Pre trans splicing gene therapy

Pre trans splicing gene therapy overview, PTST of Duchenne Muscular dystrophy

1. P re RNA trans-splicing Gene T herapy Presented by: Farah Arooj MS (Biochemistry) 2015-2017 Institute of Biochemistry & Biotechnology University of the Punjab

2. RNA splicing in which exons from two different primary RNA transcripts are joined end to end and ligated. Target Pre mRNA and other is trans splicing molecule Rare in higher eukaryotes Emerging technique for RNA repair RNA trans-splicing

3. Spliceosome-mediated pre-RNA trans-splicing Endonuclease-mediated trans-splicing Trans splicing ribozyme Three main approaches for RNA trans splicing:

4. Cis splicing: Splicing and exon ligation on the same mRNA Trans splicing: Exons from two different mRNA are ligated. Cis - splicing vs. Trans-splicing

5. Also called spliceosome mediated RNA trans splicing ( SMaRT ) Repairs the mutated part of target mRNA instead of whole gene by giving a Pre trans splicing molecule (PTM) exogenously which has the corrected coding sequence . First reported by Pattaraju et al., in 1999 in cancer cell lines Pre RNA trans splicing gene therapy

6. Uses 3 components Target mRNA Spliceosome Pre trans splicing molecule PTM Spliceosome Macromolecular enzyme complex Consists of 5 uridine rich small nuclear RNA ( snRNA ) U1, U2, U4, U5, U6 and large number of proteins. There are approximately 100,000-200,000 splicesome per cell.

7. Artificially designed RNA molecule Contains Binding domain helps binding of PTM on target Coding domain modified sequences to be added Trans splicing domain having elements for recognition of spliceosome and splice sites Enhanced 3’ UTRs Pre trans-splicing molecule

8. Cut and Paste Therapy!

9. Based on different splice sites in trans splicing domains 3’ exons replacement 5’ exon replacement Internal exon replacement 3 Mechanisms of Repair

10. Injecting the plasmid DNA encoding the PTM Viral vectors e.g. AAV, Lentiviral , retroviral Trans splicing efficiency increases with high conc. of PTM. A library of PTMs is generated. Bound with GFP and RFP Screened for efficient PTM by checking intensity of red and green fluorescence PTM delivery

11. Target pre mRNA expression level The type of PTM and its expression level Binding strength with target splice sites The ease of accessibility of the binding domain The route of PTM delivery Stem loop structures. Long binding domains Factors affecting trans splicing

12. Still in pre-clinical era SMaRT utility has been studied for various genetic diseases like cystic fibrosis, hemophilia , SCID and cancers in xenograft models and showed significant level of repair. First In vivo demonstration of SMaRT Performed in factor VIII hemophilia knockout mice. 16-26 exon were replaced by 3’ trans splicing. For 8 weeks, circulating FVIII was detected. Therapeutic applications

13. Duchenne Muscular dystrophy Muscle degeneration and premature death Caused by mutation in dystrophin gene X linked recessive disorder, affects mostly males Dystrophin anchor the cytoskeleton into muscle cells Muscle contraction disrupt sarcolemma leading to muscle weakening

14. Study on mdx mouse model Lorain, S., Peccate , C., Le Hir , M., Griffith, G., Philippi, S., Précigout , G., ... & Garcia, L. (2013). Dystrophin rescue by trans-splicing: a strategy for DMD genotypes not eligible for exon skipping approaches. Nucleic acids research, gkt621.

15. Fibroblasts microscopy Blankinship , M. J., Gregorevic , P., & Chamberlain, J. S. (2006). Gene therapy strategies for Duchenne muscular dystrophy utilizing recombinant adeno -associated virus vectors. Molecular Therapy, 13(2), 241-249.

16. Can be used in suicide gene therapy by ligating a suicide gene with the mRNA of potential gene involved in disease. Cell death was observed when tried on Epidermolysis bullosa associated squamous cell carcinoma cells

17. Small Trans gene size (corrected exons only) PTM targets the mutated gene with high specificity Natural regulation of gene Eliminating the expression of deleterious protein. Less chance of random mutagenesis. Undesired gene expression minimized as trans-splicing only occur in cells expressing the target pre-mRNA. Advantages

18. Efficient technique for repairing mutation More understanding of PTM designing and efficiency required Studies should extend to higher levels Can be used in molecular imaging In vivo drug screening Conclusion

19. Thank you!