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Cell therapy model in hemophilia B by genetically modified keratinocytes by lentiviral vectors to produce human clotting factor IX.

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Cell therapy model in hemophilia B by genetically modified keratinocytes by lentiviral vectors to produce human clotting factor IX.


IA González-Ramos, IJ Lara-Navarro, LF Jave-Suárez, JA Marchal, A Bernad, AR Jaloma-Cruz.


BACKGROUND / AIMS. Hemophilia B is a key model for gene therapy as replacement treatment. Keratinocytes are highly attractive as "target" of ex vivo gene therapy by their physiological properties and bio-safety traits as monitorable transplant. Self-inactivated (SIN), third-generation lentiviral vectors, show bio-security advantages and higher efficiency of gene expression. Ángeles Escartí (2006) proved the capacity of lentivirus-transduced keratinocyte grafts to secrete biologically active human FIX (hFIX) into the bloodstream in a FIX-deficient transgenic mouse. Dr. A. Bernad transferred us the three lentiviral vectors of hFIX, tested by Escartí, to scale the approach to porcine model. We show preliminary results of the transduction of the vectors into a human cell line DOK (dysplastic oral keratinocytes).

The aim of this study is optimizing the genetic modification of keratinocytes culture by SIN lentiviral vectors for hFIX production.


MATERIAL AND METHODS. Fibroblasts were obtained by enzymatic digestion with collagenase-dispase and trypsin-EDTA from porcine skin biopsies; the DOK cell line was kindly donated by Dr. Juan-Antonio Marchal. Plasmids of lentiviral constructs were transfected into 293-LentiX cells with the HT-Packaging-kit Clontech™ to produce lentiviral particles which in turn transduced keratinocytes to express hFIX as therapeutic gene and EGFP as marker. Transduction of DOK keratinocytes cell line was performed in 8-16 cell colonies and the efficiency was evaluated at 48 hours and after a week of culture, by EGFP incorporation and hFIX production assessed by flow cytometry. Transduced cells were harvested and sub-cultured in two conditions: with specific medium DMEM/Hydrocortisone and on a gel biomembrane composed of porcine plasma and fibroblasts.

RESULTS. The transduction efficiency, formerly evaluated on human keratinocytes by EGFP production reached >80% with the three vectors (figure 1). The amount of hFIX protein in the supernatant of the DOK cells culture was similar among the three vectors at 48 hours (p = 0.94) and after one week of transduction (p = 0.7). A higher production of hFIX was demonstrated in cultured DOK on the biomembrane at 48 hours (p = 0.38) and a significant increase was reached after one week of culture (p = 0.00).

CONCLUSIONS. Our findings did not show significant differences between the three vectors, because the in vitro model probably was not suitable to test the secretion of therapeutic protein under physiological conditions. A statistically significant difference in the increased production of supernatant hFIX in the gel-cultures, suggests that the bio-matrix provides a more similar physiological environment and promotes the hFIX secretion to the supernatant. Genetically modified keratinocytes for therapeutic gene production by lentiviral vectors is a promising model for several monogenic diseases, including hemophilia. According to our results in vitro, next step will include the graft of genetically modified skin into porcine model to test the efficiency of systemic production of hFIX transgene.




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  Escartí NA. Terapia génica cutánea ex vivo para la hemofilia B mediante trasplantes de equivalentes dermo-epidérmicos manipulados genéticamente con vectores retrovirales. Doctoral Thesis. Universidad Autónoma de Madrid. 2006.


  Gerrard AJ, Hudson DL, Brownlee GG, Watt FM. Towards gene therapy for haemophilia B using human keratinocytes. Nat Genet. 1993;3:180-183.


  Llames S, Del Rio M, Larcher F, García E., Meana A. Human plasma as a dermal scaffold for the generation of a completely autologous bioengineered skin. Transplantation. 2004;77:350–355.


  Serrano F, Del Río M, Larcher F, García M, Muñoz E, Escamez MJ, Muñoz M, Meana M, Bernad A, Jorcano JL. A comparison of targeting performance of oncoretrovoral versus lentiviral vectors on human keratinocytes. Hum Gene Ther. 2003; 14:1579-1585.




ACKNOWLEDEGEMENTS. This project has been registered in CIBO, IMSS with the number R-2014-1305-4.  AR Jaloma-Cruz was sponsored by CSL-Behring, Mexico to attend the BIC Congress.