Pre RNA trans-splicing Gene Therapy

Presented by:Farah Arooj

MS (Biochemistry)2015-2017

Institute of Biochemistry & BiotechnologyUniversity of the Punjab

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

Spliceosome-mediated pre-RNA trans-splicing

Endonuclease-mediated trans-splicing Trans splicing ribozyme

Three main approaches for RNA trans splicing:

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

Cis-splicing vs. Trans-splicing

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

Uses 3 components1. Target mRNA2. Spliceosome3. 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.

Artificially designed RNA molecule Contains 1. Binding domain helps binding of PTM on target2. Coding domain modified sequences to be added3. Trans splicing domain having elements for recognition

of spliceosome and splice sites4. Enhanced 3’ UTRs

Pre trans-splicing molecule

Cut and Paste Therapy!

Based on different splice sites in trans splicing domains 3’ exons replacement 5’ exon replacement Internal exon replacement

3 Mechanisms of Repair

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

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

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

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

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.

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.

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

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

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

Thank you!