| Hemophilia A is an X chromosome-linked recessive inheritablebleed-ing disorder with an estimated incidence of 1-2 per 10000 males.Patients wi-th Hemophilia A experience spontaneous bleeding into joints,soft tissues and other sites. Uncontrolled haemorrhagic episodes arecrippling and potentially lifethreatening. Hemophilia A is caused by adeficiency or abnormality in factorⅧ. which is one of the most attractivecandidates for gene therapy.The expression level of recombinant hFⅧis extremely low in mam-malian cells. The major factors that limit hFⅧexpression have been ide-ntified: (ⅰ) There is a 1.2kb stretch of FⅧcDNA within the FⅧcodingregion having a deleterious effect on RNA accumulation; (ⅱ) the primarytranslation product is inefficiently transported from the endoplasmic rect-iculum (ER) to the Golgi apparatus; and (ⅲ) FⅧis very sensitive to theprotease, and von Willebrand factor (vWF) are required for stable accu-mulation of FⅧ. Results of many laboratories showed that modifying thehFⅧmolecule is an efficient way to improve its expression in mamm-alian cells. The major modifications of hFⅧmolecule are as follow: 1,deletion of B domain; 2, deletion of 5'-UTR and 3'-UTR; 3, insertion ofintron of ApoA-1. Our lab had generated hFⅧ-BSP which contain themodification of hFⅧdescribed above. In this study, we modified the hFⅧ-BSP to hFⅧ-SK39 as follows: 1, Kozak box was inserted justbefore the initial code of hFⅧ-BSP, which can improve the translationefficiency of hFⅧ; 2, L303E & F309S, which can enhance the secretionof functional hFⅧ.In gene therapy, one of the most important issues is the choice ofvectors. Viral vectors are capable of efficiently delivering geneticmaterial into cells in vivo and in vitro, and about 70% of the clinical trialsare performed using viral vectors. However, their safety is of seriousconcern, pHrneo is a new type of nonviral vector previously developed byour group, which can target a foreign therapeutic gene into humanribosomal DNA (hrDNA) locus in HT1080 cells.In this study, firstly, we transfected HT1080 with pcDBSP(containing hFⅧ-BSP) and pcDSK39 (containing hFⅧ-SK39) usingelectroporation. The expression of hFⅧ-SK39 and hFⅧ-BSP in HT1080were tested. Secondly, we inserted expression cassette of hFⅧ-SK39 intopHrneo to construct targeting vector: pHrneo-SK39. Throughelectroporation of pHrneo-SK39 into HL7702 cells (human hepatocyte),we identified the homologous recombinants by PCR. The expression ofhFⅧ-SK39 and growth character of the site-specific integration cellswere tested. Finally, pHrneo-SK39 was injected into the tail vein ofBalb/c-nu mouse by high-pressure hydrodynamic injection. We tested theconcentration of hFⅧ-SK39 in Balb/c-nu mice, and examined the toxicity in the mouse li-vers after the injection of pHrneo-SK39.Our results show that①, the expression level of hFⅧ-BSP andhFⅧ-SK39 in HT1080 are 2.99±0.28 ng·10-6cells·24 h-1 and 9.90±1.89ng·10-6cells·24 h-1 respectively.②,The hFⅧ-SK39 was successfullytargeted into hrDNA locus of HL7702 by pHrneo-SK39, and theefficiency of site-specific int-egration was 1.1×10-5.③, The hFⅧ-SK39at the hrDNA locus of HL7702 was found to be able to express efficiently(4.3±0.9 ng·10-6cells·24 h-1), and the growth character ofsite-integration cells was normal.④, the expression of hFⅧ-SK39 insite-integration cells was stable.⑤, pHrneo-SK39 can express inBalb/c-nu mice, and the highest expression level is 4.56ng/ml in bloodplasm.In short, we think that pHrneo-SK39 may find uses in gene therapyfor hemophilia A.
|
PDF Full Text Request