T cell:

Because HIV preferentially targets CD4+ T cells, we propose to use HIV envelope protein to direct our exosomes to the CD4+ T cells. We prepared exosomes from 293T cells that were engineered to express a fusion of the exosomal membrane protein Lamp2b and a short peptide from the HIV envelope glycoprotein gp120. The peptide from gp120 can recognizes the host cell receptors CD4, and Lamp2b can bring the peptide to the surface of exosomes. Theoretically, by expressing this CD4+ T cell-targeting protein on the surface of exosomes, filling exosomes with siRNA drug and injecting the modified exosomes into the bloodstream, we will achieve specific siRNA delivery to the CD4+ T cells, whereas non-specific uptake of siRNA in other tissues was be avoided.

Experimental procedure

To verify the targeting ability of our designed exosomes, we first collected two kinds of exosomes: 1) exsosomes with both siRNA inside and targeting peptides gp120 on the surface. 2) exsosomes with only siRNA inside (without targeting ability).

The second step was to obtain a pool of lymphocyte (containing both CD4+ cells and CD4- cells), which were harvested from spleen and blood of C57BL/6 mice. We first determined the percentage of CD4+ T cell in all lymphocytes by flow cytometry. Then we set up two groups: 1) incubation of lymphocytes with CD4+ T-targeting exosomes for 24 hours (10 μg exosomes/100000 cells). 2) incubation of lymphocytes with non-targeting exosomes for 24 hours (10 μg exosomes/100000 cells).

Subsequently, the CD4+ T cells in each group were separated from the lymphocytes pool by magnetic bead separation techniques. Finally, we got 4 groups of lymphocytes: 1) CD4+ T cells treated with non-targeting exosomes; 2) other lymphocytes (CD4-) treated with non-targeting exosomes; 3) CD4+ T cells treated with CD4+ T cell-targeting exosomes; 4) other lymphocytes (CD4-) treated with CD4+ T cell-targeting exosomes.

Finally, qPCR analysis was used to determine the level of siRNA in these four groups.

Results

As shown in the Fig.1, flow cytometry analysis showed that CD4+ T cells accounted for 16.0% of the total lymphocytes, whereas 84.0% of lymphocytes were CD4-. (Fig.1A and 1B)

In the group of lymphocytes treated with non-targeting exosomes, the percentage of siRNA in CD4+ T cell was only 16.4%, whereas 83.6% of siRNA was delivered to CD4- lymphocytes (Fig.1C). The accordance between siRNA distribution and cell type ratio indicates that exosomes, without modification, will enter the lymphocytes randomly.

The most exciting part of this experiment is shown below. In the group of lymphocytes treated with CD4+ T cell-targeting, the percentage of siRNA in CD4+ T cell was increased to 61.9%, much higher than that of random distribution, and siRNA that was delivered to CD4- lymphocytes significantly dropped (Fig.1D). The result suggests that our CD4+ T cell-targeting exosomes successfully deliver siRNA to CD4+ T cells with high specificity and efficiency.

Fig.1 A. direct result of flow cytometry analysis of lymphocytes. B. Percentage of CD4+/- cells. C. group treated with exosomes containning siRNA only and the figure shows the siRNA distribution between CD4- T cells and CD4+ T cells. D. group treated with T-cell targeting siRNA-contained exosomes. Figure shows the siRNA distrbution between CD4- T cells and CD4+ T cells.