Basic Parts
This year our team worked to provide a library of essential parts for constructing chimeric antigen receptor T-cells, Thus we have submitted our sequences of basic parts tp IGEM biobricks registry including aiming to make it simple and modular for others to find informative sequence data about CAR construction for therapeutic purposes:
Parts you will need to Design a Second generation CAR:
You will need parts from BBa_K3244006 to BBa_K3244012 which includes a CD8 leader sequence, a scFv (single chain variable fragment) for recognition antibody against your target (In our case we have 2 scFv, 1 for estrogen receptor beta and another scFv for s.haematobium major egg antigen)-visit our modeling page for information on antibodies design-, you will also need a hinge that should be determined in terms of length in relation to the proximity of your antigen so long hinges could better adapt your CAR design for proximal cellular targets and in our case we selected CD8 Hinge. Then, after having essential extracellular components, you will need extracellular domain, in our case we have selected CD28 TM domain. Finally, you will need the intracellular domains, the case for 2nd generation CAR requires only 2 domain so we used CD3 zeta domain for cytotoxicity functions and CD28 intracellular domain for costimulatory function.
Parts you will need to design a TRUCK (T cells Redirected for Universal Cytokine Killing) 4th generation CAR:
You will need the basic parts to enable you to induce expression of specific cytokine.Thus NFAT RE (Response Element), IL-2 promoter, Il-18 CDS which we have selected over other used cytokines as Il-12 due to its potential reactive oxygen species modulation which could present an advantage against s.haematobium ova. You will also need IRES and GFP for characterization.
Parts you will need to transfect CAR to T-cells using CRISPR Cas9:
You will need a Cas9, in our case we used IDT cas9 from CRISPR Alt-products as well as True Cut Cas9 from ThermoFisher. You will also need a gRNA BBa_K3244023 for TRAC locus on T-cells to guide the cas9 to for proper cutting by cas9 and insertion by homology directed repair templates designed to include CAR sequence.
You will also need L-ARM and R-ARM for you CAR HDR insertion to fank the gRNA for proper insertion in parts BBa_K3244021, BBa_K3244022.
Parts you will need to design a siRNA cassette vector:
Part BBa_K3244024 consists of triple cassettes for U6, TOX and NR4A each consists PD-1 promoter, sense, hairpin loop, antisense and terminator. To form vector you will need RSV promoter, U6 promoter. bGh poly(A) for termination in mammalian cells. In our case we also added AmpR and AmpR promoter to compensate for the unavailability of chloramphenicol for research purposes.
Bio brick | Type | Role | Length |
Regulatory | CD8 Leader Sequence | 64 | |
Coding | scFv for ESR2 neoantigen | 744 | |
Coding | Gly Ser Linker for TanCAR-T scFvs | 75 | |
Coding | scFv for Schistosoma haematobium Major egg antigen | 723 | |
Coding | CD8 Hinge | 144 | |
Coding | CD28 Trans-Membrane Domain | 81 | |
Coding | CD28 Intracellular signaling domain | 123 | |
Coding | CD3 zeta signalling domain | 339 | |
Regulatory | NFAT-Response Element | 117 | |
Regulatory | IL-2 Promoter | 114 | |
Coding | IL-18 | 579 | |
Regulatory | IRES | 625 | |
Coding | GFP | 720 | |
Coding | BZip Leucine zipper | 150 | |
Coding | AZip Leucine zipper | 171 | |
Coding | 2ِA Cleavage | 54 | |
DNA | L-ARM for CAR-T insertion in TRAC using CRISPR | 582 | |
DNA | R-ARM for CAR-T insertion in TRAC using CRISPR | 590 | |
Regulatory | Guide RNA Scaffold for CRISPR insertion of CAR-T cell therapies | 107 | |
Coding | AmpR | 861 | |
Regulatory | AmpR Promoter | 105 | |
Regulatory | U6 Promoter | 228 | |
Coding | EGFP | 768 | |
Terminator | bGH poly(A) signal | 211 | |
Regulatory | RSV Promoter | 227 |
We have also added characterization to part BBa_K3244016
In order to reduce CAR exhaustion and prevent undesired expression,, the introduction of an internal ribosomal entry site (IRES) to lower tonic signaling and ligand-independent phosphorylation of the CAR-CD3ζ chain which has been shown to improve CAR-T cell expansion as compared to CAR-T cells not containing the IRES element.
Thus, we have added IRES to the construction of our TRUCK CAR and we also characterized part BBa_K1442023 in terms of enhancement to expression of our TRUCK CAR HDR insertion in engineered T-Cells
group | Ct (HDR1) | Ct(ACTB) | ΔCt (HDR1) | ΔΔCt(HDR1) | RQ(HDR1) |
T cell from BC Patient [engineered with IRES] | 28.51 | 30.25 | -1.74 | -3.84 | 14.3 |
T cell from BC patient [engineered without IRES] | 30.41 | 31.07 | -0.66 | -2.76 | 6.77 |
T cell BC [unengineered] | 38.26 | 36.15 | 2.11 | 0.01 | 0.99 |
This real time qPCR shows the quantitative increase in expression of the CAR insertion in schistosomiasis associated bladder cancer patient T-cells engineered with IRES in comparison to relatively lower expression in engineered T cells without IRES.
Composite Parts
Out of the basic parts we have created our main composite fragments to be synthesized using IDT and twist offers Including (please have a look at our Design page):
Putting the first circuit (siRNA) together
Part BBa_K3244024 (siRNA cassettes for optimizing CAR-T cells for solid tumors) has been composited into part BBa_K3244025 which consists of triple cassettes for U6, TOX and NR4A each consists PD-1 promoter, sense, hairpin loop, antisense and terminator. To form vector you will need RSV promoter, U6 promoter. bGh poly(A) for termination in mammalian cells. In our case we also added AmpR and AmpR promoter to compensate for the unavailability of chloramphenicol for research purposes.
Lane 2: siRNA fragments ligated Lane 3: siRNA Ligated to pSB1C3+ (10 kb Ladder lane adjusted)
Functional characterization of BBa_K3244025
We performed gene Expression analysis of cultured T cell that was isolated from BC patients and engineered by using our first circuit by Real time quantitative PCR (qPCR) using Specific oligonucleotide sequences for NR4a and PD-1. A significant downregulation of PD1 and NR4a was observed in engineered patient-derived T-cells compared to un-engineered cells (p<0.01), however, higher levels were obtained in cells derived from the Schistosoma associated BC patient, although significance was not achieved (p<0.05). On the other hand, NR4a was decreased in engineered T cells (p<0.05). The gene expression was calculated according to un-engineered T cells (negative control)
Serial | group | Ct (NR4a) | Ct(ACTB) | ΔCt (NR4a) | ΔΔCt(NR4a) | RQ(NR4a) |
1 | T cell from BC associated Schitosoma [silenced] | 38.82 | 36.26 | 2.56 | -3.59 | 0.08 |
2 | T cell from BC negative for Schitosomiasis [silenced] | 40.19 | 32.13 | 8.06 | 1.91 | 3.75 |
3 | T cell BC [control] | 36.29 | 30.14 | 6.15 | 0 | 1 |
Serial | group | Ct (PD-1) | Ct(ACTB) | ΔCt (PD-1) | ΔΔCt(PD-1) | RQ(PD-1) |
1 | T cell from BC associated Schitosoma [engineered] | 35.82 | 30.25 | 5.57 | 4.75 | 26.99 |
2 | T cell from BC negative for Schitosomiasis [engineered] | 32.08 | 26.75 | 5.33 | 4.51 | 22.783 |
3 | T cell BC [unengineered] | 38.11 | 37.29 | 0.82 | 0 | 1 |
Putting the second circuit (CAR) together:
Lane2: CAR HDR Ligated to pcDNA3.1+ (Ladder lane adjusted)
Part BBa_K3244027 which represents a composite device of 2nd Generation CAR-T, part BBa_K3244028 which represents a composite IL-18 expression construct for TRUCK CAR design and finally BBa_K3244029 which represents CRISPR Homology Directed Repair template with both L-Arm and R-Arm for IL-18-producing TRUCK CAR-T Cell.
- A statistical significance difference was achieved between the % of viability in CAR-T treated BC cells associated Schistosomiasis and unassociated cells (p<0.05). Moreover, between untreated and CAR-t treated BC cells (p<0.01). High reduction of viable BC cells that was treated by CAR-T was demonstrated in Schistosomiasis associated BC compared to unassociated cells, but no statistical significance was achieved (p>0.05).BC cells that was treated with CAR-t cells engineered for ERB2 and Schistosoma egg antigen should a significant apoptosis (54.8%) and 21% dead tumor cells.
Part Improvement:
We have worked to create an improved Glycine serine linker in part BBa_K1537017 in our new part BBa_K3244026
to adapt for our bispecific extracellular domain which should carry 2 scFv targeting 2 different antigens. This part has been modified through codon optimization -which has represented an issue for our fragment DNA synthesis due to the need for repetition of linker sequence between 4 different chains of 2 scFvs- and amino acid sequence length editing t(GGGGS)5 to act as an inter-chain linker between the 2 scFv for enhanced production and stability of the bispecific scFv.
group | Ct (EGR2ab) | Ct(ACTB) | Δ Ct (EGR2ab) | ΔΔ Ct(EGR2ab) | RQ(EGR2ab) |
T cells of BC associated Schistosomiasis patient (engineered with GSSS in BBa_K1537017 ) | 39.26 | 29.22 | 10.04 | 0.00 | 1.00 |
T-cells of BC associated Schistosomiasis patient BC associated Schistosomiasis (engineered with GSSS in BBa_K3244026) | 38.51 | 28.26 | 10.25 | 0.21 | 0.86 |
group | Ct (MEAab) | Ct(ACTB) | Δ Ct (MEAab) | ΔΔ Ct(MEAab) | RQ(MEAab) |
T cells of BC associated Schistosomiasis patient (engineered with GSSS in BBa_K1537017 ) | 40.26 | 29.22 | 11.04 | 0.15 | 0.90 |
T-cells of BC associated Schistosomiasis patient BC associated Schistosomiasis (engineered with GSSS in BBa_K3244026)" | 32.15 | 21.26 | 10.89 | 0.00 | 1.00 |