Recently, the research group led by Dr. Chen Jia, Scientific Founder of CorrectSequence Therapeutics and Professor at the School of Life Science and Technology (SLST), ShanghaiTech University, together with collaborators, made important advances in the in vivo delivery of cytosine base editors using virus-like particles. The findings were published in Nature Biotechnology under the title “Efficient in vivo cytosine base editing via virus-like particles with uracil DNA glycosylase inhibition.” By engineering both the base editor and virus-like particle (VLP) packaging strategies, the research team developed an efficient in vivo cytosine base editing system, tBE-VLP4. By delivering the transformer base editor (tBE) via VLPs, the system enabled efficient C-to-T editing in mice.
Figure 1: a. Schematic illustration of ABE- and CBE-mediated deamination of adenosine and cytidine in cells. b. Editing efficiency of tBE-VLP1 in wild-type 293FT cells and hUNG/hSMUG1 double-knockout cells.
VLPs are a promising gene-editing delivery tool. They can transiently deliver editors into cells in the form of ribonucleoprotein complexes or mRNA. Compared with AAV or plasmid delivery, VLPs can reduce off-target editing and potential safety risks associated with prolonged editor expression. Previously, VLPs have been used to deliver Cas9-sgRNA, adenine base editors (ABEs), and prime editors (PEs), but their efficiency in mediating cytosine base editing (CBE) in vivo has remained limited. CBE converts cytosine into uracil through deamination, ultimately enabling C-to-T conversion. However, endogenous cellular uracil DNA glycosylase (UNG) recognizes and excises uracil in DNA, reducing editing efficiency and increasing byproducts. Conventional AAV or plasmid delivery can continuously express UGI protein to inhibit UNG, whereas VLPs provide only a one-time, limited amount of UGI protein. The research team found that insufficient UNG inhibition limits the in vivo editing efficiency of VLP-delivered CBE.
The research team first tested VLP-delivered tBE in wild-type 293FT cells and hUNG/hSMUG1 double-knockout 293FT cells. The results showed that, compared with wild-type cells, C-to-T editing efficiency was significantly increased in hUNG/hSMUG1 double-knockout cells, while C-to-A and C-to-G byproducts were markedly reduced. This finding indicates that endogenous uracil DNA glycosylase activity is an important factor limiting the editing efficiency of VLP-CBE.
To address the core issue of insufficient UNG inhibition, the research team further carried out systematic optimization of the tBE system and VLP packaging strategy. They introduced additional RNA aptamers into tBE to enhance UGI recruitment and constructed multiple improved VLP systems.
Among them, the optimized tBE-VLP4 significantly increased the loading levels of UGI and sgRNA in VLPs and demonstrated stable and efficient cytosine base editing in 293FT, HeLa, U2OS, and monkey-derived FRhK-4 cells. Further results showed that tBE-VLP4 is also compatible with the Cas9 SpG variant and conventional CBE architecture, suggesting that the system has strong versatility and scalability.
In mouse experiments, tBE-VLP4 demonstrated favorable editing efficiency and disease-intervention effects. After a single tail-vein injection, tBE-VLP4 achieved an average C-to-T editing efficiency of 46.0% at the mPcsk9 locus in mouse liver, significantly reducing serum PCSK9 protein and total cholesterol levels. In a mouse model of hereditary tyrosinemia type I, the research team used tBE-VLP4 to target the mHpd locus, achieving a maximum editing efficiency of 64.2%. The treatment successfully reduced weight loss and le
thality in the mice and markedly alleviated liver function impairment. In addition, the research team delivered tBE-VLP4 to retinal pigment epithelial cells via subretinal injection to edit the mVegfa locus, achieving an average editing efficiency of 24.2%. In a laser-induced choroidal neovascularization model, this strategy significantly alleviated lesion severity and protected retinal function, suggesting that tBE-VLP4 is not only applicable to liver editing but also has potential for expansion into ophthalmic disease therapy.
In terms of safety, the research team detected no obvious DNA or RNA off-target editing induced by tBE-VLP4 in either in vitro or in vivo experiments. Compared with AAV or LNP-mRNA delivery methods, tBE-VLP4 showed better editing specificity.
Figure 2: a. Schematic illustration of tBE-VLP4 infection of mouse liver and retinal pigment epithelial cells. b. Editing efficiencies of tBE-VLP system at different loci in mice.
This study elucidates the key mechanism limiting the in vivo efficiency of VLP-delivered CBE and, based on this finding, establishes tBE-VLP4 as an efficient and precise in vivo cytosine base editing platform, offering a new strategy for safe and efficient in vivo gene-editing therapies. Building on this foundation, the research team will conduct studies applying the tBE-VLP4 system to in vivo editing of hematopoietic stem cells (HSCs). Compared with current ex vivo editing approaches for HSCs, in vivo HSC editing is expected to streamline therapeutic procedures, reduce manufacturing costs, and alleviate clinical burdens.
Dr. Xiaodun Mou, CEO of Correctseq, stated: "Congratulations to the scientific founder team of Correctseq on this innovative breakthrough in the in vivo delivery system! The safety and efficacy of tBE-VLP4 have been preliminarily validated in animal models, demonstrating that it can achieve precise editing by tBE in organs and tissues that are difficult to reach with LNP and AAV. We are already exploring the application of the tBE-VLP system in the treatment of stem cell-related diseases, and look forward to unveiling breakthrough progress in the near future."
Dr. Jia Chen, Scientific Founder of Correctseq, Professor at the School of Life Science and Technology of ShanghaiTech University, and Director of the Gene Editing Center, stated: "The tBE-VLP4 in vivo base editing platform represents another important research achievement driven by unmet clinical needs. It marks the expansion of our team's research endeavors into delivery science, building upon our solid foundation in base editing tool development. Moving forward, the Correctseq team will leverage the VLP delivery platform to develop in vivo base editing pipelines for hematopoietic stem cells, with applications in the in vivo treatment of diseases such as thalassemia and sickle cell disease, bringing safe, efficient, durable, and accessible novel therapeutic options to patients worldwide."
