GBA GT Executive summary cassette selection, 3m-mice ICV screen, D409V 7-group selection, TE / PRC1 / Bettina, PD mouse model 4L/PS
STIG1 — cassette selection (criterion: GBA transgene expression ≥ 1× endogenous GBA)
- Wild-type (B7) iPSC-derived DA neurons
- B7-GBA L444P/L444P iPSC-derived DA neurons (optional)
기준: GBA transgene expression ≥ 1× endogenous GBA), 4 cassettes were selected
| In vitro | 기준: ↑ endogenous GBA in iPSC-L444P DA | 기준: GBA transgene expression ≥ 1× endogenous GBA UCU on iPSC L444P DA | 기준: > 40% GlcSph reduction | D409V homo mice AAV Injection ICV |
|---|
Results 20201119: VC010, PD010, PD011, 013 세개가 80% 이상 GBA expression, GlcSph 3개가 80% 이상 reduction 보임, control 은 L444P mock → Select 1-2 lead expression cassette
- In PFF-treated mouse primary neurons (GBA 무관히 PD Model 임): AAV9-29-aSyn, GBA1 → ↓P(129)-aSyn
3m mice ICV experiment (3 groups)
| 군 | 동물 | AAV |
|---|---|---|
| 1 | GD mice | rAAV9-CBh-GFP |
| 2 | GD mice | rAAV5-CBh-GFP |
| 3 (Ctl) | GD mice | No treatment |
3.6 × 10? vg/injection site, bilateral, icv → after 3w, kill
Results: AAV5 signal was faint. ↓ (~50%) GBA activity. Readout: IHC, vg, GBA mRNA (but little distribution in striatum).
7-group D409V homozygous cassette-selection study (rAAV9, ICV)
| # | Animal | AAV | Dose | N |
|---|---|---|---|---|
| 1 | WT | Sham | 8 | |
| 2 | GD mice | Sham | 8 | |
| 3 | GD mice | AAV9-VC006 | 3.30E+10 vg/inj. site | 8 |
| 4 | GD mice | AAV9-PD010 | 3.30E+10 vg/inj. site | 8 |
| 5 | GD mice | AAV9-PD011 | 3.30E+10 vg/inj. site | 8 |
| 6 | GD mice | AAV9-PD012 | 4.80E+9 vg/inj. site | 8 |
| 7 | GD mice | AAV9-PD013 | 3.30E+10 vg/inj. site | 8 |
will start early in Oct. 3m D409V homoz mice, AAV9 으로 할거. icv, bilatrally, after 28 days kill → readout: vg, GBA Mrna, GBA protein, gba activity → Selection of 1 expression cassette
Result: 20201119: PD010, 013, ↑(×3) GBA, 다음은 GlcSph 결과겠네 (40% 이상 줄이러나?)!
right-side criteria: No apparent safety issues. For manufacturing, a feasibility study must demonstrate overall manufacturability with similar yields to AAV9.
MOI definition
MOI: the number of infected viral particles per cell, Plaque forming units (pfu) of virus used for infection / number of cells.
Final PRC1 submission
- 20210325_PRC_Narrative_GBA_PD_GT_final.docx
- 20210325_PPT_PRC_slide deck_GBA-PD GT_submission.pptx
TE: Transgene product definition
| delivery | expression | activity | Interaction | |
|---|---|---|---|---|
| FDA guidance (2020 neurod) | the amount of transgene product expressed in the targeted tissue protein that is expressed by a GT product → -expression and function of the gene expression product | Activity of a GT product, GT product activity, bioactivity, biological activity, functional activity | ||
| EMEA (2018) | transgene product (e.g. expressed proteins or genomic signatures). |
question: AAV, Transgene 이 product 인가? mRNA, protein 도 product 인가? 위 예 보면 guide document 들 보고, rna/protein 은 뭘로 칭하는지 파악하자. Endpoint 쪽 보자, prc 문서 소제목을 아예 바꾸자. 다른 더 general 한 fda guidance 보자, Transgene product 는 a foreign protein 만을 지칭함.
In addition to that I would like to share high level schedule toward PRC1: GBA PD gene therapy Portfolio entry schedule 20210106.pptx (#3 slide). This is a link to the folder: PRC-PE preparation
Target engagement markers for gene therapy can be viewed as markers that demonstrate in vivo target gene expression (ie GBA PET and fluid biomarkers that reflect the change in the amount of target gene expression (ie GBA mRNA and protein) and the activity (ie GBA activity and the lipid substrate levels).
GBA protein assay (Meeting with Bettina 20201021)
(this is in outlook’s > GBA>RDTM)
| Reactivity | RD DDU’s antibody: recognize (wt 당연이겠지), D409 (but not mouse, js: but should be ok as we will look at transgene expression), does not recognize mouse gba. L444P 는 인식여부 언급 기억 안 남. 웬지 (GBA 가 이겠지?) CSF 에서 aggregate 되더라. Js: mouse 는 mutation 여부 상관없이 미인식이므로 어차피 우리는 transgene 만 measure |
| Availability | Old lot: not much left. New lot: test by mid Nov → pass along the information to us → will be sharing with us (we have to let her know how much we need!) |
GD mouse model — GbaD409V/D409V KI homo
accumulation of GlcSph is rapid and we can assess our constructs in one month
PE schedule (4L/PS, CBE+A53T, CBE+Line 83)
| PE | ICV | CBE | aSyn | ||
|---|---|---|---|---|---|
| 4L/PS | 3-4w | NA | Zhu, Y.H. et al., Mol. Gen. Metab., 2011 at 10-12 wk, Large α-syn particles (≥5 μm) were observed in cerebral cortex. | ||
| CBE+A53T G | Kuo, Y.M. et al., Hum. Mol. Gen., 2010. At 6w ↑ aSyn 9-10w, preclinical 은 확실히 아닌데 prodromal 인지 Symptomatic 인지 불명. 11-12w | 11-12w | |||
| CBE+Line 83 |
PD mouse model (4L/PS mice), young mice (4w 에 weaning)
| Group | N Number | Mice | Material | Route |
|---|---|---|---|---|
| 1 | 12 | 4L/PS(+/+)-NA | Sham (buffer) | i.c.v. |
| 2 | 12 | 4L/PS(-/-)-NA | Sham (buffer) | i.c.v. |
| 3 | 12 | 4L/PS(-/-)-NA | AAV-PD010 | i.c.v. |
| 4 | 12 | 4L/PS(-/-)-NA | AAV-PD013 | i.c.v. |
Beam walk test, rotarod, wire suspension. 14w 에 kill, CSF collection (js: serial sampling 가능하구나!).
D409V/D409V KI homo + GBA GT
| field | content |
|---|---|
| material | We are going to test two prioritized cassettes in candidate AAV serotypes. (prc1) |
| AAV | PD010, PD011, PD012, PD013 (rAAV9) |
| Readout | AAV genome, GBA protein expression, GBA activity, and glucosylsphingosine (GlcSph) |
| CSF schedule | Baseline CSF will be collected before study start (day -5) (maybe canceled). 8 weeks after treatment: (~3 μL) (maybe canceled). 18 weeks after treatment (~3 μL), before necropsy (see 5.6). Protein (left cortex and hippocampus) - GCase (ELISA or GCase activity assay) - alpha-Syn (soluble vs insoluble, monomer vs HMW) - phospho-alpha-Syn - Iba1/Gfap (for neuroinflammation) |
| Criteria | - GBA protein expression level in mouse brain: e.g. restoration to at least WT level or above; preferred levels to be 2-5× above normal expression - Reduction of GlcSph (preferred levels to be comparable to WT mouse level) |
| Goal | Select 1 lead expression cassette - Obtain in vivo POC that GBA gene therapy would show efficacy on GBA-PD patients - Estimate the needed expression levels to show the efficacy in patients |
Uncertain Spans
| location | transcription | uncertainty |
|---|---|---|
| 3m mice ICV / dose | 3.6 × 10<sup>?</sup> vg/injection site | the exponent on the 10 is too small to read on this capture; preserved verbatim with ?. |
| TE / FDA expression cell | the row pairs the amount of transgene product expressed in the targeted tissue with protein that is expressed by a GT product → -expression and function of the gene expression product | the source visually splits this across two adjacent cells; preserved as a single FDA guidance cell with both clauses. |