Therapeutic applications of trans-splicing

Abstract

Background

RNA trans-splicing joins exons from different pre-mRNA transcripts to generate a chimeric product. Trans-splicing can also occur at the protein level, with split inteins mediating the ligation of separate gene products to generate a mature protein.

Sources of data

Comprehensive literature search of published research papers and reviews using Pubmed.

Areas of agreement

Trans-splicing techniques have been used to target a wide range of diseases in both in vitro and in vivo models, resulting in RNA, protein and functional correction.

Areas of controversy

Off-target effects can lead to therapeutically undesirable consequences. In vivo efficacy is typically low, and delivery issues remain a challenge.

Growing points

Trans-splicing provides a promising avenue for developing novel therapeutic approaches. However, much more research needs to be done before developing towards preclinical studies.

Areas timely for developing research

Increasing trans-splicing efficacy and specificity by rational design, screening and competitive inhibition of endogenous cis-splicing.

Introduction

In order to become fully functional mature messenger RNA (mRNA), a pre-mRNA molecule needs to undergo various post-transcriptional modifications. One of these is splicing, whereby introns are removed from the pre-mRNA transcript(s) and the remaining exons are joined together. The predominant form of splicing in humans is cis-splicing, in which a single pre-mRNA transcript produces a single mRNA molecule (Fig. 1). However, the spotlight has increasingly been cast on the role of RNA trans-splicing in human health and disease. RNA trans-splicing involves the joining of exons from more than one pre-mRNA transcript to form a single chimeric mRNA molecule (Fig. 1), and was initially proposed as a possible mechanism involved in transcription of trypanosome variant surface glycoprotein.^{1,  2} It was subsequently observed in in vitro cell-free systems,^{3,  4} before being shown to occur naturally in trypanosomes,^{5,  6}  Caenorhabditis elegans⁷ and other lower eukaryotes. Further studies have described trans-splicing in viruses,^8–12 bacteriophages¹³ and prokaryotes,^{14,  15} suggesting an early evolutionary origin for this form of RNA processing. In vertebrates, RNA trans-splicing occurs far less frequently, but it has been observed in many species including Homo sapiens. In humans, RNA trans-splicing is a physiological phenomenon seen in genes coding for certain enzymes, transcription factors, cancer biomarkers, gene expression regulators and more (reviewed by Lei et al.¹⁶).

Fig. 1.

Schematic depiction of different types of pre-mRNA splicing. (a) Cis-splicing. Exons from a single pre-mRNA molecule are ligated together to generate a mature linear transcript. (b) Intergenic trans-splicing. Exons from different genes are joined to produce a chimeric molecule. (c) Intragenic trans-splicing. Exons from different pre-mRNAs of the same gene are spliced together, resulting in exon duplication and sense-antisense fusion. (d) SL trans-splicing. SL exon is spliced onto multiple genes of a polycistronic pre-mRNA, resulting in a number of mature transcripts containing a common 5′ sequence.

RNA trans-splicing can be categorized as intergenic or intragenic, depending on the pre-mRNA source (Fig. 1). In intergenic trans-splicing, exons from separate genes are joined together to produce a chimeric mRNA transcript, while in intragenic trans-splicing, the exons which are spliced together originate from different pre-mRNAs of the same gene (whether they are identical transcripts of the same strand, resulting in exon duplication, or complementary transcripts of different strands, resulting in sense-antisense fusion). Spliced leader (SL) trans-splicing is a special form of trans-splicing, widespread in lower eukaryotes but not yet observed in vertebrates, in which an SL sequence is joined to the 5′-end of multiple pre-mRNAs¹⁷ (Fig. 1).

On a post-translational level, polypeptides can undergo protein splicing, where protein sequences with autocatalytic activity (inteins) excise themselves from the precursor polypeptide and ligate the flanking regions (exteins) with a peptide bond. Some inteins—known as split inteins—are derived from two separate genes, one encoding the N-terminus and the other encoding the C-terminus, whose products undergo protein trans-splicing to generate a single mature protein.¹⁸

In this article, we review the latest development of RNA and protein trans-splicing technology as potential therapeutic strategies for human diseases.

Trans-splicing therapeutic strategies and techniques

Research has predominantly focused on two main therapeutic strategies: (a) using RNA trans-splicing to repair mutations at the mRNA level, and (b) inducing cell death by introducing trans-splicing constructs encoding toxins, apoptotic factors or suicide genes into target cells. Other therapeutic strategies include using trans-splicing to produce therapeutic proteins and to generate probes for direct molecular imaging of gene expression.

Therapeutic techniques used to carry out these strategies include spliceosome-mediated RNA trans-splicing (SMaRT), ribozyme-mediated trans-splicing and at the protein level, split intein-mediated trans-splicing. First developed by Puttaraju et al.¹⁹ in 1999, SMaRT exploits the cell’s own splicing machinery to trans-splice therapeutic coding sequences into endogenous pre-mRNA targets. The therapeutic coding sequences are delivered by artificially engineered pre-mRNA trans-splicing molecules (PTMs). In order to successfully produce the desired product, the trans-splicing reaction must be induced in preference to the cis-splicing reaction, and the specificity of the trans-splicing reaction must be maintained. Thus, in addition to the coding domain, the PTM must also be containing a splicing domain (an artificial intron, containing functional splice sites and spliceosomal recognition sites, which regulates the splicing reaction catalysed by the spliceosome) and a binding domain that hybridizes specifically to an intron within the target pre-mRNA. Only the coding domain is retained in the mature mRNA product.

PTMs can be introduced into the target pre-mRNA by 3′-, 5′- or internal-exon replacement (3′ER, 5′ER or IER, respectively; Fig. 2). 3′ER, the most commonly described SMaRT approach in literature, corrects the distal exons of a target gene by trans-splicing the therapeutic coding sequence downstream of a 5′ splice site. 5′ER was first described in cystic fibrosis (CF) research using the ΔF508 CFTR minigene system²⁰ and involves trans-splicing the coding sequence upstream of a 3′ splice site in the target gene. IER involves a double trans-splicing reaction within the same pre-mRNA target, and as such requires a PTM with two binding domains and two splicing domains to insert the coding sequence in the middle of the target gene. Due to the relative difficulty of designing PTMs for IER and the low repair efficacy of this approach, research into the therapeutic applications of IER has been limited compared to 3′ER and 5′ER, although there have been a few IER studies in minigene^21–23 and endogenous RNA systems.²⁴

Fig. 2.

Schematic depiction of different types of exon replacement. * indicates a mutation within the targeted exon.

RNA trans-splicing can also be carried out by non-spliceosomal systems. Group I intron ribozymes have been designed to cleave a target pre-mRNA transcript upstream of the sequences to be replaced, and subsequently catalyse a trans-splicing reaction to attach therapeutic sequences to the 3′ end (reviewed by Lee et al.²⁵). Because ribozyme-mediated RNA trans-splicing is limited to 3′ exon replacement, this is an obvious limitation of this technique compared to SMaRT.

Protein trans-splicing techniques use split inteins to separately deliver gene fragments, which are translated to polypeptides that trans-splice themselves together to reconstitute the full-length protein within target cells (Fig. 3), thus overcoming the limited packaging capacity of gene therapy vectors such as adeno-associated viruses (AAV).²⁶ Split intein-mediated trans-splicing can also be used as a platform for biotechnological development, including protein purification, protein labelling, protein cyclization, protein engineering and design of molecular biosensors.^27–29

Fig. 3.

Schematic depiction of protein trans-splicing mediated by split inteins. The DNA sequence coding for the desired product is split into two pieces, and the N-intein and C-intein coding sequences added to each gene fragment. The two genetic constructs are co-delivered to target cells and undergo transcription and translation to produce two precursor polypeptides. The N-intein and C-intein self-assemble, excise themselves out of the polypeptide, and ligate the flanking regions together to generate the mature full-length protein.

Therapeutic applications in disease

Both RNA- (Table 1) and protein- (Table 2) mediated trans-splicing have been explored in a variety of diseases.

Table 1.

Summary of the applications of RNA trans-splicing

(a) Repairing mutations
Application	Disease	Target gene	Mutation	Models  in vitro/vivo
Repairing mutations	Cancer	p1673	-	vitro
	Cancer: BPL	p5375–77	-	vitro + vivo
		CD2278,  79	-	vitro + vivo
	Cystic fibrosis	CFTR20,  30–33	Recessive	vitro + vivo
	Duchenne muscular dystrophy	Dystrophin22,23,45	Recessive	vitro + vivo
	Dysferlinopathies	DYSF38,  47	Recessive	vitro + vivo
	Dystrophia myotonica type 1	DMPK40,  41	Dominant	vitro
	Epidermolysis bullosa	COL7A124,  65,  66 COL17A121,  67 Plectin168 Keratin1469,  70	Recessive	vitro
	Frontotemporal dementia with parkinsonism	Tau-162,63	Recessive	vitro
	Haemophilia A	FVIII49	Recessive	vitro + vivo
	Huntington’s disease	Huntingtin60,61	Dominant	vitro
	Hypertrophic cardiomyopathy	Cardiac myosin-binding protein C55	Dominant	vitro
	GNE myopathy	GNE39	Recessive	vitro
	L-CMD	LMNA46	Dominant	vitro + vivo
	Leber congenital amaurosis type 10, Joubert syndrome	CEP29058,  59	Recessive	vitro + vivo
	Myotonia congenita	ClC-142	Dominant or recessive	vitro
	SCID	DNA-PKcs64	Recessive	vitro
	Sickle cell anaemia, beta-thalassaemia	β-globin50–53	Recessive	vitro
	Spinal muscular atrophy	SMN234–37	Recessive	vitro + vivo
	X-linked immunodeficiency with hyper IgM	CD40L56	Recessive	vitro
(b) Inducing cell death
Application		Target	Models in vitro/vivo
Inducing cell death	Inducing apoptosis	CHIKV101	vitro
		DENV99,100	vitro
	Intracellular toxin delivery	HCV97,98	vitro
		MMP981	vitro
	Suicide gene therapy (HSV-tk/GCV system)	AFP90	vitro
		AIMP292	vitro
		CEA91	vitro
		Ct-OATP1B396	vitro + vivo
		hCKAP293	vitro + vivo
		hTERT25,  83–89	vitro + vivo
		HIV103	vitro
		HPV-16102	vitro
		KRAS94	vitro + vivo
		SLCO1B395	vitro + vivo
(c) Other applications
Application
Generating therapeutic proteins71,  104,  105
Molecular imaging25,106

Table 2.

Summary of the applications of split intein-mediated protein trans-splicing

Application	Disease	Target gene	Mutation	Models in vitro/vivo
Repairing mutations	Amyotrophic lateral sclerosis	SOD143	Dominant	vitro + vivo
	Becker muscular dystrophy	Dystrophin44	Recessive	vitro + vivo
	Haemophilia A	FVIII48	Recessive	vitro
	Stargardt disease	ABCA459	Recessive	vitro + vivo
Biotechnological development 27–29,  107–116
Facilitating delivery of other gene therapies72

Genetic diseases

Trans-splicing techniques have been most enthusiastically embraced in the development of therapy for genetic diseases, particularly in the correction of monogenic recessive mutations at the mRNA level, since even partial correction may be able to achieve a heterozygous non-disease phenotype. CF was one of the first genetic diseases targeted using RNA trans-splicing. As a monogenic autosomal recessive disorder whose most common disease-causing mutation, ΔF508 CFTR, has been extensively investigated, CF has proved an attractive target for gene therapy. In in vitro disease models, both 3′ER and 5′ER SMaRT have been found to be effective at correcting the ΔF508 mutation.^{20,  30–32} Song et al.³² used a segmental RNA trans-splicing approach by co-transfection of two AAV vectors to deliver the 5′ and 3′ halves of CFTR cDNA, which resulted in expression of full-length CFTR mRNA and protein as well as correction of CFTR Cl⁻ conductance. In in vivo disease models, Liu et al.³³ found that 3′ER SMaRT corrected CFTR Cl⁻ conductance to 22% of normal function.

Trans-splicing approaches have also been investigated for the treatment of inheritable neuromuscular disease. An optimized 3′ER SMaRT system, where the RNA trans-splicing construct was co-expressed with antisense RNA that competitively inhibited endogenous downstream splice sites and thus increased trans-splicing efficacy, has been used to functionally correct aberrant survival motor neuron-2 (SMN2) expression in mice with spinal muscular atrophy and increase their lifespan.^34–37 SMaRT 3′ER has also been used to correct dysferlin (DYSF),³⁸ bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE)³⁹ and dystrophin myotonica protein kinase (DMPK)⁴⁰ mutations. In addition, RNA trans-splicing ribozymes have been developed to target DMPK⁴¹ and a mutant canine skeletal muscle chloride channel (ClC-1), which causes myotonia congenita.⁴² A split intein-based trans-splicing system has been used to deliver the Streptococcus pyogenes Cas9 protein into a mouse model of amyotrophic lateral sclerosis in order to introduce a nonsense-coding substitution in the mutated superoxide dismutase 1 (SOD1) gene, resulting in reduced expression of mutant SOD1 in vivo and improved therapeutic outcomes.⁴³

The dystrophin gene has been targeted using split intein protein trans-splicing in vitro and in vivo in order to treat Becker muscular dystrophy,⁴⁴ and it has also been targeted using RNA trans-splicing 3′ER and IER to treat Duchenne muscular dystrophy (DMD).^{22,  23,  45} However, while in vitro dystrophin rescue has been seen,^{22,  23} only 1% of dystrophin transcripts were repaired in vivo²³, and when Koo et al.⁴⁵ used a novel triple-AAV RNA trans-splicing vector system to deliver the full-length dystrophin coding sequence to skeletal muscle of DMD mice, only low levels of protein were expressed. Similarly, RNA trans-splicing efficacy was extremely low in vivo when using 5′ER SMaRT to target the Lamin A/C (LMNA) gene mutated in autosomal dominant LMNA-related congenital muscular dystrophy (L-CMD).⁴⁶ While 3′ER SMaRT was effective at repairing DYSF and titin RNA expression in vivo, the corresponding protein was not sufficiently expressed.⁴⁷ Moreover, undesired proteins were translated from the PTM due to both the presence of a previously unmapped open reading frame (ORF), as well as cis-splicing within the PTM itself giving rise to a number of unexpected gene products with toxic potential.

Further studies demonstrated that haematological disorders could be targeted and repaired by trans-splicing including haemophilia A, sickle cell anaemia (SCA) and beta-thalassaemia. In vitro split intein protein trans-splicing⁴⁸ and in vivo 3′ER SMaRT⁴⁹ have been used to correct Factor VIII (FVIII) RNA and protein production. In mouse models, sufficient functional FVIII was produced to correct the haemophilia A phenotype. Ribozyme-mediated RNA trans-splicing^{50,  51} and 5′ER SMaRT^{52,  53} have been used to convert the mutated SCA β-globin to anti-sickling γ-globin. However, repair efficacy has been low, perhaps because normal β-globin splicing is very efficient⁵⁴ so RNA trans-splicing constructs cannot outcompete endogenous splice sites for the splicing machinery. Splicing-impaired targets such as the beta-thalassaemia splice mutants investigated by Kierlin-Duncan and Sullenger⁵² show higher repair efficiencies of up to 36%.

A variety of other genes and genetic conditions have been investigated using in vivo mouse model SMaRT studies, including cardiac myosin-binding protein C in hypertrophic cardiomyopathy (reviewed by Prondzynski et al.⁵⁵), CD40L in X-linked immunodeficiency with hyper IgM,⁵⁶ CEP290 associated with Leber congenital amaurosis type 10 and Joubert syndrome,⁵⁷ and rhodopsin in autosomal dominant retinitis pigmentosa.⁵⁸ Moreover, Tornabene et al.⁵⁹ demonstrated that split intein protein trans-splicing improved CEP290 and ABCA4 expression (associated with Stargardt disease) in vitro and in vivo mouse and pig retina, as well as improving the retinal phenotype of mouse models. They also showed that split intein protein trans-splicing can be used to reconstitute marker proteins in 3D human retinal organoids. Furthermore, in in vitro disease models, aberrant huntingtin in Huntington’s disease has been corrected using 5′ER SMaRT,^{60,  61} mutant tau-1 in frontotemporal dementia with parkinsonism has been corrected using 3′ER SMaRT,^{62,  63} and DNA protein kinase, catalytic subunit (DNA-PKcs) deficiency in severe combined immunodeficiency (SCID) has been corrected using 3′ER SMaRT delivered by a non-viral delivery system.⁶⁴ Moreover, RNA, protein and functional correction of genes involved in epidermolysis bullosa—including collagen type VII (COL7A1),^{24,  65,  66} collagen type XVII (COL17A1),^{21,  67} plectin1⁶⁸ and keratin14^{69,  70}—has been achieved in vitro using 3′,^{65,  67} 5’^{66,  68–70} and IER SMaRT.^{21,  24}

Besides mutation repair, other strategies include using trans-splicing to express therapeutic proteins in vivo. Wang et al.⁷¹ demonstrated that therapeutic proteins such as disease-specific antibodies and functional FVIII can be produced by RNA trans-splicing on to highly abundant transcripts, in this case albumin pre-mRNA. Another interesting avenue of research is in exploiting the ability of protein trans-splicing to overcome the packaging limitations of gene therapy vectors: Truong et al.⁷² investigated a split-Cas9 system, delivered via a recombinant AAV vector, and found that its nuclease activity was reconstituted efficiently in target cells.

Cancer

Since the vast majority of cancers show multifactorial aetiologies and involve mutations in multiple genes, cancer has been a less popular target for trans-splicing techniques compared to genetic disease. Nonetheless, single gene repair has been shown to have some efficacy: mutated tumour suppressors such as p16⁷³ and p53, the latter of which is among the most frequently mutated genes in human cancer,⁷⁴ have been targeted for correction and restoration of function. Both RNA trans-splicing ribozyme^{75,  76} and SMART techniques have been used to repair mutant p53 transcripts, with He et al.⁷⁷ demonstrating that SMART-mediated 3′ER is capable of correcting transcripts and restoring wildtype function in vivo, suppressing the growth of hepatocellular carcinoma xenograft tumours in mice. Beside tumour suppressor defects, SMART-mediated repair of the CD22ΔE12 defect found in paediatric B-precursor leukaemia (BPL) has been shown to reduce in vitro and in vivo clonogenicity of cells derived from BPL patients.^{78,  79}

Another approach is to use trans-splicing to deliver intracellular toxins, thus inducing cancer cell death. Using segmental trans-splicing—a technique in which 5′ and 3′ segments of the pre-mRNA of a toxin gene are encoded in separate vectors, delivered to target cells, and then trans-spliced together by the cell’s own spliceosome to generate a functional toxin—Nakayama et al.⁸⁰ inhibited tumour growth in vivo by inducing expression of Shigatoxin1A1. Using 3′ER SMART, Gruber et al.⁸¹ targeted the matrix metalloproteinase-9 (MMP9) transcript linked to epidermolysis bullosa-associated squamous cell carcinoma, inserting the toxin streptolysin O into the MMP9 gene to trigger cell death.

RNA trans-splicing has also been used in suicide gene therapy, where genes which convert non-toxic compounds into active metabolites are delivered to cancer cells. One such suicide system is the Herpes simplex virus thymidine kinase-ganciclovir (HSV-tk/GCV) system, in which the HSV-tk enzyme catalyses the phosphorylation of the prodrug ganciclovir into an active cytotoxin that causes cell death by DNA replication chain termination and possibly other mechanisms (Fig. 4). To avoid off-target effects, HSV-tk must be specifically expressed only in the cancer cells. This has been achieved by trans-splicing HSV-tk onto mRNA transcripts of genes that are overexpressed in cancer cells, such as the human telomerase reverse transcriptase catalytic subunit (hTERT).⁸² RNA trans-splicing group I intron ribozymes containing the HSV-tk gene as a 3′ exon have been designed to specifically target hTERT (reviewed by Lee et al.²⁵), with the incorporation of tissue-specific promoters such as the liver-specific phosphoenolpyruvate carboxykinase and apolipoprotein E endowing further specificity in vitro⁸³ and in vivo^84–86. Specificity has been further honed by developing hTERT-targeting RNA trans-splicing ribozymes regulated by hypoxia response elements,⁸⁷ and by liver-specific microRNAs, the latter of which produced efficient hepatocellular carcinoma regression in vivo⁸⁸. Kim et al.⁸⁹ reported an hTERT-targeting RNA trans-splicing ribozyme regulated exogenously by theophylline in vitro, pointing to a further level of control over the expression of therapeutic transgenes.

Fig. 4.

Mechanism of action of the HSV-tk/GCV suicide system. HSV-tk is delivered to and specifically expressed in target cells, followed by application of the inactive prodrug ganciclovir (GCV). HSV-tk and other endogenous enzymes phosphorylate GCV to produce the activated drug, which induces DNA replication chain termination and consequently cell death.

Besides hTERT, HSV-tk has been trans-spliced via ribozymes onto a number of other targets, including alpha-fetoprotein (AFP),⁹⁰ carcinoembryonic antigen (CEA),⁹¹ the splicing variant AIMP2-DX2,⁹² human cytoskeleton-associated protein 2 (hCKAP2) in vivo⁹³ and the KRAS-G12V mutant in vivo⁹⁴. In addition, SMART has been used in vivo to trans-splice HSV-tk onto SLCO1B3, a marker gene in EB-SCC⁹⁵, and Ct-OATP1B3, a cancer-specific transcript found in a number of tumours.⁹⁶

Infectious diseases

Ribozyme-mediated 3′ER has been used to trans-splice the diphtheria toxin (DTA) chain onto hepatitis C (HCV) mRNA and induce apoptosis in transfected human cells.⁹⁷ In order to specifically target HCV-infected cells, Nawtaisong et al.⁹⁸ modified apoptosis-inducing genes by replacing their endogenous cleavage sites with the HCV NS5A/5B cleavage recognition site, such that the apoptotic cascade would only be initiated in the presence of HCV protease in infected cells, and trans-spliced the modified constructs onto HCV mRNA using ribozyme-mediated 3′ER. This not only cleaves and destroys the target viral genome, but also induces cytotoxicity in infected cells, thus producing a two-pronged inhibition of viral replication in vitro. However, rapid simultaneous killing of HCV-infected cells may not be therapeutically desirable since it may cause widespread liver cell death.

Group I introns have also been used to target and suppress dengue virus (DENV) replication in mosquito cells by trans-splicing the pro-apoptotic Bax C-terminal domain onto DENV mRNA.^{99,  100} DENV shares a mosquito vector with the Chikungunya (CHIKV) virus, and cases of co-infection are rising; the same group engineered the DENV-targeting group I ribozyme so that it would also target the highly conserved NS1 region in the CHIKV genome,¹⁰¹ demonstrating for the first time the capability of a single dual-acting ribozyme to trans-splice two different RNA sequences.

Besides its applications in anticancer therapy, the HSV-tk/GCV suicide system has been used to induce selective killing of HPV-16¹⁰² and HIV-infected¹⁰³ human cells in vitro. Ingemarsdotter et al.¹⁰³ performed extensive bioinformatic analyses to identify target HIV splice sites and design HIV binding domains, and found that trans-splicing constructs targeting HIV donor site D4 for 3′ER and HIV acceptor sites A5, A7 and A8 for 5′ER successfully reduced the viability of HIV-expressing cells in the presence of ganciclovir, with the constructs targeting D4 and A8 being most efficient. Current HIV treatment inhibits viral replication but does not eradicate the viral reservoir that resides latently in memory T cells. Latency does not equate to total transcriptional inactivity for HIV. Thus, RNA trans-splicing techniques that selectively kill HIV-expressing cells may potentially contribute to a cure strategy which targets both actively replicating virus and latently infected cells.

Other therapeutic applications

SMaRT has been used to generate antibody-reporter enzyme fusion proteins¹⁰⁴ and to develop modular protein expression systems that allow the production of different antibody formats from the same phage display vector without clone reformatting.¹⁰⁵ Both SMaRT and RNA trans-splicing ribozymes have been used as molecular imaging tools, as reviewed by Wally et al.¹⁰⁶ and Lee et al.,²⁵ respectively.

Split intein protein trans-splicing has been widely used in biotechnological development, as reviewed by Sarmiento et al.,²⁷ Li²⁸ and Wood et al.²⁹ Protein trans-splicing has been used in protein labelling and ligation, such as in segmental isotopic labelling, where specific segment(s) within a protein are selectively labelled by stable isotopes in order to reduce nuclear magnetic resonance (NMR) signal overlap.¹⁰⁷ It has also been used to label proteins within living cells,^108–110 as well as in purification of recombinant proteins for research,^111–114 and in the purification of toxic proteins.¹¹⁵ Moreover, split inteins have been used as biosensors^{27,  29} and in targeting AAV vectors to specific cell types.¹¹⁶ Unmodified AAV vectors are not selective, so Muik et al.¹¹⁶ used split intein trans-splicing to couple targeting ligands to AAV capsid protein VP2, resulting in specific gene delivery into target receptor-positive cell types in vitro and in vivo.

Conclusion

Trans-splicing techniques have been used for a wide array of therapeutic applications targeting cancer, genetic and infectious diseases, and demonstrate numerous advantages compared to more traditional therapeutic approaches. Since only the therapeutic sequence needs to be delivered, rather than the full gene, trans-splicing techniques can overcome vector packaging limitations; segmental RNA trans-splicing and intein-mediated protein trans-splicing take this a step further by enabling even smaller gene fragments to be delivered separately and assembled within target cells. Off-target effects are reduced because RNA trans-splicing can only occur in cells where the target pre-mRNA is expressed. By correcting mutated mRNA to wild type, RNA trans-splicing not only induces therapeutic gene expression but also ablates disease-associated gene expression. In addition, it allows endogenous regulation of the transcript to be maintained, which is particularly important when constitutive expression has negative consequences: for example, gene transfer has been successful in correcting CD40L deficiency in HIGM1 model mice, but subsequently resulted in development of lymphoproliferative disease.¹¹⁷ A later trans-splicing study, on the other hand, found no evidence of lymphoproliferative disease after CD40L correction, emphasizing the importance of preserving regulated expression.⁵⁶

However, there are still some significant limitations. Firstly, non-specific trans-splicing, activation of cryptic splice sites and undesired splicing within the trans-splicing constructs themselves can all lead to off-target effects.⁴⁷ Furthermore, although functional and phenotypic correction has been achieved in some cases, trans-splicing efficacy in vivo is typically low. This makes it insufficient for repair of dominantly inherited diseases, and risks the escape of tumour cells from anticancer treatment as well as the development of escape mutants when used in antiviral therapy. Some diseases will require sustained expression of repaired mRNA, and in vivo delivery issues are still challenging. The use of viral vectors to deliver RNA trans-splicing constructs may have some limitations, as there are risks of insertional mutagenesis, cytopathic effects and undesired immune responses to the viral vector.¹¹⁸ Non-viral delivery methods that could feasibly be adapted for clinical practice include the biolistic gene gun approach investigated by Peking et al.,⁶⁶ where non-viral minicircle plasmids expressing 5’ PTM were delivered into murine skin and efficiently corrected mutant COL7A1 in vivo. This could potentially provide a pain-reduced alternative to local intradermal injections, which is of particular relevance to patients with epidermolysis bullosa.

Off-target effects may be reduced by screening trans-splicing constructs to exclude sequence homologies, as well as by more specific delivery to target cells. There are also several strategies that can be pursued to increase trans-splicing efficacy. Screening methods, such as the in vivo rescue assay described by Olson and Müller,¹¹⁹ have been developed to select the most efficient trans-splicing constructs, and in silico techniques have been used to aid rational design of RNA trans-splicing constructs.^{102,  103} Poddar et al.¹⁰² developed bispecific PTMs, targeting multiple disease markers, which increased the efficacy of the HSV/tk-GCV suicide system at triggering cell death, suggesting an avenue for future improvement of RNA trans-splicing activity and specificity. Moreover, antisense oligonucleotides have been used to competitively inhibit endogenous cis-splicing and thus improve trans-splicing efficacy.^{21,  34–37,  120} Overall, trans-splicing technology is a tool of great therapeutic potential with the capacity to target a diverse spectrum of disease, but there is still much progress to be made before trans-splicing therapy is ready to reach the clinic.

Data Availability Statement

No new data were generated or analysed in support of this review.

Conflict of Interest

Authors claim no conflict of interest.