Abstract

The experimental setup to establish the ASTERISK workflow for glioma treatment, including the integration of Nanopore-Sequencing technology and application of our intraoperative mRNA sensing and kill switch, we present the experiments conducted down below including optimized protocols we contributed to iGEM and the synthetic biology community.

Introduction

Our mRNA-based sensing and kill switch for tumor cell specific detection and apoptosis based on Mechanism proposed by Gayet et al. 2023. To conceptualize ASTERISK, we used several standard protocols from the cloning and in vitro transcription toolbox of iGEM. We choose Gibson assembly as our primary cloning strategy, but also perform restriction enzyme cloning according to BioBrick RFC 10 standard. After establishing 6 level 0 constructs in total, we assembled 5 different level 1 and 24 level 2 vectors. You can find comprehensive information regarding the underlying cloning scheme and the design of the performed experiments in our Overview. All sequencing results performed through Sanger Sequencing and Nanopore Sequencing are listed in the Sequences section.

Prior to start, we conceptualize the whole project, collecting protocols and start with basic preparations and Research. We have had to work our way up from scratch, starting with designing the desired mRNA sensing and kill switch including sensor target site. We set the strategy for cloning our DNA insert into the plasmids by employing the Gibson assembly technique as well as BioBrick assembly methods. Based on our Research, we listed all experiments conducted as a part of our wet lab experience structured as our Engineering Cycle.

However, during the experiments, we adjust our initial protocols to optimize the individual reactions to our preferences. Based on the experiences of previous iGEM Teams, ourselves and literature searches, we are capable of presenting optimized abläufe for each reaction and adding useful tips for upcoming iGEM Teams or scientists, which can be visited in the Protocols Section. Therefore, the material and methods part provides detailed step-by-step guidance to enable an easy and efficient reproduction of our experiments. Changes to protocols for certain experiments are referred to in the Results section. An overview of the Parts used in this project listed in the Part section. For detailed information, visit our Part Pages site. For detailed insights, our laboratory Notebook provides the timeline and workflow performed within the two months of lab work. Moreover, you find an overview of abbreviations for our new constructs and strains, as they are mentioned on our wiki pages.

Goals

    Improve the workflow for designing and constructing the vectors needed for the in vitro transcription of tumor-specific mRNA sensing and kill switches:
    • Excess efficiency of ADAR-based A-to-I-Editing mechanism and cell-specific translation with glioma-specific alterations as the target transcript
    • Test and verify accuracy of our software tool for sensor sequence design
    • Design and construct entry vector pASTERSIK for sensor sequences
    • Establish HEK293 cells with constitutive IDH1 R132H point mutations and EGFR amplification
    • Analyze differences between UTR variants ASTERISK-HBA1 and ASTERISK-HBB
    • Perform in vitro transcription to synthesize therapeutic mRNA sensors
    • Characterize therapeutic mRNA in cell lines and primary tumor tissue in vivo
    • Check compatibility of therapeutic mRNA and different transfection alternatives
    • Design pASTERISK_Shuttle to contribute to our dual strategy for in vitro transcription and in vivo synthesis

Strategy 1: Proof-of-concept of the DART VADAR mechanism

Strategy 1
  • Selektion and outgrowth of E. coli DH5alpha strains containing pDART VADAR and pDV06 in LBAmp50 of Gayet et al. provided by AddGene
  • Plasmid Preparation of E. coli to perform isolation and cleanup of pDART VADAR and pDV06
  • Co-transfection of pDART VADAR and pDV06 in HEK278 cells with Lipofectamine 3000 according to Gayet et al. 2023
  • Analyze transfection and translation efficiency with FACS and CLSM

Strategy 2: Utelizing the DART VADAR mechanism to target glioma specific alteration


Strategy 2
  1. PART I
    • Based on target sequencing, design and scoring of short (120 - 140 bp) sensor sequences (for brief details view our Software section)
    • Design sensor sequence synthesis and primer pairs for amplification
    • Perform amplification of sensor sequences via PCR
    • Perform gel electrophoresis to assess length of amplicons and amplification efficiency
    • Perform gel extractions and PCR cleanup of amplified sensor sequences
  2. PART II
    • Selektion and outgrowth of E. coli DH5alpha strains containing pDV06.2 in LBAmp50 of Gayet et al. provided by AddGene
    • Plasmid Preparation of E. coli to perform isolation and cleanup of pDV06.2
    • Linearize pDV06.2 via restriction enzyme XY in entry region
    • Perform phosphatase treatment to inhibit re-ligation
    • Perform gel electrophoresis to assess digestion efficiency
    • Perform gel extractions and PCR cleanup of linearized pDV06.2
  3. PART III
    • Assembly pcDNA5 for constitutive expression of IDH1 including characteristic R132H mutation
    • i) Design IDH1wt and IDH1mt sequence synthesis and primer pairs for amplification
    • ii) Perform amplification of IDH1wt and IDH1mt via PCR
    • iii) Perform gel electrophoresis to assess amplification efficiency
    • iv) Perform gel extractions and PCR cleanup of amplified sequences
  4. PART IV
    • Integrate amplified sensor sequences into digested pDV06.2 via preferred cloning strategy: overlap with homology arms of the entry vectors for Gibson assembly or restriction enzyme cloning via X and Y
    • Transfection of pDV06.2IDH1mt and pDV06.2EGFR in E. coli DH5alpha
    • Perform colony PCR to identify positive clones with integrated sensor sequence
    • Selektion and outgrowth of E. coli DH5alpha strains containing pDV06.2IDH1mt and pDV06.2EGFR
    • Plasmid Preparation of E. coli to perform isolation and cleanup of pDV06.2IDH1mt and pDV06.2EGFR
    • Co-Transfection of pDV06.2IDH1mt and pDV06.2EGFR with corresponding pcDNA5IDH1 and pCMVEGFR in HEK278 cells with Lipofectamine 3000 according to Gayet et al. 2023
    • Analyze transfection and translation efficiency with FACS and CLSM

Strategy 3: Construction of pASTERISK for in vitro transcription of therapeutic mRNA


Strategy 3
  • Design shuttle plasmid for dual-strategy: testing plasmids transfection and mRNA production via IVT
  • Selektion and outgrowth of E. coli DH5alpha strains containing p137 and p148 in LBAmp50 provided by AG Müller
  • Plasmid Preparation of E. coli to perform isolation and cleanup of p137 and p148
  • Perform single digestion of entry vector p148 with restriction enzyme SpeI
  • Perform phosphatase treatment to inhibit re-ligation
  • Perform gel electrophoresis to assess digestion efficiency
  • Perform double digestion of p137 with restriction enzyme XbaI and SpeI
  • Perform amplification of Poly-A-Signal
  • Perform gel extractions and PCR cleanup of amplified Poly-A-Signal

Strategy 4: Construction of pASTERISK_Shuttle for dual strategic usage


Strategy 4
  1. PART I - Preparation of Backbone
    1. Transfection of E. coli DH5alpha with pSB1C3 (= standard vector tool of the distribution kit of iGEM and backbone for pASTERISK)
    2. Selektion and outgrowth of E. coli DH5alpha pSB1C3+ strains in LBCm36 media
    3. Plasmid Preparation of E. coli to isolate and clean up pSB1C3
    4. Perform double digestion of entry vector pSB1C3 with restriction enzyme EcoRI and PstI
  2. PART II - Preparation of Fragments
    1. Design Fragment 1 to 3 and simulate Gibson assembly in silico
    2. Order Fragment gene block synthesis provided by IDT and primer for amplification PCR provided by Sigma
    3. Evaluate amplification efficiency by performing gel electrophoresis of amplified PCR products
    4. Test overlap extension PCR for pre-assembly of Fragment 1 - 3
    5. Perform gel extraction or PCR cleanup
    6. Measure concentration and quality of extractions using Nanodrop
  3. PART III - Gibson assembly for pASTERISK
    1. Perform Gibson assembly and transfection of pASTERISKHBA/HBB
    2. Cultivation on LBCm36 plates and incubation with standard conditions
    3. Colony PCR to assess the length of the integrated insert
    4. Outgrowth of pASTERISK-HBA1/HBB+ clones in selection media LBCm36
    5. Selektion and verification of positive pASTERISK-HBA1/HBB assemble via colony PCR and random restriction enzyme digestion
    6. Plasmid Preparation of pASTERISKHBA/HBB to isolate and cleanup pASTERISK
    7. Perform Sanger Sequencing and Nanopore Sequencing to evaluate assembly, verify orientation, and complete assembly in silico
  4. PART IV - in vitro transcription for therapeutic mRNA synthesis
    1. Amplification of insert to multiply the template for IVT procedure
    2. PCR cleanup of amplified products to reduce background activity and increase the efficiency of IVT
    3. Perform DNase I treatment to reduce DNA contamination within the sample
    4. RNA cleanup of IVT products
    5. Perform Poly-Adenylation via Poly(A)-Polymerase
    6. Perform RNase treatment to reduce degradation of RNA and increase yield
    7. RNA cleanup of adenylated RNA products
    8. Capping reaction for final preparation of therapeutic RNA for eukaryotic usage
    9. RNA cleanup and verification of length in denaturing gel electrophoresis
    10. Gel extraction and PCR cleanup
  5. PART V - Test Nucleotide Modifications
    1. Changing nucleotides to increase therapeutic effect, for example, increase stability, increase translation rate, or decrease decay
  6. PART VI - Lipid Nanoparticle (LNP) Synthesis
    1. Production of LNPs
    2. Testing of compatibility
    3. Comparison against Lipofectamine and Mirus for glioma treatment

Our Protocols