The major challenges of gene therapy for hemophilia A using adeno-associated virus (AAV) vectors are reducing vector doses and the long-term maintenance of stable factor VIII (FVIII). In this study, we developed engineered human B-domain-deleted FVIIIs (FVIIISQ) with enhanced secretion and coagulation potential. Intracellular accumulation was markedly reduced in some engineered FVIIISQ, resulting in reduced unfolded protein responses. The administration of AAV vectors carrying engineered FVIIISQ to hemophilia A mice resulted in ∼8-fold higher FVIII activity and 4-fold higher FVIII antigen levels compared with wild-type FVIIISQ administration. The specific FVIII activity of the engineered FVIIISQ was 3.6 times higher than that of the wild-type FVIIISQ, and its binding to activated coagulation factor IX was significantly enhanced, which is supported by the structural analysis. In macaques, the administration of AAV5 vector carrying the engineered FVIIISQ without CpG sequences resulted in a supraphysiological increase in plasma FVIII activity at a dose one-thirtieth that of valoctocogene roxaparvovec (2 × 1012 vector genome per kg). The engineered FVIIISQ may thus provide stable, long-term therapeutic efficacy in AAV-mediated hemophilia A gene therapy even at low doses.

Self-renewal and differentiation are at the basis of hematopoiesis. Although it is known that tight regulation of translation is vital for hematopoietic stem cells' (HSC) biology, the mechanisms underlying translation regulation across the hematopoietic system remain obscure. Here, we reveal a novel mechanism of translation regulation in the hematopoietic hierarchy, which is mediated by rRNA methylation dynamics. Using ultralow-input ribosome profiling, we characterized cell-type-specific translation capacity during erythroid differentiation. We found that translation efficiency (TE) changes progressively with differentiation and can distinguish between discrete cell populations, as well as define differentiation trajectories. To reveal the underlying mechanism, we performed comprehensive mapping of the most abundant rRNA modification, 2'-O-methyl (2'OMe). We found that, such as TE, 2'OMe dynamics followed a distinct trajectory during erythroid differentiation. Genetic perturbation of individual 2'OMe sites demonstrated their distinct roles in modulating proliferation and differentiation. By combining CRISPR screening, molecular, and functional analyses, we identified a specific methylation site, 28S-Gm4588, which is progressively lost during differentiation, as a key regulator of HSC self-renewal. We showed that low methylation at this site led to translational skewing, mediated mainly by codon frequency, which promoted differentiation. Functionally, HSC with diminished 28S-Gm4588 methylation exhibited impaired self-renewal capacity ex vivo, and loss of fitness in vivo in bone marrow transplants. Extending our findings beyond the hematopoietic system, we also found distinct dynamics of 2'OMe profiles during differentiation of non-HSC. Our findings reveal rRNA methylation dynamics as a general mechanism for cell-type-specific translation, required for cell function and differentiation.