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Co-expression of the complement regulatory proteins human DAF and CD59 with an IRES-mediated dicistronic mammalian

Li Xu1, Huanjie Shao, Wenlan Wu

  • 1State Key Laboratory of Virology, College of Life Science, Wuhan University, P.R. China.

International Journal of Molecular Medicine
|August 4, 2005
PubMed

Insights

Combining human complement regulatory proteins, decay accelerating factor (DAF/CD55) and protectin CD59, via a novel vector, effectively protects cells against complement-mediated damage. This strategy shows promise for preventing hyperacute rejection in xenotransplantation.

Area of Science:

  • Biotechnology
  • Immunology
  • Gene Therapy

Background:

  • Human complement regulatory proteins (hCRPs), including decay accelerating factor (DAF/CD55) and protectin CD59, are crucial for preventing host complement-mediated damage.
  • Hyperacute rejection in xenotransplantation is a major barrier, often mediated by complement activation.

Purpose of the Study:

  • To develop a strategy for co-expressing DAF and CD59 in non-human cells to enhance protection against human complement.
  • To evaluate the efficacy of a dicistronic expression vector utilizing the encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES) for co-delivering hCD59 and hDAF.

Main Methods:

  • Construction of a dicistronic mammalian expression vector (pcDNA3-CD59IRESDAF).
  • Stable transfection of NIH/3T3 cells and confirmation of co-expression using RT-PCR, Western blotting, and immunofluorescence microscopy.
  • Assessment of cellular protection via human complement-mediated cytolysis assays.

Main Results:

  • The EMCV IRES facilitated efficient co-expression of hCD59 and hDAF on the cell surface.
  • Co-expressed DAF and CD59 provided significantly greater protection against complement-mediated cytolysis compared to individual proteins.
  • The pcDNA3-CD59IRESDAF vector demonstrated effective control of complement activation.

Conclusions:

  • Internal ribosomal entry site (IRES)-containing polycistronic vectors enhance multi-gene delivery efficiency and effectiveness.
  • The pcDNA3-CD59IRESDAF construct holds therapeutic potential for controlling complement activation and preventing hyperacute rejection in clinical gene therapy applications.
  • Co-expression of DAF and CD59 is a superior strategy for combating complement-mediated damage in xenotransplantation.

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