../components/makeSources";
export default function EngPegsources(){
return (
<div>
- <BibtexParser bibtexSources={bibtexSources} />
+ <BibtexParser bibtexSources={bibtexSources} start={18} />
</div>
);
}