Enhanced long-term transduction and multilineage engraftment of human hematopoietic stem cells transduced with tyrosine-modified recombinant adeno-associated virus serotype 2

Abstract

The search for the ideal stem cell gene therapy vector continues as recognized problems persist. Although recombinant adeno-associated virus serotype 2 (rAAV2) mediates gene transfer into hematopoietic stem cells, identified restrictions to transgene expression reduce overall efficiency. Studies have shown that transduction efficiencies are significantly improved by preventing early proteasomal degradation after mutation of surface-exposed tyrosine residues on the capsid to phenylalanine. Here, we report that transduction of human cord blood CD34(+) stem cells by tyrosine-modified rAAV2 is significantly enhanced both in vitro and in vivo. Serial long-term in vivo bioluminescent imaging of immune-deficient recipients after xenotransplantation of CD34(+) cells transduced with tyrosine-modified rAAV2-luciferase revealed that modification of rAAV2 capsids led to a significant increase in the transduction of human CD34(+) cells, without adversely affecting engraftment capacity, or the ability to undergo multilineage differentiation and self-renewal. Together with observations of sustained high-level transgene expression in vivo and efficient persistence of rAAV genomes in human hematopoietic cells, these results suggest that, because of their ability to bypass restrictions to transduction, tyrosine-modified rAAV vectors, particularly Y500F, Y700F, Y444F, and Y704F, represent highly promising candidates for therapeutic evaluation for diseases of human hematopoietic stem cells.

FIG. 1.

In vitro transgene expression in human cord blood CD34⁺ cells after transduction with AAV2-self-complementary EGFP packaged in wild-type and tyrosine-modified AAV2 capsids. EGFP expression was determined by flow cytometric analysis. Data shown represent CD34⁺ cells pooled from three separate CB samples per group.

FIG. 2.

Engraftment of human CB CD34⁺ cells transduced with tyrosine-modified AAV2. Engraftment was determined in NOD/SCID mice transplanted with tyrosine-modified rAAV2 encoding either self-complementary enhanced green fluorescent protein or single-stranded luciferase. Mice were transplanted with transduced CD34⁺ cells pooled from one to five blood samples. (A) Human cell engraftment in NOD/SCID mice as determined by the frequency of CD45⁺ cells in the marrow. Each point represents an individual xenograft recipient. Forty mice were analyzed. (B) Frequency of human hematopoietic lineages derived from transplanted CD34⁺ cells at 12–22 weeks posttransplantation. Error bars represent standard errors of the mean. The total number of mice (n) analyzed for CD34, CD33, CD19, CD14, and glycophorin A lineages were 25, 24, 23, 13, and 18, respectively.

FIG. 3.

(A) Whole body luciferase expression in xenograft recipients of rAAV-luciferase-transduced human CD34⁺ cells. Representative mice from different time intervals posttransplantation are shown for each capsid type. (B) Serial in vivo bioluminescent imaging showing luciferase expression in NOD/SCID mice transplanted with human CD34⁺ cells transduced with tyrosine-modified rAAV. Serial whole body imaging was performed biweekly after luciferin administration. Results are shown for three to five mice per group. Each mouse was transplanted with cells pooled from one to five CB samples. Luciferase expression is depicted as flux (photons/sec). ***Luciferase expression for the tyrosine-modified groups relative to wild-type rAAV2 is significant to p < 0.0001; **significant to p < 0.001; *significant to p < 0.005.