Liver gene therapy with intein‐mediated F8 trans ‐splicing corrects mouse haemophilia A
2022; Springer Nature; Volume: 14; Issue: 6 Linguagem: Inglês
10.15252/emmm.202115199
ISSN1757-4684
AutoresFederica Esposito, Hristiana Lyubenova, Patrizia Tornabene, Stefano Auricchio, Antonella Iuliano, Edoardo Nusco, Simone Merlin, Cristina Olgasi, Giorgia Manni, Marco Gargaro, Francesca Fallarino, Antonia Follenzi, Alberto Auricchio,
Tópico(s)RNA Interference and Gene Delivery
ResumoArticle2 May 2022Open Access Source DataTransparent process Liver gene therapy with intein-mediated F8 trans-splicing corrects mouse haemophilia A Federica Esposito Federica Esposito orcid.org/0000-0002-8521-4627 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Hristiana Lyubenova Hristiana Lyubenova orcid.org/0000-0002-5590-3835 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Contribution: Data curation, Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Patrizia Tornabene Patrizia Tornabene orcid.org/0000-0002-0700-3007 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Contribution: Investigation, Methodology, Writing - review & editing Search for more papers by this author Stefano Auricchio Stefano Auricchio Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Contribution: Validation, Investigation, Writing - review & editing Search for more papers by this author Antonella Iuliano Antonella Iuliano orcid.org/0000-0001-8541-8120 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Contribution: Formal analysis, Writing - review & editing Search for more papers by this author Edoardo Nusco Edoardo Nusco Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Contribution: Resources, Investigation, Writing - review & editing Search for more papers by this author Simone Merlin Simone Merlin orcid.org/0000-0001-5209-3921 Department of Health Sciences, University of Piemonte Orientale “Amedeo Avogadro”, Novara, Italy Contribution: Resources, Supervision, Writing - review & editing Search for more papers by this author Cristina Olgasi Cristina Olgasi orcid.org/0000-0002-5738-213X Department of Health Sciences, University of Piemonte Orientale “Amedeo Avogadro”, Novara, Italy Contribution: Resources, Supervision, Writing - review & editing Search for more papers by this author Giorgia Manni Giorgia Manni orcid.org/0000-0002-5697-2648 Department of Medicine and Surgery, University of Perugia, Perugia, Italy Contribution: Data curation, Investigation, Writing - review & editing Search for more papers by this author Marco Gargaro Marco Gargaro orcid.org/0000-0003-0645-9800 Department of Medicine and Surgery, University of Perugia, Perugia, Italy Contribution: Resources, Supervision, Writing - review & editing Search for more papers by this author Francesca Fallarino Francesca Fallarino orcid.org/0000-0002-8501-2136 Department of Medicine and Surgery, University of Perugia, Perugia, Italy Contribution: Resources, Supervision, Writing - review & editing Search for more papers by this author Antonia Follenzi Antonia Follenzi orcid.org/0000-0001-9780-300X Department of Health Sciences, University of Piemonte Orientale “Amedeo Avogadro”, Novara, Italy Contribution: Resources, Supervision, Writing - review & editing Search for more papers by this author Alberto Auricchio Corresponding Author Alberto Auricchio [email protected] orcid.org/0000-0002-0832-2472 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Medical Genetics, Department of Advanced Biomedical Sciences, Federico II University, Naples, Italy Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Federica Esposito Federica Esposito orcid.org/0000-0002-8521-4627 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Hristiana Lyubenova Hristiana Lyubenova orcid.org/0000-0002-5590-3835 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Contribution: Data curation, Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Patrizia Tornabene Patrizia Tornabene orcid.org/0000-0002-0700-3007 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Contribution: Investigation, Methodology, Writing - review & editing Search for more papers by this author Stefano Auricchio Stefano Auricchio Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Contribution: Validation, Investigation, Writing - review & editing Search for more papers by this author Antonella Iuliano Antonella Iuliano orcid.org/0000-0001-8541-8120 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Contribution: Formal analysis, Writing - review & editing Search for more papers by this author Edoardo Nusco Edoardo Nusco Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Contribution: Resources, Investigation, Writing - review & editing Search for more papers by this author Simone Merlin Simone Merlin orcid.org/0000-0001-5209-3921 Department of Health Sciences, University of Piemonte Orientale “Amedeo Avogadro”, Novara, Italy Contribution: Resources, Supervision, Writing - review & editing Search for more papers by this author Cristina Olgasi Cristina Olgasi orcid.org/0000-0002-5738-213X Department of Health Sciences, University of Piemonte Orientale “Amedeo Avogadro”, Novara, Italy Contribution: Resources, Supervision, Writing - review & editing Search for more papers by this author Giorgia Manni Giorgia Manni orcid.org/0000-0002-5697-2648 Department of Medicine and Surgery, University of Perugia, Perugia, Italy Contribution: Data curation, Investigation, Writing - review & editing Search for more papers by this author Marco Gargaro Marco Gargaro orcid.org/0000-0003-0645-9800 Department of Medicine and Surgery, University of Perugia, Perugia, Italy Contribution: Resources, Supervision, Writing - review & editing Search for more papers by this author Francesca Fallarino Francesca Fallarino orcid.org/0000-0002-8501-2136 Department of Medicine and Surgery, University of Perugia, Perugia, Italy Contribution: Resources, Supervision, Writing - review & editing Search for more papers by this author Antonia Follenzi Antonia Follenzi orcid.org/0000-0001-9780-300X Department of Health Sciences, University of Piemonte Orientale “Amedeo Avogadro”, Novara, Italy Contribution: Resources, Supervision, Writing - review & editing Search for more papers by this author Alberto Auricchio Corresponding Author Alberto Auricchio [email protected] orcid.org/0000-0002-0832-2472 Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy Medical Genetics, Department of Advanced Biomedical Sciences, Federico II University, Naples, Italy Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Federica Esposito1, Hristiana Lyubenova1, Patrizia Tornabene1, Stefano Auricchio1, Antonella Iuliano1, Edoardo Nusco1, Simone Merlin2, Cristina Olgasi2, Giorgia Manni3, Marco Gargaro3, Francesca Fallarino3, Antonia Follenzi2 and Alberto Auricchio *,1,4 1Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy 2Department of Health Sciences, University of Piemonte Orientale “Amedeo Avogadro”, Novara, Italy 3Department of Medicine and Surgery, University of Perugia, Perugia, Italy 4Medical Genetics, Department of Advanced Biomedical Sciences, Federico II University, Naples, Italy *Corresponding author. Tel: +39 081 19230605; E-mail: [email protected] EMBO Mol Med (2022)14:e15199https://doi.org/10.15252/emmm.202115199 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Liver gene therapy with adeno-associated viral (AAV) vectors is under clinical investigation for haemophilia A (HemA), the most common inherited X-linked bleeding disorder. Major limitations are the large size of the F8 transgene, which makes packaging in a single AAV vector a challenge, as well as the development of circulating anti-F8 antibodies which neutralise F8 activity. Taking advantage of split-intein-mediated protein trans-splicing, we divided the coding sequence of the large and highly secreted F8-N6 variant in two separate AAV-intein vectors whose co-administration to HemA mice results in the expression of therapeutic levels of F8 over time. This occurred without eliciting circulating anti-F8 antibodies unlike animals treated with the single oversized AAV-F8 vector under clinical development. Therefore, liver gene therapy with AAV-F8-N6 intein should be considered as a potential therapeutic strategy for HemA. Synopsis Liver directed AAV-intein mediated protein trans-splicing results in stable therapeutic levels of F8 in mice thus representing a novel therapeutic strategy for haemophilia A. Split-intein mediated protein trans-splicing (PTS) allows reconstitution of large proteins via adeno-associated viral (AAV) vectors in mouse liver. The highly secreted and active F8-N6 (N6) variant has been adapted to fit into AAV intein vectors. A single systemic injection of AAV-N6 intein targets liver of hemophilic mice resulting in stable therapeutic levels of F8 without eliciting anti-F8 antibodies at the vector doses used. The paper explained Medical issue With an incidence of 1 in 5,000 males, haemophilia A (HemA) is the most common inherited bleeding disorder which is caused by the deficiency of coagulation factor VIII (F8). The size of F8 coding sequence exceeds the cargo capacity of adeno-associated viral (AAV) vectors; therefore, current HemA gene therapy clinical trials use AAV with oversize genomes. Results We tested if intein-mediated protein trans-splicing (PTS) allows to reconstitute F8 in mouse liver transduced by AAV, thus overcoming the limitations imposed by the vector transfer capacity. We show that AAV-intein reconstitute F8 to therapeutic levels in HemA mice and this occurs without the development of anti-F8 antibodies at the vector doses tested. Clinical impact Liver gene therapy with AAV-intein represents a potential therapeutic strategy for HemA using relatively low doses of vectors with defined genomes which do not elicit anti-F8 antibodies. Introduction Haemophilia A (HemA) is the most common inherited X-linked recessive coagulation disorder caused by the partial or complete deficiency of coagulation F8. F8 activity levels are inversely proportional to bleeding risk; severely affected patients (about 50% of all cases) have circulating protein levels of < 1% (Antonarakis et al, 1995; White et al, 2001; Bowen, 2002). Levels of F8 activity between 1 and 5% result in a moderate phenotype, levels between 5 and 50% give a mild phenotype, and levels above 50% are associated with normal haemostasis (White et al, 2001). The current management of HemA involves prophylactic administration of recombinant or plasma-derived F8. Lifelong intravenous infusions are required as often as 2–3 times weekly in severely affected patients. The chances of developing neutralising anti-F8 antibodies (inhibitors) remain high with about 30% of patients having to discontinue treatment (Cafuir & Kempton, 2017). This is potentially overcome by the recently approved bispecific antibody Emicizumab, which has significantly broadened the treatment options for HemA, allowing treatment of even younger patients which was previously unfeasible with standard care (Mahlangu et al, 2018; Oldenburg et al, 2018). Emicizumab is injected subcutaneously every 1–4 weeks with almost no bleeding occurring in the majority of patients. However, the management of spontaneous bleeds on Emicizumab still requires standard infusions (Butterfield et al, 2019). Regardless of the various existing treatment options, the possibility of curing HemA rather than managing the disease remains the hope of many patients. In the last two decades, gene therapy for HemA has been under extensive investigation after it was observed that even modest improvements in the F8 levels (by 1–2%) can significantly reduce the risk of spontaneous bleeding events and the need for F8 replacement infusions (Manco-Johnson et al, 2007). Adeno-associated viral (AAV) vectors have emerged as the most promising in vivo gene therapy approach for HemA, because of their excellent safety profile and their ability to direct long-term transgene expression from post-mitotic tissues such as the liver (Nathwani et al, 2004, 2017). However, HemA poses a great challenge to AAV gene therapy because of the size (7 kb) of the F8 coding sequence (CDS) to be transferred which exceeds the canonical AAV cargo capacity of ~4.7 kb. For this reason, all of the AAV-based products under clinical investigation consist of B-domain-deleted (BDD) versions of the F8 transgene, which are ~4.4 kb in size (Makris, 2020). However, using a transgene of this size leaves limited space in the vector for the necessary regulatory elements, thus restricting the choice of promoters and polyA signals. Moreover, all these vector genomes are on the verge of AAV’s normal cargo capacity and are thus at risk of being improperly packaged as a library of heterogeneous truncated genomes. Despite the ability of such oversize vectors to successfully express large proteins, their long-term efficiency and safety are yet to be confirmed (Grieger & Samulski, 2005; Dong et al, 2010; Hirsch et al, 2010; Wu et al, 2010). As an alternative, different groups have explored strategies based on co-delivery of dual AAV vectors to reconstitute F8. Each vector encodes for one of the two chains, which should then re-associate and produce the biologically active heterodimer F8. However, the main drawback of this approach is the apparent chain imbalance, which derives from less efficient secretion of the heavy chain than the light one. This results in the production of higher amounts of inactive protein compared with full-length F8 (Burton et al, 1999; Scallan et al, 2003; Chen et al, 2009; Zhu et al, 2012). More recently, protein trans-splicing (PTS) has been evaluated to reconstitute large proteins via AAV vectors. PTS is used by unicellular organisms across all three domains of life to reconstitute large proteins from shorter precursors that include split-inteins (intervening proteins) at their extremities. Following translation, split-inteins mediate their association and self-excision from the host protein in an independent process that does not require any energy supply (Mills et al, 2014; Li, 2015). PTS has been used with limited success to reconstitute F8 (Chen et al, 2007), while we have recently shown that AAV-intein-mediated PTS in the retina results in therapeutic levels of protein reconstitution which in some instances match those achieved by single AAV vectors (Tornabene et al, 2019). Encouraged by these results, we explored the efficiency of liver gene therapy with AAV-intein vectors to reconstitute the large and highly active F8-N6 variant (N6) (5 kb) (Miao et al, 2004; Ward et al, 2011). Results AAV-intein-mediated protein trans-splicing in mouse liver We assessed the potential of split-intein-mediated protein trans-splicing in liver by comparing the efficiency of adeno-associated viral (AAV) vector intein to that of a single AAV vector. To do so, we used the reporter enhanced green fluorescent protein (eGFP) whose coding sequence (CDS) fits well within a single AAV. The full-length eGFP CDS was divided into two separate halves at Cysteine 71 and fused to either the N- and C-terminal halves of the DnaE split-inteins from Nostoc punctiforme (Npu; Iwai et al, 2006) (Fig 1A). These were cloned under the human thyroxin binding globulin (TBG) liver promoter (Yan et al, 2012) and separately packaged into AAV8 vectors that efficiently target liver. A single AAV8 vector carrying the full-length eGFP CDS under the same TBG promoter was produced for the in vivo comparison. Five-week-old C57/BL6 mice were injected retro-orbitally with either the single or the two AAV-intein vectors at the dose of 5 × 1011 GC of each vector per animal. Livers were harvested 4-week post-injection (4 wpi) and Western blot (WB) analysis of liver lysates followed by quantification of the eGFP bands showed that AAV-intein reconstitute about 76% of the full-length eGFP protein produced by the single vector (Fig 1B and C). Figure 1. Intein-mediated protein trans-splicing in liver Schematic representation of the enhanced green fluorescent protein (eGFP) intein constructs and of the intein-mediated protein trans-splicing. ITR—inverted terminal repeats; Prom—promoter; 5’eGFP—5’eGFP coding sequence (CDS); n-intein—N-terminal of DnaE intein; star symbol—3xFlag tag; PolyA—short synthetic polyadenylation signal; c-intein—C-terminal of DnaE intein; 3’eGFP—3’ eGFP CDS. Western blot analysis of liver lysates (100 µg) shows that intein-mediated protein trans-splicing efficiently reconstitutes full-length eGFP. Single AAV: n = 5; AAV-intein: n = 5. Arrows indicate both full-length eGFP and excised inteins. Quantification of eGFP protein bands. Values are reported as mean ± SEM. Each dot represents the eGFP protein band quantification from animals injected with either single AAV n = 5 or AAV-intein n = 5. Source data are available online for this figure. Source Data for Figure 1 [emmm202115199-sup-0003-SDatafig1.xlsx] Download figure Download PowerPoint In vitro characterisation of human F8 variants After assessing that AAV-intein transduce efficiently liver, we set up to select the best F8 transgenic variant to be expressed via AAV-intein vectors. To this end, we compared the wild-type F8 coding sequence (CDS) to 3 commonly used B-domain-deleted (BDD, which lack F8 amino acids from 740 to 1649 (Miao et al, 2004) versions (Figs 2A and EV1 for exact amino acid differences). Specifically, the 3 BDD constructs carry different codon-optimised linkers in the place of the B domain, which are designed to promote efficient F8 secretion by mimicking some of the natural F8 post-translational modifications (Miao et al, 2004): F8-N6 (N6) contains 11 amino acids from the modified SQ activation peptide (SQm) from Ward et al (2011), followed by the N6 human B domain spacer involving 6 (N)-linked glycosylation sites (Miao et al, 2004); F8-SQ (SQ) contains the original SQ linker described by Sandberg et al (2001). This variant is available for clinical use as a replacement recombinant F8 product (ReFacto, Wyeth Pharma; Toole et al, 1986) and is also under investigation in more than one AAV gene therapy clinical trial (Butterfield et al, 2019). The F8-V3 variant (V3) consists of a small 17-aa peptide, which contains the original 6 (N)-linked glycosylation triplets from the N6 inserted into and flanked by the SQ linker (McIntosh et al, 2013). This variant has been previously described as another small version of BDD F8, able to achieve high levels of F8 activity in mice and non-human primates (McIntosh et al, 2013) as well as in human subjects (Nathwani et al, 2018). Figure 2. Comparison of human F8 variants in vitro Schematic representation of the four different F8 variants that were cloned into an AAV plasmid: wild-type F8; N6 containing 11 amino acids of the modified SQ amino acid linker (SQm) followed by the human N6 B domain; SQ containing the SQ amino acid linker; V3 containing the V3 peptide in the middle of the SQ linker. ITR—inverted terminal repeats; CMV—cytomegalovirus promoter; star symbol—3xFlag tag; PolyA—short synthetic polyadenylation signal; Ntds—nucleotides; SP—signal peptide. Details on the exact amino acid differences in the B domain can be found in Fig EV1. Western blot analysis of lysates of HEK293 cells 72-h post-transfection with the various F8 variants. Neg—non-transfected cells. Chromogenic assay of F8 activity in the medium of transfected cells is reported as International Units/decilitre (IU/dl). Data are presented as mean ± SEM. Each dot within the same group corresponds to a biological replicate: Neg n = 5; Wild-type n = 8; N6 n = 8; SQ n = 7; V3 n = 6. Significant differences between groups were assessed using the Kruskal–Wallis test followed by the post hoc analysis: Nemenyi’s All-Pairs Rank Comparison Test. The Kruskal–Wallis test P = 2.88e-05. **indicates the significant difference between the N6 and the Wild-type groups: P ≤ 0.01. *indicates the significant difference between the V3 and the Wild-type groups: P ≤ 0.05. All P-values are reported in Appendix Table S1. Source data are available online for this figure. Source Data for Figure 2 [emmm202115199-sup-0004-SDatafig2.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Amino acid sequence alignment underlining the differences in the B domain of the F8 variants The amino acid (aa) sequences of the B domain (or substitution linkers corresponding to it) of the F8 variants were aligned using the Clustal Omega software. a2 acidic linker of the F8 protein spans from aa position 720 to 740; the entire B domain or its substitutions are shown between aa 741 and 1659; a3 acidic linker spans from aa 1660 to 1702. Full-length, wild-type F8; N6, BDD F8-N6; SQ, BDD F8-SQ; V3, BDD F8-V3. Download figure Download PowerPoint All four variants were independently cloned into an AAV backbone plasmid under the control of the CMV promoter, including both a short synthetic polyadenylation signal (Levitt et al, 1989) and a triple flag tag (3xFlag) to allow for easy detection of the proteins. The constructs were tested by transient transfection in the human embryonic kidney cell line 293 (HEK293). WB analysis of the cell lysates 72-h post-transfection (hpt) revealed bands of the expected size (Fig 2B). To detect the biological activity of each variant, cells were cultured for 12 h following transfection, after which they were kept in serum-free medium until the time point of 72 hpt when F8 activity was measured by chromogenic assay on media of transfected cells. All variants produced detectable F8 activity levels (Fig 2C). Wild-type F8 had fairly low mean levels of activity of 8.4 International Unit/decilitre (IU/dl), followed by SQ with 27.4 IU/dl, V3 with 57.6 IU/dl and N6 with the highest mean levels of 71.6 IU/dl. There was a significant difference in potency of the wild-type F8 and both the N6 and V3 constructs, which was determined by the Kruskal–Wallis rank-sum test; this difference was more significant for the N6 (**P ≤ 0.001) than for the V3 variant (*P ≤ 0.05). In addition, enzyme-linked immunosorbent assay (ELISA) analysis of media from transfected cells showed that N6 is the most secreted F8 variant (Fig EV2). Based on this, we selected N6 as the variant to be tested by AAV-intein in comparison with one of the traditional single AAV replacement gene therapy, which is under clinical investigation (NCT03001830). Click here to expand this figure. Figure EV2. F8 antigen quantification in the media of transfected HEK293 cells Enzyme-linked immunosorbent assay (ELISA) for F8 antigen performed on medium of HEK293 cells transfected with plasmid encoding for each hF8 variant (n = 3 biological replicates). F8 antigen levels are reported as International Units/millilitres (IU/ml). Data are represented as mean ± SEM. Source data are available online for this figure. Download figure Download PowerPoint AAV-intein-mediated protein trans-splicing efficiently reconstitutes N6 in vitro To test the efficiency of intein-mediated N6 protein trans-splicing (PTS), we split the large coding sequence (CDS) into two fragments, that is, the 5’ and 3’ half, fused, respectively, to the N- and C-terminal halves of the DnaE split-inteins from Nostoc punctiforme (Npu; Iwai et al, 2006). The split CDS was cloned into two separate AAV plasmids which included the same regulatory elements as above, together with a 3xFlag to detect both N6 halves as well as the full-length protein and excised inteins. The splitting point was selected within the B domain, which is known to be dispensable for F8 expression and procoagulant activity (Pipe, 2009), thus aiming to preserve the integrity of the other more critical protein domains. To optimise the chosen splitting position, the intrinsic amino acid residue requirements for efficient protein trans-splicing with the Npu inteins were also considered. Specifically, the main prerequisite is the presence of an amino acid containing either a thiol or hydroxyl group (Cysteine, Serine or Threonine) as the first residue in the 3’ half of the CDS (Shah et al, 2013; Cheriyan et al, 2014). The intein set was designed within the N6 linker (Ser962, considering the signal peptide) of the N6 variant. Moreover, to assess whether F8 activity in the medium of transfected cells was specifically due to the reconstitution of the full-length N6 after PTS, a set of N6 flanked by heterologous split-inteins was also designed. In this set, the N-terminal of the 5’ half of N6 CDS was fused to the split N-inteins DnaB from Rhodothermus marinus (Rma; Zhu et al, 2013; Tornabene et al, 2019) while the C-terminal of the 3’half was fused to the split C-intein DnaE. Both intein sets were tested by transient transfection into HEK293 cells. Seventy-two-hour post-transfection, cell lysates and medium were harvested, and N6 expression was evaluated by WB (Fig 3A and B). Both full-length N6 protein of the expected size (~190 kDa in cell lysate and ~170 kDa in the medium) and the excised DnaE inteins (~18 kDa) were detected only when the Npu inteins set was used. Quantification of the single halves after PTS shows that the 5’(~123 kDa) and 3’(~95 kDa) half are fivefold and fourfold more abundant than the full-length N6, respectively. Figure 3. In vitro F8-N6 (N6) intein expression and activity Western blot (WB) of protein lysates of HEK293 cells 72-h post-transfection (n = 3; biological replicates) with either Npu inteins or Heterologous (N-intein DnaB + C-intein DnaE) split-inteins. I + II, N6 split-intein proteins; I+II (Het.), heterologous split-intein proteins; I, 5’N6 coding sequence (CDS)-N-DnaE protein; I (N-int B), 5’N6 CDS-N-DnaB. II, C-DnaE-3’N6 CDS protein. Excised inteins (~12 kDa) are present only in the down part of the blot when I + II (Npu inteins) are provided. Arrows indicate the full-length N6 protein, single halves and excised inteins. WB of medium of the transfected cells showing the secreted proteins (n = 3; biological replicates). Arrows indicate the full-length N6 protein as well as single halves and excised inteins. Chromogenic assay performed on the medium of transfected cells to detect F8 activity levels reported as International Units/decilitre (IU/dl). Data are presented as mean ± SEM. Each dot within each group represents a different biological replicate (n): I + II n = 3; I + II (Het.) n = 3; I (N-int E) n = 3; I (N-int B) n = 3; II (C-int E) n = 3; Neg n = 4. Significant differences between groups were assessed using Kruskal–Wallis rank-sum test Kruskal−Wallis, P = 0.013. *indicates the significant difference between the I + II and the I (N-int E) groups: P ≤ 0.05. ***indicates the significant difference between the I + II and the Neg groups: P ≤ 0.001. All P-values are reported in Appendix Table S1. Source data are available online for this figure. Source Data for Figure 3 [emmm202115199-sup-0005-SDatafig3.xlsx] Download figure Download PowerPoint The activity levels of the secreted F8 in the medium were found to be ~60 IU/dl on average, while single halves as well as the heterologous intein set exhibited little to no activity (Fig 3C). N6 codon optimisation increases F8 expression and activity in vitro To further improve the efficiency of the N6 split-inteins, we codon-optimised the N6 CDS (CodopN6), as this has been previously reported to improve F8 levels (Ward et al, 2011; McIntosh et al, 2013). A fourfold increase in CodopN6 protein expression and secretion was observed by WB compared with the non-codon-optimised N6 split-inteins (Fig 4A and B). Moreover, cells expressing CodopN6 had higher F8 activity levels (~200 IU/dl) than the corresponding non-codopN6 (~70 IU/dl) as assessed by chromogenic assay (Fig 4C). In addition, to demonstrate that PTS results in precise CodopN6 reconstitution, we transfected HEK293 cells with the CodopN6 intein plasmids and immunopurified the resultant full-length N6 protein. Liquid chromatography-mass spectrometry (LC-MS) analysis showed reconstituted CodopN6 peptides with sequences at the splitting point, which were identical to full-length CodopN6 encoded by a single plasmid. Figure 4. Codon optimisation of the N6 split-intein improves F8 activity levels Western blot (WB) of protein lysates of HEK293 cells 72 hpt with the AAV-N6 split-intein plasmids and with the codon-optimised set. I + II, N6 split-intein proteins; I, 5’ N6 CDS-N-DnaE protein; II, C-DnaE-3’N6 CDS protein. (n = 3; biological replicates). Arrows indicate the full-length N6 protein, excised inteins and both single halves. Co
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