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Effects of correcting aberrant splicing of the human CD22 gene using spliceosome-mediated RNA trans -splicing technology. (A and B) RT-PCR analyses of ALL-1 cells transfected with CD22 RTM vs. control plasmids ( i.e. , EPL: empty plasmid; DYST RTM: an RTM targeting the dystrophin gene unrelated to CD22). Total RNA for the PCR was extracted 48 h after transfection with the CD22 RTM or control plasmids. A1 depicts an agarose gel that shows the RT-PCR amplification of a 415-bp RTM-specific RNA-segment amplified by RT-PCR using a CD22 exon 11 primer as forward primer and the RTM IRES primer RTM-4494-5263 as a reverse primer, thereby confirming successful transfection of ALL-1 cells with the CD22 RTM. A2 depicts an agarose gel that shows the RT-PCR amplification of a 506-bp RTM-specific RNA-segment amplified by RT-PCR using a CD22 exon 10 primer as forward primer and an RTM IRES primer as the reverse primer (hCD22E10-Fwd 5 0 -ATCCTCATCCTGGCAATCTG-3 0 /IRES-R 5 0 -AAGCGGCTTCGGCCAGTAAC-3 0 ) as a further confirmation of
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Our recent studies have demonstrated that the CD22 exon 12 deletion (CD22ΔE12) is a characteristic genetic defect of therapy-refractory clones in pediatric B-precursor acute lymphoblastic leukemia (BPL) and implicated the CD22ΔE12 genetic defect in the aggressive biology of relapsed or therapy-refractory pediatric BPL. The purpose of the present st...
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... In an attempt to overcome the limitations of prior trans -splicing technologies that utilized an RNA-only approach, we first designed a CRISPR-guided trans -splicing system using our previously reported RNA-targeting Cas13d from Ruminococcus flavefaciens XPD3002 (CasRx) (Konermann et al., 2018) . We paired the catalytically inactive form of the enzyme, dCasRx, with a guided trans-splicing module (TSM): an RNA consisting of 1) a guide containing a CasRx direct repeat (DR) and a spacer with reverse complementarity to the target sequence (Konermann et al., 2018) ; 2) a minimal intron containing a stuffer region and splice sites for spliceosomal recognition (Uckun et al., 2015) ; and 3) a cargo sequence containing the desired exon to be trans -spliced ( Fig. 1B ). We sought to test the ability of dCasRx to enhance the efficiency and specificity of the system by targeting the TSM to the target pre-mRNA in a guide RNA-programmable manner. ...
... Previous attempts at trans -splicing reported in the literature achieved success with highly-expressed and/or simplified (i.e. minigene) reporter constructs but did not comprehensively or directly characterize efficiency or transcriptome-wide specificity when targeting endogenous transcripts (Kikumori et al., 2001;Liu et al., 2002;Mansfield et al., 2000;Murauer et al., 2011;Puttaraju et al., 1999;Tockner et al., 2016;Uckun et al., 2015) . Since endogenous targets are at the core of most potential applications of trans -splicing for research and medicine, we sought to move RESPLICE beyond an artificial reporter system to characterize its ability to trans -splice into endogenous human transcripts at the molecular level. ...
... For our reporter assay, the TSM plasmid contains a CasRx or Cas7-11 direct repeat (DR), a type IIS landing pad site, a stuffer sequence (Uckun et al., 2015) , splice motifs (Uckun et al., 2015) , the 3' half of the msfGFP coding sequence, a T2A ribosomal skip sequence, and full-length mTagBFP2 in Puc19 (Addgene Plasmid #50005), driven by a SFFV promoter (from plasmid #pFu-62 derived from Addgene Plasmid #57827, gift from Becky Xu Hua Fu). TSM spacers were sub-cloned into this plasmid using Golden Gate cloning. ...
Splicing bridges the gap between static DNA sequence and the diverse and dynamic set of protein products that execute a gene’s biological functions. While exon skipping technologies enable influence over splice site selection, many desired perturbations to the transcriptome require replacement or addition of exogenous exons to target mRNAs: for example, to replace disease-causing exons, repair truncated proteins, or engineer protein fusions. Here, we report the development of R NA-guid e d trans - spli cing with C as e ditor (RESPLICE), inspired by the rare, natural process of trans-splicing that joins exons from two distinct primary transcripts. RESPLICE uses two orthogonal RNA-targeting CRISPR effectors to co-localize a trans-splicing pre-mRNA and to inhibit the cis-splicing reaction, respectively. We demonstrate efficient, specific, and programmable trans-splicing of multi-kilobase RNA cargo into nine endogenous transcripts across two human cell types, achieving up to 45% trans-splicing efficiency in bulk, or 90% when sorting for high effector expression. Our results present RESPLICE as a new mode of RNA editing for fine-tuned and transient control of cellular programs without permanent alterations to the genetic code.
... Furthermore, forced overexpression of the mutant human CD22∆E12 in transgenic mice caused fatal B-ALL, demonstrating that CD22∆E12 alone may be sufficient as a driver lesion for the leukemic transformation and aggressive in vivo growth of BCPs [7,8]. We previously reported that CD22E12 mRNA expression levels, as measured via multiprobe transcriptome profiling using the microarray platform, are selectively and significantly reduced in B-ALL cells with the CD22∆E12 splicing defect [8][9][10][11][12][13][14]. Using Western blot analysis and RT-PCR, we demonstrated that CD22 exon 12 deletion is not observed in normal human pro-B and pre-pre-B cells [7,10]. ...
... Further, our comparison of matched-pair diagnostic vs. post-induction remission bone marrow specimens from B-ALL patients showed a marked reduction of CD22∆E12 mRNA levels after chemotherapy. These findings demonstrate that normal hematopoietic cells in the remission bone marrow of CD22∆E12 + B-ALL patients do not express the aberrant CD22∆E12 mRNA associated with the CD22∆E12 genetic defect [12]. ...
... Although CD22∆E12 was detected in a high percentage of newly diagnosed and relapsed B-ALL patients [7][8][9][10][11][12][13][14], its clinical significance has yet to be deciphered. The purpose of the present study was to evaluate the clinical prognostic significance of CD22∆E12 in B-ALL. ...
Simple Summary
We previously reported that cancer cells from the most common type of childhood cancer, namely, a form of acute leukemia known as B-ALL, are characterized by an abnormality known as CD22 exon 12 deletion. The purpose of the present study was to evaluate the clinical significance of the CD22 exon 12 deletion. Our findings provide the first evidence that CD22 exon 12 deletion is associated with a poor treatment outcome in B-ALL. The reported results also support the notion that the further evaluation of the clinical potential of new strategies targeting this abnormality in B-ALL is warranted.
Abstract
We previously reported a splicing defect (CD22ΔE12) associated with the deletion of exon 12 of the inhibitory co-receptor CD22 (Siglec-2) in leukemia cells from patients with CD19⁺ B-precursor acute lymphoblastic leukemia (B-ALL). CD22ΔE12 causes a truncating frameshift mutation and yields a dysfunctional CD22 protein that lacks most of the cytoplasmic domain required for its inhibitory function, and it is associated with aggressive in vivo growth of human B-ALL cells in mouse xenograft models. Although CD22ΔE12 with selective reduction of CD22 exon 12 (CD22E12) levels was detected in a high percentage of newly diagnosed as well as relapsed B-ALL patients, its clinical significance remains unknown. We hypothesized that B-ALL patients with very low levels of wildtype CD22 would exhibit a more aggressive disease with a worse prognosis because the missing inhibitory function of the truncated CD22 molecules could not be adequately compensated by competing wildtype CD22. Here, we demonstrate that newly diagnosed B-ALL patients with very low levels of residual wildtype CD22 (“CD22E12low”), as measured by RNAseq-based CD22E12 mRNA levels, have significantly worse leukemia-free survival (LFS) as well as overall survival (OS) than other B-ALL patients. CD22E12low status was identified as a poor prognostic indicator in both univariate and multivariate Cox proportional hazards models. CD22E12low status at presentation shows clinical potential as a poor prognostic biomarker that may guide the early allocation of risk-adjusted, patient-tailored treatment regimens and refine risk classification in high-risk B-ALL.
... Beside tumour suppressor defects, SMARTmediated repair of the CD22 E12 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. ...
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.
... Use of trans-splicing to inactivate deleterious gene products or to generate functional proteins has been investigated as a potential therapeutic strategy for multiple diseases. In particular, strategies have been developed which use engineered "pre-trans-splicing molecules" (PTMs) to promote specific trans-splicing events in host cells [7][8][9][10][11][12][13][14][15][16][17][18] . PTMs are engineered RNAs that include the exons to be spliced into the target mRNA and a splice acceptor site coupled with a binding domain, typically ≥50 nucleotides long, that is complementary to an intron in the target mRNA. ...
We present a plasmid-based system in which upstream trans-splicing efficiently generates mRNAs that encode head-to-tail protein multimers. In this system, trans-splicing occurs between one of two downstream splice donors in the sequence encoding a C-terminal V5 epitope tag and an upstream splice acceptor in the 5′ region of the pCS2(+) host plasmid. Using deletion and fusion constructs of the DUX4 protein as an example, we found that this system produced trans-spliced mRNAs in which coding regions from independent transcripts were fused in phase such that covalent head-to-tail protein multimers were translated. For a cDNA of ~450 bp, about half of the expressed proteins were multimeric, with the efficiency of trans-splicing and extent of multimer expression decreasing as cDNA length increased. This system generated covalent heterodimeric proteins upon co-transfections of plasmids encoding separate proteins and did not require a long complementary binding domain to position mRNAs for trans-splicing. This plasmid-based trans-splicing system is adaptable to multiple gene delivery systems, and it presents new opportunities for investigating molecular mechanisms of trans-splicing, generating covalent protein multimers with novel functions within cells, and producing mRNAs encoding large proteins from split precursors.
... Our recent studies have provided direct evidence that CD22ΔE12 is a characteristic genetic defect of therapy-refractory clones in pediatric BPL and implicated the CD22ΔE12 genetic defect in the aggressive biology of relapsed or therapy-refractory pediatric BPL [8] . More recent studies revealed a very high incidence of CD22ΔE12 in both pediatric and adult aggressive B-lineage lymphoid malignancies [9,10] . Examination of the CD22ΔE12 index in genetically defined high-risk BPL patient subsets revealed a high incidence of CD22ΔE12 in primary BPL cells from newly diagnosed high-risk BPL patients as well [9][10][11] . ...
... More recent studies revealed a very high incidence of CD22ΔE12 in both pediatric and adult aggressive B-lineage lymphoid malignancies [9,10] . Examination of the CD22ΔE12 index in genetically defined high-risk BPL patient subsets revealed a high incidence of CD22ΔE12 in primary BPL cells from newly diagnosed high-risk BPL patients as well [9][10][11] . Our studies using quantitative real time RT-PCR on samples obtained from 114 newly diagnosed pediatric ALL patients showed a high incidence of CD22ΔE12 in pediatric BPL and confirmed the presence of this genetic defect in 100% (14/14) of infant ALL cases [7][8][9][10][11] . ...
... Examination of the CD22ΔE12 index in genetically defined high-risk BPL patient subsets revealed a high incidence of CD22ΔE12 in primary BPL cells from newly diagnosed high-risk BPL patients as well [9][10][11] . Our studies using quantitative real time RT-PCR on samples obtained from 114 newly diagnosed pediatric ALL patients showed a high incidence of CD22ΔE12 in pediatric BPL and confirmed the presence of this genetic defect in 100% (14/14) of infant ALL cases [7][8][9][10][11] . ...
Aim:
CD22ΔE12 as an oncogenic driver lesion in aggressive and drug-resistant B-precursor acute lymphoblastic leukemia (BPL) cells. The purpose of the present study was to identify the CD22ΔE12-specific signature transcriptome in human BPL cells and evaluate the clinical potential of a nanoscale formulation of CD22ΔE12-siRNA as an RNAi therapeutic against drug-resistant BPL. CD22ΔE12-siRNA nanoparticles significantly improved the event-free survival (EFS) outcome of NOD/SCID (NS) mice challenged with human BPL xenograft cells.
Methods:
Gene expression and translational bioinformatics methods were applied to examine the expression of the CD22ΔE12-specific signature transcriptome in human BPL cells in subsets of BPL patients. Survival analysis for mice challenged with BPL cells and treated with CD22ΔE12 siRNA was performed using standard methods.
Results:
Leukemia cells from CD22ΔE12-Tg mice exhibit gene and protein expression profiles consistent with constitutive activation of multiple signaling networks, mimicking the profiles of relapsed BPL patients as well as newly diagnosed high-risk patients with BCR-ABL+/Philadelphia chromosome (Ph)+ BPL as well as Ph-like BPL. A nanoscale formulation of CD22ΔE12-siRNA abrogated the in vivo clonogenicity of the leukemia-initiating leukemic cell fraction in xenograft specimens derived from patients with relapsed BPL and significantly improved the EFS outcome of NS mice challenged with drug-resistant human BPL xenograft cells.
Conclusion:
The CD22-RNAi technology is applicable to all BPL patients both high risk and standard risk. That is because CD22ΔE12 is a characteristic feature of drug-resistant leukemic clones that escape chemotherapy and cause relapse in both high risk and low risk subgroups of patients. The technology therefore has the potential (1) for prevention of relapses by selectively killing the clones that are most likely to escape chemotherapy and cause relapse as well (2) for treatment of relapses in BPL. This research project may also lead to innovative salvage regimens against other forms of CD22ΔE12-positive relapsed B-lineage leukemias and lymphomas.
... Functional RNA interference (RNAi) experiments using CD22E12-specific siRNA and its nano scale formulations both in vitro and in vivo have confirmed the causal link between CD22E12 and the stemness features as well as aggressiveness and chemotherapy resistance of human B-lineage leukemia/ lymphoma cells. Notably, forced expression of the mutant CD22E12 protein in transgenic (Tg) mice under control of the immunoglobulin enhancer Eµ that is activated in early B-cell ontogeny prior to immunoglobulin gene rearrangements caused fatal B-lineage leukemia with lymphomatous features in C57/ BL/6 mice [30,31]. This Tg mouse model recapitulated the gene expression profile of CD22E12+ human B-lineage lymphoma and leukemia cells, indicating that CD22E12 alone as a driver lesion is sufficient for malignant transformation and clonal expansion of B-lineage lymphoid cells [29,30]. ...
... This Tg mouse model recapitulated the gene expression profile of CD22E12+ human B-lineage lymphoma and leukemia cells, indicating that CD22E12 alone as a driver lesion is sufficient for malignant transformation and clonal expansion of B-lineage lymphoid cells [29,30]. Leukemia cells from CD22E12-Tg mice exhibit characteristic gene expression and protein expression profiles consistent with constitutive activation of multiple signaling networks, including the WNT, PI3-Kinase and MAPK/SYK pathways, mimicking the profiles of aggressive human B-lineage leukemia/lymphoma cells [31,32]. ...
... We recently reported that the CD22E12 defect has been detected in 97% of MCL cases [31][32][33]. As shown in Figure 1, the CD22E12 transcriptome was strongly represented in primary lymphoma cells from MCL patients. ...
... Further advantages of using SMaRT include the maintenance of endogenous regulation of the targeted gene and the possibility to reduce dominant negative protein expression [6][7][8][9][10][11]. Moreover, RNA trans-splicing is not only suitable to correct transcripts in inherited diseases, but can also be used in a suicide therapy approach, e.g., to induce toxin-mediated cell death in tumour cells, as demonstrated by our group and others [12][13][14]. ...
RNA trans-splicing is a promising tool for mRNA modification in a diversity of genetic disorders. In particular, the substitution of internal exons of a gene by combining 3′ and 5′ RNA trans-splicing seems to be an elegant way to modify especially large pre-mRNAs. Here we discuss a robust method for designing double RNA trans-splicing molecules (dRTM). We demonstrate how the technique can be implemented in an endogenous setting, using COL7A1, the gene encoding type VII collagen, as a target. An RTM screening system was developed with the aim of testing the replacement of two internal COL7A1 exons, harbouring a homozygous mutation, with the wild-type version. The most efficient RTMs from a pool of randomly generated variants were selected via our fluorescence-based screening system and adapted for use in an in vitro disease model system. Transduction of type VII collagen-deficient keratinocytes with the selected dRTM led to accurate replacement of two internal COL7A1 exons resulting in a restored wild-type RNA sequence. This is the first study demonstrating specific exon replacement by double RNA trans-splicing within an endogenous transcript in cultured cells, corroborating the utility of this technology for mRNA repair in a variety of genetic disorders.
... Presently there is an unmet need for highly targeted therapies for infant leukemias to reduce toxicity of chemo/ radiation therapy and treat the rapid development of resistant clones of cancer cells. Data mining, bioinformatics, computational modeling and simulation have increasingly become integral to development of novel hypotheses in disease progression and potentially novel therapeutic innovations that target prominent receptors in leukemias (CD19 and CD22 receptors) to eradicate cancer cells [14][15][16][17][18][19][20]. Our project attempted to link data mining efforts that identify genetic signatures in high risk patient subgroups to data explorations methods developed using R made available in Bioconductor (http://www.bioconductor.org/) by writing interactive, exploration based statistical analysis platform provided by Shiny applications (http://shiny.rstudio.com/). ...
The Precision Medicine Initiative (PMI) aims to treat and prevent disease by accounting for individual variability in genes, environment, and lifestyle for each person. The initiative announced by President Obama aims to build the PMI Cohort Program that will compile a national research cohort of one million or more U.S. participants. Through technical advances in research capability for high throughput screening of genes/gene products, data sharing technology and informed consent policies that empowers patients, the PMI will usher in a new era of medicine in which researchers, providers and patients work together to develop individualized care. Paramount to successful delivery of this initiative will require trust and participation of the public and scientists being able to share data and protect the rights of the public. It is our contention that liberal arts colleges can fully participate in PMI by: 1. Taking advantage of open source and cloud platforms to access and analyze data obtained from both basic and clinical sciences; and 2. Providing an intellectually diverse student body to survey opinions and knowledge of modern genomic technologies in order to identify areas of public distrust and issues with informed consent arising from the implementation of these technologies. Furthermore, the curriculum at liberal arts colleges will enable deep discussion and exploration of moral, ethical, legal and scientific aspects of large scale initiatives such as PMI.
... , 2015a, 2010). Because CD22ΔE12 is associated with aggressive and chemo-refractory disease, the authors sought to repair this defect by using SMaRT technology to replace the exons 10-14 of the mutant pre-mRNA with the wildtype sequence using a rationally designed RTM (Uckun et al., 2015b). Transfection of the RTM into leukemiainitiating ALL cells significantly reduced their ability to cause leukemia in a xenograft animal model (Uckun et al., 2015a). ...
... 3 Uckun et al., 2014a;Uckun et al., 2015a). The incidence of CD22E12 is also high in adult aggressive B-lineage leukemias and lymphomas (uckun et al., 2015a). ...
... Functional RNA interference experiments using CD22E12-specific siRNA and its nanoscale formulations have confirmed the causal link between CD22E12 and the stemness features as well as aggressiveness and drug resistance of B-lineage ALL cells (Uckun et al., 2014;Uckun et al., 2015b). Our most recent preliminary studies have established the "undruggable" CD22E12 genetic defect can be repaired using RNA trans-splicing molecules (RTM) (Uckun et al., 2015a). The aim of the current study was to further assess the anti-leukemic activity of CD22-RTM on clonogenic B-lineage ALL cells and to characterize a unique multifunctional nanoparticle (NP) of CD22-RTM as a novel anti-ALL nanomedicine candidate. ...
... Bioinformatics and statistical analysis of gene expression profiles. The Exon 12 Index values were calculated by subtracting the median centered expression values of the Exons 10-11 plus Exons 13-14 probes from the median centered expression values of the Exon 12 probes, as previously reported (Uckun et al., 2014a;Uckun et al., 2015a). In order to determine the representation of the leading-edge genes of the CD22E12 transcriptome in the transcriptome of primary leukemia cells from relapsed BPL patients, the RMAnormalized gene expression values for leukemia cells obtained from 49 BPL patients in relapse (GSE28460) ...
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 indicates 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.
