Bespoke Base and Prime Editing Approaches for STING-Associated Vasculopathy with Onset in Infancy (SAVI)

Introduction STING-associated vasculopathy with onset in infancy (SAVI) is a rare, severe type I interferonopathy caused by gain-of-function mutations in STING1, leading to early-onset systemic inflammation, cutaneous vasculopathy, and life-threatening interstitial lung disease. Current treatments attenuate downstream inflammation without fully addressing the pathogenic driver and carry significant adverse effects. Recently, gene editing has emerged as a paradigm-shifting approach for IEI, with substantial potential for gain-of-function disorders that require allele-specific correction. We present a highly efficient and specific base- and prime-editing strategy that supports both ex vivo and in vivo therapeutic applications. Results K562 were transduced to generate stable STING1-WT and STING1-V155M lines (K562wt, K562mut). 8 sgRNAs were screened for base editing across PAM-flexible nucleases (SpCas9-NG, SpRY) in K562mut, and sgRNA2 and sgRNA5 were selected based on on-target editing. Given the high bystander activity observed with sgRNA5, we leveraged a previously developed library-derived in silico TadA design tool to identify >15 variants for testing. The TadA variant prioritized after experimental screening (var_17) reduced bystander editing (p<0.05). To overcome bystander constraints, PEGsm was selected for prime editing after comprehensive screening. Top-performing guides and selected editor variants were delivered by electroporation as in vitro–transcribed mRNA, achieving high on-target editing (90.10% ± 2.73%) for sgRNA5 and PEGsm (PE2 and PE3) and 53.48% ± 2.02% for sgRNA2. To functionally assess editing, interferon-stimulated gene transcripts (IFIT1, ISG15, and IFI44L) were quantified by droplet digital PCR (ddPCR) before and after 2′3′-cGAMP stimulation. ISG15 and IFIT1 were significantly reduced in unstimulated K562mut-edited cells (sgRNA2, PEGsm) versus K562mut, while after stimulation, K562wt and K562mut-edited reached the K562mut plateau as expected. In healthy donor hematopoietic stem and progenitor cells (HSPCs), a surrogate sgRNA was used to benchmark editing efficiency within the target window. Surrogate sgRNA achieved a mean bystander editing rate of 75.0% ± 4.4%. Finally, peripheral blood mononuclear cells (PBMCs) were reprogrammed into patient-derived induced pluripotent stem cells (iPSCs) using non-integrating Sendai vectors. iPSCs were prime edited to generate fully corrected isogenic single-cell–derived clones. Conclusion We developed a comprehensive, bespoke base- and prime-editing platform for SAVI, enabling efficient and specific STING1 variant correction with functional normalization of interferon-stimulated genes (ISGs) expression, paving the way for future therapeutic applications. We also generated multiple fully corrected isogenic iPSC clones to support mechanistic studies of SAVI pathogenesis. Figure 1. Engineering of a STING1-V155M K562 model and functional assessment of bespoke base and prime editing. (A) Bidirectional third-generation lentiviral (LV) vector used to generate stable K562wt and K562mut lines expressing STING1-WT or STING1-V155M together with an EGFRt surface marker. (B) In vitro transcription (IVT) mRNA electroporation of top guide–editor pairs yields high A-to-G editing in K562mut, including base editing with sgRNA2/sgRNA5, a bystander-reducing TadA variant (var17) with sgRNA5, and prime editing with PEGsm in PE2 and PE3 configurations; editing was quantified by Sanger sequencing (EditR). (C) Functional readout by ddPCR showing IFIT1 and ISG15 expression (normalized to HPRT1) in K562mut across editing conditions and after STING inhibition (H-151). Statistics were computed by one-way ANOVA with multiple comparisons correction; significance is indicated, and where not otherwise specified, **** denotes p < 0.0001.