Engineering

Reneurish Engineering Overview

Reneurish aims to restore lost neuron activity in ischaemic stroke patients. To do so, we propose the use of genetically-modified neural progenitors enriched with neurotrophic factor expression. This expression is accomplished through transfecting iPSC-derived NPC with custom-designed vectors.

To fulfil our goal, we followed the Design-Build-Test-Learn (DBTL) cycle. It is a cornerstone in every synthetic biology project and Reneurish was no exception, these cycles have been fundamental to the development of our project, enabling us to design genetic circuits tailored to tackle specific needs.

We have divided our engineering strategy into three main Branches:

Branch 1: Constitutive RFP Reporter Cassete, where we tested the efficiency of two different construct designs to use mCherry as a transfection reporter. This led to Branch 1.2: Customizable Transfection Reporter Cassette, where we developed a chassis that future iGEM teams can use to customize reporters and their cellular localization.

Once we obtained the first results from Branch 1, we parallelly started with Branch 2: Time-Dependent Neurotrophic Factor Overexpression, for this we needed to determine the effects of overexpressing BDNF, the neurotrophic factor of choice, on neural activity. After verifying the positive effects of BDNF, we continued with Branch 2.2: Time-dependant promoters to regulate BDNF expression. In this stage, we are testing how different promoters regulate BDNF expression over time and how this impacts neural activity.

Finally, Branch 3 is our way of looking towards the future. This branch includes design of future steps in Reneurish, which includes AAV infection of iPSC-derived NPC, the universalisation of our cell line via CRISPR-Cas Knock-Out of HLA phenotypes, and the addition of a co-transfection cassette to test cell proliferation in order to avoid the development of teratomas.

Design and Building Strategy

We have used Type IIS cloning to assemble all constructs during the span of the project.
Type IIS restriction enzymes are a class of restriction enzymes (RE) that cut DNA a short distance away from their recognition site, rather than within the recognition sequence itself. Another interesting feature is that they generate sticky ends, which can be customizable and used to easily assemble multiple parts in a single reaction. [1]

Except in some justified cases to facilitate testing, the constructs have been transfected into Long-Term Self-Renewing Neuoepithelial Stem (lt-NES C4) derived from UBI001-A hiPSCs

These lt-NES cells are compromised to differentiate into neural progenitors, with restricted potential to become neurons, astrocytes, or oligodendrocytes. Differentiation is restricted under the exposure of WNT3A and BMP4 factors during the cortical priming process. Moreover, lt-NES cells are easily cryopreserved in liquid nitrogen with specific media for stem cells without DMSO.

This is useful for us because we can de-freeze and differentiate them into the cell type that best suits our needs. Also, lt-NES cells are capable of long-term self-renewal allowing for extended experiments. [2]

However, lt-NES cells require specific, delicate and more expensive working measures, which can difficultate culturing and maintenance. Therefore, clonings were done with XL-Blue bacteria and some of the tests were performed on HEK293T cells, which are feasibly transformed and have a rapid growth[3].

Branches

References

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[19] Hoerster, K., Uhrberg, M., Wiek, C., Horn, P. A., Hanenberg, H., & Heinrichs, S. (2021). HLA Class I Knockout Converts Allogeneic Primary NK Cells Into Suitable Effectors for “Off-the-Shelf” Immunotherapy. Frontiers in Immunology, 11. https://doi.org/10.3389/fimmu.2020.586168
[20] Kagoya, Y., Guo, T., Yeung, B., Saso, K., Anczurowski, M., Wang, C.-H., Murata, K., Sugata, K., Saijo, H., Matsunaga, Y., Ohashi, Y., Butler, M. O., & Hirano, N. (2020). Genetic Ablation of HLA Class I, Class II, and the T-cell Receptor Enables Allogeneic T Cells to Be Used for Adoptive T-cell Therapy. Cancer Immunology Research, 8(7), 926–936. https://doi.org/10.1158/2326-6066.CIR-18-0508
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BRANCH 1: Constitutive RFP Reporter Cassette

Overall, the main goal is to determine the design that best works as a transfection reporter and that will be used throughout the rest of the constructs of the projects.
It consisted of two different cycles.

Cycle 1: BDNF followed by mCherry

Design

The cell line used during the Reneurish project (UBI001-A hiPSCs cell line) has been genetically modified to express GCamP -a calcium detection system- on the GFP spectrum [4].

For this reason, when adding the transfection reporter for our constructs, we needed a reporter with a different wavelength. We chose mCherry because it is one of the most common Fluorescent Proteins, meaning there is a wide range of techniques and optimised protocols already at our disposal to test it. Additionally, mCherry's red emission spectrum is useful for multicolor imaging, as it can be used combined with other fluorescent proteins (like GFP) without significant spectral overlap.

In order to have mCherry as a transfection reporter, we wanted it to be regulated by the same promoter as BDNF in a polycistronic construct, so we used the following scheme:

Build

The construct was transfected using a Lentivirus vector into lt-NES cells before starting differentiation. We expected to start seeing expression on day 1 after transfection.

We used Lentiviral transfection to ensure we would be able to see the expression of our construct for an extended period of time. In this case, we infected lt-NES and wanted to see the expression signal up to the point where they were differentiated into neurons.
During this process, cell proliferation is notably elevated, thus we aimed to avoid episomal transfection, as this would result in exponential dilution of the expression signal. Therefore, the ability of the lentiviral vector to integrate the construct into the host genome was highly beneficial for maintaining stable expression and obtaining results.

Test

The goal of the experiments from this cycle was to know if the design fit the requirements to use RFP as a transfection reporter. The activity of the reporter was studied through fluorescence microscopy, measuring the signal from mCherry in the range of 550-650 nm wavelength. Additionally, we checked the GFP emission spectrum to make sure there were no interferences between fluorescent proteins.
For more information see Page: Results.

Learn

From this cycle we concluded:

mCherry can be used as a reporter for our cell line because it does not overlap with the GFP emission spectrum and will not interfere with the Calcium detection system.
The design of the construct worked, but it wasn’t useful as a transfection reporter as we intended it to be. We realized that since mCherry is a cytoplasmic protein, it complicated differentiating the limits between cells and identifying the transfected ones.

Why do we need to count individual cells?

On further steps of our project (Branch 3), we want to transfect a genetic circuit into lt-NES cells with AVV and be able to select only the infected cells. Thus, we need to be able to differentiate which cells express the reporter.

To solve this, we entered the next cycle with a new design.

Cycle 2: Nucleus-targeted RFP.

To solve the problems that arose during cycle 1, we thought of building a nucleus-targeted reporter.

Design

To make sure that our reporter is located in the nucleus, easing the cell counting process, we designed a Composite Part (BBa_K5316020) of H2B linked to the mCherry sequence

Why H2B?

Build

We also changed the building strategy. The purpose of this cycle was to check if the new design containing H2B succeeded at locating mCherry into the nucleus with the goal of being able to differentiate those cells which had been transfected and expressed the construct.

To do this, we realised we could simplify the testing process by not using neurons derived from lt-NES, which required Lentiviral transfection, it is not an immortal cell line and incur higher costs due to their media and growth factor requirements, instead -to be more efficient- we thought of doing the tests on HEK293T cells.

Why HEK293T cells?

Overall, doing the experiments on HEK293T cells will already give us the needed information to know if the construct works, and it will easen the transfection and data gathering process.
HEK293T cells will be transformed using lipofectamine 3000.

Test

We followed the same testing strategy as in Cycle 1, this way we will be able to compare results between cycles. The activity of the reporter was studied through fluorescence microscopy, measuring the signal from mCherry at in the range of 550-650 nm wavelength, and especially focusing on the cellular localization of the signal.
The goal of the experiments from this cycle was to know if this new design fit the requirements to use H2B-RFP as a transfection reporter.

Learn

Testing for this cycle is being carried out at this moment, results are expected to be ready during the judging session.
Based on literature reviews [5] we expect this genetic circuit to allow us to locate the transfected cells.
If results meet our expectations, we will be closing the loop for this first Branch of the project.

1. We needed a reporter that did not interfere with the calcium detection system.

2. We found mCherry successfully fit this requirement. However, the design did not serve as a transfection reporter system because it located the reporter throughout the cytoplasm rather than concentrated it in the nucleus, difficulting cell counting.

3. We redesigned the construct to target our reporter into the nucleus using H2B as a signal peptide, solving the counting cells problem.

Branch 1 consisted of the cycles aimed at testing POC0. To learn more about POC0, see: POCs Page

BRANCH 1.2: Customizable Transfection Reporter Cassette

A Part Collection for future iGEM teams

As we worked with our construct, we realised that the use of reporters targeted to specific cellular locations could be highly helpful to other iGEM teams.
This led us to initiate Branch 1.2 with the goal to create a reporter cassette that allows its users to choose both the signal peptide and the linked reporter according to their preferences and the specific requirements of their projects.

Cycle 3: Customizable Reporter Cassette

Design

Using our previous design from Cycle 2 as the foundation for Cycle 3, we implemented specific modifications to develop a chassis that allows the user to select and switch both the cellular region for reporter localization and the type of fluorescent protein they want to use
To do so, we thought of adding restriction enzyme’s recognition sites that flanked both the H2B and mCherry sequence.

The raw structure of this plasmid consists of two primary components: a signal peptide and a fluorescent protein. Each sequence is flanked by a strategically placed Type IIS restriction enzyme.
The final design on this cycle includes CMV as a constitutive promoter, followed by the H2B sequence, linked to mCherry, which are flanked by Esp3I and BsaI recognition site respectively.

Build

We needed to build both the backbone and the inserts. Since we planned to use Type IIS assembly as the cloning method, we needed to design the overhangs for each of our parts to properly fit the backbone, taking into consideration both the position and direction of each insert.

Backbone

Inserts

We generated a library of different Parts for commonly used Signal Peptides and Fluorescent Proteins, including the overhangs specifically adapted to fit the designated positions in the backbone, assuring compatibility.

Since our backbone has been designed to be compatible with the iGEM kit, we have been able to use Parts from previous iGEM teams and included them in the library. Additionally, to contribute more to the community, we have added three new Signal Peptides and two Fluorescent Proteins. This will allow teams to target new cellular locations with their reporters and to use new colours.

Fluorescent Proteins

Kit number	FP	Part name	TIIS RE	Flanking compatibility	Uses
9	eCFP	BBa_E0020	BsaI	AATG/GCTT	95
10	eYFP	BBa_E0030	BsaI	AATG/GCTT	140
16	TannenRFP	BBa_J97003	BsaI	AATG/GCTT	1
N/A	EGFP	BBa_K1071009	BsaI	AATG/GCTT	1
N/A	bfVFP	BBa_K5316012	BsaI	AATG/GCTT	-
N/A	SVFP	BBa_K5316013	BsaI	AATG/GCTT	-

Signal Peptides

Kit number	Localisation	Part name	TIIS RE	Flanking compatibility
N/A	Membrane	BBa_K5316015	Esp3I	ACGA/TCTC
N/A	Mitochondria	BBa_K5316014	Esp3I	ACGA/TCTC
N/A	Nucleous	BBa_K5316007	Esp3I	ACGA/TCTC
N/A	ER	BBa_K5316017	Esp3I	ACGA/TCTC

It’s important to emphasise that all overhangs in this design can be easily added to other reporters, making them compatible with the cassette. This means that other teams can incorporate any fluorescent protein or signal peptide of their choice, even if they aren’t already included in this library.

For more info see: Part Collection and FP Handbook

Test

Assemblies for this cycle were validated using a cloning model (Geneious Prime). The goal was to check that the overhangs were correct and they allowed the insertion of the different fragments at the desired positions.

Learn

From this cycle we concluded:

We confirmed that the assembly of H2B and mCherry into the desired positions of the backbone succeeded, meaning that we had correctly designed the Type IIS recognition site, cutting direction and overhangs.
From these assemblies we saw that the Ampicillin Part we used was not compatible with the Type IIS cloning system because it contained a BsaI recognition site in the position 772.

During the learning step of this cycle, we acquired valuable insights and thought of multiple enhancements for our design. Consequently, we engaged in multiple iterations of the same cycle to refine our approach effectively:

Cycle 3.1

Design

Derived from our learnings of the previous cycle, we needed to eliminate the BsaI recognition site to make it compatible with our cloning system.

The modification was done at the nucleotide level, so the final peptide remains the same as the unmodified ampicillin without affecting functionality.

Build

We changed GGTCTC nucleotides at position 723 for GAGATC.

Test

This modification has been proved to work because transformed E. coli have been successfully selected and grown in Amp+ media.

Learn

We learned that the modification successfully adapts the Ampicillin sequence to be Type IIS compatible

Further experiments can be conducted to confirm the success of this new part. For example, we could digest the ampicillin sequence with BsaI and run the resulting fragments on an agarose gel. We would expect to see a single band, as BsaI should not find a recognition site to cleave, leaving the fragment intact.

We also thought of adding an extra recognition site for a different Type IIS enzyme: BbsI flanking the whole cassette. This way different teams can insert the reporter cassette into their plasmids.

However, while revising the design and results for this cycle we thought of a way to improve it and make it easier for future iGEM teams to use it as a transfection reporter; instead of having mCherry at the reporter position we will add an ORF Stuffer, a new Part that has not been registered by any team before.

Cycle 3.2

Design

Changing the mCherry sequence from the backbone for an ORF stuffer. This way, other teams can easily interchange it for whatever reporter better suits the needs of their project.

What is an ORF Stuffer

Build and Test

Using the same modelling system as in previous iterations, we confirmed the correct assembly of the ORF Stuffer into our backbone and verified that it generates the correct overhangs when digested with BsaI.

Learn

Assemblies successfully work on the modelling program.

Assemblies Modelling Results

Since all assemblies worked, we decided that by default, the raw backbone will include a nuclear localization signal (H2B) and an ORF stuffer in the position designated for the fluorescent protein (FP). For the sake of our project, it was essential that the reporter was directed to the nucleus, however, the user has the flexibility to change the localization signal in the same reaction where the FP is inserted, allowing easy customization.
We realised we hadn’t checked if the changes of the different Parts, and the scars left by the digestion and ligation process, had altered the reading frame. We tackled this problem in the following iteration.

Cycle 3.3

Design

We had the following starting parameters:

After the Signal Peptide (SP), we had Esp3I’s recognition and restriction sites, which were 11 bp in length. Changing the SP would reduce Esp3I’s sites to a 4 bp scar.
Following the linker, we had BsaI’s recognition and restriction sites, which were also 11 bp in length. Changing the ORF_Stuffer to a Fluorescent Protein (FP) would reduce BsaI’s sites to a 4 bp scar.

ORF aberrations:
- The untouched backbone had 22 additional bp, changing the ORF.
- Only changing the ORF_Stuffer to a FP would result in 15 additional bp, technically maintaining the ORF. However, those 15 bp are not distributed in codons in the two scarred regions, changing the ORF for the linker but maintaining it for the FPs.
- Changing both the SP and ORF_Stuffer would result in 8 additional bp, changing the ORF.

Therefore, we needed to find a way to maintain the ORF of the linker and the FPs by only changing the ORF_Stuffer or both SP and ORF_Stuffer.

Build

After three design iterations, we came up with the perfect solution. We had to add a total of 6 bp in selected positions for the ORF to be maintained in all possible combinations:

1. 2 bp after the linker sequence but right before the BsaI site. Since the ORF_Stuffer is always changed for a FP, the BsaI restriction site is always reduced from 11 bp to a 4 bp scar. Therefore, adding 2 bp to the 4 bp scar allows for the maintenance of the ORF since there will now be 6 bp between the linker and the FP.
2. 2 bp after the Esp3I site and before the linker. There are two options:
- 2.a If the SP is changed, the scar will be 4 bp long. Therefore, adding 2 bp to the 4 bp scar allows for the maintenance of the ORF since there will now be 6 bp between the SP and the linker.
- 2.b If the SP remains unchanged, there will be the 11 bp of the unmodified Esp3I site, and the 2 additional bp we have introduced. These 13 bp change the ORF.
3. 2 bp after the H2B sequence and before the Esp3I site. This corrects the ORF aberration in the case that the SP remains untouched and the ORF_Stuffer is changed with a FP. Therefore, the number of nucleotides between the H2B and the linker is 15 (2 + 11 + 2), maintaining the ORF.

Lastly, we had to carefully choose which nucleotides we wanted to insert to minimise the effect of the additional aminoacids. In the end, we added the following residues:

FP substitution: H2B - [GVSSL] - Linker - [G] - FP
SP and FP substitution: SP - [SL] - Linker - [G] - FP

Test

Using the same modelling system as in previous iterations, Geneius, we confirmed that with the new adaptations of the design, none of the assemblies changed the reading frame.

Learn

We were able to efficiently correct the changes of the fused ORFs by inserting just 6 bp in specific locations.
We minimised the effect of the scars by choosing the aminoacids with the lowest steric hindrance
In silico testing was successful.
The next step is to translate the design to the laboratory

Finally, we obtained the perfect design that fit all the requirements needed to create the Customizable Transfection Reporter Cassette, with a library of interchangeable Signal Peptides and Fluorescent Proteins adapted for other iGEM teams to use.

BRANCH 2: Time-Dependent Neurotrophic Factor Overexpression

One of the main goals of the Reneurish project is to genetically modify neural progenitors to overexpress the neurotrophic factor BDNF and reduce the damage after an ischemic stroke event by improving neural activity regeneration.
Learn more about why we chose BDNF as the neurotrophic factor to express.

Cycle 4: BDNF followed by mCherry

Design: Overexpression of Neurotrophic factors to increase neural activity

The main goal of this cycle is to check the effects of constitutive overexpression of BDNF on neural progenitors and the consequences on the numbers of active neurons, neural connectivity, cell maturation and population interactions.

To achieve this, we put BDNF under the regulation of EF1A, a constitutive eukaryotic promoter, followed by the transfection reporter chassis from Branch 1. Moreover, we flanked this reporter cassette with LoxP sites, enabling us to remove it once approaching the clinical steps of the project.

Build

With the assistance of our PI, lt-NES cells were transfected with a Lentivirus.

Learn more about lentiviral transfection safety on Page: Safe Lab Practices.

Transfected cells were then differentiated via exposure to WNT3A and BMP4 factors at post infection.

Test

To check transfection and functionality of our construct and BDNF expression, we conducted both Immuno and histochemical assays and BDNF qPCR, respectively.
To assess different aspects affected by BDNF overexpression on lt-NES, we performed a variety of experiments:

1. Effects on differentiation: Immuno-histochemical assays and culture observation, focusing on differentiation markers such as PSD95, MAP2, NeuN, etc.
2. Effects on projection formation: Chip-cultures
3. Effects of synaptic activity: GECI calcium recordings.

Learn

Throughout the diverse experiments performed and comparing the different parameters between the control and transfected (tBDNF) cells, we could determine that overexpressing BDNF caused:

Higher cell count in comparison to control cells, without generating uncontrolled growth.
BDNF expressing cells generate more projections.
BDNF expressing cell cultures contain more active neurons than the control.
BDNF expression does not affect the formation of neuronal networks, avoiding aberrant networks.

See the results obtained from this tests on Page: results.

BRANCH 2: Time-Dependent Neurotrophic Factor Overexpression

One of the main goals of the Reneurish project is to genetically modify neural progenitors to overexpress the neurotrophic factor BDNF and reduce the damage after an ischemic stroke event by improving neural activity regeneration.
Learn more about why we chose BDNF as the neurotrophic factor to express.

Cycle 5: Time-dependant promoters to regulate BDNF expression

Design

Once we confirmed the beneficial effects of BDNF on neural activity with Cycle 4, we entered Cycle 5. In this cycle, we innovated by replacing the constitutive EF1A promoter in the design and adding pA, pB, and pC*. These new promoters are time-dependant, which will allow us to determine which one is the best to express BDNF only during the first stages of the therapy. Our goal is to compare their regulatory capacity to determine which achieves the most effective BDNF levels.

*Promoter names are censored under a patent pending system. To learn more, see Page: IP.

Additionally, these promoters are inactivated once neuronal differentiation is complete, providing an extra layer of clinical safety. See more info in Page: Clinical Safety

Build

Promoters are assembled in the same construct as in Cycle 4, where they replace EF1a. By using different flanking restriction enzymes, the promoters are integrated with the proper orientation in the vector.

Test

A double digestion was carried out to ensure that both the plasmid backbone and the promoters had the required cohesive ends. The digestion was verified through an electrophoresis gel and afterwards the ligation reaction was prepared with a insert : vector ratio of 5 : 1. To validate the ligation, a second digestion was performed using enzymes that cut once in the backbone and once within the ligated promoters, but not in the control.
This method allowed us to easily determine if the correct promoters had been inserted into the backbone:

Two bands: This indicates that the enzyme cut twice—once in the backbone and once in the promoter of interest.
One band: This indicates that the enzyme only found a single recognition site in the backbone, meaning the construct contains the control promoter.

All tested plasmids were linearized, indicating they contained the original EF1a promoter

Learned

Through the weeks spent in the laboratory, we learned the importance of meticulous preparation, validation, and optimising experimental conditions to ensure efficient and reliable lab outcomes.
With the results from Cycle 5 we learnt that:

The promoters of interest were not ligated in the vector as we have proven in the gel electrophoresis (see Page: Results, Plasmid assembly).
Unwanted DNA fragments may be the limiting factor in our assembly.
DNA gel recovery after a complete digestion may allow us to succeed assembling the plasmid.

Learn more about how we are trying to overcome this on Page: Results, Plasmid assembly

BRANCH 3: Designs for integrated future steps

From the lab bench to the patient

Branch 3 is our way of looking towards the future. This branch includes design of future steps in Reneurish, which includes AAV infection of iPSC-derived NPC, the universalisation of our cell line via CRISPR-Cas Knock-Out of HLA phenotypes, and the addition of a co-transfection cassette to test cell proliferation in order to avoid the development of teratomas.

Branch 3.1: AAV transfection

Design: Overexpression of Neurotrophic factors to increase neural activity

Using engineered cells for cell therapies requires strict safety measurements. This is why we are interested on using AAV to transfect our NPC, as they do not integrate into the cell genome and their expression disappears overtime. For more information regarding use of adeno-associated viruses for cellular therapy, See Page: Biosafety.

Build

From the very beginning, we have optimised our BDNF expression construct for use in AAV. By using an AAV-compatible backbone and introducing loxP sites to optimise sequence length for encapsulation.

LoxP removal

Our BDNF expression vector contains a reporter cassette with an mCherry-H2B fusion protein. However, our final product cannot contain fluorescence-emitting cells, which is why from day one our construct has LoxP sites flanking the DNA sequence of the reporter cassette. By designing our LoxP sites orientated in the same direction inside the plasmid, we intend to generate a deletion in our expression plasmid that removes our reporter cassette [11].

AAV serotype

Selecting the serotype of the AAV proteic capsid can help improve transfection efficiency, as well as increase cell specificity. We intend to use AAV7m8 for our transfections, as it is the best performing serotype in iPSC transfection in comparison to other AAV serotypes [12].

Cell transfection

Transfection of our cells with AAV will be performed during the differentiation (priming) stage. This ensures transfection is performed at optimal time of cell proliferation, and prevents overdilution of the adenovirus before patient transplant.

Branch 3.2: Cell universalisation

In the future, the Reneurish strain will undergo a HLA Knock-Out modification to universalise our cell line.

One of the main holdbacks of cell therapies are immunogenic rejection towards the grafted cells. For Reneurish to reach as many patients as possible, we need to find a way to overcome this issue.

In a nutshell, an immunogenic rejection towards the grafted cells occurs when host cytotoxic T cells recognise foreign HLA antigens, activating Natural Killer Cells (NKs). These, in turn, recognise the HLA marker that does not belong to the host, triggering a response to terminate the cells containing this mismatched antigen [13].

After researching different options, we have decided to universalise our cell line via CRISPR/Cas9 by eliminating the HLA genes from our cell line. This essentially creates non-immunogenic cells.

HLA-KO is of special interest in cell therapies and has been performed on numerous occasions [14, 15, 16, 17, 18] due to the possibility of evading T-cell responses.

Not only that, but a combination of Knocked-Out genes and a Knock in allows the cells to bypass recognition by Natural Killer cells [19, 20].

Design

Knock-out

According to literature, with a simple 3 gene Knock-Out we might be able to bypass allogenic detection. The genes we are interested in Knocking-Out are:

β2-Microglobulin (B2M):These genes codify proteins that allow HLA class I proteins to be presented on cell surface [21]. Eliminating the expression of this protein prevents the presentation of HLA-I molecules on cellular surface. This KO allows us to bypass cytotoxic T-cell detection.
MHC class II TransActivator (CIITA):Is the master regulator for HLA-II gene expression [22]. Knock out of this gene blocks transcription of HLA-II genes, giving a second layer to avoid an allogenic response.
T-cell Receptor Alpha Constant (TRAC):These genes codify the T-cell receptors (TCRs). We would specifically target the TCR-⍺ region to completely abolish TCR expression [14].

To Knock-Out these genes, we will design single guide RNAs (sgRNAs) for the CRISPR/Cas9 system to introduce Double Strand Breaks in the target genes. To properly knock out the genes, we will prepare two sgRNAs per gene, cutting at different points and introducing large deletions to completely Knock-Out expression.

Knock-in

In order to avoid triggering the Natural Killer response, we need to knock in the expression of polymorphic HLA-E molecules. The recognition of HLA-E by the NK cells avoids induced “missing-self” lysis of the transplanted cells.

As shown in literature, the ideal knock in is a fusion protein consisting of [20]:

HLA-E single-chain dimer (heavy chain)
B2M

The proteins are covalently fused via a flexible linker.
Knock in will be performed via lentiviral transfection to ensure the modification of the cell strain persists through future generations.

Branch 3.1: AAV transfection

Safety is key for Reneurish. This is why we want to implement a specialised cell selection method using Flow-cytometry.

After transfection of our NPC cells with AAV to express BDNF, the cells have to undergo strict testing to minimise any risk of teratoma formation. This means we need a highly efficient method capable of selecting cells individually.

This new construct will be co-transfected with our BDNF expression circuit, meaning it will also behave as our transfection reporter once we have removed the fluorescence cassette from the backbone via LoxP sites.

Design

Expression

According to a study conducted in embryonic stem cell-derived Neural Progenitors, the likelihood of teratoma formation oscillates according to the maturation stage of the transplanted cells [23]. To minimise teratoma formation, we need to transplant NPC in their Mid to Late stage of maturation, which is why we have chosen SOX2, a Neural Progenitor Cell specific promoter for this cassette.

Marker

Controlled by the SOX2 promoter will be the expression of a custom synthetic membrane protein not expressed in any other cell types [24]. This protein will be recognised by a specific fluorescent antibody so we can perform Fluorescence-Activated Cell sorting, which has purity rates of 95 to 100% [25].

By adding fluorescence to the cell surface, we are not hindering cell viability nor manipulating them, and the superficial expression of the fluorophore makes the cells compatible for the FACS system.

For clinical application, the transplanted cells need to be highly purified and strictly tested. The FACS sorting process allows us to select cells individually and not by population, diminishing risk of teratoma formation and allowing selection of BDNF-expressing cells.

For additional safety, we might implement back-to-back rounds of FACS sorting of the cells to minimise any potential error.