Transcript 基因治疗 - 复旦大学上海医学院

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
Classification of Diseases
Inherited
Diseases
Diseases
Caused by
Interaction of
Gene and
Environment
Chromosome Diseases
Multiple Gene Disease
Single Gene Diseases
Mitochondrial Disease
Congenital Disease
Tumor
Infection
Adult Diseases
Acquired
Diseases
Trauma
Suffocate
Radiation
Starvation
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
Bring down a fever
Stop pain
Depressurization
Replacement
Nutrition
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 ?
Recombinant Factor FVIII: 250U/Bottle.
Price: ￥1320
Half time of FVIII: 6-14h
Phenylketonuria, PKU
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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.
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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
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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
M Y I
Q
I
S
H
I
G
H
Met Tyr Ile Gln Ile Ser His Ile Gly His
ATG TAT ATT CAA ATT AGT CAT ATT GGT CAT
M Y I
Q
I
S
H
I
G
H
Met Tyr Ile Gln Ile Ser His Ile Gly His
ATG TAT ATT CAA ATT AGT CAC ATT GGT CAT
M Y I
Q
I
S
L
I
G
H
Met Tyr Ile Gln Ile Ser His Ile Gly His
ATG TAT ATT CAA ATT AGT CTT ATT GGT CAT
M Stop
Sense mutation
Missense mutation
Nonsense mutation
Met end
ATG TAA
M Y
Normal Sequence
I
Q
I
S
P
Y W
S
Met Tyr Ile Gln Ile Ser Pro Tye Trp Ser
ATG TAT ATT CAA ATT AGT CCA TAT TGG TCA T
Frame shift
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
Epigenetics Changes
Non-coding RNA
Type
DNA Methylation
tRNA
rRNA
siRNA
miRNA
piRNA
cis-NATs
snRNA
snoRNA
RNase P
SINES
Histone Modification
Function
mRNA decoding
Ribosome core
peptidyl transferase
Endonucleolytic mRNA cleavage
Endonucleolytic mRNA cleavage
mRNA-specific translational inhibitor
Retrotransposon silencing
Modulate transcript levels
Pre-mRNA splicing
rRNA and tRNA maturation
tRNA maturation
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
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). Science 175 (4025): 949.
 Rogers S, New Sci. 1970, p. 194
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)
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
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。
Translational Medicine
 Bench to bedside
 Bedside to bench
Questions
 What diseases can be treated by gene therapy?
 How to do a wonderful translational medicine?
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。
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