mRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy.

Large genes including several CRISPR-Cas modules like gene activators (CRISPRa) require dual adeno-associated viral (AAV) vectors for an efficient in vivo delivery and expression. Current dual AAV vector approaches have important limitations, e.g., low reconstitution efficiency, production of alien proteins, or low flexibility in split site selection. Here, we present a dual AAV vector technology based on reconstitution via mRNA trans-splicing (REVeRT). REVeRT is flexible in split site selection and can efficiently reconstitute different split genes in numerous in vitro models, in human organoids, and in vivo. Furthermore, REVeRT can functionally reconstitute a CRISPRa module targeting genes in various mouse tissues and organs in single or multiplexed approaches upon different routes of administration. Finally, REVeRT enabled the reconstitution of full-length ABCA4 after intravitreal injection in a mouse model of Stargardt disease. Due to its flexibility and efficiency REVeRT harbors great potential for basic research and clinical applications.

Evidence of constraint in the 3D genome for trans-splicing in human cells.

Fusion transcripts are commonly found in eukaryotes, and many aberrant fusions are associated with severe diseases, including cancer. One class of fusion transcripts is generated by joining separate transcripts through trans-splicing. However, the mechanism of trans-splicing in mammals remains largely elusive. Here we showed evidence to support an intuitive hypothesis that attributes trans-sphcing to the spatial proximity between premature transcripts. A novel trans-splicing detection tool (TSD) was developed to reliably identify intra-chromosomal trans-splicing events (iTSEs) from RNA-seq data. TSD can maintain a remarkable balance between sensitivity and accuracy, thus distinguishing it from most state-of-the-art tools. The accuracy of TSD was experimentally demonstrated by excluding potential false discovery from mosaic genome or template switching during PCR. We showed that iTSEs identified by TSD were frequently found between genomic regulatory elements, which are known to be more prone to interact with each other. Moreover, iTSE sites may be more physically adjacent to each other than random control in the tested human lymphoblastoid cell line according to Hi-C data. Our results suggest that trans-splicing and 3D genome architecture may be coupled in mammals and that our pipeline, TSD, may facilitate investigations of trans-splicing on a systematic and accurate level previously thought impossible.

Small Animal PET Imaging of hTERT RNA-Targeted HSV1-tk Gene Expression with Trans-Splicing Ribozyme.

Background: Trans-splicing ribozymes (TSR) are useful anticancer agents targeting cancer-specific transcripts and replacing the RNA to induce anticancer gene expression specifically and selectively in cancer cells. Similar to other gene therapy methods, it is also important to evaluate the transgene expression for target specificity and ribozyme activity. Materials and Methods: In this study, the authors performed in vivo small animal positron emission tomography (PET) imaging and biodistribution assay to evaluate human telomerase reverse transcriptase (hTERT) RNA-targeting-specific TSR, which directs the expression of herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene selectively in hTERT-positive tumors through targeted RNA replacement of the hTERT transcript. Results: The hTERT RNA-targeted HSV1-tk expression with TSR was monitored by PET imaging with 124I labeled 2'-fluoro-2'-deoxy-1-ß-D-arabinofuranosyl-5-iodouracil, which is one of the thymidine derivatives acting as substrates for HSV1-tk, in hTERT-positive tumor-bearing mice. Conclusions: Imaging of hTERT RNA-targeted HSV1-tk expression by TSR could be used in the development of advanced gene therapy using tumor-specific TSR.

ChiTaRS 5.0: the comprehensive database of chimeric transcripts matched with druggable fusions and 3D chromatin maps.

Chimeric RNA transcripts are formed when exons from two genes fuse together, often due to chromosomal translocations, transcriptional errors or trans-splicing effect. While these chimeric RNAs produce functional proteins only in certain cases, they play a significant role in disease phenotyping and progression. ChiTaRS 5.0 (http://chitars.md.biu.ac.il/) is the latest and most comprehensive chimeric transcript repository, with 111 582 annotated entries from eight species, including 23 167 known human cancer breakpoints. The database includes unique information correlating chimeric breakpoints with 3D chromatin contact maps, generated from public datasets of chromosome conformation capture techniques (Hi-C). In this update, we have added curated information on druggable fusion targets matched with chimeric breakpoints, which are applicable to precision medicine in cancers. The introduction of a new section that lists chimeric RNAs in various cell-lines is another salient feature. Finally, using text-mining techniques, novel chimeras in Alzheimer's disease, schizophrenia, dyslexia and other diseases were collected in ChiTaRS. Thus, this improved version is an extensive catalogue of chimeras from multiple species. It extends our understanding of the evolution of chimeric transcripts in eukaryotes and contributes to the analysis of 3D genome conformational changes and the functional role of chimeras in the etiopathogenesis of cancers and other complex diseases.

Intein-mediated protein trans-splicing expands adeno-associated virus transfer capacity in the retina.

Retinal gene therapy with adeno-associated viral (AAV) vectors holds promises for treating inherited and noninherited diseases of the eye. Although clinical data suggest that retinal gene therapy is safe and effective, delivery of large genes is hindered by the limited AAV cargo capacity. Protein trans-splicing mediated by split inteins is used by single-cell organisms to reconstitute proteins. Here, we show that delivery of multiple AAV vectors each encoding one of the fragments of target proteins flanked by short split inteins results in protein trans-splicing and full-length protein reconstitution in the retina of mice and pigs and in human retinal organoids. The reconstitution of large therapeutic proteins using this approach improved the phenotype of two mouse models of inherited retinal diseases. Our data support the use of split intein-mediated protein trans-splicing in combination with AAV subretinal delivery for gene therapy of inherited blindness due to mutations in large genes.

Heterodera glycines utilizes promiscuous spliced leaders and demonstrates a unique preference for a species-specific spliced leader over C. elegans SL1.

Spliced leader trans-splicing (SLTS) plays a part in the maturation of pre-mRNAs in select species across multiple phyla but is particularly prevalent in Nematoda. The role of spliced leaders (SL) within the cell is unclear and an accurate assessment of SL occurrence within an organism is possible only after extensive sequencing data are available, which is not currently the case for many nematode species. SL discovery is further complicated by an absence of SL sequences from high-throughput sequencing results due to incomplete sequencing of the 5'-ends of transcripts during RNA-seq library preparation, known as 5'-bias. Existing datasets and novel methodology were used to identify both conserved SLs and unique hypervariable SLs within Heterodera glycines, the soybean cyst nematode. In H. glycines, twenty-one distinct SL sequences were found on 2,532 unique H. glycines transcripts. The SL sequences identified on the H. glycines transcripts demonstrated a high level of promiscuity, meaning that some transcripts produced as many as nine different individual SL-transcript combinations. Most uniquely, transcriptome analysis revealed that H. glycines is the first nematode to demonstrate a higher SL trans-splicing rate using a species-specific SL over well-conserved Caenorhabditis elegans SL-like sequences.

Discovery and characterization of novel trans-spliced products of human polyoma JC virus late transcripts from PML patients.

Although the human neurotropic polyomavirus, JC virus (JCV), was isolated almost a half century ago, understanding the molecular mechanisms governing its biology remains highly elusive. JCV infects oligodendrocytes and astrocytes in the central nervous system (CNS) and causes a rare fatal brain disease known as progressive multifocal leukoencephalopathy (PML) in immunocompromised individuals including AIDS. It has a small circular DNA genome (∼5 kb) and generates two primary transcripts from its early and late coding regions, producing several predicted alternatively spliced products mainly by cis-splicing. Here, we report the discovery and characterization of two novel open reading frames (ORF1 and ORF2) associated with JCV late transcripts, generated by an unusual splicing process called trans-splicing. These ORFs result from (i) the trans-splicing of two different lengths of the 5'-short coding region of VP1 between the coding regions of agnoprotein and VP2 after replacing the intron located between these two coding regions and (ii) frame-shifts occurring within the VP2 coding sequences terminated by a stop codon. ORF1 and ORF2 are capable of encoding 58 and 72 aa long proteins respectively and are expressed in infected cells and PML patients. Each ORF protein shares a common coding region with VP1 and has a unique coding sequence of their own. When the expression of the unique coding regions of ORFs is blocked by a stop codon insertion in the viral background, the mutant virus replicates less efficiently when compared to wild-type, suggesting that the newly discovered ORFs play critical roles in the JCV life cycle.

Intein-mediated backbone cyclization of VP1 protein enhanced protection of CVB3-induced viral myocarditis.

CVB3 is a common human pathogen to be highly lethal to newborns and causes viral myocarditis and pancreatitis in adults. However, there is no vaccine available for clinical use. CVB3 capsid protein VP1 is an immunodominant structural protein, containing several B- and T-cell epitopes. However, immunization of mice with VP1 protein is ineffective. Cyclization of peptide is commonly used to improve their in vivo stability and biological activity. Here, we designed and synthesizd cyclic VP1 protein by using engineered split Rma DnaB intein and the cyclization efficiency was 100% in E. coli. As a result, the cyclic VP1 was significantly more stable against irreversible aggregation upon heating and against carboxypeptidase in vitro and the degradation rate was more slowly in vivo. Compared with linear VP1, immunization mice with circular VP1 significantly increased CVB3-specific serum IgG level and augmented CVB3-specific cellular immune responses, consequently afforded better protection against CVB3-induced viral myocarditis. The cyclic VP1 may be a novel candidate protein vaccine for preventing CVB3 infection and similar approaches could be employed to a variety of protein vaccines to enhance their protection effect.

Toward RNA Repair of Diamond Blackfan Anemia Hematopoietic Stem Cells.

Diamond blackfan anemia (DBA) is a well-known inherited bone marrow failure syndrome mostly caused by mutations in ribosomal protein (RP) genes but also rarely in the hematopoietic transcription factor gene, GATA1, or TSR2, a ribosomal protein (Rps26) chaperone gene. About 25% of patients have heterozygous mutations in the RPS19 gene, which leads to haploinsufficiency of Rps19 protein in most cases. However, some RPS19 missense mutations appear to act in a dominant negative fashion. DBA typically leads to a hypoplastic anemia that becomes apparent during the first year of life, and standard treatment includes steroids or red blood cell transfusions, each modality having attendant side effects. The only curative therapy is allogeneic stem-cell transplantation, but this option is limited to patients with a histocompatible donor. DBA-mutant embryonic, induced pluripotent, and hematopoietic stem cells all exhibit growth abnormalities that can be corrected by DNA gene transfer, suggesting the possibility of ex vivo autologous gene therapy. The authors have been interested in the application of spliceosome-mediated mRNA trans-splicing (SMaRT) technology to RNA repair of DBA stem cells. Compared with gene replacement or other RNA re-programming approaches, SMaRT has several potential advantages. First, delivery of the entire normal cDNA is unnecessary, thus minimizing the overall size of the construct for packaging into a viral delivery vector. Second, RNA transcription of the corrected gene relies on the cell's endogenous transcriptional, processing, and regulatory machinery, thereby ensuring faithful and contextual expression. Third, RNA trans-splicing employs the endogenous spliceosome enzymatic machinery present in nearly all cells. Fourth, RNA trans-splicing converts mutant transcripts into therapeutically useful mRNA, and thus may be capable of treating disorders caused by dominant negative mutations. This review critically assesses prospects for both gene and RNA repair in DBA stem cells.

The Trans-Spliced Long Noncoding RNA tsRMST Impedes Human Embryonic Stem Cell Differentiation Through WNT5A-Mediated Inhibition of the Epithelial-to-Mesenchymal Transition.

The trans-spliced noncoding RNA RMST (tsRMST) is an emerging regulatory lncRNA in the human pluripotency circuit. Previously, we found that tsRMST represses lineage-specific transcription factors through the PRC2 complex and NANOG in human pluripotent stem cells (hESCs). Here, we demonstrate that tsRMST also modulates noncanonical Wnt signaling to suppress the epithelial-to-mesenchymal transition (EMT) and in vitro differentiation of embryonic stem cells (ESCs). Our results demonstrate that disruption of tsRMST expression in hESCs results in the upregulation of WNT5A, EMT, and lineage-specific genes/markers. Furthermore, we found that the PKC inhibitors Go6983 and Go6976 inhibited the effects of WNT5A, indicating that WNT5A promotes the EMT and in vitro differentiation although conventional and novel PKC activation in hESCs. Finally, we showed that either antiserum neutralization of WNT5A or Go6983 treatment in tsRMST knockdown cells decreased the expression of mesenchymal and lineage-specific markers. Together, these findings indicate that tsRMST regulates Wnt and EMT signaling pathways in hESCs by repressing WNT5A, which is a potential EMT inducer for promoting in vitro differentiation of hESCs through PKC activation. Our findings provide further insights into the role of trans-spliced RNA and WNT5A in hESC differentiation, in which EMT plays an important role. Stem Cells 2016;34:2052-2062.

Development of Polypeptide-based Nanoparticles for Non-viral Delivery of CD22 RNA Trans-splicing Molecule as a New Precision Medicine Candidate Against B-lineage ALL.

CD22ΔE12 has emerged as a driver lesion in the pathogenesis of pediatric B-lineage acute lymphoblastic leukemia (ALL) and a new molecular target for RNA therapeutics. Here we report a 43-gene CD22ΔE12 signature transcriptome that shows a striking representation in primary human leukemia cells from patients with relapsed BPL. Our data uniquely indicate that CD22ΔE12 is a candidate driver lesion responsible for the activation of MAPK and PI3-K pathways in aggressive forms of B-lineage ALL. We also show that the forced expression of a CD22 RNA trans-splicing molecule (RTM) markedly reduces the capacity of the leukemic stem cell fraction of CD22ΔE12(+) B-lineage ALL cells to engraft and cause overt leukemia in NOD/SCID mice. We have successfully complexed our rationally designed lead CD22-RTM with PVBLG-8 to prepare a non-viral nanoscale formulation of CD22ΔE12-RTM with potent anti-cancer activity against CD22ΔE12(+) B-lineage leukemia and lymphoma cells. CD22-RTM nanoparticles effectively delivered the CD22-RTM cargo into B-lineage ALL cells and exhibited significant anti-leukemic activity in vitro.

Trans-spliced Cas9 allows cleavage of HBB and CCR5 genes in human cells using compact expression cassettes.

CRISPR/Cas9 systems have been used in a wide variety of biological studies; however, the large size of CRISPR/Cas9 presents challenges in packaging it within adeno-associated viruses (AAVs) for clinical applications. We identified a two-cassette system expressing pieces of the S. pyogenes Cas9 (SpCas9) protein which splice together in cellula to form a functional protein capable of site-specific DNA cleavage. With specific CRISPR guide strands, we demonstrated the efficacy of this system in cleaving the HBB and CCR5 genes in human HEK-293T cells as a single Cas9 and as a pair of Cas9 nickases. The trans-spliced SpCas9 (tsSpCas9) displayed ~35% of the nuclease activity compared with the wild-type SpCas9 (wtSpCas9) at standard transfection doses, but had substantially decreased activity at lower dosing levels. The greatly reduced open reading frame length of the tsSpCas9 relative to wtSpCas9 potentially allows for more complex and longer genetic elements to be packaged into an AAV vector including tissue-specific promoters, multiplexed guide RNA expression, and effector domain fusions to SpCas9. For unknown reasons, the tsSpCas9 system did not work in all cell types tested. The use of protein trans-splicing may help facilitate exciting new avenues of research and therapeutic applications through AAV-based delivery of CRISPR/Cas9 systems.

Effective delivery of large genes to the retina by dual AAV vectors.

Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, AAV's limited cargo capacity prevents its application to therapies of inherited retinal diseases due to mutations of genes over 5 kb, like Stargardt's disease (STGD) and Usher syndrome type IB (USH1B). Previous methods based on 'forced' packaging of large genes into AAV capsids may not be easily translated to the clinic due to the generation of genomes of heterogeneous size which raise safety concerns. Taking advantage of AAV's ability to concatemerize, we generated dual AAV vectors which reconstitute a large gene by either splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybrid). We found that dual trans-splicing and hybrid vectors transduce efficiently mouse and pig photoreceptors to levels that, albeit lower than those achieved with a single AAV, resulted in significant improvement of the retinal phenotype of mouse models of STGD and USH1B. Thus, dual AAV trans-splicing or hybrid vectors are an attractive strategy for gene therapy of retinal diseases that require delivery of large genes.

Use of tumor-targeting trans-splicing ribozyme for cancer treatment.

One of the major concerns with regard to successful cancer gene therapy is to enhance both efficacy and safety. Gene targeting may represent an attractive tool to combat cancer cells without damage to normal cells. Here, we introduce a tumor-targeting approach with the Tetrahymena group I intron-based trans-splicing ribozyme, which cleaves target RNA and trans-ligate an exon tagged at the end of the ribozyme onto the downstream U nucleotide of the cleaved target RNA. We develop a specific trans-splicing ribozyme that can target and reprogram human cytoskeleton-associate protein 2 (hCKAP2)-encoding RNA to trigger therapeutic transgene herpes simplex virus thymidine kinase (HSVtk) selectively in cancer cells that express the RNA. Adenoviral vectors encoding the hCKAP2-specific trans-splicing ribozyme are constructed for in vivo delivery into either subcutaneous tumor xenograft or orthotopically multifocal hepatocarcinoma. We present analyses of the efficacy of the recombinant adenoviral vectors in terms of cancer retardation, target RNA and cell specificity, and in vivo toxicity.

The design and optimization of RNA trans-splicing molecules for skin cancer therapy.

Targeting tumor marker genes by RNA trans-splicing is a promising means to induce tumor cell-specific death. Using a screening system we designed RNA trans-splicing molecules (RTM) specifically binding the pre-mRNA of SLCO1B3, a marker gene in epidermolysis bullosa associated squamous cell carcinoma (EB-SCC). Specific trans-splicing, results in the fusion of the endogenous target mRNA of SLCO1B3 and the coding sequence of the suicide gene, provided by the RTM. SLCO1B3-specific RTMs containing HSV-tk were analyzed regarding their trans-splicing potential in a heterologous context using a SLCO1B3 expressing minigene (SLCO1B3-MG). Expression of the chimeric SLCO1B3-tk was detected by semi-quantitative RT-PCR and Western blot analysis. Cell viability and apoptosis assays confirmed that the RTMs induced suicide gene-mediated apoptosis in SLCO1B3-MG expressing cells. The lead RTM also showed its potential to facilitate a trans-splicing reaction into the endogenous SLCO1B3 pre-mRNA in EB-SCC cells resulting in tk-mediated apoptosis. We assume that the pre-selection of RTMs by our inducible cell-death system accelerates the design of optimal RTMs capable to induce tumor specific cell death in skin cancer cells.

A directed approach for the identification of transcripts harbouring the spliced leader sequence and the effect of trans-splicing knockdown in Schistosoma mansoni

Schistosomiasis is a major neglected tropical disease caused by trematodes from the genus Schistosoma. Because schistosomes exhibit a complex life cycle and numerous mechanisms for regulating gene expression, it is believed that spliced leader (SL) trans-splicing could play an important role in the biology of these parasites. The purpose of this study was to investigate the function of trans-splicing in Schistosoma mansoni through analysis of genes that may be regulated by this mechanism and via silencing SL-containing transcripts through RNA interference. Here, we report our analysis of SL transcript-enriched cDNA libraries from different S. mansoni life stages. Our results show that the trans-splicing mechanism is apparently not associated with specific genes, subcellular localisations or life stages. In cross-species comparisons, even though the sets of genes that are subject to SL trans-splicing regulation appear to differ between organisms, several commonly shared orthologues were observed. Knockdown of trans-spliced transcripts in sporocysts resulted in a systemic reduction of the expression levels of all tested trans-spliced transcripts; however, the only phenotypic effect observed was diminished larval size. Further studies involving the findings from this work will provide new insights into the role of trans-splicing in the biology of S. mansoni and other organisms. All Expressed Sequence Tags generated in this study were submitted to dbEST as five different libraries. The accessions for each library and for the individual sequences are as follows: (i) adult worms of mixed sexes (LIBEST_027999: JZ139310 - JZ139779), (ii) female adult worms (LIBEST_028000: JZ139780 - JZ140379), (iii) male adult worms (LIBEST_028001: JZ140380 - JZ141002), (iv) eggs (LIBEST_028002: JZ141003 - JZ141497) and (v) schistosomula (LIBEST_028003: JZ141498 - JZ141974).

The production of male-sterile wheat plants through split barnase expression is promoted by the insertion of introns and flexible peptide linkers.

The successful use of transgenic plants depends on the strong and stable expression of the heterologous genes. In this study, three introns (PSK7-i1 and PSK7-i3 from Petunia and UBQ10-i1 from Arabidopsis) were tested for their ability to enhance the tapetum-specific expression of a split barnase transgene. We also analyzed the effects of introducing multiple copies of flexible peptide linkers that bridged the fusion domains of the assembled protein. The barnase fragments were assembled into a functional cytotoxin via intein-mediated trans-splicing, thus leading to male sterility through pollen ablation. A total of 14 constructs carrying different combinations of introns and peptide linkers were transformed into wheat plants. The resulting populations (between 41 and 301 independent plants for each construct) were assayed for trait formation. Depending on which construct was used, there was an increase of up to fivefold in the proportion of plants exhibiting male sterility compared to the populations harboring unmodified constructs. Furthermore, the average barnase copy number in the plants displaying male sterility could be reduced. The metabolic profiles of male-sterile transgenic plants and non-transgenic plants were compared using gas chromatography-mass spectrometry. The profiles generated from leaf tissues displayed no differences, thus corroborating the anther specificity of barnase expression. The technical advances achieved in this study may be a valuable contribution for future improvement of transgenic crop systems.

The cystic fibrosis gene: a molecular genetic perspective.

The positional cloning of the gene responsible for cystic fibrosis (CF) was the important first step in understanding the basic defect and pathophysiology of the disease. This study aims to provide a historical account of key developments as well as factors that contributed to the cystic fibrosis transmembrane conductance regulator (CFTR) gene identification work. A redefined gene structure based on the full sequence of the gene derived from the Human Genome Project is presented, along with brief reviews of the transcription regulatory sequences for the CFTR gene, the role of mRNA splicing in gene regulation and CF disease, and, various related sequences in the human genome and other species. Because CF mutations and genotype-phenotype correlations are covered by our colleagues (Ferec C, Cutting GR. 2012. Assessing the disease-liability of mutations in CFTR. Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a009480), we only attempt to provide an introduction of the CF mutation database here for reference purposes.

A widespread and unusual RNA trans-splicing type in dinoflagellate mitochondria.

Cytochrome oxidase subunit 3 (Cox3) is a mitochondrion-encoded core membrane protein of complex IV of the mitochondrial respiratory chain, and consists of seven trans-membrane helices. Here we show that in diverse later-branching dinoflagellates, cox3 is consistently split into two exons in the mitochondrial genome between helices six and seven. Gene exons are transcribed as two discrete oligoadenylated precursor RNAs, and these are subsequently trans-spliced to form a complete coding mRNA. This trans-splicing is highly unusual in that some of the oligoadenylated tail is incorporated at the splice site, such that a short string of adenosines links the two coding exons. This feature is consistently represented in diverse dinoflagellates, however the number of adenosines added varies according to the size of the coding gap between the two exons. Thus we observed between zero (Amphidinium carterae) and 10 (Symbiodinium sp.) adenosines added in different taxa, but the final coding sequence length is identical with the reading frame maintained. Northern analyses show that precursor cox3 transcripts are approximately equally abundant as mature cox3 mRNAs, suggesting a slow or regulated maturation process. These data indicate that the splicing mechanism in dinoflagellate mitochondria is tolerant of variations in the length of the precursor coding sequence, and implicates the use of a splicing template, or guide molecule, during splicing that controls mature mRNA length.