1、The Treatment of Genetic Diseases遺傳病的治療http:/ 藥物開發初期要投入多少錢?Pompe ？Glycogen storage disease type IIAcid maltase deficiencyautosomal recessivecause: mutation in acid alpha-glucosidaseaccumulation of structurally normal glycogen in lysosomes and cytoplasm The Treatment of Genetic Diseases Final cureEli

2、minate the symptomAmeliorate the effectNot only on the patient, but also on their familyTreatments of single gene disorders單基因遺傳病的治療Overall：powerful advances are being made, but unsatisfactory WHY?Gene not identified or pathogenesis not understood.Prediagnostic fetal damage.Severe phenotype are less

3、 amenable to intervention.Special considerations in treating genetic diseases某些遺傳病的治療，最初階段效果滿意，但 某些藥物短期對受累器官/組織效果良好，但隨著患者生命的延續，可能會暴露出更多異常某些藥物短期療效尚可，但長期效果欠佳也有某些藥物短期反應良好，但長期使用可造成嚴重副作用。治療策略治療策略 The various levels of treatment that are relevant to genetic disease, with the corresponding strategies used

4、at each level. For each level, a disease discussed in the text is given as an example目前各種治療手段比例目前各種治療手段比例 直接改變患者的表型直接改變患者的表型 手術手術cleft lip（單純性唇裂）Cleft lip & palate唇裂修復術:36月腭裂修復術:12歲牙槽突裂修復術:911歲腭咽成形術:56歲鼻唇畸形治療:1315歲頜骨發育畸形治療:18歲以后先天性馬蹄內翻足先天性馬蹄內翻足 club footClub foot手術：活動受限、手術：活動受限、關節僵硬、足部無關節僵硬、足部無力

5、。力。矯形：矯形：Ponseti法法成功率成功率90%出生后出生后7-10天開始天開始4-5次石膏次石膏“Ponseti”療法療法/chinaclubfoot/vip_ser/398190.html“潘塞緹潘塞緹”法先天性法先天性馬蹄內翻足治療手冊馬蹄內翻足治療手冊Treatment at protein levelTreatment of metabolic abnormalities- protein levelThe most successful approach.Dietary restriction/ avoidance 飲食控

6、制/ - 苯丙酮尿癥Replacement 替代 溶酶體病Diversion 轉化 尿素循環障礙Inhibition 抑制抑制Depletion 清除Enhancement 增強Phenylketonuria （PKU）苯丙酮尿癥苯丙酮尿癥 （高苯丙氨酸血癥）（高苯丙氨酸血癥）A model for dietetic and pharmacologic therapy and its consequences黃、白、丑、臭、抽信廣告，得信廣告，得“永生永生”高苯丙氨酸血癥Hyperphenylalaninemia 0510152025303540455000.511.522.533.544.5

7、1 - 360 - 180 20120010 - 20600 - 12003 - 10180 - 600NormalHyperphenylalaninemiaatypic PKUClassical PKUmg/dlmol/lHyperphenylalaninemia classification. Classical PKU : PAH (苯丙氨酸羥化酶)- PAH activity : 0-1%, PHE tolerance : 200 - 350 mg. Atypic PKU :- PAH activity : 1-3 %,PHE tolerance : 350 - 850 mg. Mod

8、erate Hyperphenylalaninemia - PAH activity : 3 - 5 %, PHE tolerance : 850 mg 400 mutations worldwidehttp:/www.pahdb.mcgill.caFirst dietetic treatmentfor an inborn error of metabolism-Low-phenylalanine diet-低苯丙氨酸飲食低苯丙氨酸（Phe）飲食對于高蛋白食物，根據Phe耐受控制攝入量：Avoidance of high protein food (milk, dairy products,

9、meat, fish, chicken, eggs, beans and nuts,.無Phe配方食物、維生素、微量元素, 低蛋白食物，少量 (如面包)無蛋白飲食，不控制Galactosemia Galactosemia 半乳糖血癥AR患病率：1 / 40 000 birth Galactose-1-phosphate-uridyl-transferase （半乳糖1磷酸尿苷酰轉移酶）(GALT)等缺乏新生兒期發病需要及時發現，及時治療癥狀可逆半乳糖血癥半乳糖血癥Galactosemia出生后一周內即發病胃腸道反應肝臟受累診斷 : spot test 酶學法診斷治療: 無乳糖/半乳糖飲食- r

10、apid recovery半乳糖血癥半乳糖血癥 后期可能出現的異常With treatment, most clinical complications can be prevented and a normal life expectancy achieved; intellectual disability, speech problems, reduced coordination, and primary amenorrhea or premature menopause can occur despite adherence to galactose-restricted diets

11、.Replacement（ 替代治療）The provinsion of essential metabolites, cofactors, or hormones whose deficiency is due to a genetic disease is simple in concept and often simple in application. Some of the most successfully treated single-gene defects belong to this category. Ex. Congenital hypothyroidism.Repla

12、cementReplacement（ 替代治療）前提： enzyme + substrateWhatHowWhere ReplacementSynthesis of thyroid hormoneReplacementTreating Congenital Treating Congenital hypothyroidism - hypothyroidism - replacementCongenital hypothyroidism （甲狀腺功能低下）thyroidhormone(甲狀腺激素) deficiency present at birth患病率：1 / 4000 If untrea

13、ted for several months after birth, can lead to growth failure and permanent mental retardation. daily dose of thyroid hormone (thyroxine) by mouthReplacementTreating Congenital Treating Congenital hypothyroidism - hypothyroidism - replacement新生兒篩查：早發現，早治療first 12 weeks of life.每日服用(levothyroxine -

14、左旋甲狀腺素).劑量：10-15g/kgdaily.The dose increases as the child grows. ReplacementEnzyme replacement腺苷脫氨酶缺乏癥adenosine deaminase (腺苷脫氨酶腺苷脫氨酶) deficiencyan accumulation of deoxyadenosinetoxic to immature lymphocytescauses a build up of dATP in all cells, which inhibits ribonucleotide reductase and prevents

15、DNA synthesis the immune system is severely compromised or completely lackingReplacementAdenosine deaminase deficiencyReplacementADA deficiency 治療胞外補充胞內作用的酶Chemically modified ADA: PEG-ADA (聚乙二醇-ADA)injectionAdvantages:less antibody responseRemains extracellularLonger half-lifeReplacementEnzyme repl

16、acement葡糖腦苷脂酶缺乏癥（戈謝氏病）ReplacementEnzyme replacement葡糖腦苷脂酶缺乏癥（戈謝氏?。﹩魏司奘杉毎?Hematologic manifestations Visceral involvement Bone disease Neurologic manifestationsFour main manifestations : Replacement戈謝氏病Replacement戈謝氏病 Gaucher DiseaseGaucher Disease - Most prevalent lysosomal disorderGlucocerebrosidea

17、se (葡糖腦苷脂酶)deficiency accumulation of glucocerebroside/glucosylceramidCNS normally is not involvedOnly alternative therapy is stem cell transplantationRich source of WT enzymesEasy to target macrophageReplacementReplacementEnzyme replacement葡糖腦苷脂酶缺乏癥（戈謝氏?。〢chieved by editing the sugar residue of the

18、 glycoprotein.The modified protein directly targeting to macrophage through mannose receptor (甘露糖受體）of cell surface.Benefits：Liver, spleen size reducedEnriched hemoglobinImproves skeletal conditionAccelerates growthReplacementEnzyme replacement therapyReplacementReplacementCost .ReplacementDiversion

19、 轉化治療轉化治療diversion therapy is the enhanced use of alternative metabolic pathways to reduce the concentration of a harmful metabolite.DiversionDiversion 轉化治療Urea cycle defectAn urea cycle disorder or urea cycle defect is a genetic disorder caused by a deficiency of one of the enzymes in the urea cycl

20、e which is responsible for removing ammonia from the blood stream. Normally, the urea is transferred into the urine and removed from the body.nitrogen accumulates in the form of ammonia, a highly toxic substance, and is not removed from the body.Diversion鳥氨酸轉氨甲酰酶DiversionUrea cycle defectAccumulatio

21、n of ammonium in bloodNEONATAL PERIOD: vomiting, seizures, respiratory distressCHILDHOOD: failure to thrive, self-injurious behavior, hyperammonemic coma or deathADULTHOOD: schizophrenia, bipolar disorderDiversionUrea cycle defect苯甲酸鈉/苯丁酸鈉DiversionDepletion （清除）Example Hemochromatosis 血色素沉著 causes t

22、he body to absorb and store too much iron.The extra iron builds up in the bodys organs and damages them. Without treatment, the disease can cause the liver, heart, and pancreas to fail.Healthy people usually absorb about 10 percent of the iron contained in the food they eat, which meets normal dieta

23、ry requirements. People with hemochromatosis absorb up to 30 percent of iron. Over time, they absorb and retain between 5-20 times more iron than the body needs.DepletionHemochromatosisDepletionDepletion （清除）Phlebotomy (放血)is a procedure that removes blood from the body in a process similar to donat

24、ing blood. Phlebotomy is the preferred method of treating most forms of hemochromatosis. Most people with hemochromatosis need regular phlebotomy throughout their lives. DepletionDepletion Ex. 2Familial hypercholesterolemia 家族性高膽固醇血癥DepletionFamilial hypercholesterolemiaDepletionDepletion works？When

25、 the cholesterol load is decreased by diverting it to other compounds or by removing it with physical methods, the liver tries to compensate for the decreased cholesterol intake by up-regulating cholesterol synthesis.DepletionCholesterol synthesis in liverInhibition & DepletionInhibitionThe phar

26、macological inhibition of enzymes is sometimes used to modify the metabolic abnormalities of inborn errors.Drug: statin (抑制素)Inhibits 3-hydroxy-3-methylgutaryl coenzyme A (HMG-CoA, 3-羥基-3-甲基戊二烯輔酶A )reductase.Inhibition & DepletionTreating Familial hypercholesterolemiaInhibition & DepletionEn

27、hancement of mutant protein function with small molecule加強治療EnhancementHomocystinuria (高胱氨酸尿)1/200,000AR。高胱氨酸尿癥是在甲硫氨酸的異化過程中缺乏胱硫醚合成酶而產生的一種遺傳病癥狀：nearsightednessblood clottingfracturedevelopmental delaylearning problems。Enhancement50 % of patients responsive to B6 胱硫醚EnhancementHigh doses of B12/B6遺傳病的

28、治療 蛋白質/酶層次AvoidanceEnhancementEnzyme replacementDiversionInhibitionDepletion基因表達層次基因表達層次Increase/reduce gene expressionModification cells by transplantationStem cell/organ transplantationGene therapyIncrease gene expressionfrom the WT or mutant locus提高受累基因mRNA的表達： transcribed from the WT locus assoc

29、iated with a dominant disease or form a mutant locus, if the mutant protein retains some function.遺傳性血管水腫(Hereditary angioedema)ADdeficiency of the C1 inhibitorC1 inhibitor is needed to control the coagulation cascade in blood clottingHereditary angioedemaHereditary angioedemaTypes:Type I: Low C1 in

30、hibitorType II: Normal C1 inhibitor, but defectedsymptoms: laryngeal edema (喉頭水腫)Treatment:Danazol （達那唑）Increases the abundance of the C1 inhibitor mRNASerious attack reducedSide effect in long-term useIncrease gene expressionfrom a locus not affectedIncrease the expression of a normal gene that com

31、pensates for the effect of mutation at another locus.Extremely promising in the management of sickle cell disease and beta-thalassemia.Drug: decitabine (地西他濱地西他濱)Inhibits methylation of -globinProduce more HbFReducing the expressiondominant mutant geneApproach: RNA interference (RNAi)Can be used to

32、degrade a specific target RNA.Short designed RNA, once introduced in the cell, it will bind to target RNA and initiate degradation.Still in early stage.In trial with Huntington DiseaseRNA interference Modification of the somatic genome by transplantationIndication:Cells or organs may be transplanted

33、 to introduce WT copies of a gene into a patient with mutations in that gene.For cell replacement, to compensate for an organ damaged by genetic disease.Stem cell transplantationFirst successful transplantslate 1960s30,000-40,000 transplants performed yearly worldwide20,000 patients have survived 5

34、yearsStem cell transplantationcan divide through mitosis and differentiate into diverse specialized cell types and can self renew to produce more stem cellsSources:ClonedFrom human donorsAllogeneic: from another personSyngeneic: from an identical twinAutologous: from the patientStem cell from human

35、donors Type: Hematopoietic （造血）Source: bone marrow, placental core blood Cancer or Lysosomal storage diseaseCorneal （角膜）Regenerating corneal epitheliumothers: embryotic （胚胎）Hematopoietic stem cellscharacterized by the presence of CD 34Seen in the umbilical cord and fetal liverHave a higher cloning e

36、fficiency and generates more progenitors than adult bone marrow.They have a huge competitive engraftment advantage relative to the adult bone marrow.Fetal liver is now used to treat fetuses having X-Linked SCID.Hematopoietic stem cellsThey are used in Providing a functional immune system in a person

37、 with SCID.Replacing a defective blood system with a functional one who has non malignant genetic disorder like sickle cell anaemia and thallasemia. Restoring the haematopoietic system in cancer patients after treatment.Other fetal cellsMesenchymal stem cells (間充質細胞）-differentiate to bone, fat and c

38、artilage like the adult counterpart.Neural stem cell they differentiate into neurons, astrocytes and oligodendrocytes. They are the main source of cells for degenerative CNS injury for replacement.Stem cell transplantation can treatHistocytic disordersInherited erythrocyte abnormalitiesInherited imm

39、une system disorders like ataxia telangectesia, DiGeorge syndrome, SCID etcPlasma cell disorderInherited disorders like Lesch Nyhan syndrome, beta Thallesemia etcInherited platelet abnormalities Inherited metabolic disorders like Mucopolysaccharidosis, Hurlers syndrome, Krabbe disease, Niemann- pick

40、 disease etcTrials underwayCardiac diseaseDiabetesMultiple SclerosisMuscular DystrophyParkinsons diseaseSpinal cord injuryStrokeComplicationsEarlyGraft RejectionHost versus graftDrug injury to marrowViral infections: CMV, HHV-6 & 8Interstitial PneumonitisDiffuse alveolar hemorrhageToo few donor

41、stem cellsARDS often caused by CMVGene TherapyDefinition The deliberate introduction of genetic material into human somatic cells for therapeutic, prophylactic or diagnostic purposesAddition of EXTRA genes or extra functionsAim is to cure disease (or at least help the patient)First introduction of g

42、ene-modified cells into a patient was in 1989First gene therapy product approved for market in 2004Still very experimental and early in its developmentGene TherapyGene Therapy - Background1990 - The first gene therapy journal published, Human Gene Therapy1990 - The first approved gene therapy clinic

43、al trial took place when Ashanthi DeSilva, a 4 year old girl with ADA-deficient Severe Combined Immunodeficiency, was given her own T cells engineered with a retroviral vector carrying a normal ADA gene 2000 - The first gene therapy cure was reported when Alain Fischer (Paris) succeeded in totally c

44、orrecting children with SCID-X1, or “bubble boy” syndrome“Bubble Boy”Gene TherapyGene Therapy - BackgroundGene TherapyGeneral concernsShort-lived nature of gene therapy. Very hard to achieve any long-term benefits without integration and even with it. Immune response. It reduces gene therapy effecti

45、veness and makes repetitive rounds of gene therapy uselessProblems with viral vectors . Toxicity, immune and inflammatory responses, also fears that viral vector may recover disease-causing abilityMultigene disorders. Most commonly occurring disorders, such as heart disease, Alzheimers disease, arth

46、ritis, and diabetes, are caused by the combined effects of variations in many genes.Gene TherapyGene Therapy 準備工作Must know & do:Identify the molecular defectA functional copy of the geneKnowledge of the pathophysiological mechanismFavorable risk-to-benefit rationAppropriate regulatory components

47、 for the transferred geneAppropriate target cellStrong evidence of efficacy and safetyRegulatory approvalGene TherapyGene TherapyGene Therapy Involves delivery of therapeutic genes into the human body to correct disease conditions created by faulty genes Two primary strategies: Ex vivo gene therapy

48、In vivo gene therapyGene TherapyEx vivo gene therapy Cells from diseased person are removed Then, they are treated in lab (using techniques similar to bacterial transformation) Finally, they are reintroduced to the patient More effective than in vivo Transfection is the introduction of DNA into anim

49、al or plant cellsGene TherapyIn vivo gene therapy Introducing genes directly into tissues or organs without removing body cells Challenge is delivery only to intended tissues Viruses act as vectors for gene delivery, but some injected directly into tissueGene TherapyDelivery of therapeutic genes The

50、rapeutic genes often called payload May require long-term expression of corrective gene Others require rapid expression for short periods of timeGene TherapyViral vectorsViral vectors use viral genome to carry therapeutic gene(s) and to infect human body cells Adenovirus (common cold) Adeno-Associat

51、ed Virus Retrovirus (HIV) Herpes simplex virus (cold sores) Viruses must be engineered so that they can neither produce disease nor spread (extremely effective at infecting human cells)Gene TherapyVector TransfectionTargeted gene therapy may result since some viruses infect certain body cells Adenov

52、iruses infect both dividing & non-dividing cells effectivelyAdeno-Associated viruses do not cause illness in humans, can infect a wide variety of cells, & integrate 95% of time in same locationRetroviruses are of interest because they insert DNA into the genome of host where it remains perma

53、nently (integration), but often, randomlyGene TherapyVector TransfectionHerpes virus (HSV-1) strain primarily affects central nervous system (CNS)May help develop treatments for Alzheimers, Parkinsons, and other genetic neurodegenerative diseasesHowever, although viral vectors may help, most human cells are not easily transfectedGene TherapyAlt