High-Efficiency Transduction of Primary Human Hematopoietic Stem/Progenitor Cells by AAV6 Vectors: Strategies for Overcoming Donor-Variation and Implications in Genome Editing

Abstract

We have reported that of the 10 commonly used AAV serotype vectors, AAV6 is the most efficient in transducing primary human hematopoietic stem/progenitor cells (HSPCs). However, the transduction efficiency of the wild-type (WT) AAV6 vector varies greatly in HSPCs from different donors. Here we report two distinct strategies to further increase the transduction efficiency in HSPCs from donors that are transduced less efficiently with the WT AAV6 vectors. The first strategy involved modifications of the viral capsid proteins where specific surface-exposed tyrosine (Y) and threonine (T) residues were mutagenized to generate a triple-mutant (Y705 + Y731F + T492V) AAV6 vector. The second strategy involved the use of ex vivo transduction at high cell density. The combined use of these strategies resulted in transduction efficiency exceeding ~90% in HSPCs at significantly reduced vector doses. Our studies have significant implications in the optimal use of capsid-optimized AAV6 vectors in genome editing in HSPCs.

Figure 1. Transduction efficiency of various capsid-modified AAV6 vectors in human hematopoietic cells.

(a) Human erythroleukemia cells, K562, were either mock-transduced, or transduced with the wild-type (WT) or various indicated capsid-modified scAAV6-CBAp-EGFP vectors at an MOI of 3,000 vgs/cell. Transgene expression was analyzed by flow cytometry 48 hrs post-transduction. (b) Primary human bone marrow-derived CD34⁺ cells from a single individual donor were transduced in triplicate with the WT or the Y705 + 731F + T492V triple-mutant (TM) scAAV6-CBAp-EGFP vectors at MOIs of 3,000 or 10,000 vgs/cell. Transgene expression was analyzed by flow cytometry 48 hrs post-transduction. (c) Mean fluorescence intensity of transgene expression. Error bars represent standard deviations (SD).

Figure 2. Transduction efficiency of AAV vectors in human hematopoietic cells at various cell densities.

(a) K562 cells were transduced at various indicated cell densities at MOIs of 3,000 or 30,000 vgs/ml with WT scAAV6-CBAp-EGFP vectors. Transgene expression was analyzed by FACS 48 hrs post-transduction. (b) K562 cells were also transduced at low (1 × 10⁶/mL) or high (1 × 10⁷/mL) cell densities with 3,000 vgs/cell of TM scAAV6-CBAp-EGFP vectors, and transgene expression and mean fluorescence intensity were determined as described above. (c) The vector genome copy numbers/cell were determined 2 hrs post-vector transduction by qPCR and data were normalized to β-actin DNA copy number. (d) K562 cells were transduced at low or high cell densities with TM-scAAV6-CBAp-Gluc vectors, and luciferase expression was determined in the culture supernatants. (e) K562 cells were transduced at low or high cell densities with QM-scAAV2-CBAp-EGFP vectors, and transgene expression and mean fluorescence intensity were determined as described above. (f) Primary human bone marrow-derived CD34⁺ cells were transduced at low or high densities with indicated AAV6 or AAV2 vectors, EGFP-expressing cells were visualized under a fluorescence microscope 48 hrs post-transduction. (g) The vector genome copy numbers/cell were determined as described above.

Figure 3. Transduction efficiency of TM-ssAAV6 and TM-scAAV6 vectors in primary human CD34⁺ cells.

(a) Primary human cord blood-derived CD34⁺ cells were either mock-transduced, or transduced at day 0 at low (0.5 × 10⁶cells/ml,) or high (1 × 10⁷ cells/ml) cell density with 20,000 vgs/cell of the indicated AAV6 vectors in serum free XVIVO20 medium. Two hrs later, cells were diluted to 5 × 10⁵cells/mL and switched to the expansion medium (IMDM + FBS + SCF + IL3 + Epo+ Dexamethasone + β-estradiol + β-mercapthoethanol). EGFP expression was determined by flow cytometry at day 4 and day 10 post-transduction. (b) Following mock-transduction, or transduction of CD34⁺  cells as described above, cells were switched to the expansion medium for 10 days, and cultured in an erythroid differentiation medium (IMDM + BSA + Insulin + Transferrin + Epo) for an additional four days. EGFP expression was determined by flow cytometry. (c,d) Vector-transduced CD34⁺ cells cultured in the differentiation medium were stained with hCD36-PE and hGlycophorin A⁻FITC and analyzed by flow cytometry for the following: non-erythroid (CD36⁻/glycoA⁻), and erythroid cells (CD36⁺/GlycoA⁺) from day 10 to day 14.