Chen, L., Zhu, B., Ru, G., et al. Re-engineering the adenine deaminase TadA-8e for efficient and specific CRISPR-based cytosine base editing. Nature Biotechnology (2022).
Cytosine base editors (CBEs) efciently generate precise C·G-to-T·A base
conversions, but the activation-induced cytidine deaminase/apolipoprotein
B mRNA-editing enzyme catalytic polypeptide-like (AID/APOBEC) protein
family deaminase component induces considerable of-target efects
and indels. To explore unnatural cytosine deaminases, we repurpose the
adenine deaminase TadA-8e for cytosine conversion. The introduction
of an N46L variant in TadA-8e eliminates its adenine deaminase activity
and results in a TadA-8e-derived C-to-G base editor (Td-CGBE) capable of
highly efcient and precise C·G-to-G·C editing. Through fusion with uracil
glycosylase inhibitors and further introduction of additional variants, a
series of Td-CBEs was obtained either with a high activity similar to that
of BE4max or with higher precision compared to other reported accurate
CBEs. Td-CGBE/Td-CBEs show very low indel efects and a background level
of Cas9-dependent or Cas9-independent DNA/RNA of-target editing.
Moreover, Td-CGBE/Td-CBEs are more efcient in generating accurate edits
in homopolymeric cytosine sites in cells or mouse embryos, suggesting their
accuracy and safety for gene therapy and other applications.
Chen, L., Zhang, S., Xue, N., et al. Engineering a precise adenine base editor with minimal bystander editing. Nature Chemical Biology (2022).
Adenine base editors (ABEs) catalyze A-to-G transitions showing broad
applications, but their bystander mutations and of-target editing efects
raise safety concerns. Through structure-guided engineering, we found
ABE8e with an N108Q mutation reduced both adenine and cytosine
bystander editing, and introduction of an additional L145T mutation (ABE9),
further refned the editing window to 1–2 nucleotides with eliminated
cytosine editing. Importantly, ABE9 induced very minimal RNA and
undetectable Cas9-independent DNA of-target efects, which mainly
installed desired single A-to-G conversion in mouse and rat embryos to
efciently generate disease models. Moreover, ABE9 accurately edited the
A5 position of the protospacer sequence in pathogenic homopolymeric
adenosine sites (up to 342.5-fold precision over ABE8e) and was further
confrmed through a library of guide RNA–target sequence pairs. Owing to
the minimized editing window, ABE9 could further broaden the targeting
scope for precise correction of pathogenic single-nucleotide variants
when fused to Cas9 variants with expanded protospacer adjacent motif
compatibility. bpNLS, bipartite nuclear localization signals
Zhang, J., Hu, Y., Yang, J., et al. Non-viral, specifically targeted CAR-T cells achieve high safety and efficacy in B-NHL. Nature (2022).
Recently, chimeric antigen receptor (CAR)-T cell therapy has shown great promise in
treating haematological malignancies1–7
. However, CAR-T cell therapy currently has
several limitations8–12. Here we successfully developed a two-in-one approach to
generate non-viral, gene-specifc targeted CAR-T cells through CRISPR–Cas9. Using
the optimized protocol, we demonstrated feasibility in a preclinical study by inserting
an anti-CD19 CAR cassette into the AAVS1 safe-harbour locus. Furthermore, an
innovative type of anti-CD19 CAR-T cell with PD1 integration was developed and
showed superior ability to eradicate tumour cells in xenograft models. In adoptive
therapy for relapsed/refractory aggressive B cell non-Hodgkin lymphoma
(ClinicalTrials.gov, NCT04213469), we observed a high rate (87.5%) of complete
remission and durable responses without serious adverse events in eight patients.
Notably, these enhanced CAR-T cells were efective even at a low infusion dose
and with a low percentage of CAR+
cells. Single-cell analysis showed that the
electroporation method resulted in a high percentage of memory T cells in infusion
products, and PD1 interference enhanced anti-tumour immune functions, further
validating the advantages of non-viral, PD1-integrated CAR-T cells. Collectively, our
results demonstrate the high safety and efcacy of non-viral, gene-specifc integrated
CAR-T cells, thus providing an innovative technology for CAR-T cell therapy.
Fu, B., Liao, J., Chen, S. et al. CRISPR–Cas9-mediated gene editing of the BCL11A enhancer for pediatric β0/β0 transfusion-dependent β-thalassemia. Nature Medicine (2022).
Gene editing to disrupt the GATA1-binding site at the +58 BCL11A erythroid enhancer could induce γ-globin expression, which is a promising therapeutic strategy to alleviate β-hemoglobinopathy caused by HBB gene mutation. In the present study, we report the preliminary results of an ongoing phase 1/2 trial (NCT04211480) evaluating safety and efficacy of gene editing therapy in children with blood transfusion-dependent β-thalassemia (TDT). We transplanted BCL11A enhancer-edited, autologous, hematopoietic stem and progenitor cells into two children, one carrying the β0/β0 genotype, classified as the most severe type of TDT. Primary endpoints included engraftment, overall survival and incidence of adverse events (AEs). Both patients were clinically well with multilineage engraftment, and all AEs to date were considered unrelated to gene editing and resolved after treatment. Secondary endpoints included achieving transfusion independence, editing rate in bone marrow cells and change in hemoglobin (Hb) concentration. Both patients achieved transfusion independence for >18 months after treatment, and their Hb increased from 8.2 and 10.8 g dl−1 at screening to 15.0 and 14.0 g dl−1 at the last visit, respectively, with 85.46% and 89.48% editing persistence in bone marrow cells. Exploratory analysis of single-cell transcriptome and indel patterns in edited peripheral blood mononuclear cells showed no notable side effects of the therapy.