This study highlights the development of a temperature-sensitive Sendai virus (ts SeV) as a novel delivery vector for CRISPR-Cas9, offering a safer and more efficient alternative to DNA viral vectors for gene editing. Unlike DNA vectors, ts SeV eliminates the risk of genome integration and demonstrates low immunogenicity. By introducing temperature-sensitive mutations in the P and L genes, viral replication is restricted at physiological temperatures (37°C), enhancing safety while maintaining efficient transduction at permissive temperatures (32°C).
The ts SeV system achieved over 90% transduction efficiency in human CD34+ hematopoietic stem and progenitor cells (HSPCs), specifically targeting the stem cell-enriched subpopulation. Edited HSPCs retained multilineage differentiation capacity, critical for clinical use. Additionally, in primary CD14+ monocytes, ts SeV achieved 90% CCR5 gene editing, significantly inhibiting HIV-1 infection.
This work underscores the ts SeV-Cas9 platform as a safe, efficient, and flexible tool for gene editing, with broad applications in treating genetic disorders and infectious diseases, particularly through ex vivo editing of sensitive cell types like HSPCs.
RESULTS
Mutants in P and L genes result in a temperature-sensitive phenotype
This study developed a temperature-sensitive Sendai virus (ts SeV-Cas9) by introducing mutations in the P (D433A, R434A, K437A) and L (L1558I, K1795E) genes, which restrict viral replication to lower temperatures (32–34°C). The ts mutations effectively limit viral growth at physiological temperatures (37°C), enhancing safety while maintaining efficient gene editing. In 293T cells, ts SeV-Cas9 demonstrated a rapid decline in viral gene expression and undetectable titers after shifting to 37°C, ensuring clearance post-editing. Despite this, the vector achieved equal or superior efficiency in mCherry gene editing at 32°C. This design provides a promising strategy to reduce cytotoxicity associated with prolonged viral infection while enabling precise and effective gene editing.
Figure 1 demonstrates the development and validation of a temperature-sensitive Sendai virus (ts SeV-Cas9) vector engineered for CRISPR-Cas9 delivery. (A) The viral genome was modified to include an eGFP-P2A-Cas9 cassette between the N and P genes and a guide RNA flanked by self-cleaving ribozymes between the P and M genes, with mutations in the P and L genes to confer temperature sensitivity. (B–D) In 293T cells, ts SeV-Cas9 showed efficient gene expression at 32°C but rapidly declined after shifting to 37°C, confirming its temperature-restricted replication. Viral titers became undetectable after 7 days at 37°C, ensuring safety. (E) The ts SeV-Cas9 system achieved efficient gene editing in mCherry-expressing cells, as indicated by eGFP positivity and loss of mCherry, demonstrating successful transduction and gene knockout. These findings highlight the ts SeV-Cas9 vector's potential for safe, temperature-controlled gene editing.
Infection by ts SeV-Cas9 elicits a minimally immunogenic phenotype
This study investigates the role of temperature-sensitive mutations in the Sendai virus (SeV) on reducing interferon responses. By introducing mutations in the P (D433A, R434A, K437A) and L (L1558I, K1795E) genes, the authors demonstrate that the P mutations (Pmut) play a critical role in dampening the innate immune response, while the L mutations primarily attenuate viral growth. Infections with temperature-sensitive SeV-Cas9 in 293T cells and primary monocyte-derived macrophages (MDMs) showed significantly reduced expression of interferon-stimulated genes (RIG-I and IFIT1) compared to wild-type SeV. Analysis revealed that wild-type SeV produced higher levels of defective viral genomes (DVGs), which are known to strongly stimulate innate immunity, whereas Pmut SeV generated fewer DVGs. This reduction in DVG production explains the dampened immune response observed with Pmut SeV. Overall, these findings highlight the dual benefit of temperature-sensitive mutations: they restrict viral replication at physiological temperatures and reduce immunogenicity, enhancing the safety profile of SeV as a gene delivery vector.
Figure 2 examines the effects of temperature-sensitive mutations in the P and L genes of Sendai virus (SeV) on innate immune responses and viral replication. Wild-type (WT) and temperature-sensitive (ts) SeV were compared in 293T cells and primary monocyte-derived macrophages (MDMs). Results showed that ts SeV significantly reduced the expression of interferon-stimulated genes (ISGs) like RIG-I and IFIT1 after shifting to 37°C (A–D). Further analysis demonstrated that the P mutations (Pmut) contributed to reduced ISG activation, independent of viral genome copies, while L mutations (Lmut) attenuated viral replication without reducing ISG expression (E–F). In BSR-T7 cells, Pmut SeV produced fewer defective viral genomes (DVGs), which are known to stimulate ISG expression, compared to WT SeV (G–J). Consequently, infection with Pmut SeV elicited significantly lower levels of IFIT1 expression (K). These findings suggest that the P mutations reduce innate immune activation primarily by lowering DVG production, contributing to the enhanced safety profile of the temperature-sensitive SeV vector.
ts SeV-Cas9 can effectively deliver either one or two guides in a single construct
This study describes the optimization of the SeV-Cas9 system for efficient gene editing. The first generation SeV-Cas9 incorporated two cassettes: eGFP-P2A-Cas9 between the N and P genes and a guide RNA (gRNA) flanked by self-cleaving ribozymes between the P and M genes. Efficient editing required precise ribozyme self-cleavage, which depended on consistent folding of the ribozyme-gRNA complex. By analyzing the RNA secondary structure and predicting base pair formation, the researchers identified a positive correlation between ribozyme folding efficiency and gene editing outcomes. Using this method, they accurately predicted efficient ribozyme-gRNA combinations. Additionally, they expanded the system to deliver two gRNAs within a single cassette, flanked by ribozymes. Testing in three cell lines showed efficient editing of both CCR5 and HPRT genes, individually and simultaneously, demonstrating the versatility and efficiency of the dual-gRNA SeV-Cas9 system for multiplex gene editing.
Figure 3 highlights the versatility of the SeV-Cas9 system in delivering diverse gRNAs and utilizing novel guide strategies. (A) The gRNA cassette includes two self-cleaving ribozymes essential for efficient editing, and RNA structural analysis predicts proper ribozyme folding and cleavage. (B) A positive correlation was observed between predicted ribozyme efficiency and measured indel frequency, confirming the importance of ribozyme folding in editing efficiency. (C) The system was expanded to deliver two gRNAs within a single cassette, separated by a GGS linker and flanked by ribozymes. (D) Testing single and dual gRNA systems targeting CCR5 and HPRT in multiple cell lines (293Ts, H441s, and Huh7) demonstrated efficient editing, both individually and in combination, showing the system’s potential for multiplex gene editing.
Efficient ts SeV-Cas9-CCR5 mediated transduction in CD34+ hematopoietic stem progenitor cells
This study demonstrates the high efficiency of ts SeV-Cas9-mediated CCR5 editing in human CD34+ hematopoietic stem and progenitor cells (HSPCs), highlighting its potential for HIV therapy. CD34+ cells derived from fetal liver (FL) and G-CSF mobilized peripheral blood (mPB) were transduced with ts SeV-Cas9-CCR5 at various MOIs (0.1–20) and cultured at 34°C for 3 days. Transduction efficiency exceeded 90% at MOIs greater than 1, with no impact from varying incubation times (1–20 h). Notably, the rare CD34+/CD38–/CD90+/CD49fhigh HSC subpopulation achieved >95% transduction efficiency. These findings underscore the robust ability of the ts SeV-Cas9 system to efficiently edit CCR5 in primary human HSPCs, including clinically relevant stem cell subsets.
On-target editing efficiency of ts rSeV-Cas9-CCR5 and its effects in CD34+ HSPC
This study demonstrates that ts SeV-Cas9-CCR5 efficiently edits CCR5 in human CD34+ HSPCs derived from both fetal liver (FL) and mobilized peripheral blood (mPB), achieving over 90% transduction efficiency and 88% CCR5 editing at an MOI of 10. Editing efficiency increased with higher MOIs, while no significant editing was observed at MOIs below 1 or without a CCR5 guide. Importantly, edited HSPCs retained their multi-lineage hematopoietic differentiation potential, though a slight reduction in colony formation was observed due to viral transduction and double-stranded breaks. Deep sequencing revealed minimal off-target effects (<0.4% in FL and <1% in mPB), confirming the precision of the ts SeV-Cas9 system. These results highlight its potential for safe and efficient ex vivo gene editing of human HSPCs.
Efficient CCR5 editing of primary human CD14+ monocytes by ts SeV-Cas9 inhibits HIV infection
This study demonstrates the efficacy of ts SeV-Cas9 in targeting and editing the CCR5 gene in primary CD14+ monocytes to inhibit HIV infection. Monocytes were infected with ts SeV-Cas9-CCR5 at an MOI of 10, resulting in efficient CCR5 editing (~80% at 10 days and ~90% at 18 days post-infection). No editing was observed in control cells infected with a virus targeting US11. In HIV infection assays using R5-tropic HIV strain JR-FL, ts SeV-Cas9-CCR5 transduced cells showed a significant reduction in HIV markers, including mCherry-positive cells and p24 accumulation, compared to controls. These findings highlight the potential of ts SeV-Cas9 as a gene-editing tool to inhibit HIV by disrupting CCR5 in primary monocytes.
Figure 5 evaluates the editing efficiency and functional impact of ts SeV-Cas9 in CD34+ HSPCs derived from fetal liver (FL) and mobilized peripheral blood (mPB). At various MOIs, CCR5 editing efficiency exceeded 90%, with indels analyzed using Synthego ICE and Illumina sequencing. Functional differentiation assays demonstrated that transduced HSPCs retained multi-lineage colony formation potential (CFU-E, CFU-G, CFU-GM, CFU-GEMM, BFU-E, CFU-M) with a slight reduction in total colony counts, likely due to transduction and double-stranded breaks. Additionally, off-target analysis at the top five predicted sites revealed minimal editing (<1%), confirming the high specificity of ts SeV-Cas9-CCR5 in CD34+ HSPCs.
DISCUSSION
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Full URL Link:
A temperature-sensitive and less immunogenic Sendai virus for efficient gene editing | Journal of Virology
