Part Collection 1: CAAR Collection

Overview

This collection (which ranges from part number BBa_K5310000 to BBa_K5310018) includes nineteen successfully submitted basic parts that can be used to produce four new composite parts (BBa_K5310050 to BBa_K5310053), four novel chimeric autoantibody receptors, that bind MS-specific antibodies and produce cell responses. Parts BBa_K5310000 and BBa_K5310006-11 are components of both receptors. The two receptors differ in the modular extracellular domain. Receptor 1 includes part BBa_K5310001 between the Igk signal peptide and the linker sequence, while receptor 2 incorporates BBa_K5310003.

The collection ends with the most important parts BBa_K5310060 and BBa_K5310061, a modular extracellular part and the modular body of the CAAR. The body, made up of CD8a hinge/TM- CD28- CD137- CD3z is proposed as the most valuable asset of our collection. With its MoClo compatibility, any extracellular part design can be incorporated. This way, by exchanging the immunogenic domains in the extracellular part and using BBa_K5310061 as the body, we can shift the focus to new autoantibodies produced in MS or even autoimmune diseases.

Structural Domains

The transmembrane (TMD) domain of the receptor (encoded by BBa_K5310008) is an important structural component mediating external ligand binding and intracellular signalling. CD8a -TMD is a known, well-performing domain that is derived from CD8, a membrane-bound glycoprotein involved in T cell antigen recognition ¹. Although weaker than the CD28 alternative, it has been known to make CAR-T cells safer and more durable ².

The hinge domain (HD) (encoded by BBa_K5310007) is essentially a spacer amino acid sequence linking the autoantibody recognition domain with the transmembrane region. The extracellular component has more freedom of movement and can bind to its target more easily, enhancing flexibility as well as treatment success ³. Much like its TM counterpart, the CD8a hinge domain is empirically used due to its established and consistent efficiency ⁴.

Apart from their optimal length and tertiary structure, the endogenous origin of CD8a-TMD/HD facilitates receptor expression in T-cells during manufacturing ⁵.

Igk signal peptide (encoded by BBa_K5310000) is a short amino acid sequence present at the N-terminus of many newly synthesised proteins. It facilitates the entry of the protein into the endoplasmic reticulum (ER), where it is processed and directed to its destination, either within the cell membrane or outside the cell. Once the protein enters the ER, the signal peptide is typically cleaved off by signal peptidases.

Signalling Domains

CD3-zeta (encoded by BBa_K5310011) is the cytoplasmic domain used in all approved CAR T-cell therapies. Deriving from the transmembrane domain of TCRs, it contains Immunoreceptor Tyrosine-based Activation Motifs (ITAMs), a sequence necessary for signal transduction ⁶. When the ligand recognises the receptor, tyrosine residues in ITAMs are phosphorylated by a member of the Src tyrosine kinase family. ITAMs then attract ZAP-kinases which are also phosphorylated and activated, leading to downstream signalling. This results in Ras activation, Ca2+ mobilisation, actin cytoskeleton reorganisation and transcription factor activation which ultimately leads to T-cell activation ⁷.

It has been demonstrated in literature that the main activation domain benefits from the existence of co-stimulatory sequences that amplify signalling and prolong CAR T-cell survival. While this started with a single co-stimulatory endodomain (2nd generation), it was quickly established that a combination (3rd generation) produces better results. The domains used almost exclusively are CD28 (encoded by BBa_K5310009) and 4-1BB (encoded by BBa_K5310010) ⁸.

4-1BB belongs to the TNF receptor family and promotes T-cell proliferation via a pathway called ncNF-κB (non-canonical nuclear factor κB). Upon ligand-induced trimerization of 4-1BB, TNF receptor–associated factors (TRAF) are recruited, shifting ubiquitination from NIK to TRAF3. NIK, which is normally ubiquitinated and degraded, can now accumulate and trigger a signalling cascade that ends with the activation of important lymphocyte development genes ⁹.

CD28 is an intrinsic T-cell protein that provides a complementary signal, assisting activation and minimising depletion. It has key signalling motifs, notably YMNM and PYAP that initiate the P13K/Akt/mTOR and PDK1/Akt pathways respectively, both of which are involved in the cell cycle and cytokine production ¹⁰.

Autoantibody Binding Domains

As a new take on the established Chimeric Antigen Receptors, this domain consists of an immunodominant epitope of an autoimmunity-triggering protein. Therefore, it recruits and binds with circulating autoantibodies and autoreactive lymphocytes ¹¹.

For MS, we selected two autoantibody binding domains, amino acids 35-55 of Myelin Oligodendrocyte Glycoprotein (encoded by BBa_K5310001 and employed in the CAAR composite parts BBa_K5310050 and BBa_K5310051) and amino acids 83-99 of Myelin Basic Protein (encoded by BBa_K5310003 and employed in the CAAR composite parts BBa_K5310052 and BBa_K5310053) ^{12 13}.

Part Collection 2: CRISPR/dCas9-VPR Collection

Overview

This collection (which ranges from part number BBa_K5310027 to BBa_K5310036) includes specific sgRNAs designed to facilitate the targeted activation of the FOXP3 gene through a CRISPR/dCas9-VPR platform. The utility of these sgRNAs lies in their ability to guide the dCas9-VPR nuclease to precise locations within the FOXP3 gene, promoting targeted epigenetic modifications that convert cytotoxic T cells into regulatory T cells (T-regs). This transformation is crucial for enhancing immune regulation and safety in the context of NeuroMuSceteer.

sgRNAs Structure

The structure of our selected sgRNAs is meticulously designed to ensure optimal targeting and effectiveness in modifying the FOXP3 gene, as analyzed in detail in the Design Page. Utilizing CRISPick tool to select specific epitopes within the FOXP3 gene and focusing on regions that are essential for its function in T-reg development, the targets sequences as well as their complementary sequences were identified, ensuring high specificity for the desired modifications.

Through CRISPR-Cas9-mediated editing process the successful binding of the sgRNA -in their correct orientation (5' to 3')- to the target DNA is achieved. The already incorporated BsmBI restriction enzyme sequences at both ends of the sgRNAs facilitate subsequent cloning and insertion into the plasmid backbone, allowing for precise and efficient insertion of the sgRNAs into the pAW12.lentiguide.GFP plasmid, and therefore setting the stage for effective transduction into K562 cells ¹⁴.

The designed sgRNAs will effectively guide the CRISPR machinery, leading to successful FOXP3 activation. This is expected to increase the yield of T regulatory cells in our cell population tenfold, before we transduct them to express the chimeric autoantibody receptor.

Part Collection 3: miRNAs and HCR Collection

Overview

This collection contains the seven new basic parts (ranging from BBa_K5310020 to BBa_K5310027) that were developed for the miRNA detection and release mechanism of NeuroMusceteer.

The mechanism requires two different structures to be successful:

1. a T-shaped structure that recognizes 2 miRNA disease biomarkers and unfolds, releasing an initiator molecule
2. an HCR hairpin set that is activated by the initiator and undergoes a series of hybridisations-base pair separations, resulting in the release of two therapeutic miRNAs

T-structure

As described on our design page, the biomarker miRNAs selected have to be highly expressed in pathogenic cells to ensure that the initiator triggers the Hybridisation Chain Reaction with specificity. For remyelination in multiple sclerosis, we target cells of the oligodendroglial lineage which were found to have abnormally high levels of miR-125a-3p and miR-146a-5p in patients.

The T-structure consists of three arms (T1, T2, T3) held together in a stable structure. A sequence of T1 (BBa_K5310020) is attached to T2, but is also complementary to miR-125a-3p. When the hairpin enters the target cells the miR binds to it and the whole T1 arm is released, leaving an exposed strand in the T2 arm (BBa_K5310021). T2 is now able to bind to the complementary miR-146a-5p and be released along with T3 (BBa_K5310022), the aforementioned initiator for the hybridisation process.

Hybridisation Chain Reaction Hairpins

This set includes four hairpins (H1, H2, H3, H4) that are stable and inactive without the initiator. H1 and H2 are bound with miRNA-219-a-5p and miR-338-3p respectively, which were both found to induce oligodendroglia regeneration and remyelination. When released, the initiator binds to a complementary sequence in H1 (BBa_K5310023) and causes it to unfold and hybridise with H2 (BBa_K5310024). H2 then becomes linear, and in turn binds to H3 (BBa_K5310025). The unfolded H3 can attach to H4 (BBa_K5310026) as well as H1, causing the release of miR-219-5p. H4 in turn is hybridised with H2 to release miR-338-3p and with H1 to start a new reaction cycle.