Lentiviral-Mediated RNAi Knockdown of SBDS in Murine Hematopoietic Progenitors Impairs Their Engraftment Potential and Granulocytic Differentiation.

Shwachman-Bodian Diamond Syndrome (SDS) is an autosomal recessive disorder characterized by congenital neutropenia, pancreatic exocrine insufficiency and bony abnormalities. About 90% of SDS patients have mutations in the novel SBDS gene. The SBDS protein is conserved from archaea to mammals; however, the cellular functions of SBDS, as well as its contribution to the molecular pathogenesis of this disease, are not yet known. In this study, we developed an RNAi knockdown model to investigate the consequences of a reduction in SBDS protein on hematopoiesis.

Three lentiviral RNAi constructs directed against SBDS were generated and tested in BAF3 cells, a murine pro-B cell line. SBDS protein reduction of 78 ± 11%, 63 ± 14%, and 27 ± 6% was observed. The most efficient lentiviral SBDS RNAi construct was used to transduce primary murine hematopoietic cells, with knockdown similar to that observed in BAF3 cells. In addition, empty lentiviral and Cathepsin G (CG) RNAi lentiviral constructs were studied to control for non-specific effects of lentivirus infection and/or induction of the RNAi machinery. In each case, the lentiviral vector contained a yellow fluorescent protein (YFP) expression cassette to track transduced cells.

Murine hematopoietic progenitors (Kit+, Lin-) were transduced with these lentiviral vectors and transplanted into irradiated syngeneic recipients. Initially, a high multiplicity of infection (MOI) was used, resulting in a transduction efficiency of ~75%. Interestingly, 5/5 animals transplanted with SBDS RNAi transduced cells died by 19 days post-transplantation due to engraftment failure, compared to 0/5 animals transplanted with empty lentivirus transduced cells. To study the long-term consequence of SBDS knockdown, cells were transduced at a lower MOI, resulting in a transduction efficiency of ~15%. Engraftment of SBDS RNAi cells (N=8) was modestly (3-fold), yet consistently, reduced compared to both empty lentivirus (N=8) and CG RNAi (N=5) controls. However, once engraftment of SBDS RNAi cells was established, the percentage of transduced (YFP+) cells remained stable for at least 3 months. Moreover, there was no evidence of lineage skewing, as a similar percentage of YFP+ cells was observed in all hematopoietic lineages. Importantly, the SBDS knockdown effect was maintained in YFP+ cells recovered from mice 3 months post-transplantation (~70% reduction in SBDS expression).

Neutropenia is the most common hematopoietic abnormality in patients with SDS. Thus, we also characterized the effect of reduced SBDS function on granulocytic differentiation. Specifically, the ability of SBDS RNAi transduced Kit+, Lin- progenitors to generate hematopoietic colonies and differentiate into mature granulocytes was assessed. No defect in the ability of transduced progenitors to form CFU-GM or CFU-G was observed. However, a modest, yet significant, delay in neutrophil maturation was observed. Mature neutrophils constitute only 34.6 ± 7.3% of SBDS RNAi granulocyte cultures by day 7, compared to 59.7 ± 6.9% and 60.1 ± 12.9% in empty lentivirus and CG RNAi cultures, respectively.

In summary, we established an RNAi model for SDS and have shown that a 70% reduction in SBDS protein levels in hematopoietic progenitors is sufficient for (i) impaired engraftment upon bone marrow transplantation and (ii) modest delay in neutrophil maturation in vitro. These findings provide the first experimental evidence that reduced SBDS function may drive the hematopoietic defects seen in SDS patients.