Circular RNA (circRNA) is a class of single-stranded RNA with a covalently closed loop structure, lacking both the 5' cap and poly-A tail found on linear messenger RNA. This closed architecture makes circRNA resistant to exonuclease degradation, a primary mechanism by which cells break down conventional mRNA.
For cell therapy applications, this stability translates into extended protein expression. Linear mRNA typically produces detectable protein for 24 to 48 hours before degradation reduces expression below functional thresholds. Circular RNA sustains expression for days, providing a wider manufacturing and therapeutic window without requiring permanent genomic integration.
This positions circRNA in a unique middle ground: longer-lasting than mRNA, but fully transient. Researchers gain extended functional windows for CAR expression, cytokine signaling, and other therapeutic payloads while avoiding the insertional mutagenesis risks associated with viral vectors or DNA-based approaches.
Portal mechanoporation delivers circRNA into primary immune cells through controlled mechanical membrane disruption, without viral vectors, lipid nanoparticles, or electrical current.
Cells pass through precisely engineered pores in a MicroBooster™ cartridge. The controlled mechanical stress creates transient openings in the cell membrane. CircRNA in the surrounding solution enters the cytosol through these openings, and the membrane self-heals within seconds.
This delivery method is cargo-agnostic. The same platform and workflow delivers mRNA, circRNA, proteins, CRISPR ribonucleoproteins, and small molecules. For circRNA specifically, mechanoporation offers a critical advantage: no modified nucleotides are required. Lipid nanoparticle and electroporation-based delivery of RNA therapeutics often require chemical modifications such as pseudouridine to reduce innate immune sensing via endosomal pathways. Because circRNA is inherently less immunogenic than linear mRNA and mechanoporation bypasses endosomal immune sensors entirely, the combination eliminates the need for chemical base modifications.
Portal has demonstrated circRNA delivery to T cells and monocytes, with the mechanoporation platform validated for intracellular delivery across T cells, NK cells, B cells, PBMCs, and monocytes using multiple cargo types.
Portal offers two instrument scales for circRNA delivery to primary T cells, both using the same mechanoporation principle with consistent performance metrics.
Scale
Instrument
Consumable
Cell Capacity per Run
The Gateway system is a benchtop instrument designed for research workflows. For clinical manufacturing, the MilliBooster cartridge integrates with clinical cell processing systems, including the Fresenius Kabi Lovo Cell Processing System via a simple sterile weld or luer lock connection, enabling processing of 1B+ cells with validated GMP compatibility.
No specialized reagents are required beyond the circRNA constructs themselves. Mechanoporation eliminates the need for viral vectors, lipid nanoparticles, modified nucleotides, or electroporation buffers.
Multiplexed delivery in one step: For co-delivery of multiple constructs, simply mix the circRNA molecules together before loading. No sequential transfections are required. Portal achieved 76% dual positive co-expression (CD19 CAR + mbIL-12) in activated T cells using this single-step approach (MGH collaboration).
Scale-up note: Results generated on the Gateway translate to the MilliBooster with consistent viability and delivery efficiency. Minimal optimization is required when scaling up.
Circular RNA delivered by Portal mechanoporation sustained GFP expression for up to 10 days, as demonstrated in Portal's circRNA application note, significantly outlasting linear mRNA expression.
Parameter
Linear mRNA
Circular RNA (circRNA)
Structure
Linear, 5' cap + poly-A tail
Covalently closed loop
Primary degradation pathway
Exonuclease cleavage
Resistant to exonucleases
Typical expression duration
24 to 48 hours
Up to 10 days
Modified bases required
Often required for LNP/electroporation delivery
Not required — circRNA's closed structure is inherently less immunogenic, and mechanoporation bypasses endosomal sensing
Genomic integration risk
None
None
This extended expression window matters for cell therapy manufacturing. Many therapeutic applications, including CAR-T production and cytokine-enhanced TIL therapy, require sustained protein expression over multiple days of ex vivo culture and expansion. CircRNA delivered by mechanoporation provides this duration without the permanence or safety concerns of viral integration.
Yes. Portal mechanoporation delivered two circular RNA constructs simultaneously into activated T cells in a single processing step, achieving 75+% dual positive expression.
In collaboration with Massachusetts General Hospital, activated T cells received both CD19 CAR circRNA and membrane-bound IL-12 (mbIL-12) circRNA via mechanoporation. Flow cytometry confirmed that 75+% of treated cells expressed both the CD19 CAR and mbIL-12, with 15.6% expressing CAR alone.
Condition
CD19 CAR+ / mbIL-12+ (Dual Positive)
CAR+ Only
Control (no cargo)
0%
0%
Mechanoporation boost
75+%
15.6%
This single-step multiplexed delivery eliminates the need for sequential transfections, which reduce viability and complicate manufacturing workflows. The stoichiometry of each construct is tunable by adjusting the ratio of circRNA molecules in the input mixture, giving researchers precise control over co-expression levels.
CircRNA-engineered CAR-T cells achieved 95% cytotoxicity at a 3:1 effector-to-target ratio, demonstrating that mechanoporation-delivered circular RNA produces fully functional therapeutic proteins.
In the MGH collaboration, T cells co-expressing CD19 CAR and mbIL-12 from circRNA delivery were tested against target tumor cells at increasing effector-to-target (E:T) ratios.
E:T Ratio
No Cargo Control
circRNA CAR-T (CD19 CAR + mbIL-12)
0:1
Minimal
Minimal
1:3
~0%
~5 to 10%
1:1
~0%
~60 to 70%
3:1
~0%
~95%
Cytotoxicity increased proportionally with the number of effector cells, consistent with antigen-specific CAR-mediated killing rather than nonspecific toxicity. The no-cargo control showed negligible killing at all ratios, confirming that the cytotoxic activity was attributable to the delivered circRNA constructs.
In a separate preclinical study from the MGH collaboration, presented at ESMO 2022, T cells engineered with mbIL-2 and mbIL-12 via mechanoporation demonstrated 2.5-fold enhanced proliferation and a 4-fold increase in central memory phenotype — distinct from the CD19 CAR + mbIL-12 co-delivery experiment above.
Preclinical Metric
Control
mbIL-2 + mbIL-12 (mechanoporation)
Fold Change
Human cell count (Day 5)
~25,000
~65,000
2.5x
Central memory phenotype (CD62L+CD69-)
~10%
~43%
4x
Effector phenotype
Maintained
Maintained
No loss
The 2.5-fold proliferation increase (65,000 vs. 25,000 cells at Day 5) indicates that membrane-bound cytokine signaling from mbIL-2 and mbIL-12 provides sustained autocrine stimulation that enhances T cell expansion.
The 4-fold enrichment in central memory T cells (43% vs. 10% CD62L+CD69-) is particularly significant. Central memory T cells are associated with long-term persistence, self-renewal capacity, and superior antitumor efficacy based on published research. This phenotype shift was achieved through transient expression via mechanoporation, without permanent genetic modification.
Yes. Portal has demonstrated generation of multi-engineered CAR-T cells directly from whole blood in under 10 minutes, without apheresis, cell isolation, or preprocessing.
In this workflow, whole blood is processed directly through the Portal mechanoporation system with CD19 CAR circRNA and mbIL-2 mRNA. The mechanoporation selectively affects immune cells within the mixed blood population, leaving other blood components unaffected.
Metric
Untreated Whole Blood
Portal-Processed Whole Blood
Double positive (CAR+ IL-2+)
15.7%
52.9%
CD19 CAR+ cells
0.17%
52.9% (311x increase)
Processing time
N/A
Under 10 minutes
Cell isolation required
N/A
None
The 311-fold increase in CAR-positive cells from whole blood starting material, combined with 52.9% double positive co-expression, demonstrates that complex multi-cargo cell engineering is achievable without the traditional manufacturing infrastructure of apheresis machines, cell separators, and cleanroom facilities. This opens a path toward point-of-care CAR-T cell manufacturing, where engineered cells could be produced at the bedside without centralized manufacturing infrastructure.
Back