Therapeutic IVT RNA 개발
VectorBuilder는 백신, 유전자 대체, CAR (chimeric antigen receptor) 및 유전자 편집을 포함하여 RNA 치료제 개발을 위한 포괄적인 CRO 서비스를 제공합니다. VectorBuilder 전문가팀은 치료용 디자인 및 공정 개발에 대한 주요 고려사항을 이해하고, VectorBuilder의 노하우를 활용하여 RNA 치료제의 효능, 안전성 및 제조 가능성을 향상시킬 수 있습니다. RNA 치료제의 대량 생산을 원하시면 VectorBuilder의 CDMO 서비스를 확인해 보세요.
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IVT Vector
Design & Cloning
Design & Cloning
IVT RNA Production
LNP Encapsulation
Quality Control (QC)
Functional Validation
IVT 벡터 디자인 & 클로닝
- 최적의 발현을 위한 독자적인 서열 최적화
- 120 nt (and longer) polyA tail의 강력한 클로닝 및 전사
- UTR optimization
- UTR engineering
- Coding sequence optimization
- PolyA integrity
- PolyA length and GOI expression
Figure 1. UTR optimization for improved mRNA expression. Different 5' and 3' UTR combinations were tested regulating Gaussia luciferase expression in vitro. HEK293T cells were seeded on 12-well plates at a density of 2.3x105 cells per well. Cells were transfected with 1 ug of mRNA per well. At 6 h, 24 h, 48 h, 72 h, and 96 h post-transfection, Gaussia luciferase activities were measured from cell culture medium.
Figure 2. Incorporation of a miR-122 targeting site into the 3’ UTR reduces liver-specific expression of IVT mRNA. Mice were i.v. injected with 10 ug of LNP-mRNA coding for HiExpressTM Firefly Luciferase, with or without a miR-122 targeting site in the 3’ UTR. Two different configurations with different positions of the miR-122 targeting site were tested (design A + B). 6 hours post-injection mice were sacrificed, organs were dissected, and luminescence was measured. miR-122 is highly and specifically expressed in the liver and the inclusion of the miR-122 targeting site led to reduced IVT mRNA expression in liver tissue.
Figure 3. Codon optimization increases mRNA expression in vitro and in vivo. (A) Expression of HiExpress™ Firefly Luciferase mRNA and other luciferase mRNA in HEK293T cells. Cells grown on a 12-well plate were transfected with 0.5 ug of mRNA per well and luciferase activity was measured at 6 h, 24 h, 48 h, and 72 h post-transfection. (B) Luciferase activity measured in adult C57BL/6 mice injected intramuscularly with 30 ug of LNP-mRNA at 6 h, 24 h, and 48 h post-injection. FLuc WT indicates wild-type firefly luciferase. FLuc WT (co) indicates codon-optimized wild-type firefly luciferase. FLuc2 indicates Luc2 firefly luciferase.
Figure 4. PolyA tail size analysis. PolyA tails were cleaved from IVT mRNA using ribonuclease T1 and isolated by oligo dT affinity chromatography. (A) Isolated polyA tails analyzed by Urea-PAGE gel electrophoresis. (B) Isolated polyA tails analyzed by LC-MS. Deconvoluted spectrum was generated from 120 pmol of polyA tails with an expected size of 120 nt. (C) Size distribution of polyA tails with an expected size of 120 nt. Error bars represent standard deviation from triplicates. Weighted average length is 123 nt.
Figure 5. The influence of polyA length and structure on translational efficiency. HEK293T cells were transfected with IVT EGFP mRNA with the same Cap1 and UTRs but different polyA tail lengths as listed above.
IVT RNA 생산
- 벡터 클로닝부터 LNP encapsulation 까지 최단 5주 소요
- ug에서 gram 스케일로 최대 10,000 nt mRNA 및 saRNA, 5,000 nt circRNA 합성
- Co-transcription 또는 enzyme method를 통한 높은 capping 효율 (최대 99%)
- 다양한 변형 뉴클레오티드 옵션: m1Ψ, m5C, 5moU 등
- 불순물을 효율적으로 제거하는 독자적인 정제 공정
- 종합적인 QC 패널
- 대량 IVT RNA 제조를 위해 생산시간을 단축하는 cell-free IVT template DNA 생산 제공
- mRNA integrity
- Capping efficiency
- Modified nucleotide
- dsRNA removal
Figure 6. Denaturing agarose gel result indicating high integrity IVT RNA >10,000 nt.
Figure 7. IVT mRNA capping efficiency analyzed by LC-MS. Highly efficient capping (>99%) can be achieved either using (A) co-transcriptional or (B) enzymatic approaches.
Figure 8. Modified nucleotides increase mRNA expression and decrease dsRNA impurities. (A) Expression of firefly luciferase in HEK293T cells. mRNA was generated with or without modified nucleotides, N1-Methylpseudouridine (m1Ψ) and 5-Methylcytosine (m5C). Cells were grown on 12-well plates and transfected with 1 ug of mRNA per well. Luciferase activities in HEK293T cells at 6 h, 24 h, and 48 h post-transfection were measured. Error bars indicate standard deviations. (B) Equal amounts (750 ng per dot) of magnetic bead-purified EGFP IVT mRNA with or without nucleotide modification (m1Ψ) was assessed by dot blot assay for dsRNA impurities.
Double-stranded RNA (dsRNA) is a by-product of IVT and a major trigger of undesired immunogenicity. The dot blot result demonstrates that extra purification steps (e.g. IP-PR) may be necessary to achieve an ultra-purification scale with very low levels of dsRNA.
Figure 9. dsRNA removal efficiency of different purification processes. Equal amounts (1500 ng per dot) of hSpCas9 IVT mRNA purified by different processes was assessed by dot blot assay to estimate dsRNA impurities. Abbreviations: HIC, Hydrophobic interaction chromatography; IP-RP, Ion-pair reversed-phase liquid chromatography.
LNP Encapsulation
자세한 내용은 LNP Encapsulation 서비스 페이지를 참조하세요.
품질관리 (QC)
VectorBuilder는 IVT RNA에 대한 종합적인 QC 분석을 제공합니다. 기본 QC 항목 (√ 표시)은 항상 수행되며, 옵션 QC 항목은 개별 프로젝트 요구사항에 따라 수행됩니다.
Attribute | QC assay | Research-grade | GMP-like | |
---|---|---|---|---|
Identity | mRNA sequence | Sanger sequencing | √ | √ |
mRNA length | Denaturing agarose gel electrophoresis | √ | √ | |
Capillary gel electrophoresis (CGE) | Optional | √ | ||
General/physical property | mRNA concentration | UV spectrophotometry | √ | √ |
RiboGreen assay | Optional | √ | ||
Appearance | Visual inspection | Optional | √ | |
Potency | Gene expression | In vitro translation followed by Western blot | Optional | Optional |
Cell transfection | Optional | Optional | ||
Safety | Sterility | Bioburden test | Optional | √ |
Mycoplasma | Culture method | Optional | √ | |
qPCR | Optional | Optional | ||
Endotoxin | Kinetic chromogenic assay (KCA) | Optional | √ | |
Purity | mRNA integrity | Denaturing agarose gel electrophoresis | √ | √ |
Capillary gel electrophoresis (CGE) | Optional | √ | ||
A260/280 | UV spectrophotometry | √ | √ | |
Capping efficiency | LC-MS | Optional | √ | |
PolyA analysis | LC-MS | Optional | √ | |
Residual dsRNA | Dot blot assay | Optional | √ | |
Residual plasmid DNA | qPCR | Optional | √ | |
Residual protein | NanoOrange assay | Optional | √ | |
Residual solvents | Gas chromatography | Optional | Optional | |
Circularization efficiency (for circRNA) | Denaturing agarose gel electrophoresis | √ | Optional | |
Capillary gel electrophoresis (CGE) | Optional | √ |
기능 검증
VectorBuilder의 과학자 및 기술팀은 다음을 포함하여 치료 목적에 맞게 IVT RNA 디자인을 최적화하기 위한 기능적 연구를 통해 서포트할 수 있습니다.
- in vitro 및 in vivo에서 다양한 벡터 구성요소 (UTR, coding sequence, polyA, Kozak등)의 서열 최적화 효과 평가
- Antigen presentation, 항체 발현, CAR 발현, CRISPR 등 다양한 응용 분야에 대한 IVT RNA 테스트
- 설치류 및 NHP를 포함한 동물 모델을 사용하여 LNP 유전자 전달 효능 및 안전성 평가