In vitro and in vivo expression of human coagulation factor IX mediated by nonviral transfection of three plasmid vectors coding a factor IX gene : 혈액응고인자 IX 유전자를 함유한 세 가지 플라즈미드의 비바이러스성 유전자 전달을 통한 체외와 체내에서의 응고요소 IX의 발현

국문 초록 (Abstract)

B형 혈우병은 X 염색체에 위치한 혈액응고인자 IX 유전자의 손상에 의해 혈액 응고인자가 생성되지 못하여 생기는 유전병으로 현재는 혈소판 농축액을 주입하여 일시적인 치료를 하고 있다. 그러나 이런 치료방법은 바이러스 감염 등의 부작용을 일으킬 수 있어 상대적으로 안전하고 경제적인 치료방법으로 여겨지는 유전자 치료 방법을 연구하고 있다. B 형 혈우병 유전자 치료는 응고인자 IX의 혈액내 수치를 최소 치료 농도인 5%이상의 발현을 목표로 하고 있다.
우선 세 가지 유전자발현 벡터 pAAVCMV-hFIX, pFLAG-hFIX, pCIneo-hFIX 를 제조하고, 이 세 가지 벡터의 효율을 비교하기 위해 세 가지 양이온지질 DOTAP, DMDK, DMEK와 여러 비율로 섞어서 293 cell에 유전자를 전달하고 최적의 조건을 확립하였다. 또한 같은 유형의 실험을 HepG2 cell과 Chang cell에서도 시행하였다. 그 결과 체외 유전자 전달에서는 DMDK 와 DMEK를 이용한 pAAVCMV-hFIX의 발현이 181 ng/㎖까지 이르러 가장 높게 발현되었으며, 같은 조건에서 HepG2 cell과 Chang cell에서 보다 높게 발현되었다. 이 수치는 DOTAP 을 이용했을 경우보다 1.8배 높게 발현된 수치였다. 같은 실험조건에서 벡터만을 비교했을 경우는 pAAVCMV-hFIX가 pFLAG-hFIX보다 1.37배, pCIneo-hFIX보다 1.17배 높게 발현되었다. 종합적으로 볼 때 pAAVCMV-hFIX을 DMDK나 DMEK 을 이용하여 유전자 전달했을 경우 가장 우수한 체외 유전자 발현이 관찰되었다. 발현된 응고인자 IX의 단백질 기능을 확인키 위해 aPTT 실험을 하였다. pAAVCMV-hFIX, pFLAG-hFIX, pCIneo-hFIX에서 발현된 응고인자를 응고인자 IX 결핍 혈장에 섞었을 때 혈장응고가 각각 68.8초, 65.7초, 64.9초에 일어남에 따라 정상기능의 응고인자 IX이 발현됨을 알 수 있었다.
마우스체내에서의 발현을 확인하기 위해 각 벡터를 Hydrodynamics 방법으로 주사하여 유전자 발현율을 비교하였다. 그 결과 유전자 전달 24시간 후에 pAAVCMV-hFIX, pFLAG-hFIX, pCIneo-hFIX vector는 각각 2250ng/㎖, 750 ng/㎖, 1860 ng/㎖의 응고인자 IX을 발현하였다. 체외유전자 전달에서와 마찬가지로 pAAVCMV-hFIX이 가장 높은 발현을 보였으며 5일째까지도 1170 ng/㎖의 발현을 유지하였다. 체내와 체외에서 발현된 factor IX은 SDS-PAGE와 Western blot analysis로 정성분석 하였고, ELISA로 정량하였다. Hydrodynamic 유전자 전달로 인한 간 손상여부를 분석하기 위해 ALT, AST, Total bilirubin 측정을 하였는데 일시적인 ALT, AST수치 상승만을 보이고 5일 내에 정상으로 돌아와 비교적 안전함을 확인하였다.
세 가지 유전자 vector를 비교한 결과 pAAVCMV-hFIX이 가장 우수한 것으로 확인되었으며 이러한 실험결과들은 혈우병에 대한 유전자 치료의 기초 자료로 이용될 수 있을 것이다.

다국어 초록 (Multilingual Abstract)

Gene therapy for hemophilia B is an attractive treatment because the current method of repeated infusion of purified factor IX concentrates has a high risk of viral infection and also is very expensive and inconvenient.The goal of the hemophilia B gene therapy is to introduce a continuous supply of factor IX at a therapeutic level. In this thesis, the efficiencies of three plasmid vectors containing human factor IX gene (pAAVCMV-hFIX, pFLAG-hFIX, and pCIneo-hFIX) transferred with three different cationic liposomes (DOTAP 1,2-dioleoyloxyproryl-3-N,N,N-trimethylammonium chloride, DMDK dimyristyl aspartyl lysine, and DMEK dimyristoyl-glutamyl-lysine) were compared to each other. Under the optimized in vitro transfection conditions for 293 cells, transfection of pAAVCMV-hFIX with DMDK liposomes vector was able to produce approximately 181 ng factor IX/106 cells (3.6% of normal factor IX level) at 5 days post transfection. At the same conditions, pFLAG-hFIX and pCIneo-hFIX were expressed 132 ng factor IX/106 cells ,155 ng factor IX/106 cells, respectively. The DMDK and DMEK liposomes exhibited a similar gene transfection efficiency in 293 cells and Chang cells. The expression level of human factor IX transfected with the DMEK or DMDK liposomes was 1.8-fold higher than that with DOTAP-based liposomes. Among the DNA vectors, pAAVCMV-hFIX exhibited the highest expression of human factor IX which was 1.37-fold and 1.17-fold higher than pFLAG-hFIX and pCIneo-hFIX, respectively. The factor IX protein expressed by three different vectors showed clotting activity which was assayed by an aPTT test. When 100 ㎕ culture medias transfected with pAAVCMV-hFIX, pFLAG-hFIX, or pCIneo-hFIX in 293 cells was mixed with 100 ㎕ of factor IX-deficient human plasma, their clotting times were shorten to 68.8, 65.7, and 64.9 seconds. Furthermore, to evaluate in vivo expression efficiencies of the three plasmids containing human factor IX gene, the DNA vectors were administerred to C57BL/6 mice by the hydrodynamics method. The pAAVCMV-hFIX, pFLAG-hFIX, or pCIneo-hFIX administerred by the hydrodynamics method was able to express the human factor IX at the levels of 2250 ng factor IX/㎖, 750 ng factor IX/㎖, and 1860 ng factor IX/㎖24 hours post injection, respectively. The expression levels were equivalent to 15∼45% of the factor IX level in normal blood. However, 5 days later, only the mice transfected with pAAVCMV-hFIX maintained the expresseion level at 1170 ng factor IX/㎖ while the mice transfected with the other vectors showed substantially diminished their gene expression. The expression of factor IX protein was maintained only for a week. This may be due to its degradation by anti-human factor IX antibodies induced by stimulation of the expressed human factor IX. Anti-human factor IX antibodies were detected by an indirect ELISA and its level was 100-200 ng/㎖ in all three vectors at 5 days post transfection and maintained for 28 days. This hydrodynamics gene transfer of hFIX plasmid DNA into liver elicited neither transgene specific cytotoxic effect nor long-term toxicity. In summary, among the tested DNA vectors and cationic liposomes, the combination of pAAVCMV-hFIX and DMDK or DMEK liposomes was a primary choice for further preclinical experiment of hemophilia B gene therapy.

목차 (Table of Contents)

• LIST OF FIGURE and TABLE = iii
• ABBREVIATION = iv
• ABSTRACT = v
• I. INTRODUCTION = 1
• II. MATERIALS AND METHODS = 4
• 1. Constructions of hFIX-plasmids = 4
• 1-1. Human factor IX gene = 4
• 1-2. pAAVCMV-hFIX plasmid vector = 4
• 1-3. pFLAG-hFIX plasmid vector = 5
• 1-4. pCIneo-hFIX plasmid vector = 5
• 1-5. Transformation and preparation of plasmid = 7
• 2. Cell culture = 7
• 3. Preparation of cationic liposomes and DNA/liposome complex = 7
• 4. Gene transfer into 293, HepG2, and Chang cells = 8
• 5. Detection of expressed factor IX in vitro = 9
• 6. Quantitative analysis of in vitro expressed human factor IX by ELISA = 10
• 7. Blood clotting activity of the expressed human factor IX protein = 11
• 8. In vivo gene transfection = 11
• 9. Measurement of anti-human factor IX antibodies induced by foreign gene transfer = 12
• 10. Liver cytotoxicity test = 13
• III. Results = 14
• 1. In vitro human factor IX expression mediated by a variety of lipoplex = 14
• 2. Identification of the expressed human factor IX in vitro = 15
• 3. Biological activity test of the expressed human factor IX in media = 19
• 4. In vivo human factor IX expression = 19
• 5. Induction of anti-human factor IX antibodies in transfected mice = 22
• 6. Transient cytotoxic effect = 25
• IV. Discussion = 28
• V. Conclusion = 35
• VI. References = 37
• 국문요약 = 49