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Persistence of HIV-1 in cellular reservoirs results in lifelong infection, with cure achieved only in rare cases through ablation of marrow-derived cells. We report on optimization of an approach that could potentially be aimed at eliminating these reservoirs, hijacking the HIV-1 alternative splicing process to functionalize the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) cell suicide system through targeted RNA trans-splicing at the HIV-1 D4 donor site. AUG1-deficient HSVtk therapeutic pre-mRNA was designed to gain an in-frame start codon from HIV-1 tat1. D4-targeting lentiviral vectors were produced and used to transduce HIV-1-expressing cells, where trans-spliced HIV-1 tat/HSVtk mRNA was successfully detected. However, translation of catalytically active HSVtk polypeptides from internal AUGs in HSVtkΔAUG1 caused GCV-mediated cytotoxicity in uninfected cells. Modifying these sites in the D4 opt 2 lentiviral vector effectively mitigated this major off-target effect. Promoter choice was optimized for increased transgene expression. Affinity for HIV-1 RNA predicted in silico correlated with the propensity of opt 2 payloads to induce HIV-1 RNA trans-splicing and killing of HIV-1-expressing cells with no significant effect on uninfected cells. Following latency reversing agent (LRA) optimization and treatment, 45% of lymphocytes in an HIV-1-infected latency model could be eliminated with D4 opt 2/GCV. Further development would be warranted to exploit this approach.

Background: Epidermolysis bullosa (EB) is a clinically-heterogeneous genodermatosis with severe manifestations in the skin and other organs. The significant burden this condition places on patients justifies the development of gene therapeutic strategies targeting the genetic cause of the disease., Methods: Emerging RNA and DNA editing tools have shown remarkable advances in efficiency and safety. Applicable both in ex vivo - and in vivo settings, these gene therapeutics based on gene replacement or editing are either at the pre-clinical or clinical stage., Results: The recent landmark FDA approvals for gene editing based on CRISPR/Cas9, along with the first FDA-approved redosable in vivo gene replacement therapy for EB, will invigorate ongoing research efforts, increasing the likelihood of achieving local cure via CRISPR-based technologies in the near future., Conclusions: This review discusses the status quo of current gene therapeutics that act at the level of RNA or DNA, all with the common aim of improving the quality of life for EB patients.

Persistence of HIV-1 in cellular reservoirs results in lifelong infection, with cure achieved only in rare cases through ablation of marrow-derived cells. We report on optimization of an approach that could potentially be aimed at eliminating these reservoirs, hijacking the HIV-1 alternative splicing process to functionalize the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) cell suicide system through targeted RNA trans -splicing at the HIV-1 D4 donor site. AUG1-deficient HSVtk therapeutic pre-mRNA was designed to gain an in-frame start codon from HIV-1 tat1 . D4-targeting lentiviral vectors were produced and used to transduce HIV-1-expressing cells, where trans -spliced HIV-1 tat / HSVtk mRNA was successfully detected. However, translation of catalytically active HSVtk polypeptides from internal AUGs in HSVtk ΔAUG1 caused GCV-mediated cytotoxicity in uninfected cells. Modifying these sites in the D4 opt 2 lentiviral vector effectively mitigated this major off-target effect. Promoter choice was optimized for increased transgene expression. Affinity for HIV-1 RNA predicted in silico correlated with the propensity of opt 2 payloads to induce HIV-1 RNA trans -splicing and killing of HIV-1-expressing cells with no significant effect on uninfected cells. Following latency reversing agent (LRA) optimization and treatment, 45% of lymphocytes in an HIV-1-infected latency model could be eliminated with D4 opt 2/GCV. Further development would be warranted to exploit this approach., Competing Interests: C.K.I. and A.M.L.L. are inventors on a patent application relating to parts of this work and have management roles with shareholdings in Spliceor Ltd. A.B.B. formerly consulted for Spliceor Ltd. F.K.-M. is an employee of Cambridge Design Partnership (Cambridge, UK) but contributed to the work exclusively in a personal capacity. S.H. contributed to the work while affiliated exclusively with the University of Cambridge but is now an employee of Gerson Lehrman Group., (© 2024 The Author(s).)

Spliceosome-mediated RNA trans-splicing enables correction or labeling of pre-mRNA, but therapeutic applications are hampered by issues related to the activity and target specificity of trans-splicing RNA (tsRNA). We employed computational RNA structure design to improve both on-target activity and specificity of tsRNA in a herpes simplex virus thymidine kinase/ganciclovir suicide gene therapy approach targeting alpha fetoprotein (AFP), a marker of hepatocellular carcinoma (HCC) or human papillomavirus type 16 (HPV-16) pre-mRNA. While unstructured, mismatched target binding domains significantly improved 3′ exon replacement (3’ER), 5′ exon replacement (5’ER) correlated with the thermodynamic stability of the tsRNA 3′ end. Alternative on-target trans-splicing was found to be a prevalent event. The specificity of trans-splicing with the intended target splice site was improved 10-fold by designing tsRNA that harbors secondary target binding domains shielding alternative on-target and blinding off-target splicing events. Such rationally designed suicide RNAs efficiently triggered death of HPV-16-transduced or hepatoblastoma-derived human tissue culture cells without evidence for off-target cell killing. Highest cell death activities were observed with novel dual-targeting tsRNAs programmed for trans-splicing toward AFP and a second HCC pre-mRNA biomarker. Our observations suggest trans-splicing represents a promising approach to suicide gene therapy.

Antiviral strategies targeting hijacked cellular processes are less easily evaded by the virus than viral targets. If selective for viral functions, they can have a high therapeutic index. We used RNA trans-splicing to deliver the herpes simplex virus thymidine kinase-ganciclovir (HSV-tk/GCV) cell suicide system into HIV-producing cells. Using an extensive in silico bioinformatics and RNA structural analysis approach, ten HIV RNA trans-splicing constructs were designed targeting eight different HIV splice donor or acceptor sites and were tested in cells expressing HIV. Trans-spliced mRNAs were identified in HIV-expressing cells using qRT-PCR with successful detection of fusion RNA transcripts between HIV RNA and the HSV-tk RNA transcripts from six of ten candidate RNA trans-splicing constructs. Conventional PCR and Sanger sequencing confirmed RNA trans-splicing junctions. Measuring cell viability in the presence or absence of GCV expression of HSV-tk by RNA trans-splicing led to selective killing of HIV-producing cells using either 3′ exon replacement or 5′ exon replacement in the presence of GCV. Five constructs targeting four HIV splice donor and acceptor sites, D4, A5, A7, and A8, involved in regulating the generation of multiple HIV RNA transcripts proved to be effective for trans-splicing mediated selective killing of HIV-infected cells, within which individual constructs targeting D4 and A8 were the most efficient.

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.

In recent years, RNA trans-splicing has emerged as a suitable RNA editing tool for the specific replacement of mutated gene regions at the pre-mRNA level. Although the technology has been successfully applied for the restoration of protein function in various genetic diseases, a higher trans-splicing efficiency is still desired to facilitate its clinical application. Here, we describe a modified, easily applicable, fluorescence-based screening system for the generation and analysis of antisense molecules specifically capable of improving the RNA reprogramming efficiency of a selected KRT14-specific RNA trans-splicing molecule. Using this screening procedure, we identified several antisense RNAs and short rationally designed oligonucleotides, which are able to increase the trans-splicing efficiency. Thus, we assume that besides the RNA trans-splicing molecule, short antisense molecules can act as splicing modulators, thereby increasing the trans-splicing efficiency to a level that may be sufficient to overcome the effects of certain genetic predispositions, particularly those associated with dominantly inherited diseases. [ABSTRACT FROM AUTHOR]

To date the Simian Virus 40 (SV40) is the only proven example of a virus that recruits the mechanism of RNA trans-splicing to diversify its sequences and gene products. Thereby, two identical viral transcripts are efficiently joined by homologous trans-splicing triggering the formation of a highly transforming 100 kDa super T antigen. Sequences of other viruses including HIV-1 and the human adenovirus type 5 were reported to be involved in heterologous trans-splicing towards cellular or viral sequences but the meaning of these events remains unclear. We computationally and experimentally investigated molecular features associated with viral RNA trans-splicing and identified a common pattern: Viral RNA trans-splicing occurs between strong cryptic or regular viral splice sites and strong regular or cryptic splice sites of the trans-splice partner sequences. The majority of these splice sites are supported by exonic splice enhancers. Splice sites that could compete with the trans-splicing sites for cis-splice reactions are weaker or inexistent. Finally, all but one of the trans-splice reactions seem to be facilitated by one or more complementary binding domains of 11 to 16 nucleotides in length which, however occur with a statistical probability close to one for the given length of the involved sequences. The chimeric RNAs generated via heterologous viral RNA trans-splicing either did not lead to fusion proteins or led to proteins of unknown function. Our data suggest that distinct viral RNAs are highly susceptible to trans-splicing and that heterologous viral trans-splicing, unlike homologous SV40 trans-splicing, represents a chance event.

Genetic disorders affecting the skin, genodermatoses, constitute a large and heterogeneous group of diseases, for which treatment is generally limited to management of symptoms. RNA-based therapies are emerging as a powerful tool to treat genodermatoses. In this review, we discuss in detail RNA splicing modulation by antisense oligonucleotides and RNA trans-splicing, transcript replacement and genome editing by in vitro-transcribed m RNAs, and gene knockdown by small interfering RNA and antisense oligonucleotides. We present the current state of these therapeutic approaches and critically discuss their opportunities, limitations and the challenges that remain to be solved. The aim of this review was to set the stage for the development of new and better therapies to improve the lives of patients and families affected by a genodermatosis. [ABSTRACT FROM AUTHOR]

Mutations in the COL7A1 gene lead to malfunction, reduction or complete absence of type VII collagen (C7) in the skin's basement membrane zone (BMZ), impairing skin integrity. In epidermolysis bullosa (EB), more than 800 mutations in COL7A1 have been reported, leading to the dystrophic form of EB (DEB), a severe and rare skin blistering disease associated with a high risk of developing an aggressive form of squamous cell carcinoma. Here, we leveraged a previously described 3'-RTMS6m repair molecule to develop a non-viral, non-invasive and efficient RNA therapy to correct mutations within COL7A1 via spliceosome-mediated RNA trans -splicing (SMaRT). RTM-S6m, cloned into a non-viral minicircle-GFP vector, is capable of correcting all mutations occurring between exon 65 and exon 118 of COL7A1 via SMaRT. Transfection of the RTM into recessive dystrophic EB (RDEB) keratinocytes resulted in a trans -splicing efficiency of ~1.5% in keratinocytes and ~0.6% in fibroblasts, as confirmed on mRNA level via next-generation sequencing (NGS). Full-length C7 protein expression was primarily confirmed in vitro via immunofluorescence (IF) staining and Western blot analysis of transfected cells. Additionally, we complexed 3'-RTMS6m with a DDC642 liposomal carrier to deliver the RTM topically onto RDEB skin equivalents and were subsequently able to detect an accumulation of restored C7 within the basement membrane zone (BMZ). In summary, we transiently corrected COL7A1 mutations in vitro in RDEB keratinocytes and skin equivalents derived from RDEB keratinocytes and fibroblasts using a non-viral 3'-RTMS6m repair molecule.

Spliceosome-mediated RNA trans-splicing has become an emergent tool for the repair of mutated pre-mRNAs in the treatment of genetic diseases. RNA trans-splicing molecules (RTMs) are designed to induce a specific trans-splicing reaction via a binding domain for a respective target pre-mRNA region. A previously established reporter-based screening system allows us to analyze the impact of various factors on the RTM trans-splicing efficiency in vitro. Using this system, we are further able to investigate the potential of antisense RNAs (AS RNAs), presuming to improve the trans-splicing efficiency of a selected RTM, specific for intron 102 of COL7A1. Mutations in the COL7A1 gene underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa (DEB). We have shown that co-transfections of the RTM and a selected AS RNA, interfering with competitive splicing elements on a COL7A1-minigene (COL7A1-MG), lead to a significant increase of the RNA trans-splicing efficiency. Thereby, accurate trans-splicing between the RTM and the COL7A1-MG is represented by the restoration of full-length green fluorescent protein GFP on mRNA and protein level. This mechanism can be crucial for the improvement of an RTM-mediated correction, especially in cases where a high trans-splicing efficiency is required. [ABSTRACT FROM AUTHOR]

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. Highlights: The marker gene SLCO1B3 can be targeted by RNA trans‐splicing molecules (RTMs).Accurate RNA trans‐splicing leads to the fusion of SLCO1B3 and HSV‐thymidine kinase.Screening system accelerates the construction of RTMs with improved functionality.The expression of the SLCO1B3‐HSV‐tk fusion protein induces apoptosis in target cells. [ABSTRACT FROM AUTHOR]

Introduction: The genodermatosis epidermolysis bullosa (EB) is a monogenetic disease, characterized by severe blister formation on the skin and mucous membranes upon minimal mechanical trauma. Causes for the disease are mutations in genes encoding proteins that are essential for skin integrity. In EB, one of these proteins is either functionally impaired or completely absent. Therefore, the development and improvement of DNA and RNA-based therapeutic approaches for this severe blistering skin disease is mandatory to achieve a treatment option for the patients., Areas Covered: Currently, there are several forms of DNA/RNA therapies potentially feasible for EB. Whereas some of them are still at the preclinical stage, others are clinically advanced and have already been applied to patients. In particular, this is the case for a cDNA replacement approach successfully applied for a small number of patients with junctional EB., Expert Opinion: The heterogeneity of EB justifies the development of therapeutic options with distinct modes of action at a DNA or RNA level. In addition, splicing-modulating therapies, based on RNA trans -splicing or short antisense oligonucleotides, especially designer nucleases, have steadily improved in efficiency and safety and thus likely represent the most promising gene therapy tool in the near future.

Abstract: RNA repair or reprogramming is a new avenue for human gene therapy. Unlike conventional gene therapy, in which exogenous cDNAs are introduced into cells, RNA repair approaches, which are based on spliceosome-mediated pre-mRNA trans-splicing, trans-splicing ribozymes, and tRNA-splicing endonuclease, allow the correction of endogenous RNA species. Recently published accounts that in vivo phenotypic correction of a variety of inherited diseases can be achieved by RNA repair are encouraging. Nevertheless, the science of RNA repair for treatment of human diseases is just beginning and faces several scientific and technical challenges that must be addressed and surmounted. In this review, we summarize recent advances in spliceosome-mediated pre-mRNA trans-splicing. We also provide an update on the progress of this emerging technology toward the development of molecular therapy and diagnosis for human diseases and discuss the outstanding issues and challenges confronting RNA therapeutics. [Copyright &y& Elsevier]

The humoral immune response propels the production of a diversified pool of antibodies with high affinity and selectivity for the eliciting antigen. Their isolation entails either B-cell cloning or the linking of authentic pairs of variable region genes encoding them. We hypothesized that targeted RNA trans-splicing (TS) inside the B-cell nucleus could be harnessed as a novel means to link both variable region genes and reconstitute genuine immune B-cell specificities. This could be accomplished by a special targeting gene harboring a peptide linker exon flanked by sequences capable of targeting both heavy (HC) and light chain (LC) transcripts. Following sequential trans-splicing reactions, the resulting RNA in each cell would encode the two variable regions, joined by the peptide linker. In this study, we examined genetic components and configurations required for the separate trans-splicing steps and for the combined two-step reactions. Using a model antibody, we show that in transiently transfected cells, we can target variable region exons through both their acceptor and donor splice sites, precisely joining an exon encoding a synthetic linker and the complementary variable region so as to form a single-chain Fv. We also demonstrate the accurate formation of single-chain Fv transcript as a result of trans-splicing of RNA synthesized from two chromosomal genes expressed by a stably transfected B-cell hybridoma. Our attempts to link the two variable region genes via a synthetic linker exon through sequential trans-splicing events were only successful with regard to both ends of the linker and to the 3′ end of the light chain, but repeatedly resulted in a deletion at the 5′ end of the joined heavy chain transcript. The implications of our findings on the potential application of trans-splicing for the isolation of useful antibodies are discussed. [Copyright &y& Elsevier]

Mutations within the COL7A1 gene underlie the inherited recessive subtype of the blistering skin disease dystrophic epidermolysis bullosa (RDEB). Although gene replacement approaches for genodermatoses are clinically advanced, their implementation for RDEB is challenging and requires endogenous regulation of transgene expression. Thus, we are using spliceosome-mediated RNA trans -splicing (SMaRT) to repair mutations in COL7A1 at the mRNA level. Here, we demonstrate the capability of a COL7A1 -specific RNA trans -splicing molecule (RTM), initially selected using a fluorescence-based screening procedure, to accurately replace COL7A1 exons 1 to 64 in an endogenous setting. Retroviral RTM transduction into patient-derived, immortalized keratinocytes resulted in an increase in wild-type transcript and protein levels, respectively. Furthermore, we revealed accurate deposition of recovered type VII collagen protein within the basement membrane zone of expanded skin equivalents using immunofluorescence staining. In summary, we showed for the first time the potential of endogenous 5' trans -splicing to correct pathogenic mutations within the COL7A1 gene. Therefore, we consider 5' RNA trans -splicing a suitable tool to beneficially modulate the RDEB-phenotype, thus targeting an urgent need of this patient population.

Spliceosome-mediated RNA trans-splicing enables correction or labeling of pre-mRNA, but therapeutic applications are hampered by issues related to the activity and target specificity of trans-splicing RNA (tsRNA). We employed computational RNA structure design to improve both on-target activity and specificity of tsRNA in a herpes simplex virus thymidine kinase/ganciclovir suicide gene therapy approach targeting alpha fetoprotein (AFP), a marker of hepatocellular carcinoma (HCC) or human papillomavirus type 16 (HPV-16) pre-mRNA. While unstructured, mismatched target binding domains significantly improved 3' exon replacement (3'ER), 5' exon replacement (5'ER) correlated with the thermodynamic stability of the tsRNA 3' end. Alternative on-target trans-splicing was found to be a prevalent event. The specificity of trans-splicing with the intended target splice site was improved 10-fold by designing tsRNA that harbors secondary target binding domains shielding alternative on-target and blinding off-target splicing events. Such rationally designed suicide RNAs efficiently triggered death of HPV-16-transduced or hepatoblastoma-derived human tissue culture cells without evidence for off-target cell killing. Highest cell death activities were observed with novel dual-targeting tsRNAs programmed for trans-splicing toward AFP and a second HCC pre-mRNA biomarker. Our observations suggest trans-splicing represents a promising approach to suicide gene therapy., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Sickle cell disease results from a point mutation in exon 1 of the β-globin gene (total 3 exons). Replacing sickle β-globin exon 1 (and exon 2) with a normal sequence by trans-splicing is a potential therapeutic strategy. Therefore, this study sought to develop trans-splicing targeting β-globin pre-messenger RNA among human erythroid cells. Binding domains from random β-globin sequences were comprehensively screened. Six candidates had optimal binding, and all targeted intron 2. Next, lentiviral vectors encoding RNA trans-splicing molecules were constructed incorporating a unique binding domain from these candidates, artificial 5' splice site, and γ-globin cDNA, and trans-splicing was evaluated in CD34 + cell-derived erythroid cells from healthy individuals. Lentiviral transduction was efficient, with vector copy numbers of 9.7 to 15.3. The intended trans-spliced RNA product, including exon 3 of endogenous β-globin and γ-globin, was detected at the molecular level. Trans-splicing efficiency was improved to 0.07-0.09% by longer binding domains, including the 5' splice site of intron 2. In summary, screening was performed to select efficient binding domains for trans-splicing. Detectable levels of trans-splicing were obtained for endogenous β-globin RNA in human erythroid cells. These methods provide the basis for future trans-splicing directed gene therapy.

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.

To date the Simian Virus 40 (SV40) is the only proven example of a virus that recruits the mechanism of RNA trans-splicing to diversify its sequences and gene products. Thereby, two identical viral transcripts are efficiently joined by homologous trans-splicing triggering the formation of a highly transforming 100 kDa super T antigen. Sequences of other viruses including HIV-1 and the human adenovirus type 5 were reported to be involved in heterologous trans-splicing towards cellular or viral sequences but the meaning of these events remains unclear. We computationally and experimentally investigated molecular features associated with viral RNA trans-splicing and identified a common pattern: Viral RNA trans-splicing occurs between strong cryptic or regular viral splice sites and strong regular or cryptic splice sites of the trans-splice partner sequences. The majority of these splice sites are supported by exonic splice enhancers. Splice sites that could compete with the trans-splicing sites for cis-splice reactions are weaker or inexistent. Finally, all but one of the trans-splice reactions seem to be facilitated by one or more complementary binding domains of 11 to 16 nucleotides in length which, however occur with a statistical probability close to one for the given length of the involved sequences. The chimeric RNAs generated via heterologous viral RNA trans-splicing either did not lead to fusion proteins or led to proteins of unknown function. Our data suggest that distinct viral RNAs are highly susceptible to trans-splicing and that heterologous viral trans-splicing, unlike homologous SV40 trans-splicing, represents a chance event.