31/05/2025 - Sarepta Therapeutics Inc.: P1422 Baine et al SRP 9003 RNAscope

‌#1422

In Situ Biodistribution and Localization of Bidridistrogene Xeboparvovec (SRP-9003) in LGMD2E/R4 Mice After 1 Year of Follow-up

Stephen Baine, Young Seo, Stephen Fasul, Jenna Greve, Chrissy Hopkins,

Akansha Pradhan, Jasmine Wu, Alex Haile, Sarah Lewis, Louise Rodino-Klapac, Rachael Potter

Sarepta Therapeutics, Inc., Cambridge, MA, USA

Introduction

• Limb-girdle muscular dystrophy type 2E/R4

  Figure 1 SRP-9003 Construct

  rAAVrh74

  Methods

• The administration of SRP-9003 and necropsy timepoints

  Figure 2 Study Design

  Administration of SRP-9003

  Necropsy day

  Conclusions

  (LGMD2E/R4) is caused by pathogenic variants in the β-sarcoglycan (SGCB) gene and results

  in a loss of functional SGCB protein, progressive muscle degeneration, and shortened lifespan1,2

  ITR

  MHCK7 PROMOTER

  INTRON

  hSGCB cDNA

  pA ITR

  Efficient skeletal

  for the mouse model (n=5) are described in Figure 2

  pe

  • Subcellular localization of vector genomes and mRNA were assessed using RNAsco , which employs an in situ hybridization (ISH) technique to detect targeted nucleic acid

    IV administration of SRP-9003:

    e

    7.41×1013 vg/kga

    This study demonstrates the applicability of RNAscop to

• Bidridistrogene xeboparvovec (SRP-9003, rAAVrh74.MHCK7.hSGCB) is an investigational

  Optimized for expression in

  skeletal and cardiac muscle5-7

  Full-length hSGCB

  gene transfer

  and cardiac muscle

  transduction8

  levels at single molecule resolution within tissues and cells9,10

  pe

• The ISH probes used for RNAsco were designed to be

  1 year durability

  determine spatial,

  adeno-associated virus (AAV)-based gene therapy designed to restore functional SGCB expression in muscle and improve clinical

  outcomes in patients with LGMD2E/R4 (Figure 1)3

• The maintenance of SRP-9003 vector DNA and transgene mRNA up to 120 days following systemic administration in LGMD2E mice has been previously demonstrated4

  cDNA=complementary DNA; hSGCB=human β-sarcoglycan; ITR=inverted terminal repeat; MHCK7=myosin heavy-chain muscle creatine kinase promoter; pA=polyadenylation; rAAVrh74=recombinant adeno-associated virus serotype rh74.

  Objective

  To evaluate the in situ spatial biodistribution, cellular tropism, transgene expression, and durability of SRP-9003 up to 1 year following intravenous (IV) administration in LGMD2E/R4 mice

  non-overlapping for two-plex ISH

• Two-plex ISH allowed for the evaluation of subcellular localization of vector genome DNA and hSGCB RNA for spatial analysis

• Droplet digital polymerase chain reaction (ddPCR), reverse transcription-droplet digital polymerase chain reaction (RT-ddPCR), and immunofluorescence (IF) methods are described in the Supplemental Methods

  DAY

  1 14 28 60 120 240 365

  aDose is calculated based on a linearized plasmid standard.3 IV=intravenous; vg=vector genomes.

  cellular organization,

  and molecular trafficking of SRP-9003

  Results indicate the durability of SRP-9003 to generate stable SGCB mRNA and protein

  Results

  SRP-9003 Biodistribution

• SRP-9003 vector DNA levels (ddPCR) and mRNA expression (RT-ddPCR) were maintained through Day (D) 365 in skeletal and heart muscle, indicating vector durability over extended periods in LGMD2E/R4 mice (Figure 3)

• Considering preclinical evidence from 12-week pharmacology studies, SRP-9003 biodistribution at D365 was expected11

• No significant differences were seen between D60 and any subsequent timepoint in vector DNA and mRNA expression in all tissues shown here (Figure 3)

• In off-target organs such as the liver and kidney, detectable SRP-9003 vector DNA and RNA were also present up to D365, but minimal mRNA expression was detected in the kidney (Figure 3)

  Detection of SRP-9003 In Situ and Durable SGCB Expression

  pe

• In skeletal muscle, tibialis anterior (TA), vector DNA signal (RNAsco ) gradually declined from D1 to D240, but transgene mRNA signal was stable between D14 and D240 (Figures 4, 5)

• In the heart, vector DNA signal gradually declined from D1 to D120 but stabilized at D240 and D365 (Figures 4, 5)

• A durable transgene mRNA signal was achieved in the heart by D14 and was maintained to D365 post injection (Figures 4, 5)

  e

• No RNAscop mRNA signal was detected in the kidney over the course of the study (data not

  Figure 3 SRP-9003 Vector DNA and Transgene mRNA in Skeletal Muscle and Organs Following 365 Days of Treatment

  SRP-9003 Vector DNA Levels (ddPCR)

  SRP-9003 hSGCB mRNA Levels (RT-ddPCR)

  Figures 4, 5 Detection of SRP-9003 In Situ Using RNAsco : TA and HRT

  pe

  e

  
  
  

  SRP-9003 Detection in TA Using RNAscop SRP-9003 Detection in HRT Using RNAscope

  Representative RNAscopeTM images of the tibialis anterior (TA) and heart (HRT) muscles from SGCB-/- mice treated with SRP-9003 showing continued hSGCB mRNA expression from 1 day to 365 days post treatment. Laminin (shown in white) represents the fiber membrane. Vector genomes are shown in green, and mRNA is shown in red. Scale bar = 25 μm.

  hSGCB=human β-sarcoglycan; SEM=standard error of the mean.

  Figure 6 SGCB Membrane-Localized Protein Restoration Is Sustained

  SGCB Protein Expression (IF) SGCB Expression (PPF) in TA

  100

  %SGCB+ Fibers

  80

  60

  40

  20

  expression over extended periods of up to 1 year in a mouse model of

  LGMD2E/R4

  These outcomes show that D60 represents

  an appropriate time frame to evaluate expression in clinical biopsies, and the data demonstrate a consistent expression through at least 1 year

  of treatment

  e

  These data may be useful to inform future application of RNAscop for biodistribution evaluation of AAVrh74 gene therapies

  QR

  code

  The QR code is intended to provide scientific information for individual reference, and the information should not be altered or reproduced in any way.

  shown)

  0

  Day 1

  14 28

  60 120 240

  365

• SGCB protein expression (percent positive fibers [PPF]) maximized at D240 and remained elevated up to D365 in TA (Figure 6)

Acknowledgments & Disclosures

Representative histograms quantifying SRP-9003 vector DNA and transgene mRNA over duration of study. Note: Error bars represent standard error of the mean. Individual data points are represented as circles (male) and triangles (female). Ap3d1=adaptor-related protein complex 3 subunit delta 1; ddPCR=droplet digital polymerase chain reaction; HRT=heart; hSGCB=human β-sarcoglycan; KID=kidney; LIV=liver; RT-ddPCR=reverse transcription-ddPCR; TA=tibialis anterior; VGC=vector genome copies.

References

Representative immunofluorescence (IF) images of tibialis anterior (TA) muscle in SGCB-/- mice treated with SRP-9003 show restoration of β-sarcoglycan (SGCB) to the cell membrane and sustained expression through 365 days post treatment. Laminin (green) represents the fiber membrane, SGCB (red) represents β-sarcoglycan expression, and the yellow merge represents localization of β-sarcoglycan to the fiber membrane.

Scale bar = 100 μm. Bars represent the mean ± SEM. No statistical significance testing was performed on the percent positive fiber (PPF) data. SEM=standard error of the mean.

This study was funded by Sarepta Therapeutics, Inc. Editorial support was provided by

Kimberly Fischer, PhD, of Eloquent Scientific Solutions and was funded by Sarepta Therapeutics, Inc. SB, YS, SF, JG, CH, AP, JW, AH, SL, LR-K, RP: All authors are employees of Sarepta Therapeutics, Inc., and may own stock/options in the company.

1. Murphy AP, Straub V. J Neuromuscul Dis. 2015;2(suppl 2):S7-S19. 2. Bouchard C, Tremblay JP. J Clin Med. 2023;12:4769. 3. Mendell JR, et al. Nat Med. 2024;30:199-206. 4. Seo Y, et al. Presented at: 2024 American Society of Gene & Cell Therapy (ASGCT) 27th Annual Meeting; May 7-11, 2024; Baltimore, MD. 5. Salva MZ, et al. Mol Ther. 2007;15:320-29. 6. Pozsgai ER, et al. Mol Ther. 2017;25:855-69. 7. Wang B, et al. Gene Ther. 2008;15:1489-99. 8. Chicoine LG, et al. Mol Ther. 2014;22:338-47. 9. Zhao J, et al. Mol Ther Methods Clin Dev. 2020;18:856-68. 10. Wang F, et al. J Mol Diagn. 2012;14:22-29. 11. Griffin DA, et al. Hum Gene Ther. 2021;32:390-404.

‌#1422

In Situ Biodistribution and Localization of Bidridistrogene Xeboparvovec (SRP-9003) in LGMD2E/R4 Mice After 1 Year of Follow-up

Stephen Baine, Young Seo, Stephen Fasul, Jenna Greve, Chrissy Hopkins,

Akansha Pradhan, Jasmine Wu, Alex Haile, Sarah Lewis, Louise Rodino-Klapac, Rachael Potter

e

pe

The QR code is intended to provide scientific information for

individual reference, and the QR

information should not be altered

or reproduced in any way. code

Sarepta Therapeutics, Inc., Cambridge, MA, USA

Methods (cont)

Supplemental Methods

• Droplet digital polymerase chain reaction (ddPCR)

  ‒ DNA isolates collected were analyzed by ddPCR for presence of vector genomes using a vector-specific primer probe set designed to amplify a sequence in the myosin heavy-chain muscle creatine kinase promoter (MHCK7)

  ‒ Beta-actin was used as reference gene to quantify the amount of genomic DNA

• Reverse transcription-droplet digital polymerase chain reaction (RT-ddPCR)

  ‒ RNA isolates were collected and analyzed by RT-ddPCR using a transgene-specific primer probe set designed to amplify a sequence in the hSGCB transgene

  ‒ A second set of primer and probes against adaptor-related protein complex 3 subunit delta 1 (Ap3d1) was used as reference

• Immunofluorescence (IF)

  ‒ IF assay was performed to assess hSGCB protein expression

  ‒ The percent of hSGCB-positive fibers was determined using HALO IA algorithms (Indica Labs, Albuquerque, NM) for each image for percent positive fibers (PPF) analysis

• RNAscop

  ‒ Visualization of subcellular localization of vector genomes and hSGCB RNA following IV injection of SRP-9003 occurred using two-plex ISH

  ‒ One probe detected AAVrh74-vector DNA only (antisense strand) and a second probe captured hSGCB transgene mRNA (sense strand)

  ‒ Quantitative analysis of vector genome and hSGCB RNA was performed using fluorescence in situ hybridization (FISH) and FISH-IF algorithm

  Limitations

• Limited ability/not able to differentiate from double-stranded DNA and episomal AAV DNA (Advanced Cell Diagnostics, Inc.) using RNAsco

• Lack of denaturing step (heat) to preserve tissue integrity. Future evaluations will examine heating step in similar tissues

• The assay is unable to detect integrated AAV-vector DNA

Attachments

• Original document
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Disclaimer

Sarepta Therapeutics Inc. published this content on May 31, 2025, and is solely responsible for the information contained herein. Distributed via Public Technologies (PUBT), unedited and unaltered, on May 31, 2025 at 11:48 UTC.