pre trans splicing gene therapy
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Pre RNA trans-splicing Gene Therapy
Presented by:Farah Arooj
MS (Biochemistry)2015-2017
Institute of Biochemistry & BiotechnologyUniversity of the Punjab
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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
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Spliceosome-mediated pre-RNA trans-splicing
Endonuclease-mediated trans-splicing Trans splicing ribozyme
Three main approaches for RNA trans splicing:
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Cis splicing: Splicing and exon ligation on the same mRNA Trans splicing: Exons from two different mRNA are ligated.
Cis-splicing vs. Trans-splicing
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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
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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.
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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
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Cut and Paste Therapy!
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Based on different splice sites in trans splicing domains 3’ exons replacement 5’ exon replacement Internal exon replacement
3 Mechanisms of Repair
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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
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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
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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
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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
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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.
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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.
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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
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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
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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
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Thank you!