What is the difference between gene therapy and gene splicing

The proof of concept of SMaRT feasibility has already been established in several models of genetic diseases caused by recessive mutations. In such cases, in fact, the repair of only a portion of the mutant mRNA pool may be sufficient to obtain a significant therapeutic effect.

However in the case of dominant mutations, the target cell must be freed from the majority of mutant mRNA copies, requiring a highly efficient trans-splicing reaction. Expression of each construct could be controlled by a different promoter to enhance specificity. However, unless at least one of the constructs also trans- spliced with a host cell transcript, the link to endogenous regulation provided by trans -splicing would be lost.

Restoration of function and correction of phenotype in animal models and cell lines are important components in preclinical development. In this study, the PTM targeted intron 9 and replaced any mutation within the coding sequence of exons 10—24, a region of 3. The first correction of a knock out KO animal by trans -splicing was achieved by Chao et al 13 using a hemophilia A mouse model with an insertion in exon 16 of the mouse factor VIII gene that eliminates all factor VIII activity.

A PTM designed to base-pair with intron 15 and trans- splice the coding sequence of exons 16—26 was delivered i. Over the 8-week study period, plasma levels of factor VIII reached a mean of PTMs have been delivered in vivo using a wide range of vectors including naked or complexed plasmids, adenovirus, retrovirus, lentivirus, and adeno-associated virus AAV.

PTM levels generally follow the expected stability and expression profile of the chosen delivery system. There are several reports on the adverse effects of undesirable ectopic gene expression, including CFTR.

A previous study found that CD40L-KO mice developed T-lymphoproliferative disease within 9 months of receiving bone marrow cells transduced ex vivo with a retrovirus that expressed full-length CD40L gene. Undesired ectopic expression of CD40L has been shown to cause T-cell lymphoproliferation or lymphomas. Treated mice produced both IgM and IgG in response to antigen stimulation and markedly attenuated Pneumocystis carinii proliferation following respiratory challenge.

Mice evaluated 3—12 months post-treatment displayed no evidence of lymphoproliferation, demonstrating the safety advantage conferred by trans- splicing. Trans- splicing technologies have a variety of applications, depending on the sequence trans- spliced into the target.

Trans- spliced reporter genes may be used to follow the process of trans- splicing itself or to identify target gene expression within living cells. Numerous studies to develop ribozyme- or spliceosome-mediated RNA trans- splicing have used the repair of lacZ , green fluorescent protein or other reporter genes to quantify the efficiency of trans- splicing in cells. No significant bioluminescent activity was detected in these mice following intraveneous injection of the luciferase substrate.

Following injection of the luciferase substrate, optical images were generated by detecting luciferase activity in target cells located in subcutaneous tumors and deeper tissues, such as the liver. This was the first study to visualize real-time gene expression in live animals by RNA trans- splicing. It represents an important step towards the universal imaging of an endogenous pre-mRNA target, with potential applications in research and diagnostics. The coding domain of the PTM can be easily changed to encode a reporter more suitable for molecular imaging in humans by PET, or to produce a protein that acts therapeutically.

Trans- splicing ribozymes were originally derived from Group I self-splicing introns by Sullenger and Cech Figure 3b. They do not require splicing or other factors and are active in relatively simple buffered solutions containing guanosine or in Escherichia coli. Such conditions have been utilized to map the most accessible and reactive ribozyme sites in a target mRNA. Selected ribozymes are individually tested for efficiency and specificity in the more complex environment of human cells.

However, demonstration of therapeutic potential by trans- splicing ribozymes in animal models and clinical application will likely require improvement in the efficiency and specificity of the reaction in vivo. Shin et al 21 recently developed a ribozyme that repairs p53 mRNA by trans- splicing at a site upstream of the AUG start codon. This ribozyme is capable of repairing any mutation in the coding sequence of p The repaired transcripts produced functional p53 protein in a human ovarian cancer cell line, demonstrated by luciferase expression driven by a presponsive promoter and an increase in apoptotic cells.

This study was the first demonstration of protein activity generated by the translation of a ribozyme-repaired endogenous mRNA in human cells, a significant early milestone in the development of trans- splicing ribozymes for therapeutic application.

Loss of p16 function is the second most common gene alteration in human tumors. A trans- splicing strategy to repair mutations in the coding sequence of p16 in a pancreatic cancer cell line has been demonstrated. These reports highlight the importance of regulating the expression of p53 and p16 and the link to endogenous regulation conferred by trans- splicing. Most mutant p53 proteins exert a dominant-negative effect. Each trans- spliced mRNA lowers the production of mutant p53 while simultaneously increasing levels of wild-type p53 protein.

Trans- splicing ribozymes have also found application in targeting infectious diseases, such as hepatitis caused by the hepatitis C virus HCV. As ribozymes function in the cytoplasm, they can be used to reprogram such targets. Ryu and Lee 24 used an extended internal guide sequence to improve specificity in targeting a conserved region of the HCV internal ribosome entry site IRES. Trans- splicing removed two in frame upstream stop codons in the ribozyme, enabling the viral IRES to drive the expression of trans- spliced luciferase or diphtheria toxin subunit A sequences.

Several possible explanations for this variability were offered, including heterogeneity of the cell cycle, variable delivery and expression of the ribozyme, and colocalization target and ribozyme. Sullenger et al 31 have reported progress towards improving ribozyme activity in mammalian cells when expressed by pol II promoters, circumventing the difficulty they encountered in expressing long ribozymes from pol III promoters.

Neither of these additions are required for trans- splicing activity when mammalian cells are transfected with in vitro transcribed ribozymes. These new studies document steady progress towards improving the efficiency and specificity of trans- splicing ribozymes. In vivo delivery and demonstration of therapeutic potential in animal models are important milestones that remain to be achieved by trans- splicing ribozymes. A third form of RNA trans -splicing has recently been described.

Upon encountering a complementary target mRNA, the target and targeting RNAs form a specific bulge—helix—bulge structure that is specifically recognized and cleaved by the endonuclease 28 , 32 Figure 3b. The cut RNAs are subsequently joined by an as yet unidentified endogenous ligase. Proteins can also be engineered to trans -splice into each other. A variety of lower organisms express proteins which undergo cis -splicing-mediated by inteins.

An intein is a protein sequence that can splice itself out of the precursor protein in which it is embedded. Like ribozymes, inteins do not naturally mediate trans- splicing. Inteins can be split into N- and C-terminal fragments. Split inteins may be incorporated into different proteins or peptides to perform protein trans- splicing, joining two separate precursors into a new chimeric molecule. Intein mediated trans- splicing has been demonstrated in mammalian cells.

Figure 4 Kinsella et al 33 have engineered a retroviral library coding for randomized 4-mer peptides linked by split inteins to generate a library of cyclic polypeptides in human cells. These peptides were subsequently screened for ability to block the IL-4 signaling pathway in human B cells. Disruption of this pathway is a potential therapeutic approach to lower IgE levels in conditions such as allergy and asthma.

Li et al 34 have also adapted trans- splicing inteins to overcome the size limitations of AAV-based gene delivery, splitting the 6. These two half genes were separately packaged into AAV vectors.

Cotransduction of these two vectors produced full-length Becker form dystrophin protein in the muscle of dystrophic mice that was correctly localized in myofibers and prevented central nucleation pathology. Intein-based trans -splicing has also been used to visualize protein—protein interactions in vivo by reconstituting a reporter molecule. Paulmurugan et al 35 imaged the joining of the N- and C-terminal portions of split luciferase proteins that were trans -spliced by inteins incorporated into two strongly interacting proteins, MyoD and Id.

However, this technique is limited to trans -splicing between proteins engineered with intein sites.

Intein-mediated protein trans-splicing. Protein 2 is connected to the C-terminal half of DnaE DnaE-c and the remaining C-terminal portion of firefly luciferase. The design requirements for a trans- splicing RNA to efficiently and specifically reprogram a defined pre-mRNA target are not fully understood. Improvements in specificity and efficiency were initially accomplished by rational design, construction and testing of candidate trans- splicing molecules one at a time.

This approach is untenable when considering the vast numbers of potential designs. A better method to identify optimal trans- splicing constructs would be able to rapidly select the best performers from a large pool of variants in human cells.

Ayre et al 36 described a yeast-based screen to identify efficient ribozymes by trans- splicing a transcription factor that conferred a growth advantage. In that study, the library consisted of 13 mixed ribozymes. This is a significant level of heterogeneity, which should be reduced before screening libraries with large populations of variant ribozymes.

The intensity of fluorescence correlates with the efficiency of trans- splicing. The PTMs selected from the screen typically demonstrated five- to fold greater efficiency in trans- splicing to target than the best rationally designed PTMs. High-capacity PTM screen. A randomized PTM library is constructed by sonicating a chosen target intron, then cloning the resulting fragments into the binding domain region of PTM expression plasmids.

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