Week 2 HW: DNA Design

Building the GD2-SynNotch–GPC2 CAR-T Genetic Circuit

This week, I translated the therapeutic concept proposed in Homework 1 into defined genetic parts and three buildable lentiviral constructs.

GD2 sensing → SynNotch activation → GPC2-CAR + EGFP expression → GPC2 recognition → T-cell activation + mbIL-15 support

1. 🧬 Define the expression target

1.a What molecules am I expressing?

For my project, I will express two synthetic receptors (GD2-SynNotch and GPC2-CAR), one membrane-tethered cytokine (IL-15), and a fluorescent reporter (EGFP).

: GD2-SynNotch
Recognizes GD2 on the neuroblastoma cell surface and releases Gal4-VP64 to activate the downstream response cassette.: GPC2-CAR Recognizes glypican-2 (GPC2) following SynNotch priming. Its activation triggers T-cell cytotoxicity.; Membrane-bound IL-15 (mbIL-15) Provides localized cytokine support to improve T-cell survival and persistence. Its expression is activation-inducible through NFAT signaling.; EGFP Serves as a fluorescent reporter of SynNotch-induced transcription and is expressed from the same inducible transcript as the GPC2-CAR through an IRES.

The current CAR response vector also retains constitutive tBFP expression as a marker of vector expression. 1.b Sequence sources GD2-SynNotch. The design is based on Moghimi et al. (2021), using the E101K-mutated anti-GD2 scFv. The remaining domains use the established Notch1 regulatory core and Gal4-VP64 architecture. Moghimi et al. (2021) | Addgene #79125 GPC2-CAR. I selected the humanized hCT3-1 anti-GPC2 scFv (VL–linker–VH), reported in patent US20250281538A1, SEQ ID NO: 12. It is incorporated into a second-generation CAR containing a CD8α hinge/transmembrane region, 4-1BB and CD3ζ. GPC2 antibody patent | Addgene #79123 Membrane-tethered IL-15. The cytokine module is based on Hurton et al. (2016), using human IL-15 connected to IL-15Rα through a flexible linker. Reference proteins: IL-15 NP_000576.1 and IL-15Rα NP_002180.1. Hurton et al. (2016) EGFP. Enhanced green fluorescent protein derived from Aequorea victoria, NCBI GenBank U55763.1. 1.c Host / chassis The chassis is primary human T lymphocytes, including CD4+ and CD8+ T cells. For the proposed autologous therapy, these cells would be obtained from the patient and genetically modified ex vivo. 1.d Where do the products go?

Membrane-associated proteins

GD2-SynNotch, GPC2-CAR and mbIL-15 require entry into the secretory pathway through a signal peptide and contain an appropriate transmembrane or membrane-anchoring region.

Cytoplasmic reporters

EGFP and tBFP remain intracellular and therefore do not require signal peptides.

🧩 Design the genetic parts I organized the system into three functional constructs, each with a different regulatory role.
Construct 1 — Constitutive GD2 sensor
Expression: Constitutive Architecture: PGK promoter → 5′ UTR → Kozak → CD8α signal peptide → GD2 scFv (14G2a-E101K) → Notch1 regulatory core → Gal4-VP64 → Stop The constitutive PGK promoter maintains expression of the GD2 sensor. After GD2 recognition, the SynNotch regulatory core releases Gal4-VP64, which activates the downstream Gal4-responsive cassette. Reference backbone: Addgene #79125, modified by replacing the original anti-CD19 scFv with the selected anti-GD2 scFv.
Construct 2 — SynNotch-inducible effector
Expression: Inducible by GD2-SynNotch activation Architecture: Gal4 UAS → minimal CMV → GPC2-CAR → IRES → EGFP → poly(A) CAR: CD8α signal peptide → GPC2 scFv → CD8α hinge → CD8 TM → 4-1BB → CD3ζ Gal4-VP64 released by activated SynNotch binds the Gal4 UAS and induces GPC2-CAR expression. EGFP is translated from the same transcript through an IRES and reports activation of the response cassette. The current vector also contains a separate constitutive PGK → tBFP unit. Reference backbone: Addgene #79123.
Construct 3 — Activation-dependent cytokine support
Expression: Activation-inducible through NFAT signaling Architecture: 3× NFAT-responsive elements → minimal IL-2 promoter + Kozak → IgE signal peptide → human IL-15 → flexible linker → IL-15Rα membrane-anchoring component → Stop Following T-cell activation, NFAT can activate transcription of the mbIL-15 cassette. Membrane tethering is intended to provide localized cytokine support rather than constitutive soluble cytokine secretion. Reference backbone: pRRLSIN.cPPT.PGK-GFP.WPRE — Addgene #12252, with the original expression cassette replaced by the NFAT-inducible mbIL-15 cassette.
2.d Genetic-part summary Part type Selected part Source / role Constitutive promoter PGK Addgene #79125; constitutive SynNotch expression Inducible regulatory element Gal4 UAS + minimal CMV Addgene #79123; activated by Gal4-VP64 Activation-responsive promoter 3× NFAT + minimal IL-2 promoter Activation-dependent mbIL-15 expression Translation initiation 5′ UTR + Kozak Translation initiation in human cells Antigen-binding domains GD2 scFv; GPC2 hCT3-1 scFv Tumor-antigen recognition Signaling domains Notch core; 4-1BB; CD3ζ Synthetic transcriptional and CAR signaling Targeting / membrane domains CD8α signal peptide, hinge/TM; IL-15Rα anchor Membrane localization Translational control IRES EGFP expression from the inducible CAR transcript Reporter EGFP; tBFP Inducible response reporter; constitutive vector marker Lentiviral context WPRE / LTRs / backbone elements Retained from the selected vectors

🧬 Exact sequence record

The detailed imported/selected sequence parts used for the three constructs are kept here so that the main page remains readable. Par type Sequence SynNotch-GD2 Backbone used is PGK promoter → 5′ UTR → Kozak → Signal peptide (CD8)→ GD2 scFv (14G2a) → Notch1 regulatory core → Gal4-VP64 → Stop codon → 3′ UTR → Poly(A) signal HumanCD8 derived signal peptide MALPVTALLLPLALLLHAARP Atggctctgccagtcaccgccctcctgctgccgctcgcccttctgcttcatgctgctaggcca GD2 scFv (14G2a) VL – linker – VH

DVVMTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELK GGGGSGGGGSGGGGS EVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMKYWGQGTSVTVSS

Notch1 regulatory core

atcctggactacagcttcacaggtggcgctgggcgcgacattcccccaccgcagattgaggaggcctgtgagctgcctgagtgccaggtggatgcaggcaataaggtctgcaacctgcagtgtaataatcacgcatgtggctgggatggtggcgactgctccctcaacttcaatgacccctggaagaactgcacgcagtctctacagtgctggaagtattttagcgacggccactgtgacagccagtgcaactcggccggctgcctctttgatggcttcgactgccagctcaccgagggacagtgcaaccccctgtatgaccagtactgcaaggaccacttcagtgatggccactgcgaccagggctgtaacagtgccgaatgtgagtgggatggcctagactgtgctgagcatgtacccgagcggctggcagccggcaccctggtgctggtggtgctgcttccacccgaccagctacggaacaactccttccactttctgcgggagctcagccacgtgctgcacaccaacgtggtcttcaagcgtgatgcgcaaggccagcagatgatcttcccgtactatggccacgaggaagagctgcgcaagcacccaatcaagcgctctacagtgggttgggccacctcttcactgcttcctggtaccagtggtgggcgccagcgcagggagctggaccccatggacatccgtggctccattgtctacctggagatcgacaaccggcaatgtgtgcagtcatcctcgcagtgcttccagagtgccaccgatgtggctgccttcctaggtgctcttgcgtcacttggcagcctcaatattccttacaagattgaggccgtgaagagtgagccggtggagcctccgctgccctcgcagctgcacctcatgtacgtggcagcggccgccttcgtgctcctgttctttgtgggctgtggggtgctgctgtcccgcaagcgccggcggatgaagctgctgagcagcatc Gal4-linker-VP64-stop codon atgaagctgctgagcagcatcgagcaggcctgtgacatctgccggctgaagaaactgaagtgcagcaaagaaaagcccaagtgcgccaagtgcctgaagaacaactgggagtgccggtacagccccaagaccaagagaagccccctgaccagagcccacctgaccgaggtggaaagccggctggaaagactggaacagctgtttctgctgatcttcccacgcgaggacctggacatgatcctgaagatggacagcctgcaggacatcaaggccctgctgaccggcctgttcgtgcaggacaacgtgaacaaggacgccgtgaccgacagactggccagcgtggaaaccgacatgcccctgaccctgcggcagcacagaatcagcgccaccagcagcagcgaggaaagcagcaacaagggccagcggcagctgacagtgtctgctgctgcaggcggaagcggaggctctggcggatctgatgccctggacgacttcgacctggatatgctgggcagcgacgccctggatgattttgatctggacatgctgggatctgacgctctggacgatttcgatctcgacatgttgggatcagatgcactggatgactttgacctggacatgctcggatcatga

CAR – GPC2

Backbone used is Addgene #79123 Gal4 UAS → Minimal promoter CMV → 5′ UTR → Kozak → Signal peptide (CD8) → GPC2 scFv (VL–linker–VH) → CD8α hinge → Transmembrane domain → 4-1BB → CD3ζ → Stop codon → IRES → EGFP CDS → Stop codon → 3′ UTR → Poly(A) signal The same vector contains a separate constitutive expression unit: PGK promoter → 5′ UTR → Kozak → tBFP CDS → Stop codon → Poly(A) signal Gal4 UAS → Minimal promoter CMV → 5′ UTR → Kozak ggagcactgtcctccgaacgtcggagcactgtcctccgaacgtcggagcactgtcctccgaacgtcggagcactgtcctccgaacggagcatgtcctccgaacgtcggagcactgtcctccgaacgactagttaggcgtgtacggtgggaggcctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctctcgacattcgttggatccttgacttgcggcc GPC2 scFv (VL–linker–VH)

DVVMTQSPLSLPVTPGEPASISCRASSSVNYIYWYLQKPGQSPQLWIYYTSNLAPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQFSSSPSTFGQGTKLEIK RSRGGGGSGGGGSGGGGSLEMA QVQLVQSGAEVKKPGASVKVSCKASRFTFTDYNIHWVRQAPGQGLEWIGYINPNNGDIFYKQKFNGRVTLTADKSTSTAYMELSSLTSEDTAVYYCVRSSNIRYTFDRFFDVWGQGTLVTVSS → CD8α hinge accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgat → Transmembrane domain CD8 + junction SL Atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc tcccta

→ 4-1BB aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg → CD3ζ → ( add Stop codon) Agagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgcctcctcgc*** MbIL15

Backbone used is

https://www.addgene.org/12252/sequences/ NFAT-responsive elements → Minimal IL-2 promoter → 5′ UTR → Kozak → Signal peptide → Human IL-15 → Flexible linker → IL-15Rα membrane-anchoring component → Stop codon → 3′ UTR → Poly(A) signal

NFAT responsive elements  (3X , 2 Forward 1 reverse)

ggaggaaaaactgtttcatacagaaggcgtggaggaaaaactgtttcatacagaaggcgtacgccttctgtatgaaacagtttttcctcc Filler + minimal IL2 promoter + Kozak tcgaggacattttgacacccccataatatttttccagaattaacagtataaattgcatctcttgttcaagagttccctatcactctctttaatcactactcacagtaacctcaactcctgcccaagcttggcattccggtactgttggtaaagccacc Signal peptide (IgE) MDWTWILFLVAAATRVHS

→ Human IL-15 (NM_000585.5) without sig pep aactgggtgaatg taataa gtgatttgaa aaaaattgaa gatcttattc aatctatgca tattgatgct actttatata cggaaagtga tgttcacccc agttgcaaag taacagcaat gaagtgcttt ctcttggagt tacaagttat ttcacttgag tccggagatg caagtattca tgatacagta gaaaatctga tcatcctagc aaacaacagt ttgtcttcta atgggaatgt aacagaatct ggatgcaaag aatgtgagga actggaggaa aaaaatatta aagaattttt gcagagtttt gtacatattg tccaaatgtt catcaacact tcttga

→ Flexible linker (SG3(SG4)3SG3SLQ (26 amino acids)) agcggcggaggatctggcggaggtggaagcggaggcggaggaagcggtggcggaggtagtggcggaggatcacttcag

→ IL-15Rα membrane-anchoring component without sig pep a tcacgtgccc tccccccatg tccgtggaac acgcagacat ctgggtcaag agctacagct tgtactccag ggagcggtac atttgtaact ctggtttcaa gcgtaaagcc ggcacgtcca gcctgacgga gtgcgtgttg aacaaggcca cgaatgtcgc ccactggaca acccccagtc tcaaatgcat tagagaccct gccctggttc accaaaggcc agcgccaccc tccacagtaa cgacggcagg ggtgacccca cagccagaga gcctctcccc ttctggaaaa gagcccgcag cttcatctcc cagctcaaac aacacagcgg ccacaacagc agctattgtc ccgggctccc agctgatgcc ttcaaaatca ccttccacag gaaccacaga gataagcagt catgagtcct cccacggcac cccctctcag acaacagcca agaactggga actcacagca tccgcctccc accagccgcc aggtgtgtat ccacagggcc acagcgacac cactgtggct atctccacgt ccactgtcct gctgtgtggg ctgagcgctg tgtctctcct ggcatgctac ctcaagtcaa ggcaaactcc cccgctggcc agcgttgaaa tggaagccat ggaggctctg ccggtgactt gggggaccag cagcagagat gaagacttgg aaaactgctc tcaccaccta tga

Stop codon → 3′ UTR → Poly(A) signal

EGFP atggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg 661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc 721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg 781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc 841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag 901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag 961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac 1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac 1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc 1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg 1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc 1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag 1321 ctgtacaagt ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc 1381 gcgggcccgg gatccaccgg atctagataa

3. 🧱 Assemble the constructs in Kernel I assembled the three constructs in Kernel using the selected lentiviral backbones and imported genetic parts. The final maps were checked for part order, orientation and coding-region continuity.

Sequence checks:

✓ Intended 5′→3′ part order
✓ Coding regions in the intended reading frame
✓ Start and stop positions checked
✓ No unintended internal stop codons retained in the final CDSs

Construct 1 — GD2-SynNotch

Kernel schematic of the GD2-SynNotch construct.

Construct 2 — GPC2-CAR–IRES–EGFP

Kernel schematic of the Gal4-inducible GPC2-CAR–IRES–EGFP construct.

Construct 3 — NFAT-inducible mbIL-15

Kernel schematic of the NFAT-inducible membrane-bound IL-15 construct.

🔎 Optimize and quality-check 4.a Codon optimization I codon-optimized the protein-coding regions of the three constructs for expression in Homo sapiens. Regulatory regions such as the PGK promoter, Gal4 UAS, NFAT-responsive elements, minimal promoters, IRES, WPRE and LTRs were left unchanged because their nucleotide sequences are functionally important. The optimization changed synonymous codon usage while preserving the intended protein products. The optimized CDS versions were retained as the proposed sequences for synthesis.

Codon optimization of the coding regions for human-cell expression.

4.b Sequence verification by BLAST I used BLASTp to verify the identity and architecture of the encoded proteins.

GD2 scFv: strong full-length matches to antibody variable-region sequences, with approximately 77–81% identity and E-values down to 2.3 × 10⁻¹²⁵.

Complete GPC2-CAR: highly significant matches to other engineered CAR proteins, with approximately 70–74% identity across most or all of the 497-aa sequence and E-values approaching zero.

GPC2 hCT3-1 scFv: direct comparison with the selected hCT3-1 reference confirmed 100% amino-acid identity.

mbIL-15 fusion: the strongest hit was human IL-15Rα, with 100% identity over residues 159–395 and an E-value of 9.15 × 10⁻¹⁶⁸, consistent with the engineered IL-15–linker–IL-15Rα fusion.

The complete CAR and mbIL-15 sequences are engineered fusion proteins, so they are not expected to be identical to one natural protein.

Representative BLASTp verification results.

4.c Biosecurity screening I ran Kernel’s DNA biosecurity screen on each complete final construct. All three constructs passed without a reported biosecurity flag. The screen evaluates submitted DNA sequences for matches associated with sequences of biosecurity concern. A clear result supports the required sequence-level security check, but it does not assess the biological or clinical safety of the engineered T cells themselves.

Kernel DNA biosecurity screening of the final constructs.

🧪 From design to build 5.a How would I build it? I would reuse the three lentiviral backbones and synthesize only the new or modified DNA modules rather than complete plasmids. Each backbone would be PCR-linearized while excluding the sequence being replaced. The new fragment would be synthesized with homologous terminal overlaps and joined to the backbone by Gibson/HiFi assembly.

PCR-linearized backbone → synthetic insert with homologous overlaps → Gibson / HiFi assembly → bacterial clone → sequence verification

: GD2-SynNotch: replace the original anti-CD19 scFv with the GD2 scFv.: GPC2-CAR: replace the inducible payload with the GPC2-CAR–IRES–EGFP cassette.; mbIL-15: replace the original pRRLSIN expression cassette with the NFAT/minimal IL-2–mbIL-15 cassette. Gibson assembly is suitable because it enables seamless joining of overlapping DNA fragments without depending on restriction sites (Gibson et al., 2009).

Proposed assembly strategy for the three lentiviral constructs.

5.b What does synthesis constrain? The main constraints are sequence length, repeated regions and synthesis errors. My design contains repeated regulatory elements, particularly the NFAT sites and Gal4 UAS, as well as repetitive linker sequences. These regions may be harder to synthesize or assemble accurately. If a complete cassette is difficult to synthesize, I would divide it into smaller fragments and assemble them together with the backbone. DNA synthesis is not inherently error-free, so the assembled plasmids would be sequence-verified before use. Error-correction approaches can reduce mutations in de novo synthesized DNA (Carr et al., 2004).

Main practical constraint

The repetitive NFAT-responsive region is the part most likely to complicate synthesis or assembly. If needed, I would order the regulatory region and mbIL-15 coding region as separate fragments rather than forcing the whole cassette into one synthesis fragment.

5.c Prototype vs. scale

Prototype

At the prototype stage, each construct would be assembled by PCR linearization and Gibson assembly, transformed into bacteria, sequence-verified and expanded to obtain plasmid DNA for small-scale lentiviral production and T-cell testing.

After the design is fixed

The constructs would not need to be resynthesized for every batch. Sequence-verified plasmid clones could be established as master bacterial stocks and expanded whenever additional plasmid DNA is required.

Larger / clinical scale

Scale-up would therefore focus mainly on controlled plasmid production, reproducible lentiviral-vector manufacturing and patient-specific T-cell processing rather than repeated de novo synthesis of the genetic constructs.

6. 🧠❓ What do I not know yet?

❓ Will the GD2-SynNotch gate be sufficiently specific and tightly controlled? The main uncertainty is whether GD2 recognition will induce enough Gal4-VP64 activity to express the GPC2-CAR while maintaining low background expression in the absence of GD2. I would compare EGFP/CAR expression after exposure to GD2-negative cells and cells expressing different GD2 levels. Synthetic transcriptional circuits can show behaviors that are difficult to predict from individual components alone, so this will need empirical tuning rather than sequence design alone (Elowitz & Leibler, 2000).
🧬 Will the three constructs function efficiently together in the same T cell? Because the system uses separate lentiviral constructs, not every T cell may receive and express all three modules at appropriate levels. I would measure expression of each module and compare different vector-delivery conditions, then test whether cells containing the complete circuit respond differently from cells missing one component.
⚖️ Will NFAT-inducible mbIL-15 remain sufficiently activation-dependent? NFAT can also be activated through endogenous T-cell signaling, so mbIL-15 may not be exclusively triggered by the GPC2-CAR. I would compare mbIL-15 expression in resting cells, after CAR stimulation and after independent TCR stimulation, together with proliferation and activation/exhaustion markers. More generally, maintaining functional separation between synthetic regulatory modules is an important consideration in engineered logic circuits (Stanton et al., 2014). The main challenge is therefore not simply whether each individual genetic module works, but whether all three modules behave together as a predictable and controllable T-cell circuit.