Inborn errors of immunity (IEIs) are rare genetic disorders caused by mutations in genes critical for immune cell function, development, or signaling pathways. Gene therapy represents a potential curative treatment for these conditions, with lentiviral vectors (LVs) emerging as a promising tool in this field. However, one of the significant limitations related to the use of LVs is their cargo capacity of around 8 kilobases. This prevents their application for the treatment of conditions requiring the delivery of larger DNA sequences and the stable expression of the transgene in actively proliferating cells. Recent studies have shown that protein trans-splicing is a powerful tool for expanding the cargo capacity of AAV vectors upon co-infection of a host cell with different vectors, each expressing a split-intein–flanked portion of the full-length protein. The aim of our study is to translate the same technology to the platform of LVs. We developed dual-intein LVs, each expressing either the N- or the C-terminal half of the EGFP reporter protein fused to the N- and C-terminal halves of the DnaE split-inteins from Nostoc punctiforme, under the control of EF1A promoter. We co-infected HEK293 cells and observed the reconstitution of a functional full-length protein of the proper size. This demonstrates that trans-splicing applied to LVs is as efficient as for AAVs.
This system could be helpful to design a gene therapy approach for specific IEIs, including LRBA deficiency and ataxia-telangiectasia (A-T), caused by mutations in genes larger than 8 kb. We are currently developing dual-intein LVs expressing either the N- or the C-terminal half of the ATM protein, mutated in A-T patients, that will be tested in vitro for their ability to reconstitute a functional protein of the correct size and for the ability to correct the mutant phenotype.
Dual-intein LVs reconstitute EGFP in vitro. Spontaneous fluorescence in HEK293 cells detected 72 hours post-infection. I: HEK293 cells infected with EGFP LV at (A) MOI 5 and (B) MOI 3; II: HEK293 cells co-infected with the dual-intein EGFP LVs at (A) MOI 2.5 each vector and (B) MOI 1.5 each vector; III: HEK293 cells co-infected with the dual-intein EGFP LVs at (A) MOI 5 each vector and (B) MOI 3 each vector. The image is representative of N = 3 independent experiments.
Dual-intein LVs reconstitute EGFP in vitro. Spontaneous fluorescence in HEK293 cells detected 72 hours post-infection. I: HEK293 cells infected with EGFP LV at (A) MOI 5 and (B) MOI 3; II: HEK293 cells co-infected with the dual-intein EGFP LVs at (A) MOI 2.5 each vector and (B) MOI 1.5 each vector; III: HEK293 cells co-infected with the dual-intein EGFP LVs at (A) MOI 5 each vector and (B) MOI 3 each vector. The image is representative of N = 3 independent experiments.
Western blot analysis performed on HEK293 cell lysates infected with dual-intein EGFP LVs. Cell lysates from cells transduced with: 1. EGFP full-length LVs at MOI = 5; 2. dual-intein N-term EGFP LV at MOI = 5; 3. dual-intein C-term EGFP LV at MOI = 5; 4. dual-intein N-term and C-term EGFP LVs at MOI = 2.5 each vector; 5. dual-intein N-term and C-term EGFP LVs at MOI = 5 each vector; 6. untreated cells. Black arrows indicate the correct spliced EGFP produced from co-infection; blue and red arrows indicate the single halves produced by LVs expressing for the N-term and C-term EGFP, respectively; red boxes indicate the spliced intein.
Western blot analysis performed on HEK293 cell lysates infected with dual-intein EGFP LVs. Cell lysates from cells transduced with: 1. EGFP full-length LVs at MOI = 5; 2. dual-intein N-term EGFP LV at MOI = 5; 3. dual-intein C-term EGFP LV at MOI = 5; 4. dual-intein N-term and C-term EGFP LVs at MOI = 2.5 each vector; 5. dual-intein N-term and C-term EGFP LVs at MOI = 5 each vector; 6. untreated cells. Black arrows indicate the correct spliced EGFP produced from co-infection; blue and red arrows indicate the single halves produced by LVs expressing for the N-term and C-term EGFP, respectively; red boxes indicate the spliced intein.
Schematic representation of dual-intein EGFP pLenti containing the nucleotides surrounding the ATM splitting point. In the pLenti encoding for the EGFP N-term half ATM CDS corresponds to nucleotides 3826…3852. In the pLenti encoding for the C-term EGFP, ATM CDS corresponds to nucleotides 3853…3879 and comprises the triplet encoding for Cys1286. nt: nucleotides.
Schematic representation of dual-intein EGFP pLenti containing the nucleotides surrounding the ATM splitting point. In the pLenti encoding for the EGFP N-term half ATM CDS corresponds to nucleotides 3826…3852. In the pLenti encoding for the C-term EGFP, ATM CDS corresponds to nucleotides 3853…3879 and comprises the triplet encoding for Cys1286. nt: nucleotides.
Western blot analysis performed on HEK293 cell lysates transfected with dual-intein pLenti EGFP plasmids with the additional ATM nucleotides surrounding the splitting point. Cell lysates from cells transfected with: 1. pLenti EGFP full-length; 2. dual-intein pLenti N-term; 3. dual-intein pLenti C-term EGFP; 4. dual-intein pLenti N-term + dual-intein pLenti C-term with additional nucleotides from ATM CDS; 5. untransfected cells. Black arrow indicates EGFP full length with additional amino acids from ATM CDS; red and blue arrows indicate the N- and the C-term products, respectively.
Western blot analysis performed on HEK293 cell lysates transfected with dual-intein pLenti EGFP plasmids with the additional ATM nucleotides surrounding the splitting point. Cell lysates from cells transfected with: 1. pLenti EGFP full-length; 2. dual-intein pLenti N-term; 3. dual-intein pLenti C-term EGFP; 4. dual-intein pLenti N-term + dual-intein pLenti C-term with additional nucleotides from ATM CDS; 5. untransfected cells. Black arrow indicates EGFP full length with additional amino acids from ATM CDS; red and blue arrows indicate the N- and the C-term products, respectively.
