gene therapy 基因治疗 复旦大学分子医学教育部重点实验室...
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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
Two Lectures
General Information of Gene Therapy
Strategy and methods of Gene Therapy
Inherited Diseases
Acquired Diseases
Diseases Caused by Interaction of Gene and Environment
Chromosome Diseases
Multiple Gene Disease
Single Gene Diseases
Mitochondrial Disease
Trauma
Suffocate
Radiation
Starvation
Congenital Disease
Tumor
Infection
Adult Diseases
Classification of Diseases
Types of Treatment
急则治其标，缓则治其本，标本俱急者，标本同治。
Antibiotics
Nutrition Depressurization
Replacement
Bring down a fever
Stop pain
Pathogen Treatment Symptom Treatment
Therapeutic Effects
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.
Why Gene Therapy ?
Half time of FVIII: 6-14h
Recombinant Factor FVIII: 250U/Bottle.
Price: ￥ 1320
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.
AIDS
per 100,000 inhabitants
Chromosome Diseases
Aneuploidy
21, 18, 13, X-triploid syndrome, Turners syndrome (X), Klinefelters syndrome (XXY),
Multy X syndrome, XYY syndrome.
Structure abnormal
William syndrome, Cat cry syndrome (5p-), 22q11 microdeletion syndrome, DiGeorge
syndrome, Miller-Dieker syndrome, Smith-Magenis syndrome, Kallmann syndrome, X-
linked ichthyosis, Wolf-Hirschhorn syndrome(4p-).
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%).
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
Mitochondria Diseases
Genetic Code
Sense mutation
Frame shift
Normal Sequence M Y I Q I S H I G H
Met Tyr Ile Gln Ile Ser His Ile Gly HisATG TAT ATT CAA ATT AGT CAT ATT GGT CAT
M Y I Q I S H I G HMet Tyr Ile Gln Ile Ser His Ile Gly HisATG TAT ATT CAA ATT AGT CAC ATT GGT CAT
M Y I Q I S L I G HMet Tyr Ile Gln Ile Ser His Ile Gly HisATG TAT ATT CAA ATT AGT CTT ATT GGT CAT
M StopMet endATG TAA
M Y I Q I S P Y W SMet Tyr Ile Gln Ile Ser Pro Tye Trp SerATG TAT ATT CAA ATT AGT CCA TAT TGG TCA T
Missense mutation
Nonsense mutation
Single Nucleotide Polymorphisms (SNPs) 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.
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
Epigenetics Changes
DNA Methylation
Histone Modification
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
miRNAs
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。
Major Developments in Gene Therapy
James D. Watson and Francis Crick. Genetic
implications of the Structure of deoxyribonucleic
acid. Nature, 1953;171:964
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.
1970s
Friedmann, T.; Roblin, R. (1972). Science 175 (4025): 949.
Rogers S, New Sci. 1970, p. 194
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)
1980s
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.
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.
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.
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.
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.
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
Clinical Trial World Wide
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。
中国的基因治疗
Translational Medicine
Bench to bedside
Bedside to bench
Questions
What diseases can be treated by gene therapy?
How to do a wonderful translational medicine?
