Assesment of Investigational Cellular and Gene Therapy Products (2013) 안전성 평가의 일환으로 백터에 대한 반응이 있는 permissiveness/susceptibility한 동물모델에서의 개념증명(POC, proof-of-concept) 시험 또는 독성시험에서 bio-distribution 및 shedding 에 대한 평가를 실시하도록 하고
| transport | ||
| Target engagement | How much? | How long? |
| biodistribution | 특히 생식상기 | |
| shedding | 제 3 자 감염 | |
- Vector
- What’s the capacity (ie the amount of DNA a vector can carry)?
- Entry into cells
- AAV 의 표면의 a single protein 이 cell-surface molecule (eg. Heparin sulfate)에 bind → endosome 능에 싸여서 cell 내 들어가기도
- Immune
- In recent human trials, CD8+ immune cells have recognized the AAV infected cells as compromised and killed these cells accordingly. This action appears to be triggered by part of the capsid or outer coat of the type 2 virus
Immunosupressnats
| IS in Intrathecal / Intracisternal AAV Programs | |
|---|---|
| AAVRH10-miRNA-SOD1 ALS EA |
AAVRH10-miRNA-SOD1, intrathecal. Patient #1 22yo July 2017 dosing. Patient #2 56yo Sept 2017 dosing 4.2e14 vgs Patient #1: week 4 post-dosing developed pain syndrome and liver inflammation. Week 10 Sural n. sensory AP absent. Week 16. MRI revealed some enhancing DRGs (responsive to rescue IS). On pathology, DRG neuron loss evident. Patient #2 initiation of triple IS (Rituximab, Prednisone, Sirolimus) prevents immune-mediated AEs |
| NINDS/Taysha GAN |
scAAV9/JeT-GAN, intrathecal dosing. Age: 5yo to 99yo IS: IV methylprednisone x2 weeks starting D-3-2. Prednisone x4 months after dosing. 1 month taper. Pulse sirolimus D-9-2. maintenance sirolimus to month 12. 1 month taper. Additionally for CRIM- patients tacrolimus D0 to month 6. 1 month taper Asymptomatic CSF pleocytosis in all patients, responsive to IS, resolving by M12 in majority |
| Lacerta Late-Onset Pompe |
AAV9 Intramuscular dosing with re-administration 4-months after 1°. 4.6e13 vg per TA muscle. Age: 18yo to 50yo. Study Initiated 2014. Evaluating re-administration of AAV9 IS: Rituximab (750 mg/m²) d-21 and d-7, 375 mg/m² @D, 375 mg/m² d-7 to 2nd dose) + Sirolimus (target trough 2-4 ng/mL. d-7 to M4 after 2nd dose (8 months total) |
| Novartis/AveXis SMA2 |
Zolgensma, AAV9 Intrathecal dosing. 6.3e13 vg, 1.2e14 vg, 2.4e14 vg. Age: 6mo to 5yo October 2019. Partial hold (high dose, low and mid dose enrollment complete) for animal findings showing DRG mononuclear infiltrate, sometimes accompanied by neuronal loss. Hold lifted 2021 IS Prednisolone |
| One prophylactic IS approach does not fit all and should be tailored depending on RoA, gene therapy, patient age, CRIM status and indication, etc. | |
Life-cycle
Figure 1. AAV genome map. (A) Capsid crystal structure. (B) AAV gene map. The positions of the three promoters (p5, p19, p40) as well as the seven protein coding regions of the AAV have been highlighted.
| Mrna 생성 | Protein 생성 | ||
|---|---|---|---|
| administration | AAVs do not replicate in the cytosol! multiplication 은 없다. Ie 넣어준 vg 그대로 1:1로 만들어지는 것. | An adenovirus introduces the DNA into the nucleus of the cell, but the DNA is not integrated into a chromosome. | |
| where | In nucleus | In cytosol | |
| AAVs typically take between 2 and 4 wk to show noticeable expression in vivo (32). This is due to AAVs being nonintegrating and requiring time to replicate (이것 이상함, AAVs don't' replicate!) in the cytosol before sufficient expression levels are observable (33). |
Localization
- Shwann cells only?
- 몇 %의 cell이 transduced?
- Voretigene: ?
- CMT1X in Shy: >50% of all Schwann cells in sciatic nerve (4552–4554 | PNAS | April 26, 2016 | vol. 113 | no. 17)
- 몇 %의 cell이 transduced?
- Nerve?
- Biodistribution
- Clinical
- transient and low levels of vector DNA were detected in tear and occasional serum samples.
- Clinical
- Vector가 전신적으로 분포해도 괜찮나?
- 전신분포 사례
Manufacturing
에 그림 Spark Therapeutics Briefing Document: October 12, 2017 FDA Advisory Committee Meeting
local vs systemic
- leg만 교정한다해도, 결국 전신에 발병할텐데?
| Plasmid construction | ||
|---|---|---|
| AAV packaging (=? Transfection) | human embryonic kidney 293 cells (HEK293) or HEK293-T, HeLa producer cell lines, HeLaS3 cell line, Baculovirus production system (Glybera), HERPES SIMPLEX TYPE I SYSTEM, AAV-293 cells | AAV vector components and therapeutic gene are transfected, usually as separate expression cassettes from different plasmids→ Expression cassettes are expressed within the cell resulting in viral proteins and a genomic ssDNA containing the expression cassette for the therapeutic gene(s). In the AAV system, viral particles containing the transgene assemble in the cytoplasm |
| Harvesting AAV particles | freeze/thaw the cell pellet to release the AAV virus. | |
| AAV purification | ||
| AAV titer detection | ||
| Quality control of AAV |
| (transgene product) ②Dose | Dose | PK | PD | Efficacy | |||||
|---|---|---|---|---|---|---|---|---|---|
| VG | mRNA | protein | Protein's activity | Substrate (eg GlcCer) | lysosome | Asyn | |||
| Drug = Vector = Capsid +VG | |||||||||
| Affected by | Capsid | − | Distribution → cell | − | Transcription rate | • | Translation | − | − |
Uncertain Spans
- “특히 생식상기” — biodistribution 행 셀, 가운데 글자가 시각적으로 ‘상’ / ‘장’ 사이에서 모호함. ‘생식기/생식 장기’ 도메인 의미일 가능성이 있으나 글자 그대로 전사.
- “에 그림 Spark Therapeutics Briefing Document…” — Manufacturing 섹션 첫 줄. 앞쪽에 추가 단어가 있었을 가능성 있으나 행 시작이 한글 ‘에 그림’으로 보여 그대로 전사.
- 하단 PK/PD 매트릭스 표의 ‘Affected by’ 행에서 ’−’ 와 ’•’ 마커가 셀 위치마다 확실하지 않음. 시각적으로 가까운 셀에 매핑.
- 하단 PK/PD 매트릭스 표 column 정렬 — ‘Distribution → cell’ 가 mRNA column 인지 protein column 인지, 그리고 ‘Transcription rate’가 어떤 PD column 인지 photo 하단 잘림으로 완전히 확정 불가. ‘Drug = Vector = Capsid +VG’ row label 의 row 위치도 column-aligned 하지 않을 수 있음.