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Electroporation for Non-viral Gene Delivery

Any Cell. Any Molecule. Any Scale. ®

The ExPERT family of MaxCyte Instruments

Rapid : 최대 2x10¹⁰ cell 에 30분 내로 bulk transfection 가능
Scalable : Small scale electroporation 을 통한 조건 Optimization 후 efficiency와 cell viability에 영향을 미치지 않고 동일한 조건으로 Large scale electroporation 가능

Instrument
Touch Screen ○ ○ ○
LED Indicator ○ ○ ○
Static EP (up to 7x10⁸ cells) ○ ○ ○
Flow EP (up to 2x10¹⁰ cells) ○ ○
Barcode Reader ○
21 CFR Enabled ○
FDA Master File ○
cGMP ○
Recommended Application Initial Stage of Research Stable cell line
Protein production		 Cell therapy

치료제 개발 방법에 따른 임상단계 진입까지 소요되는 시간
• GMP Viral Process: 약 2년 (제조까지 12-18개월 대기 + 안전성 평가 6-9개월 소요)
• Non-Viral Process with MaxCyte technologies: 1년 이내

Field Application Service of MaxCyte®
• 다양한 연구 분야에 대한 수년간의 경험을 바탕으로 최적의 실험 프로토콜 및 맞춤형 솔루션을 제공

Stable Cell Line Development

  1. Bulk transfection using commonly used cells (e.g. CHO cells, HEK cells)
  2. Co-transfection with Reporters, Targets and Accessory plasmids
  3. 활용 예:
    • Luciferase-Based Reporter Assay
    • Screening GPCRs
    • Screening Voltage-Gated Ion Channels

Stem Cell Engineering

ico_chk01  Gene Knock-in in Human iPSCs
• 타사 electroporator 대비, 높은 Knock-in efficiency 확인

 

ico_chk01  Non-Viral Engineering of Human iPSCs 
• Stable integration of the transgene: 실험 후 14일째까지 높은 expression 유지
• 낮은 effector:target ratio (2.5:1) 에도 50% 이상의 cytolytic activity 관찰

Gene Editing

ico_chk01  CRISPR Workflow for Drug Discovery in T Cells
▶ SLICE (sgRNA lentiviral infection with Cas9 protein electroporation) screening workflow

• 4 days post EP, 80% 이상의 Efficiency 확인
• 타사 electroporator 대비, 높은 수준의 Cell viability 관찰

 

ico_chk01  Large-Scale Engineering of Extracellular Nanoparticles
▶ NanoMEDIC: The engineered extracellular nanovesicle containing a CRISPR-Cas9 RNP complex

 

• NanoMEDIC 을 형성하는 anchoring protein과 Cas9 protein Co-transfection
• CRISPR/Cas9 을 포함하는 Extracellular vesicle을 세포 & animal model에 주입

 

ico_chk01  HIV-1 Vaccine
▶ Enhancing Efficacy Through CHO Cell CRISPR Engineering

• CRISPR engineering 을 통한 CHO MGAT1- cell line 구축
• Transfection transfection을 통한 Recombinant gp120 production
→ 99% 이상의 oligomannose glycan을 포함하는 rgp120 protein
→ bN-mAbs 결합력 증가

 

▶ Overcoming Viral Vector Toxicity: SB-728 mRNA for CCR5 disruption

• CCR5: HIV-1 감염을 일으키는 coreceptor
따라서, CCR5 발현 억제는 HIV-1 에 대한 저항성을 높일 수 있음

• > 90% viabilities
• ~ 73% bi-allelic gene disruption

Cell Therapy

ico_chk01  T cell engineering: TIL, CAR-T

• > 80% Viable cells expressing reporter gene
• Maintenance of CD4:CD8 ratio & T cell proliferation

 

• > 80% Viable cells expressing reporter gene
• Cells electroporated using small-scale versus large-scale MaxCyte electroporation had similar gene disruption frequencies
• PD-1 gene disruption resulted in a 75% reduction of PD-1 expression on CD3+ TIL following anti-CD3/CD28 bead stimulation

 

ico_chk01  NK cell engineering: CAR-NK

1) Viral transduction for CAR-NK generation: Cytotoxicity against B-cell malignancies.
2) Non-viral engineering using MaxCyte technology
• Significant improvements in efficiency and viability
• Clinically-feasible, FDA-approved avenue to overcome manufacturing challenges

 

• >80% Efficiency & >85% Viability Using mRNA Electroporation with MaxCyte
• Significant Enhancement of In Vitro CD19-specific Effector Functions
• Rapid and Clinical-scale Manufacturing compared to retroviral infection