AAV Mediated Gene Therapy For Hemophilia A By Combined Human FⅧ Heavy Chain And Rat FⅧ Light Chain

ObjectiveGene therapy for hemophilia A（HA）has the potential possibility for overcoming the disadvantages of the traditional replacement therapy.Gene therapy can reduce the risks arisen from high titer viral vectors（AAV）by developing a high-activity FⅧ,which is better for lowering the immunoreaction and reducing the development of neutralizing antibodies（inhibitors）.Codon optimized FⅧcDNA has exhibited the enhanced activity of FⅧ.Besides,it has been demonstrated that FⅧproteins from other species（such as porcine and canine）could be more efficient in expression or secretion than that of human FⅧ（hFⅧ）.However,the comparison of the activity of rat FⅧ（rFⅧ）and hFⅧhas not been evaluated.Based on the high homology between the rFⅧand hFⅧin nucleotide and amino acids（61%,51%）,we aimed to find a new FⅧcassete with higher expression efficiency by substitution in different amino acids and point mutation in hFⅧsequence,and offer basic research data for AAV-delivered FⅧgene therapy for HA.MethodshFⅧ-BDD-SQ,rFⅧ-BDD SQ,hHC,hLC,rHC,rLC genes were constructed into plasmids expression vectors with CB promoter（CMV enhancer with humanβ-actin promoter,ubiquitous expression）and ApoE-hAAT promoter（liver-specific expression）respectively.The plasmids expressing FⅧby single chain strategy or HCs combined with LCs by dual chain strategy were transfected into 293T and HepG2 cells,then the FⅧactivity（by APTT assay）and antigen（by ELISA and western blot）in the medium were measured.The plasmids were hydrodynamically injected into HA mice,then the FⅧactivity and antigen in the plasma were measured.The AAV vectors containing the hHC,hLC,rHC and rLC with ApoE-hAAT promoter were packaged and purified separately.Then the AAV vectors carrying the HC and LC genes were mixed at the ratio of 1:1,and were injected into the HA mice through tail vein.The HA mice were randomly grouped into high dose group(2×10¹³vg/kg)and mediate dose group(8×10¹²vg/kg).The long-term expression of FⅧin the plasma was then monitored every other week,and the safety of AAV mediated gene therapy for HA was evaluated.ResultsThe present data demonstrated that rFⅧactivity was greater than that of hFⅧin vitro and in vivo;and,the results from the 293T and HepG2 cells showed that rLC combined with hHC（hHC+rLC）increased the activity compared with the combination of hLC and hHC（hHC+hLC）.In ELISA and western blot experiments,the expression levels of hHC+rLC and hHC+hLC were comparable between the groups.The specific activity（the activity caliberated by the protein quantity）of hHC+rLC was about 4-5 folds higher than that of hHC+hLC.In addition,the rLC did not potentially enhance the thermal stability of the hHC+rLC protein.In vivo,data from the hydrodynamically injected HA mice showed the elevated activity and specific activity of hHC+rLC compared with hHC+hLC,and the specific activity of hHC+rLC was about 4.5-fold higher than that of hHC+hLC.Notably,the HA mice underwent the AAV-delivered hHC+rLC treatment,regardless of the mediate or high doses,all exhibited a significantly higher FⅧactivity than those treated with the AAV-delivered hHC+hLC,and the activity had a significant statistical difference between hHC+rLC and hHC+hLC of high dose group.The long-term therapeutic effects of the AAV-delivered hHC+rLC treatment lasted about 36 weeks.The liver function of mice was basically normal and one of the mice in high dose group developed inhibitor of FⅧ.ConclusionWe demonstrated in vitro and in vivo that rFⅧactivity was greater than that of hFⅧ,and rLC combined with hHC increased the activity and specific activity of FⅧcompared with hLC+hHC combination;the AAV-delivered FⅧcould exert a long-term therapeutic effect by sustaining the expression and maintain the activity of FⅧin HA mice;the difference in amino acids between rLC and hLC may play a leading role in elevated FⅧactivity.This study offers basic research data for AAV-delivered FⅧgene therapy for HA by finding a new FⅧcassete with higher delivery efficiency and may be of help to offer another choice for the development of the novel optimized FⅧsequence contributing to higher human FⅧactivity.