基因治疗基因治疗

张咸宁zhangxianning@zju.edu.cn

Tel ： 13105819271; 88208367 Office: C303, Teaching Building

2015/09

Learning Objectives

1. Traditional managements （传统的治疗方法）

2. Gene therapy（基因治疗）

Wilson disease: Cu toxicity, AR

Wilson SAK. Brain, 1912; 34:295-507

Wilson disease:Before/After （青霉胺） therapy

Gene therapy

The medical procedure involves either replacing, manipulating, or supplementing nonfunctional genes with healthy genes.

OR

“Everyone talks about the human genome,

but what can we do with it?”

Impact of the Genome Project on Medicine

•Facilitate identification of genes associated with complex disorders i.e. Cardiovascular disease, cancer•provides more therapeutic targets-in turn enhances our ability to treat cause of disease instead of symptoms•bioinformatics, array technology, proteomics -enable a systems approach to biomedical research

Monogenic Diseases Which May Be Candidates For Gene Therapy

Sickle cell anemia/Thal Bone MarrowCongenital immune deficiencies Bone MarrowLysosomal storage and metabolic Bone Marrow ---------------------------------------------------------------Cystic fibrosis Lung -

airways ---------------------------------------------------------------Muscular dystrophy Muscle ---------------------------------------------------------------Hemophilia A or B LiverUrea cycle defects LiverFamilial hypercholesterolemia Liver

Types Of Conditions That May Be Treated By Gene Therapy

Monogenic Diseases (>1 000 known)

Cancer, Leukemia

Infectious (AIDS, Hep C)

Cardiovascular

Neurologic

Gene Delivery Can Be: I. Ex vivo（离体） – gene into isolated cells

II. In vivo（体内） – gene directly into patient

a) Systemic injection +/- targeted localization +/- targeted expression

b) Localized 1) Percutaneous 5) Bronchoscope 2) Vascular catheter 6) Endoscope 3) Stereotactic 7) Arthroscope

4) Sub-retinal

General considerations for the use of somatic gene therapy (approved in 1988)

1. Compensate for a mutation resulting in the

loss of function

examples of monogenic disorders:

cystic fibrosis, hemophilia

General considerations for the use of gene therapy

1. Compensate for a mutation resulting in the

loss of function

examples of monogenic disorders:

cystic fibrosis, hemophilia

stage of the research:

http://clinicaltrials.gov/ct2/results?term=gene+therapy+cystic+fibrosis

General considerations for the use of gene therapy

1. Compensate for a mutation resulting in the

loss of function

2. Replace or inactivate a dominant mutant gene

General considerations for the use of gene therapy

1. Compensate for a mutation resulting in the

loss of function

2. Replace or inactivate a dominant mutant gene e.g.: Huntington disease (expanded CAG repeat)

? Ribozymes or siRNA to degrade mRNA

General considerations for the use of gene therapy

1. Compensate for a mutation resulting in the

loss of function

2. Replace or inactivate a dominant mutant gene example: Huntington disease (expanded CAG repeat)

? Ribozymes or siRNA to degrade mRNA state of research – no open studies for Huntington

General considerations for the use of gene therapy

1. Compensate for a mutation resulting in the

loss of function

2. Replace or inactivate a dominant mutant gene3. Pharmacologic gene therapy

example: cancer

General considerations for the use of gene therapy

1. Compensate for a mutation resulting in the

loss of function

2. Replace or inactivate a dominant mutant gene3. Pharmacologic gene therapy Yet, it is important to note that there is not yet a

single FDA-approved use of gene therapy!

Minimal requirements that must be met（基因治疗试验的最低要求） :

• Identification of the affected gene

• A cDNA clone encoding the gene

Minimal requirements that must be met （基因治疗试验的最低要求） :

• Identification of the affected gene

• A cDNA clone encoding the gene

• A substantial disease burden and a favorable risk-benefit ratio

• Sufficient knowledge of the molecular basis of the disease to be confident that the gene transfer will have the desired effect

Minimal requirements that must be met（基因治疗试验的最低要求） :

• Identification of the affected gene• A cDNA clone encoding the gene• A substantial disease burden and a favorable risk- benefit ratio• Sufficient knowledge of the molecular basis of the disease to be confident that the gene transfer will have the desired effect

• Appropriate regulation of the gene expression: tissue specific and levels• Appropriate target cell with either a long half life or high replicative potential• Adequate data from tissue culture and animal studies to support the use of the vector, regulatory sequences, cDNA and target cell

Minimal requirements that must be met（基因治疗试验的最低要求） :• Identification of the affected gene• A cDNA clone encoding the gene• A substantial disease burden and a favorable risk-benefit ratio• Sufficient knowledge of the molecular basis of the disease to be confident that the gene transfer will have the desired effect• Appropriate regulation of the gene expression: tissue specific and levels• Appropriate target cell with either a long half life or high replicative potential• Adequate data from tissue culture and animal studies to support the use of the vector, regulatory sequences, cDNA and target cell

• Appropriate approvals from the institutional and federal review bodies.

Gene therapy• In most gene therapy studies, a "normal"

gene is inserted into the genome to replace an "abnormal," disease-causing gene.

• A carrier molecule called a vector must be used to deliver the therapeutic gene to the patient's target cells. Currently, the most common vector is a virus that has been genetically altered to carry normal human DNA.

Gene Transfer MethodsNon-viral: Expression plasmid or other nucleic acid

(mRNA, siRNA).Challenge: Naked DNA or RNA does not enter cells.

a) Transfer into cells using physical methods such as direct micro-injection or electroporation.

b) complex to carrier to allow cross of cell membrane

liposomes,

cationic lipids,

dextrans,

cyclohexidrins

(aka nanoparticles)

Gene Transfer Methods

Viral vectors = viruses that have been

adapted to serve as gene delivery vectors

include: retrovirus Lenti-virus adenovirus adeno-associated virus (AAV)herpes virus

Characteristics of the Ideal Vector for Gene Therapy

• Safe• Sufficient capacity for size of therapeutic DNA• Non-Immunogenic• Allow re-administration• Ease of manipulation• Efficient introduction into target cells/tissues• Efficient and appropriate regulation of expression

•Level, tissue specificity, transient, stable?

Types of viral vectors

• Retrovirus

• Lenti-virus

• Adenovirus

• Adeno-Associated virus (AAV)

• Herpes virus

In vivo and ex vivo gene therapy

Two strategies for introducing foreign genes into patients

In vivo gene therapyGene therapy vector +

therapeutic gene

Advantages: cells and organs not available ex vivo (lining of the lung)

Disadvantages: virus could spread to other cells/tissues Less control over titer and conditions of exposure

Two strategies for introducing foreign genes into patients

Ex vivo gene therapyStem cells

Gene therapy vector +

Normal gene

Advantages:More controlled infectionhigher titer virus

Disadvantages:technically difficult

Types of viral vectorsstable/transient infect non-dividing cells

• Retrovirus stable no

• Lenti-virus stable yes

• Adenovirus transient yes

• Adeno-Associated virus ? yes

• Herpes virus transient yes

Use of retroviral vectors to introduce therapeutic genes into cells

Severe Combined Immunodeficiency Syndrome (SCID)——adenine deaminase (ADA) deficiency

Severe Combined Immune Deficiency (SCID)

SCID is popularly known as “bubble baby disease” after a boy with SCID was kept alive for more than a decade in a germ-free room.

SCID is a fatal disease, with infants dying from overwhelming infection due to the congenital absence of a functioning immune system.

More than a dozen genes have been found to be able to cause human SCID.

The first “SCID gene” to be identified in humans is ADA, which makes an enzyme needed forImmune cells to survive.

Severe Combined Immunodeficiency Disease (SCID) is due to a defective gene for Adenosine Deaminase (ADA). A retrovirus, which is capable of transferring it's DNA into normal eukaryotic cells (transfection), is engineered to contain the normal human ADA gene. Isolated T-cell stem line cells from the patient are exposed to the retrovirus in cell culture, and take up the ADA gene. Reimplantation of the transgenic cells into the patient's bone marrow establishes a line of cells with functional ADA, which effecitvely treats SCID.

Ex vivoSomatic Therapy for SCID

ADA deficiency (SCID): Ashanti de

Silva ， 1990

Father of GT: Anderson WF, 1990

Clinical Trial of Stem Cell Gene Therapy for Sickle Cell Disease

β-Globin LCRβAS3 Globin

WPRERRE cPPT

ψ

SIN LTR

SIN LTR

HS2 HS3 HS4

Bone Marrow Harvest Isolate Stem CellsAdd Normal

Hemoglobin Gene

Myeloablate with Busulfan (16 mg/kg)

Transplant Gene-Corrected Stem Cells

FreezeCertify

Follow: Safety Efficacy

Risks of Gene Therapy

1.Adverse reaction to vector or gene

1999/9/17: reaction to an adenovirus caused death

of 18-yo man, Jesse Gelsinger, Arizona, the first victim of gene therapy. OTC (ornithine transcarbamylase) important for metabolism of N

Injection of viral particles triggered massive inflammatory response in an individual with mild form of disease being treated with drugs and diet.

Subsequent FDA audit revealed protocol and IRB violations.

Risks of Gene Therapy

2. Activation of harmful genes by viral promoters/enhancers stably integrated into the genome.

2002 retrovirus-induced leukemia

Children with otherwise fatal X-linked SCID injected with ex vivo HSC modified by introduction of the g-c chain cytokine receptor in 2000 (affects lymphocyte maturation)

Initial immune function was good

2/11 patients developed leukemia-like disorder at 2 years.

Clonal analysis shows insertion and activation of LMO2 gene.

FDA-cannot be used as first line therapy if BMT is an option

What factors have kept GT from becoming

an effective treatment for genetic disease?

• Short-lived nature of gene therapy

• Immune response

• Problems with viral vectors • Multigene disorders

Patient

Tissue Sample (e.g. skin biopsy)

Gene Addition or Gene Correction

De-Differentiation to Induced Pluripotent Stem cells (iPS)

Differentiation to Hematopoietic Stem Cells (HSC)

Autologous Transplant

Gene therapy using Autologous HSC Made from Induced Pluripotent Stem Cells

Gene Therapy

Current Future

Experimental Proven

Limited Scope Curative

High Tech Off the Shelf

Gene Therapy Clinical Trials Worldwide (updated list of all

gene therapy protocols)www.wiley.com/legacy/wileychi/genmed/clinical/