| Gene-modified pig skin(GMPS) is made by transferred CTLA4Ig gene into viability pig skin using adenovirus vector(Ad5) in vitro, which express CTLA4Ig protein on recipient wound surface locally, blockade of costimulatory pathway of CD28-B7, induce T-cell anergy or apoptosis and even the antigen-specific immune tolerance , thus decrease immune rejection and prolong the survival of grafts, and avoid administration of immunosuppressive agents render the burn patients-- which usually have complicated immune dysfunction-- to be at high risk for various infections. So it is an ideal biologic dressings for clinic. However, pig skin is relatively difficult to transfer in vitro with Ad5-based vectors, a widely used viral vector, we should improve therapeutic efficacy of GMPS throμgh increasing transfer efficiency.The improvement of Ad gene transfer efficiency has become an area of intense investigation. Pig skin is relatively difficult to transfer since the skin functions as a barrier against invasion by chemicals and pathogens. The efficient transduction of pig skin requires a high multiplicity of infection (MOI) and long time, which results in some side effect, and the transfer efficiency is not to improved with titer when it reaches to a threshold. Therefore, the improvement of Ad gene transfer efficiency has significance for GMPS production and developing therapeutic strategies for the treatment of skin disease.The main events following an adenovirus infection of pig skin can be divided into three steps. (1) Diffusion:The first phase is adenovirus diffuse into the surrounding of skin cells overcome cutaneous barrier. This phase can be limited by permeability barrier of pig skin including horny layer, tight junctions and desmosome etc. (2) Adhesion:Gene transfer efficiency may also be influenced by electrostatic repulsion between the negatively charged cell surface and the net negatively charged Ad particles in this step. (3) Integration:This step usually proceeds through endocytosis and the efficiency of Ad-mediated gene transfer depends mainly on the density of expressed high affinity coxsackie virus and adenovirus receptor (CAR) as well as on the integrins of theανβ3/ανβ5 family on the cell surface. However, skin is relatively difficult to be transduced by Ad5 because they express low levels of CAR.The present paper was focuses on improve the Ad-mediated gene transfer efficiency of pig skin through overcome important limiting factors of three steps of adenovirus infection process, in order to decrease the amount of recombinant virus particles, reduce production time and improve GMPS quality and efficiency.In the first part:A new adenoviral vector system (Ad5/F35) was developed by substitution of a human B adenovirus serotype 35 to overcome the limitations of Ad5 tropism and achieve high transfer efficiency of skin tissue which expresses low levels of CAR. This new vector utilizes the ubiquitous CD46 molecule as receptor for its entry into host cells, and can efficiently transduce a wide variety of cell types, such as CAR-positive or -negative cells. To optimize gene transfer efficiency, we compared the exogenous gene transfer efficiency in pig skin by Ad5F35 vector to that of the widely used adenovirus vector Ad5. As a result, the best transfection condition for Ad5F35 is 5×108(TCID50)×1h,then is 3×108(TCID50)×2h The transduction efficiency were 1.49 and 1.73 times respectively to that of Ad5 at the same conditions. However pig skin treatment by higher infectious titer adenovirus with long time can lead to some side effect and decrease transduction efficiency. Thus, our results demonstrate that Ad5F35 is an effective viral carrier for gene delivery to the pig skin, but we need to carry out subsequent research using Ad5F35 to increase transfer efficiency and decrease side effect.In the second part:we investigate whether the application of polycation like DEAE- dextran,protamine,Hexadimethrine bromide may substantially improve the Ad-mediated gene transfer into skin cells. We hypothesize that polycation serve as bridges between the negatively charged cell surface and the virus and allow us to reduce the amount of recombinant virus particles necessary for an efficient transgene transfer and expression, and in this way considerably reduce the side effect caused by higher infectious titer. The results indicate that gene transfer efficiency has been augmented by addition of DEAE-dextran,protamine,Hexadimethrine bromide respectively, and 500μg/mL DEAE-dextran showed the best transduction efficiency, maximally enhanced transgene transfection in skin cells by 19.3-fold.In the third part: azone were added to adenovirus to enhance skin permeability by altering the structure of the horny layer, and combine transfection and cryopreservation together to short time of production. The result showed that 1% azone can reduce 90min of production and enhance transgene transfection by 2.9-fold, and adding 500μg/mL DEAE-dextran together can increase to 10.2-fold and remain the same viability of control.In conclusion, Ad5F35-CTLA4Ig added 1% azone and 500μg/mL DEAE-dextran can enhanced gene transfer efficiency by 10.2-fold and reduce 90min in production of GMPS which has better quality to that of control.
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