SAREPTA THERAPEUTICS, INC. In Situ Biodistribution and Localization of
Bidridistrogene Xeboparvovec (SRP-9003)
in LGMD2E/R4 Mice
Young Seo, Stephen Fasul, Chrissy Hopkins, Akansha Pradhan, Jasmine Wu, Alex Haile, Rachael Potter, Sarah Lewis, Stephen Baine, Louise Rodino-Klapac
Sarepta Therapeutics, Inc., Cambridge, MA
Presented at the 2024 American Society of Gene & Cell Therapy (ASGCT) 27th Annual Meeting Baltimore, MD, May 7-11, 2024
Disclosures
This study was funded by Sarepta Therapeutics, Inc., and all authors are employees of Sarepta Therapeutics, Inc., and may own stock/options in the company.
Limb-girdle muscular dystrophy 2E/R4
• LGMD2E/R4 is caused by mutations in the |
SGCB gene |
α2 | Laminin 2 | |
β1 | Υ1 | |
Sarcoglycan | ||
complex | αDG | Dystroglycans |
Extracellular matrix
- Mutations cause loss of SGCB protein |
and sarcoglycan components of the |
DAPC |
• In patients, the loss of functional SGCB |
Sarcolemmaδαγ
Syn
β
CT
βDG
CR
DYSFANO5
protein leads to progressive muscle |
degeneration and shortened lifespan1-3 |
Dystrophin
F-actin
Image adapted from Fairclough RJ, et al. Nat Rev Genet. 2013;14(6):373-8 and Zhao J, et al. Hum Mol Genet. 2016;25(17):3647-53.
ANO5=anoctamin 5; CR=cysteine-rich domain; CT=c-terminus;DAPC=dystrophin-associated protein complex; DG=dystroglycan; DYSF=dysferlin; LGMD2E/R4=limb-girdle muscular dystrophy type 2E; SGCB=β-sarcoglycan; Syn=syntrophin. 1. https://rarediseases.info.nih.gov/diseases/6907/limb-girdle-muscular-dystrophy [Accessed Feb 5, 2024]. 2. Murphy AP, Straub V. J Neuromuscul Dis. 2015;22:S7-19.3. Bouchard C, Tremblay JP. J Clin Med. 2023;12:4769.
SRP-9003 (bidridistrogene xeboparvovec)
SRP-9003 directly addresses the root cause of LGMD2E/R4 (SGCB)1,2 | rAAVrh74 |
MHCK7 PROMOTER | INTRON | hSGCB cDNA | pA |
ITR | ITR | ||
Optimized for expression in | Full-length hSGCB | Efficient skeletal and cardiac | |
skeletal and cardiac muscle3-5 | gene transfer | muscle transduction6 |
cDNA=complementary DNA; hSGCB=human β-sarcoglycan; ITR=inverted terminal repeat; LGMD2E/R4=limb-girdle muscular dystrophy type 2E; MHCK7=myosin heavy chain enhancer promoter; rAAVrh74=recombinant adeno-associated virus serotype rh74; pA=polyadenylation; SGCB=β-sarcoglycan.
- McNally EM. The Sarcoglycans. Austin, TX: Landes Bioscience; 2000-2013.2. Gao Q, McNally EM. Compr Physiol. 2015;5:1223-39.3. Salva MZ, et al. Mol Ther. 2007;15:320-29.4. Pozsgai ER, et al. Mol Ther. 2017;25:855-69.5. Wang B, et al. Gene Ther. 2008;15:1489-99.
- Chicoine LG, et al. Mol Ther. 2014;22:338-47.
Study design and objective
For IM administration, necropsy timepoints were D1, D7, D14, D21, and D28
- Objective: POC for RNAscopeTM assay
- SRP-9003dose: 3×1010 total vg
For IV evaluation, necropsy timepoints were D1, D14, D28, D60, D120, and D365a
- Objective: Durability, spatial distribution
- SRP-9003dose: 7.41×1013 vg/kg
SRP-9003
Bidridistrogene xeboparvovec
28-day POC
D1 D7 D14 D21 D28
Intramuscular (IM)
1-year durability
Intravenous | D1 | D14 | D28 | D60 | D120 | D365 |
(IV)
Necropsy day (D)
Objective: Determine spatial distribution of SRP-9003 in situ
aD365 data have not been reached.
D=day; IM=intramuscular; IV=intravenous; POC=proof of concept; vg=vector genomes.
RESULTS
SRP-9003 - IM Administration
RESULTS
SRP-9003 biodistribution: transduction confirmation (IM)
In TA muscle, SRP-9003 vector DNA peaked on D1 and stabilized through D28 following IM SRP-9003 administration
SRP-9003Vector DNALevels (ddPCR)
40 | |
Nucleus | 30 |
20 | |
per | 10 |
7 | |
Copies | 6 |
3 | |
5 | |
4 |
2
1
0
1 | 7 | 14 | 21 | 28 | WT 28 SGCB |
-/- | |||||
Days | Saline Control |
In TA muscle, mRNA levels were maximized on D14 and stabilized through D28 following IM SRP-9003 administration
SRP-9003 hSGCB mRNA Levels (RT-ddPCR)
50 | |
AP3D1 | 40 |
30 | |
20 | |
to | 20 |
SGCB | 15 |
Ratio | 10 |
5 | |
0 |
1 | 7 | 14 | 21 | 28 | WT 28 SGCB |
-/- | |||||
Days | Saline Control |
Note: error bars represent standard error of the mean.
AP3D1=adaptor-related protein complex 3 subunit delta 1; D=day; ddPCR=droplet digital polymerase chain reaction; hSGCB=human β-sarcoglycan; IM=intramuscular; mRNA=messenger RNA; RT-ddPCR=reverse transcription droplet digital polymerase chain reaction; SGCB=β-sarcoglycan; TA=tibialis anterior; WT, wild type.
RESULTS
Detection of SRP-9003 in situ using RNAscopeTM (IM)
SRP-9003 Detection in Muscle Post IM Injection Using RNAscopeTM
VG | mRNA mRNA VG DAPI Merge | FISH-IF |
-
Robust nuclear SRP-9003 transduction was observed as early as 1 day post IM injection in skeletal muscle, with SRP- 9003 transgene mRNA levels peaking
14 days post IM injection
Probe 1: Vector DNA (vg)
Probe 2: Transgene mRNA
Nuclear label: DAPI
Membrane marker: Laminin
D1
D7
D14
D21
D28
D=day; DAPI=4′,6-diamidino-2-phenylindole;FISH-IF=fluorescence in situ hybridization with immunofluorescence; IM=intramuscular; mRNA=messenger RNA; VG=vector genomes.
RESULTS
SGCB expression post SRP-9003 injection (IM)
- SRP-9003led to robust SGCB expression as early as D14 in skeletal muscle, with stabilization by D28
- These data support sustained SGCB protein expression 28 days following IM injection
Immunofluorescent Staining of SGCB Protein Expression (PPF) Following IM Injection
D1 | D7 | D14 | D21 | D28 |
Annotation
SGCB
HALO-MASK
D=day; IM=intramuscular; mRNA=messenger RNA; PPF, percent positive fibers; SGCB=β-sarcoglycan.
RESULTS
SRP-9003 - IV Administration
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Sarepta Therapeutics Inc. published this content on 10 May 2024 and is solely responsible for the information contained therein. Distributed by Public, unedited and unaltered, on 10 May 2024 13:05:24 UTC.
