21세기 바이오 혁명

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4 1865, 1990, HGP 1953, & DNA 1977, & DNA 2001, 99% 1983, PCR 2003,

5 How many genes in the human genome? cdna sequencing Genomic sequencing Structural Genomics What is the role of each gene? Tissue distribution Disease-related gene Gene function Functional Genomics Unknown Genes Known Genes Human Genes 50,000~100,000 Finding of novel functional genes Gene Therapy Target

6 1865, Mendel 1950 s, Franklin 1953, Watson & Crick ( ) (DNA ) (DNA ) 1963, Sanger 1966 (DNA ) ( ) 1993, Mullis (PCR) 1990 ( ) 1980 s, International team ( ) 1972, Berg (DNA ) 1994, FDA 1995, OJ Simpson ( ) (DNA ) 1997, Dolly ( ) 1998, Clinton/Lewinsky (DNA ) 2000, Craig ( )

7 ? ( ) ( ), FDA : "Gene therapy is a medicinal intervention based on modification of the genetic material of living cells

9 : 80,, : 89,, -, TNF - TNF ( ) : 90,, - 4 (Adenosine deaminase ), T, ADA (, ) : 99, - : - ( FDA/NIH ) : 00, Introgen Aventis (p53 3 )...

10 (1989 NCI, ) - TNF-α -, (tumor necrosis factor-α): T effector 1. (TIL) 2. TNF-α cdna 3. IL-2, 4. TNF-α

11 (1990 ) - 4 (Adenosine deaminase ), T ADA -, ADA T, ADA (adenosine deaminase): T adenosine, deoxyadenosine IL-2, 3. ADA cdna 4. ADA

12 1. 2. ( ) 3. (Ex vivo / In vivo) 1. (Gene replacement) 2. (Gene correction) 3. (Gene addition)

13 CFTR Factor VIII A Factor IX E1A p53 B AC6 VEGF Interleukin-2

14 (Physicochemical method), CaPO 4, (microinjection), (electroporation), (liposome), (polymer), (ligand), (antibody) (Biological method),,,,,

15 Ex vivo (indirect method) (, ), In vivo (direct method) Ex vivo In vivo Ex vivo In vivo

17 ,,,,,, HIV,,,,,,,,,,,

18 Protocols by disease 3494 Patients by disease (69%, 2392 ) (12%, 408 ) (9%, 309 ) (10%, 385 ) The Journal of Gene Medicine, John Wiley & Sons 2002

19 (4%, 150 ) (specificity) (efficiency) (specificity), (magnitude), (duration) (immunogenicity) (manufacturing) Patients by vector (6%) (22%) (50%, 1755 ) (72%) (18%, 644 ) The Journal of Gene Medicine, John Wiley & Sons 2002

20 ( / ) I, II / / I II /

22 (viral vector) - DNA virus : Adenovirus, Adeno-associated virus -RNA virus: Retrovirus, Lentivirus (non-viral vector) - : (Naked DNA injection) (Electroporation) (Gene gun) - : - (Lipid-DNA complex : Lipoplex) - (Polymer-DNA complex : Polyplex)

23 (viral vector) (non-viral vector),, Transfection,

24 Naked DNA injection ( ) ( ),,, / / / (1% ) Electroporation ( ), DNA (DNA ),, 1000 Gene gun ( ), 10~20%,

26 85% transfection virus, DNA / /

27 mrna transcription mrna translation cytoplasmic expression

28 RNA genome DNA (60-70% ) : : DNA (8 kb) Transmembrane envelope protein Core protein Core shell protein RNA

29 DNA non-enveloped virus :,, : GenVec coronary artery disease (CAD)

30 (Adeno-associated virus), adenovirus, vaccinia herpes virus helper virus, DNA 5 kb / (helper virus) (Herpes simplex virus, HSV) DNA,, (Lentivirus) (Onyx virus) p53,

32 Transfection complex 1. Endocytosis ( )? 1. Endocytosis 2. Plasma membrane fusion Endosome 2. Plasma membrane fusion DNA release transcription RNA Nucleus nuclear localiztion endosomal release mrna translation Therapeutic Protein Intracellular Effect degradation Lysosome ( ) Local Effect Systemic Effect

34 Lipid based system - Liposomes (cationic / ph-sensitive / fusogenic liposome / stealth liposome / thermosenstive liposome / immunoliposome) - Emulsions - Solid-lipid nanoparticle Polymers Plasmid DNA DNA condensate (poly-l-lysin) DNA/poly-L-lysin complex Delivery system (liposome) or Ligand or Antibody Targeted delivery system Targeted DNA /delivery system Complex

35 , ( ) : : (DNA ), :, / Phospholipids ( ) Plasmid DNA DNA : Transfection transfection : : / : / / ph / /

36 (1) Conventional : (2) Stealth :,, (3) Targeted : ligand (4) Cationic :, DNA

37 (Sterically-stabilized liposome) 100,,

38 ligand MAb Antibody coupling covalent coupling of whole Ig hapten binding Fab surface adsorption PEG spacers

39 DOSPA : 2,3-dioleyloxy-N-2-(sperminecarboxamido)ethyl-N,N-dimethyl- 1-propanammonium DC-Chol : 3-b-[N-(N,N -imethylamminoethane)carbamoyl]cholesterol DOTAP : 1,2-dioleyloxypropyl-N,N,N-trimethylammonium chloride DOTMA : N-(2,3-dioleyloxypropyl)-N,N,N-trimethylammonium chloride DOSPA O O CH 3 N CH 3 N H O H 3 N NH 2 N H 2 NH 3 O DC-Chol O N H N Transfast O O H 3 C N I O O OH OH DOTAP O O H 3 C N I O O OH OH O N Cl DOTMA O

40 Lipoplex (Lipid-DNA complex, -DNA ) DNA, DNA Lipoplex, DNA

41 Tissue or cells Hepatocyte Mouse colon tumor, Fibrosarcoma Liposome Liposome composition Species Genes composition Species Genes EPC/Chol Mice, Rat Asialofetuin DC-Chol Mice pcmv-luc Interferon-g Various organ Lipofectamine TM Balb/C mice Alkaline Phophatase Porcine artery GAP-DLRIE/DOPE Yorkshire Pig pcmv-cat Lung DC-Chol/DOPE GLB43,73,84,221,2 53/DOPE Mice β-galactosidase Lung Sera DOTIM/Chol Female CD1 mice G-CSF, pm75.6, D 5 W Various tissue Myoblast (Muscle cell) DDAB/DOPE/PE G-PE TMAG/DOPE Female CD1 mice New born rat Luciferase prsv-luc prsv-lacz Neuron DOGS Adult female Sprague- Dawley rat pcaggsbpv

42 Self assembled polyplex Polymer ( ) DNA DNA, Polyplex (Polymer-DNA complex) DNA DNA Polyplex, DNA, (Poly-L-lysine) (Polyethylamine) + PEI (polycation) self assembly DNA (polyanion)

43 Untargeted Polyplexes (Adv. Drug Del. Rev. Vol. 54, 2002) Targeted Complexes Polyplex 1. Condensed DNA 2. Endosomolytic peptide 3. Nuclear targeting peptide 4. Hydrophilic shield 5. Cleavable cross-linker 6. Targeting element 7. Erythrocyte

44 Targeting ligand PEG /DNA receptor-mediated

45 (5 mm), gap (30-500nm), endocytoisis Nano Nanosphere Polycation (Gelatin chitosan) DNA, DNA,, Nanosphere, endocytosis transfection Transfection

47 : : 8~12 ( ) :,,,,, : / / ( ) ( / ) Topipotent cell? Self renewal ( / ) differentiation Pluripotent cell differentiation (, ) Self renewal Multipotent cell ( / )

49 1. (isolation), (identification), (proliferation) 2. 3., / (Embryonic stem cell, Pluripotent cell) Vector (Multipotent cell) Somatic cell ( ) / / MHC compatibility

50 Stem cell Gene Gene therapy application Embryonic ( ) Hematopoietic ( ) Any? Phox MGMT Globins Theoretically unlimited Chronic granulomatous disease Bone marrow protection from chemothe. Thallesemias, sickle cell disease Mesenchymal ( ) Many Collagen Erythropoietin Insulin Prodution of secreted therapeu. proteins Osteogenesis imperfecta Anemia Diabetes Neural ( ) Muscle ( ) Speculated Dystropin Parkinson's disease, other neurological Muscular dystrophy,,,, (multipotency)

51 (embryonic stem cell) (adult stem cell) / 3. de novo DNA methylation

53 Cytokine -2 T,,, /,

54 : : ( / )

55 p53 1. p53 -, 50% Apoptosis( ) 3. ( - )

57 ,, AIDS

58 ( ) ( ) ( ) ( ) ( )

59 : 96~ 02, KIPRIS DB, 93

60 C12N ( ) 46% A61K ( ) 41% 5% CO7C ( CO7K ( ) 4% ) 4%

61 5% 7% 16% 13% 21% 38% -.

62 (4%) (57%) (39%)

68 Drug / Transfection / /,,,,,, Delivery via Fixed Shape particles Limited distribution receptor Protein Gene? / Nucleus???? /

69 / / / / ( )

71 KR US EPO JP DNA,,,

72 C12N ;,, ; ; ; C12N 15/00 C12N 15/10.. C12N 15/12... DNA RNA,, DNA RNA,, A61K, A61K 47/00, A61K 48/00 ;

73 ?, /?,,, ( ) - ( ) ?

74 , (?) :,,,

75 ,,,,,,,,, ( 96 2 )

76 ~ ~,,,,,

77 A Y ~ Y,, A ~ Y A A pharmaceutical composition comprising compound A Y,,, (A method for treating ~) A A - compound ~, composition~, use for manufacturing A 2 - use for manufacturing ~

78 ,, :, ( : 5 mg/day) ( :, ) :, (Ex vivo / In vivo) ( :,,, ) : / / ( :, LD 50 )

79 ( ),,,,,, ( ),

분 기 보 고 서 (제 47 기) 사업연도 2014년 01월 01일 2014년 03월 31일 부터 까지 금융위원회 한국거래소 귀중 2014년 5월 30일 제출대상법인 유형 : 면제사유발생 : 주권상장법인 해당사항 없음 회 사 명 : 파미셀 주식회사 대 표 이 사 : 김

분 기 보 고 서 (제 47 기) 사업연도 2014년 01월 01일 2014년 03월 31일 부터 까지 금융위원회 한국거래소 귀중 2014년 5월 30일 제출대상법인 유형 : 면제사유발생 : 주권상장법인 해당사항 없음 회 사 명 : 파미셀 주식회사 대 표 이 사 : 김 목 분 기 보 고 서...1 대표이사 등의 확인...2 I. 회사의 개요...3 1. 회사의 개요...3 2. 회사의 연혁...4 3. 자본금 변동사항...7 4. 주식의 총수 등...9 5. 의결권 현황...10 6. 배당에 관한 사항 등...10 II. 사업의 내용...12 III. 재무에 관한 사항...30 IV. 감사인의 감사의견 등...34 V. 이사의