Byterna Therapeutics Advances Scarless Circular RNA-Based CAR-T Platforms for Enhanced Anti-Tumor Efficacy
Byterna is a biopharmaceutical
CAR-T Cell Therapy Researcher
Yuanma Cyclization System |Design Circular RNA Precursors Based on Multi-Stem-Loop Structures with Sequence Endogeneity
Byterna Therapeutics Cyclization Strategy |Design circular RNA precursors based on the endogenous single stem-loop structure within the sequence.
In the previous three issues, we provided a detailed introduction to Byterna Therapeutics' unique circular RNA platform based on the endogenous secondary structure of the sequence-to-be-cyclized. In this issue, let's explore how Byterna Therapeutics uses circular RNA technology to construct CAR-T platforms both in vitro and in vivo.
In Vitro CAR-T Platform Based on Seamless Circular RNA
Expression Intensity and Timing of CAR
Based on the Hi-Scarless-PIE circularization strategy, construct anti-CD19 CAR-circRNA. Electroporate anti-CD19 CAR-circRNA into human primary T cells and detect CAR transfection efficiency and expression efficiency. On day 1, day 2, and day 4 after electroporation, measure the proportion of CD19-positive cells in the CD19-circRNA group.The proportions were 83%, 84%, and 32%, while the distribution of CD19-positive cells in the linear CD19-mRNA group.For 75%, 60%, 1%.Compared with linear CD19-mRNA, CD19-circRNA in T cellsExpressed for a longer duration in China, with a higher density of CAR molecules on the T cell surface.
In addition, the researchers also prepared BCMA-circRNA/GPRC5D-circRNA and electroporated T cells.The duration and intensity of CAR expression were both superior to those of the linear control group.
Tumor Cell Killing Activity of CAR-T
Researchers co-cultured CAR-T cells prepared with CD19-circRNA electroporation with target cells at different effector-to-target ratios to assess the in vitro cytotoxicity of CAR-T cells. Nalm‑6(Human B Lymphoblastic Leukemia Cells)and Raji cells(Human Burkitt's Lymphoma Cells)Surface high expression of CD19 protein, while K562 cells(Chronic Myeloid Leukemia Cell Line in Humans)The surface does not express the CD19 protein.
The First Day After ElectroporationWhen CAR-T cells were co-cultured with tumor cells, the CD19-circRNA group and the linear CD19-mRNA group targeted CD19-positiveSimilar tumor cell killing ability；The Fourth Day After ElectroporationThe killing ability of CAR-T cells co-cultured with tumor cells on Nalm-6 in the CD19-circRNA groupSignificantly stronger thanLinear CD19-mRNA group, while the linear CD19-mRNA group targets Raji cellsDid not exhibitKilling ability. Meanwhile, during the process of killing Nalm-6 and Raji cells, the CD19-circRNA group showed greater efficacy than the linear CD19-mRNA group.Induce higher levels of cytokines（IFN‑ γ/TNF‑α/IL2）。
In general,Circular mRNA-based anti-CD19 CAR T cells significantly and specifically kill CD19-positive target cells in vitro, with cytotoxic activity significantly higher than that of linear mRNA-based CAR-T cells.
Dynamic Changes of CAR-T in Mice
After injecting CAR-T cells into the tail vein of mice, the linear CD19-mRNA group and the CD19-circRNA groupCAR-T cells were detected in the peripheral blood on both Day 1 and Day 3., wherein the proportion of CAR-T cells detected in the CD19-circRNA group was approximately85％(Day 1)And about 55%(Day 3), significantly higher than the linear CD19-mRNA group(Day 1 is approximately 42%, Day 3 is approximately 20%)On the 5th day, the level of CAR-T cells detected in the peripheral blood was comparable to that of the blank control group.
The research results indicate that the baseCAR-T cells based on CD19-circRNA exhibited significantly longer survival in mice compared to CAR-T cells based on linear CD19-mRNA.
CAT's Specific Cytotoxic Activity in Mice
Divide the Nal-6-Luc mouse models with the same fluorescence intensity into three groups:UTD Blank Control Group, Low-Dose Cyclical Group, High-Dose Cyclical Group. The treatment regimen was as follows: UTD group, each mouse was injected with 3×10^6 activated but non-transfected T cells with CD19-CAR mRNA; low-dose group, each mouse was injected with 1×106 A cirRNA‑CD19 CAR‑T cell, high-dose group each mouse was injected with3×10^6cirRNA‑CD19 CAR‑T cells. CAR‑T cells were administered every 4 days.Intravenous InjectionAdministration, a total of three administrations(The dosing times were on the 3rd, 6th, and 9th days after inoculation with Nal-6-Luc cells.)。
After the first dose treatment, the fluorescence signal of Nalm-6-luc cells in the high-dose group was significantly reduced compared to the control group. After all three treatments were completed, in vivo fluorescence imaging on days 10, 17, 24, and 31 showed a significant reduction of Nalm-6-luc cells in the CAR-T treated mice, with no detectable fluorescence signal in several mice.Indicating that the Nalm-6-luc cells in these mice have been completely eliminated.. The imaging results on Day 31 showed that, despite the time elapsed since the last dose(Day 9)It has been 22 days, and cirRNA-CD19 CAR-T cells can still significantly eliminate Nalm-6-luc cells in mice without recurrence of CD19 target cells.
The experimental results demonstrate that,CAR-T cells prepared based on scarless circular RNA survive longer in mice, significantly kill and eliminate target cells, maintain the killing and elimination effects for a long time, and no regrowth of target cells is observed in mice.(Recurrence)。
In Vivo Cytotoxic Activity of Linear and Circular CAR-T Groups
Similarly, Nal-6-Luc mice models with the same fluorescence intensity were divided into three groups: UTD control group, linear group, and circular group. The treatment regimen consisted of injecting each mouse in the UTD group with 3×10^6 T cells activated but not transfected with CD19-CAR mRNA.Linear GroupEach mouse was injected with linear3×106CD19-mRNA CAR-T Cells,High-Dose Cyclical GroupEach mouse was injected with 3×10^6 cirRNA-CD19 CAR-T cells. Subsequently, CAR-T cells were intravenously administered once every 5 days for a total of four administrations.(Dosing on Day 3, Day 8, Day 13, and Day 18)。
Compared with the UTD control group,The linear group significantly inhibited the fluorescence signal of Nalm-6-luc cells, but no clearance of the fluorescence signal in Nalm-6-luc cells was observed.; The circular group significantly inhibited the fluorescence signal of Nalm-6-luc cells, and after the 14th day, it achieved clearance of the fluorescence signal of Nalm-6-luc cells. By the end of the experiment, no re-emergence of Nalm-6-luc cells was observed in the circular group.(Recurrence)。
The survival time analysis results of mice showed that,Circular group mice at the end of the experiment(Day 40 after Nalm-6-luc cell inoculation)，None of the mice died, while all the mice in the UTD group and the linear group died. The survival rate of the circular group was significantly higher than that of the linear group.
InDay 12, take miceDetection of CAR-T Cell Proportion in Peripheral Blood, the proportion of CAR-T cells in the linear group's peripheral blood is approximately18％, while the proportion of CAR-T cells in the peripheral blood of mice in the circular group was approximately40％，significantly higher than the linear group. In addition, it alsoDay 20TakeDetection of Memory T Cell Proportion in Peripheral Blood, Using CD45RA and CD62L double positivity as markers, the proportion of memory T cells in the peripheral blood of mice in the linear group was detected to be about 5%, while the proportion of memory T cells in the peripheral blood of mice in the circular group was about 33%, significantly higher than that in the linear group.
In Vivo CAR-T Platform Based on Seamless Circular RNA
Preparation of T Cell-Targeting Lipid Nanoparticles
CD5-tLNP Transfection Effect on T Cells
CD5-tLNP encapsulates Luc-transfected T cells,The transfection efficiency can reach 50%~60%.
CD5-tLNP Delivers CD19-circRNA to Produce CAR-T In Vivo
Compared with the control group, the fluorescence intensity of Nalm-6-luc cells in mice was significantly reduced in the tLNP-CD19-CAR-circRNA administration group, demonstrating thattLNP‑CD19‑CAR‑cmRNA can exert cytotoxic activity against CD19-positive target cells in mice.
Summary
Byterna Therapeutics has developed in vitro and in vivo CAR-T platforms based on seamless circular RNA. Compared to linear mRNA, circular RNA encoding CAR can achieve more persistent and higher-density expression. Correspondingly, T cells based on circular circRNA-CAR exhibit more enduring cytotoxic activity. Byterna Therapeutics uses electroporation to prepare circular RNA-based CAR-T cells in vitro, which, when injected into a mouse model of hematologic tumors, can completely inhibit tumor growth and maintain a tumor-free signal over the long term. To establish an in vivo CAR-T platform, Byterna Therapeutics constructed a lipid nanoparticle delivery system conjugated with CD5 antibody molecules, enabling specific targeting of T cells and significantly suppressing tumor growth in mice.According to the recent financing announcement of Byterna Therapeutics, it will focus on the in vivo CAR-T platform based on circular RNA in the future, and achieve breakthrough results in two directions: circRNA production technology and targeted antibody-conjugated LNP technology.
References
Method and Use of Cell Engineering Modification with Scarless Circular mRNA, Application Publication Number 119662682 A.
