High-efficiency transduction of primary human hematopoietic stem cells and erythroid lineage-restricted expression by optimized AAV6 serotype vectors in vitro and in a murine xenograft model in vivo

Abstract

We have observed that of the 10 AAV serotypes, AAV6 is the most efficient in transducing primary human hematopoietic stem cells (HSCs), and that the transduction efficiency can be further increased by specifically mutating single surface-exposed tyrosine (Y) residues on AAV6 capsids. In the present studies, we combined the two mutations to generate a tyrosine double-mutant (Y705+731F) AAV6 vector, with which >70% of CD34(+) cells could be transduced. With the long-term objective of developing recombinant AAV vectors for the potential gene therapy of human hemoglobinopathies, we generated the wild-type (WT) and tyrosine-mutant AAV6 vectors containing the following erythroid cell-specific promoters: β-globin promoter (βp) with the upstream hyper-sensitive site 2 (HS2) enhancer from the β-globin locus control region (HS2-βbp), and the human parvovirus B19 promoter at map unit 6 (B19p6). Transgene expression from the B19p6 was significantly higher than that from the HS2-βp, and increased up to 30-fold and up to 20-fold, respectively, following erythropoietin (Epo)-induced differentiation of CD34(+) cells in vitro. Transgene expression from the B19p6 or the HS2-βp was also evaluated in an immuno-deficient xenograft mouse model in vivo. Whereas low levels of expression were detected from the B19p6 in the WT AAV6 capsid, and that from the HS2-βp in the Y705+731F AAV6 capsid, transgene expression from the B19p6 promoter in the Y705+731F AAV6 capsid was significantly higher than that from the HS2-βp, and was detectable up to 12 weeks post-transplantation in primary recipients, and up to 6 additional weeks in secondary transplanted animals. These data demonstrate the feasibility of the use of the novel Y705+731F AAV6-B19p6 vectors for high-efficiency transduction of HSCs as well as expression of the b-globin gene in erythroid progenitor cells for the potential gene therapy of human hemoglobinopathies such as β-thalassemia and sickle cell disease.

Figure 1. Transduction efficiency of WT and tyrosine-mutant scAAV6 vectors in human hematopoietic cells.

Approximately 5×10³ K562 cells were either mock-infected, or infected with 5×10³ vgs/cell of WT or various tyrosine-mutant scAAV6-CBAp-EGFP vectors, and transgene expression was determined 48 hrs post-infection using a Zeiss fluorescence microscope (Panel A), and Accuri C6 flow cytometer (Panel B) (Original magnification, x100) Approximately 1×10⁴ primary human CD34⁺ cells were either mock-infected, or infected with 2×10⁴ vgs/cell of WT or various tyrosine-mutant scAAV6-CBAp-EGFP vectors under identical conditions, and transgene expression was determined 72 hrs post-infection by fluorescence microscopy (Panel C) (Original magnification, x200), and quantified by fluorescence-activated cell sorting (FACS) using a BD FACS Aria Flow Cytometer followed by processing with software FCS Express 4 (Panel D). *Y705+731F-scAAV6 vs. WT-scAAV6 vectors, p<0.01.

Figure 2. Transcriptional potential of CBAp, HS2-βp, and B19p6 promoters in human hematopoietic cells.

Approximately 1×10⁴ cells were either infected with DM scAAV6 vectors (K562 cells) or WT scAAV6 vectors (human CD34⁺ cells) expressing the EGFP gene under the control of the three different promoters at 5×10³ vgs/cell (K562 cells) or 2×10⁴ vgs/cell (human CD34⁺ cells), respectively. Transgene expression in K562 cells (Panels A and B) and human CD34⁺ cells (Panels C and D) was determined 72 hrs post-infection by fluorescence microscopy and quantitated by the flow cytometry as described above. The original image magnifications were 100× (Panel A) and 200× (Panel B). *DM-scB19p6-EGFP vs. DM-scAAV6-HS2-βp-EGFP vectors, p<0.01.

Figure 3. Transcriptional potential of CBAp, HS2-βp and B19p6 promoters in human erythroleukemia cells following erythroid differentiation.

Equivalent numbers of mock-treated, or Epo-induced erythroid-differentiated K562 cells were infected with 5×10³ vgs/cell of scAAV6-Gluc vectors, and transgene expression was determined 18 hrs post-infection (A). Fold changes in transgene expression from the three promoters were calculated from untreated vs. Epo-treated groups (B).

Figure 4. Transcriptional potential of CBAp, HS2-βp and B19p6 promoters in primary human CD34⁺ and CD36⁺ human cells following erythroid differentiation.

Approximately 1.5×10⁴ CD34⁺ cells, and ∼2×10⁴ primary human CD36⁺ erythroid progenitor cells were cultured with or without Epo (3 U/ml) for various indicated times, and infected with 1×10⁴ vgs/cell scAAV6-Gluc vectors under identical conditions. Transgene expression levels were determined at 18 hrs post-infection at each time-point. Gluc activity at various time-points was normalized to the group without Epo-induction, and the normalized absolute values are shown as average ± standard deviation from triplicates for CD34⁺ cells (Panel A), and for CD36⁺ cells (Panel C). Fold changes in transgene expression following erythroid-differentiation were calculated by dividing the normalized Gluc activities by the initial activity on Day 0 (Panels B and D).

Figure 5. Bioluminescence imaging of mice transplanted with human CD34⁺ cell in vivo.

NSGmice transplanted with mock-infected, or various indicated scAAV6 vector-infected primary human CD34⁺ cells was acquired by a Xenogen IVIS® Imaging System 6-weeks post-transplantation. Images of representative animals from each group are shown (Panel A). The luminescence signal intensity was quantified as photons/second/cm²/steridian (p/s/cm²/sr) using the Living Image® software (Panel B).

Figure 6. Relative levels of transgene expression from HS2-βp and B19p6 promoters in primary human CD34⁺ cells following xenotransplantation in NSG mice.

Approximately 1×10⁶ primary human CD34⁺ cells were either mock-infected, or infected with 2×10⁴ vgs/cell of WT-scAAV6-B19p6-Gluc, Y705+731F DM-scAAV6-HS2-βbp-Gluc, or Y705+731F DM-scAAV6-B19p6-Gluc vectors under identical conditions, and engrafted into NSG mice as described under Materials and Methods. Gluc activity was measured 3 weeks (Panel A) and 12-weeks (Panel B) post-engraftment in peripheral blood using a luminometer. Total relative light units (RLU) per second were calculated, and results are presented as mean ± s.d., with P<0.001 as calculated by student’s t-test.

Figure 7. Transgene expression in various human hematopoietic lineages 12-weeks post-transplantation of human CD34⁺ cells in primary recipient NSG mice.

Bone marrow cells were harvested and human lineage-specific cells were sorted and Gluc activity in the sorted cell populations, was determined as described above. *p<0.034 (erythroid vs. B cells) and *p<0.037 (erythroid vs. monocytes).

Figure 8. Bioluminescence imaging of mice following secondary transplantation.

Whole bone marrow cells from NSG mice transplanted with mock-infected, or DM scAAV6-B19p6-Gluc vector-infected primary human CD34⁺ cells were harvested 12-weeks post-primary transplantation, and transplanted into secondary recipient mice. Six-weeks post secondary transplantation, mice were subjected to whole-body bioluminescence imaging in vivo as described above.