Page 105 - 2022_02-Haematologica-web

Gene therapy of DBA
none of the currently available treatments is curative, and the treatments are often accompanied by serious compli- cations.1,2,10 Hematopoietic stem cell transplantation is currently the sole curative option for the treatment of DBA. This treatment is, however, limited by the availabil- ity of suitable donors and the potential for serious immunological complications.11 Gene therapy, using gene-corrected hematopoietic stem cells, would be a potential alternative therapeutic strategy, as highlighted in our previous studies.12-16 For clinical applications using this approach, the efficiency of transgene expression and safety aspects, including potential insertional mutagene- sis, should be addressed.17-19 Our group recently demon- strated that correction of Rps19-deficient BM cells using lentiviral vectors containing a clinically relevant promoter could rescue BM failure and defects in erythroid develop- ment, while exhibiting limited risk of insertional mutage- nesis.12
In our previous study, we utilized a lentiviral vector containing the RPS19 gene as well as a GFP marker. We have subsequently designed a clinically applicable single gene lentiviral self-inactivating (SIN) vector for the clinical development of gene therapy for Rps19-deficient DBA patients. This vector harbors a codon-optimized human RPS19 cDNA driven by the short human elongation fac- tor 1a promoter and lacks a fluorescent marker. In this study, we demonstrate that this vector can rescue the anemia and lethal BM failure observed in mouse models of Rps19-deficient DBA, with a low-risk insertion profile and no evidence of clonal expansion associated with vec- tor integration near cancer-associated genes. We also observed the rescue of impaired erythroid differentiation in human RPS19-deficient CD34+ cord blood cells treated with this vector. Our results demonstrate the feasibility and preclinical efficacy for treatment of RPS19-deficient DBA using a clinically applicable SIN lentiviral vector, which opens the possibility for the development of clini- cal gene therapy for RPS19-deficient DBA patients.
Methods
Lentiviral vector constructs
The SIN lentiviral vector is derived from the pRRL.PPT.PGK. vector backbone.20 A codon-optimized human RPS19 cDNA was designed as described previously21 and inserted downstream of the EFS promoter. Lentiviral vectors were produced by the Vector Unit at Lund University as previously described.12
Mice and transplantations
Mice were maintained at the Lund University animal facility and all animal experiments were approved by the Lund University animal ethics committee. The homozygous doxycy- cline-inducible Rps19-deficient mouse model used in the study was established as previously described.15 A detailed description of transplantations is provided in the Online Supplementary Methods.
Transduction
c-kit+ or lineage negative (Lin–) cells isolated from BM of trans- genic mice were enriched by using CD117 or Lin– microbeads and magnetic-activated cell sorting separation columns (all from Miltenyi Biotec) according to the manufacturer’s protocol. After enrichment, cells were pre-stimulated for 24 h in StemSpan serum-free expansion medium (Stem Cell Technologies), supple-
mented with penicillin/streptomycin (Gibco), murine stem cell factor (100 ng/mL; PeproTech), and human thrombopoietin (50 ng/mL; PeproTech) in six-well plates at the concentration of 0.5x106 cells/mL. For transduction, retronectin-coated (20 ng/mL; Takara) 12-well plates were preloaded with the viral vec- tors (multiplicity of infection [MOI]=5-10), followed by seeding of 0.5x106 cells into each well filled with 1 mL pre-stimulation medium.
Flow cytometry
A complete description of all antibodies used is listed in the Online Supplementary Methods.
Human primary cord blood cells and erythroid differentiation
Human cord blood samples were obtained from the maternity wards of Helsingborg General Hospital and Skåne University Hospital in Lund and Malmö, Sweden, after informed, written consent according to guidelines approved by the regional ethical committee. Mononuclear cells were separated through density- gradient centrifugation. CD34+ cells were magnetically isolated according to the manufacturer’s description (Milteny Biotec, cat. n. 130-046-702). Cells were cultured in serum-free expansion medium (Stem Cell Technologies), supplemented with human stem cell factor, thrombopoietin, and FLT3-ligand at 100 ng/mL from Peprotech. Full descriptions of transduction and erythroid differentiation are provided in the Online Supplementary Methods.
Other experimental details
Full descriptions of the quantitative reverse transcriptase poly- merase chain reaction (qRT-PCR), determination of transduction efficiency and vector copy number measurements are provided in the Online Supplementary Methods.
Insertion site analysis
Whole BM cells were isolated at 16 weeks after transplanta- tion. Genomic DNA was isolated from the BM of flushed femo- ra using the DNA Blood & Tissue kit (Qiagen). The vector- genome junction was amplified using the INtegration Site PIpeline for paIRed-End reaDs (INSPIIRED) workflow as described by Sherman and colleagues.24
Statistical analysis
t-tests and one-way analysis of variance with the Tukey mul- tiple-comparison test were used to determine statistical signifi- cance. Computations were performed using GraphPad Prism (version 6; GraphPad Software).
Results
High transduction efficiency of the EFS-RPS19 vector
We first studied the transduction efficiency of the vec- tor in BM progenitor cells isolated from our established Rps19-deficient DBA mouse model.15 This model contains the Rps19-targeting shRNA expressed under a doxycy- cline-responsive promoter located downstream of the collagen A1 gene (Figure 1A). Experimental animals were bred to be either heterozygous (D/+) or homozygous (D/D) for the shRNA to generate two models with inter- mediate or severe Rps19 deficiency (Figure 1B). Rps19 mRNA expression was reduced by approximately 50%, and a trend toward more efficient knockdown in D/D mice compared to D/+ mice was seen, as shown in our previous studies.15 Upon induction with doxycycline,
haematologica | 2022; 107(2)
447