Part:BBa_K3335000

We use KIBRA to control exosome secretion

KIBRA as an adaptor-like protein that stabilizes Rab27a, which in turn controls exosome secretion. Rab27a is stabilized by interacting with KIBRA, which prevents ubiquitination and degradation via the ubiquitin-proteasome pathway. KIBRA controls exosome secretion via inhibiting the proteasomal degradation of Rab27a[1].

In our experiment, we used KIBRA to promote cell release of exosomes containing siRNA, thus increasing the efficiency of cell production and release of exosomes and improving the killing of tumor cells.

[1]Lin Song et al,.KIBRA controls exosome secretion via inhibiting the proteasomal degradation of Rab27a.Nature Communication,2019.

Contribution: NJU-China 2020

As previously reported, CMV-hSDC4-STEAP3-NadB can strongly promote exosome release. To develop the more efficient booster part for our strategy, we designed another two parts (CMV-KIBRA and CMV-nSMase2) to find out the most efficient design for our project. We tested all the three designs in vitro. The expression of each mRNA was confirmed by RT-qPCR after transfected to HEK293T cells.

Figure 1. KIBRA, NadB, nSMase2 mRNA relative expression in HEK293T cell (vs GADPH)

To further verify the expression of KIBRA and nSMase2 at protein level, we used Western Blotting experiment to prove that KIBRA and nSMase2 was indeed overexpressed in HEK293T cells.

Figure 2. (A)Western Blotting result shows that nSMase2 is overexpressed in HEK293T cell. (B) Western Blotting result shows that KIBRA is overexpressed in HEK293T cell.

Figure 3. Total amounts of exosomes (shown as total protein) secreted by HEK293 cells with the introduction of nSMase2, KIBRA and hSDC4-STEAP3-NadB.

After confirming the correct expression of these three genes, we further compared their ability to promote exosome secretion. To simplify the process, we first detected the total concentration of the exosomes produced by cells transfected each part. The result indicated that overexpression of KIBRA generates the highest amount of exosomes among these three groups. So we chose KIBRA as the candidate for further characterization.

KIBRA promotes exosome release

To further evaluate the effeciency of KIBRA in promoting exosome release, HEK293T cells were transfected with the KIBRA part, and the exosomes in cell culture medium were characterized. Nanoparticle tracking analysis (NTA) revealed that higher amount of exosomes was secreted in KIBRA-overexpressing group than the control group, while similar size distribution peaking at 124-127 nm was observed in both groups. The result verified that the exosome booster part can significantly promote exosome release without changing the natural size of exosomes produced.

Figure 4.(A) Averaged FTLA Concentration / Size for Exosome (B) Intensity / Size graph for Exosome

Figure 5. NTA analysis for Exosome

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 335
  Illegal BglII site found at 2999
  Illegal BamHI site found at 246
  Illegal BamHI site found at 442
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 435
  Illegal BsaI site found at 2598
  Illegal BsaI.rc site found at 1920
  Illegal BsaI.rc site found at 2644