Gene Therapy 基因治疗

1. Gene Therapy 基因治疗 复旦大学分子医学教育部重点实验室 复旦大学出生缺陷研究中心 复旦大学儿童发育与疾病转化医学研究中心 马端

2. Two Lectures • General Information of Gene Therapy • Strategy and methods of Gene Therapy

3. Classification of Diseases Diseases Caused by Interaction of Gene and Environment Inherited Diseases Acquired Diseases Chromosome Diseases Multiple Gene Disease Single Gene Diseases Mitochondrial Disease Trauma Suffocate Radiation Starvation Congenital Disease Tumor Infection Adult Diseases

4. Types of Treatment 急则治其标，缓则治其本，标本俱急者，标本同治。 Pathogen Treatment Symptom Treatment Antibiotics Nutrition Bring down a fever Stop pain Depressurization Replacement

5. Therapeutic Effects

6. What is Gene Therapy? • Gene therapy is an experimental treatment that involves introducing genetic material into a person’s cells to fight or prevent disease.

7. Why Gene Therapy ? Recombinant Factor FVIII: 250U/Bottle. Price: ￥1320 Half time of FVIII: 6-14h

8. Phenylketonuria, PKU • Autosomal recessive metabolic genetic disorder. • A mutation in the gene of phenylalanine hydroxylase (PAH, 苯丙氨酸羟化酶) which metabolize the phenylalanine to the tyrosine. • When PAH activity is reduced, phenylalanine accumulates and is converted into phenylpyruvate. • Early cases of PKU were treated with a low-phenylalanine diet.

10. AIDS per 100,000 inhabitants

11. Chromosome Diseases • Aneuploidy 21, 18, 13, X-triploid syndrome, Turners syndrome (X),Klinefelters syndrome (XXY), Multy Xsyndrome, XYYsyndrome. • Structure abnormal Williamsyndrome, Cat cry syndrome (5p-), 22q11 microdeletion syndrome, DiGeorge syndrome, Miller-Dieker syndrome, Smith-Magenis syndrome, Kallmann syndrome, X-linked ichthyosis, Wolf-Hirschhorn syndrome(4p-).

12. Multiple Genetic Diseases • Schizophrenia (80%), Congenital asthma (80%), Cleft lips and Palate(76%), Juvenile Diabetes (75%), Congenital dislocation of the hip (70%), Coronary heart disease (65%), Hypertension (62%), Idiopathic epilepsy (57.4%), CHD (heritability: 55%) . • Hydrocephalus (49.6%), Peptic (37%), Senile diabetes (35%).

13. Monogenetic Diseases • Hereditary deafness • α & β Thalassemia • Hemophilia A, B, C • Factor V deficiency • Glucose -6-phosphate dehydrogenase deficiency • Sickle-cell anemia • Familial hypercholesterolemia • Hereditary optic neuropathy • Congenital absence of gamma globulin • Severe Myopia • Red-green blindness • Congenital glaucoma • Polycystic kidney disease • Fragile X chromosome syndrome

14. Mitochondria Diseases

15. Genetic Code

16. M Y I Q I SH I G H Met Tyr Ile Gln Ile Ser His Ile Gly His ATG TAT ATT CAA ATT AGT CAT ATT GGT CAT Normal Sequence M Y I Q I SH I G H Met Tyr Ile Gln Ile Ser His Ile Gly His ATG TAT ATT CAA ATT AGT CAC ATT GGT CAT Sense mutation M Y I Q I SL I G H Met Tyr Ile Gln Ile Ser His Ile Gly His ATG TAT ATT CAA ATT AGT CTT ATT GGT CAT Missense mutation M Stop Met end ATG TAA Nonsense mutation M Y I Q I SPYW S Met Tyr Ile Gln Ile Ser Pro Tye Trp Ser ATG TAT ATT CAA ATT AGT CCA TAT TGG TCA T Frame shift

18. Single Nucleotide Polymorphisms (SNPs) • SNP is a DNA sequence variation occurring when a single nucleotide -A, T, C, or G - in the genome differs between members of a species. • Disease SNP • Susceptibility SNP • Diagnostic SNP • Phenotype SNP • Drug associated SNP

19. Epigenetics Changes Non-coding RNA Type Function tRNA mRNA decoding rRNA Ribosome core peptidyl transferase siRNA Endonucleolytic mRNA cleavage miRNA Endonucleolytic mRNA cleavage mRNA-specific translational inhibitor piRNA Retrotransposon silencing cis-NATs Modulate transcript levels snRNA Pre-mRNA splicing snoRNA rRNA and tRNA maturation RNase P tRNA maturation SINES Initiation or eIF4-dependent translational inhibitor DNA Methylation Histone Modification

20. miRNAs

22. 以铜为镜，可以正衣冠； • 以古为镜，可以知兴替； • 以人为镜，可以知得失。

23. Major Developments in Gene Therapy James D. Watsonand Francis Crick. Genetic implications of the Structure of deoxyribonucleic acid. Nature, 1953;171:964

24. 1970s • In 1972, Friedmann and Roblin authored a paper in Science titled “Gene therapy for human genetic disease?” • They cite Rogers S for proposing that exogenous ‘good‘ DNA be used to replace the defective DNA in those who suffer from genetic defects. • Friedmann, T.; Roblin, R. (1972). Science175 (4025): 949. • Rogers S, New Sci. 1970, p. 194

25. 1980s • First unapproved gene therapy – 1980, Dr. Martin Cline, bone marrow cells of two patients with beta-thalassemia. UCLA • First human somatic gene therapy –1989, four year old Ashanti • Desilva with SCID (Adenosine deaminase deficiency)

26. 1990s • Researchers at Case Western Reserve University created tiny liposomes 25 nanometers that can carry therapeutic DNA through pores in the nuclear membrane. • Sickle cell disease is successfully treated in mice. • Claudio Bordignon working at the Vita-Salute San Raffaele University, Milan, Italy performed the first procedure of gene therapy using hematopoietic stem cells as vectors to deliver genes intended to correct hereditary diseases. • 1999: death of Jesse Gelsinger in a gene-therapy experiment resulted in a significant setback to gene therapy research in the United States.

27. 2000s • 2000: First complete gene therapy cures 10 children with X-SCID in France • 2001: Researchers at St. Barnabas Hospital in New Jersey had used ooplasmic transfer to enable several women with impaired fertility to bear children. • 2003: Researchers in UCLA inserted genes into the brain using liposomes coated in a polymer. The transfer of genes into the brain is a significant achievement because viral vectors are too big to get across the blood-brain barrier. This method has potential for treating Parkinson's disease. • RNA interference may be a new way to treat Huntington's disease.

28. Scientists at the NIH have successfully treated metastatic melanoma using killer T cells genetically retargeted to attack the cancer cells. • 2006: An international group of scientists announced the successful use of gene therapy to treat two adult patients for a disease affecting myeloid cells. • 2006: A team of scientists in Milan, Italy reported a breakthrough for gene therapy in which they developed miRNA to prevent the immune system from rejecting a newly delivered gene. • 2006: Preston Nix from the University of Pennsylvania reported the treatment of HIV that uses a lentiviral vector for delivery of an antisense gene against the HIV envelope.

29. 2007: Moorfields Eye Hospital and University College London's Institute of Ophthalmology announced the world's first gene therapy trial for inherited retinal disease. They researched the safety of the subretinal delivery of recombinant AAV carrying RPE65 gene, and found it yielded positive results, no apparent side-effects. • 2009: Nature reported that researchers at the University of Washington and University of Florida were able to give trichromatic vision to squirrel monkeys using gene therapy. • 2009: Science reported that researchers succeeded at halting a fatal brain disease, adrenoleukodystrophy using lentiviral vector. • 2011: A man was cured of HIV by repeated Hematopoietic stem cell with double-delta-32 mutation which disables the CCR5 receptor This cure required complete ablation of existing bone marrow which is very debilitating.

30. Problems in Gene Therapy • Short-lived nature of gene therapy: Problems with integrating therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent gene therapy from achieving any long-term benefits. • Immune response: The risk of stimulating the immune system in a way that reduces gene therapy effectiveness is always a possibility. • Problems with viral vectors: toxicity, immune and inflammatory responses, gene control, targeting issues, cause disease. • Multigene disorders: some of the most commonly occurring disorders, such as heart disease, high blood pressure, Alzheimer's disease, arthritis, and diabetes, are caused by the combined effects of variations in many genes. • Chance of inducing a tumor: If the DNA is integrated in the wrong place in the genome, for example in a tumor suppressor gene, it could induce a tumor.

31. Bright Horizons • Mark A.K. Gene-based therapies: the road ahead. Nat Rev Genet. 2011;12(5):316-28. • Mingozzi, F. & High, K. A. Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Nature Rev. Genet. 2011;12:341–355 • Davidson, B. L. & McCray, P. B. Jr. Current prospects for RNA interference‑based therapies. Nature Rev. Genet. 2011;12:329–340 • Naldini, L. Ex vivo gene transfer and correction for cell-based therapies. Nature Rev. Genet. 2011;12: 301–315 • Lu, Q. L. et al. The status of exon skipping as a therapeutic approach to duchenne muscular dystrophy. Mol. Ther. 2011;19:9 • Urnov, F. D. et al. Genome editing with engineered zinc finger nucleases. Nature Rev. Genet. 2010;11:636–646 • Janowski, B. A. & Corey, D. R. Switching on progesterone receptor expression with duplex RNA. Mol. Endocrinol. 2010; 24:2243–2252 • Zhou, J. & Rossi, J. J. Aptamer-targeted cell-specific RNA interference. Silence, 2010; 1:4

32. Clinical Trial World Wide

38. 中国的基因治疗 • 1987: 薛京伦,卢大儒。血友病B，1991年治疗4例，17年缓解，逆转录病毒。 • 2003年获第二个临床批件。重组AAV-2人凝血因子IX注射液。 • 2004: 彭朝晖，重组人p53腺病毒注射液（今又生），头颈鳞癌。 • 世界上第一个基因治疗上市产品，深圳赛百诺基因技术有限公司。 • 2005：第一个具有溶瘤作用的重组人5型腺病毒（H101）与化疗结合治 疗难治性晚期鼻咽癌（安柯瑞），上海三维生物技术有限公司。

39. Translational Medicine • Bench to bedside • Bedside to bench

40. Questions • What diseases can be treated by gene therapy? • How to do a wonderful translational medicine?

41. 以铜为镜，可以正衣冠； • 以古为镜，可以知兴替； • 以人为镜，可以知得失。 duanma@yahoo.cn THANK YOU FOR YOUR ATTENTION