©Laboratory for Molecular Therapy www.eb-haus.org

Type VII collagen correction by RNA trans-splicing

Patricia Peking

(ERA-NET E-RARE-2)

©Laboratory for Molecular Therapy www.eb-haus.org

Outline

• 3‘ RNA trans-splicing technology (SMaRT) to correct COL7A1 mutations

• 3‘ trans-splicing in a cell culture model system

• Ex vivo approach in a xenograft mouse model

• Conclusion

Patricia Peking, PhD.

©Laboratory for Molecular Therapy www.eb-haus.org

Target pre-mRNA 1 2 3 4 5 6

SMaRT: Spliceosome-mediated RNA trans-splicing

Patricia Peking, PhD.

©Laboratory for Molecular Therapy www.eb-haus.org

Target pre-mRNA 1

Cis-splicing

1 2 3 4 53 6

2 3 4 5 6

Mutant mRNA

SMaRT: Spliceosome-mediated RNA trans-splicing

Patricia Peking, PhD.

©Laboratory for Molecular Therapy www.eb-haus.org

Target pre-mRNA 1

Cis-splicing

AG

BD

WT cDNA

1 2 3 WT4

1 2 3 4 53 6

2 3 4 5 6

3’ RTM

Mutant mRNA

3’ Trans-splicing

Reprogrammed mRNABD= binding domainRTM= RNA trans-splicing molecule

SMaRT: Spliceosome-mediated RNA trans-splicing

Patricia Peking, PhD.

©Laboratory for Molecular Therapy www.eb-haus.org

• Preservation of the endogenous regulation

• Small transgene size

• One molecule > correction of many COL7A1 mutations possible

• Constructed to correct mutations located 5’, 3’ or internal

• Intervention at RNA level

SMaRT Advantages

Patricia Peking, PhD.

©Laboratory for Molecular Therapy www.eb-haus.org

Murauer et al. (2011). JID Peking et al. (2017). NAR

• Covering ~ 40 % of all DEB causing mutations

• SIN lentiviral vector (LV-RTM-S6m)

• RDEB keratinocyte line: c.6527dupC in exon 80 in COL7A1

Patricia Peking, PhD.

Establishment of stably corrected keratinocytes

©Laboratory for Molecular Therapy www.eb-haus.org

Analysis of single cell clones

IF: anti human type VII collagen: Western blot: anti human type VII collagen

Peking et al. (2017). NAR

Patricia Peking, PhD.

©Laboratory for Molecular Therapy www.eb-haus.org

Safety profiling

Peking et al. (2017). NAR

Patricia Peking, PhD.

Sequence VCN

normal kc 0.03

LV-RTM-S6 1.34

LV-RTM-woBD 1.35

C47 1.25

Vector copy number: sqPCR Trans-splicing COL7A1: PCRRTM integration: Southern blot

©Laboratory for Molecular Therapy www.eb-haus.org

Transplantations onto immuno-deficient mice

• Transplantation of skin sheets derived from RTM S6 corrected keratinocytes

• Analysis 12 weeks after transplantation

Peking et al. (2017). NAR

50µm

Patricia Peking, PhD.

DapiC7

©Laboratory for Molecular Therapy www.eb-haus.org

Conclusion

• Establishment of trans-splicing corrected RDEB keratinocytes using a safe SIN

lentiviral vector

• Long term type VII collagen expression at basement membrane zone in

xenograft mouse model

Patricia Peking, PhD.

©Laboratory for Molecular Therapy www.eb-haus.orgPatricia Peking, PhD.

Acknowledgements

EB House AustriaJulia Reichelt

Eva MurauerUlrich KollerStefan HainzlThomas Kocher Roland WagnerBernadette LiembergerOliver MarchNina Lackner

Johann Bauer

Collaborators in E-RARE-2

Marjon PasmooijUniversity Medical Center GroningenGroningen, The Netherlands

Alexander NyströmUniversity Medical Center FreiburgFreiburg, Germany

Annemieke Aartsma-RusLeiden University Medical CenterLeiden, The Netherlands

Marta Garcia, Fernando LarcherCIEMAT, Madrid, Spain

Axel SchambachMHH, Hannover, Germany

Gabriele BrachtlSpinal Cord Injury and Tissue Regeneration Center Salzburg

Franz ZimmermannDepartment of Pediatrics, University Hospital Salzburg

Andrea ZurlDepartment of Ophthalmology and Optometry, University Hospital Salzburg

Funding:

Internal Collaborators