circRNA CAR-T Solutions

Alfa Cytology provides a dedicated, end-to-end circRNA CAR-T service platform that unifies circular RNA design, CAR construct engineering, delivery optimization, functional testing, and in vivo validation. By leveraging the inherent stability and tunability of circRNAs, the platform helps address common CAR-T bottlenecks—exhaustion, limited persistence, infiltration in solid tumors, and TME suppression—within a single, streamlined workflow.

Introduction to circRNA CAR-T

circRNA CAR-T encodes the CAR (or adjunct regulators) on a circular, non-integrating RNA that supports cap-independent, re-doseable, and durable expression, lowering exhaustion and enabling fine dose/schedule control. Beyond serving as the CAR payload, circRNAs can sponge miRNAs, scaffold RNA-binding proteins, and coordinate ribosome/protein recruitment, thereby stabilizing effector and metabolic programs, tuning checkpoint tone (e.g., PD-1/PD-L1) and key signaling axes (JAK/STAT, MAPK, NF-κB), and improving function within hostile TMEs—yielding sustained, controllable anti-tumor activity suited to both solid and hematologic settings.

Mechanisms of circRNA-Enhanced CAR-T Cells

circRNA-Encoded CAR Expression

Cap-independent Translation: A circularized CAR ORF uses IRES/m⁶A-driven ribosome entry, enabling efficient translation without a 5′ cap and sustaining output despite exonucleases.
Stability → Steady Surface Density: The covalently closed loop resists degradation, yielding more stable CAR surface levels that support multi-round killing and delay exhaustion.

Intracellular Molecular Regulation

miRNA Sponging: circRNAs sequester inhibitory miRNAs, easing PD-1/PD-L1–driven dysfunction and supporting persistence.
RBP Sponging/Scaffolding: circRNAs organize RNA-binding proteins/translation machinery to stabilize pro-effector transcription and survival programs.
Translational Control: coordinated ribosome/protein recruitment yields steady output of effector and metabolic genes that sustain function.

Pathway & Phenotype Tuning

Checkpoint Attenuation: dampens PD-1 (T cells) / PD-L1 (tumor) programs to preserve activity under chronic stimulation.
Signal Rewiring: modulation of JAK/STAT, MAPK, NF-κB helps balance activation vs. memory and reduces apoptosis propensity.

TME Remodeling & Infiltration

Macrophage Polarization: shifts M2 → M1 to relieve immunosuppression.
NK Synergy & Chemotaxis: improves NK support and adjusts chemokine/ECM cues, enhancing CAR-T homing, entry, and retention in solid tumors.

CircRNA CAR-T vs Traditional CAR-T

Feature	Traditional CAR-T	circRNA CAR-T
mRNA Type	Linear mRNA	Circular RNA
Structure	5′-capped, 3′-poly(A) tailed, linear molecule	Covalently closed loop, no 5′ cap or 3′ tail
Stability	Prone to exonuclease degradation	Resistant to exonuclease degradation
Persistence	Shorter in vivo persistence	Prolonged expression and activity
Exhaustion	Tendency for early exhaustion	Reduced exhaustion due to sustained CAR expression
Transgene Expression	Temporary, declines over time	Stable, continuous expression
Gene Delivery	Viral or non-viral vectors	Viral/non-viral vectors; also compatible with mRNA delivery workflows
Immunogenicity	Higher: IVT linear mRNA can contain impurities and structural motifs that trigger innate sensing	Lower: optimized circRNA chemistries/processes reduce innate pathway activation and unnecessary inflammation
Applications	Hematologic cancers, solid tumors	Solid tumors; programs needing resistance to exhaustion and persistent activity in TME

What We Provide?

To turn circRNA’s unique strengths into actionable data, we provide an end-to-end development workflow—from construct design and circularization QC through non-viral loading, expression-kinetics profiling, TME-relevant assays, and in-vivo proof-of-mechanism—tailored to your target, cell source, and program milestones.

Mechanism & Study Design

Define whether the circRNA encodes the CAR or a regulatory add-on, set primary readouts (expression kinetics, function, phenotype), and plan dose/schedule consistent with circRNA’s re-doseable profile.

circRNA Construction & Circularization QC

Engineer junction/backbone and translation elements; verify true circularization by RNase-R enrichment plus divergent/convergent RT-PCR and junction-spanning sequencing, then confirm in-cell translation.

Ex-Vivo Loading & Expression Kinetics

Optimize non-viral electroporation into primary T cells to balance viability and expression; quantify CAR surface density and durability over time by flow and standardized protein/RNA assays.

In-Vitro Function & Phenotype

Measure cytotoxicity and cytokines, assess exhaustion/memory markers, and evaluate checkpoint tuning (e.g., PD-1 on T cells; PD-L1 on target cells) under repeated stimulation.

TME-Relevant Assays

Use 3D spheroids/ECM and chemotaxis systems for trafficking/infiltration; include macrophage M2→M1 polarization and NK co-culture assays to profile circRNA-mediated TME support.

In-Vivo Proof-of-Mechanism & Reporting

Run fit-for-purpose models to track tumor control, CAR-T persistence, and TIL phenotypes; deliver a decision-ready package (QC, raw/processed data, methods) suitable for internal reviews.

Applications of circRNA CAR-T

Solid Tumors

Non-integrating, extended CAR expression sustains activity in hostile TMEs (e.g., neuroblastoma, glioblastoma, pancreatic) and supports convenient repeat dosing.

Multiple Myeloma (MM)

Prolonged CAR expression improves persistence against myeloma targets (e.g., BCMA or others), helping maintain tumor control in relapse-prone settings.

Hematologic Cancers

Durable, re-doseable expression enhances the longevity of CD19/CD22/CD20/CD38 programs for refractory leukemias and lymphomas.

Antigen Escape Prevention

The RNA-based, modular format enables rapid re-targeting (swap the circRNA payload and re-dose) and maintains effective activity even when antigen density fluctuates.

Why Choose Us?

Expertise
Leverage a senior team of T-cell and RNA engineers with deep experience in cancer immunology and translational assay design.
circRNA × CAR-T Focus
Purpose-built methods for circular CAR expression and regulatory circRNA co-modules within one coordinated program.
Integrated Program
One team manages design, delivery, in-vitro profiling, and in-vivo validation—reducing hand-offs and rework.
Reproducibility
Multi-donor primary T-cell runs with batch comparability and orthogonal readouts to confirm key findings.
Solid-Tumor Realism
Orthotopic/PDX and matrix-rich TME models to demonstrate infiltration, persistence, and activity under suppressive conditions.

FAQs

Is the platform limited to circular CAR expression?

No. It supports circular CAR expression and regulatory circRNA modules within the same program.

Can I start with in-vitro only?

Yes. Many programs begin with expression/viability/function and then proceed to in-vivo once acceptance criteria are met.

How do you choose the delivery method?

We run side-by-side feasibility across LNP/electroporation/viral routes and select a primary plus a de-risking backup based on viability, uniformity, and function.

What about safety?

We integrate innate-sensor screens, off-target transcriptomics, and optional kill-switch designs, plus comprehensive control arms.