Bioteknologi, minggu 14, terapi gen.pdf

8/16/2019 Bioteknologi, minggu 14, terapi gen.pdf

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BIOTEKNOLOGI F RM SI

Dr. Oeke Yunita, M.Si., Apt.


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What Are Genetic Disorders?

Thee categories of genetic disorders: Single gene disorders caused by a

mistake in a single gene. Sickle cell,cystic fibrosis and Tay-Sachs diseaseare examples.

Chromosome disorders caused by anexcess or deficiency of the genes.Down syndrome is caused by an extracopy of a chromosome, but noindividual gene on the chromosome isabnormal.

Multifactorial inheritance disorders

caused by a combination of small variations in genes. Heart disease,most cancers and Alzheimer's diseaseare examples.

Sickle Cell

Disorder


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Gene therapy is the genetic alteration ofcells to correct the effects of a disease-

causing mutation.

GENE THERAPY


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GENE THERAPY


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INCORPORATION OF CLONED DNAINTO HUMANS AND OTHER ANIMALS

- Design animal model for human

disease.

Transgenic animals (germ line)

Fertilized OVA

Micro-inject

cloned DNA

New gene incorporated

Into germ line DNA

Implant in foster mother

- Offspring are transgenic- New gene inserted is a transgene• SCID (severe combined immuno-

deficiency)

• Cystic fibrosis; CFRT gene.

Cloned DNA fragments

Gene therapy (Somatic)

- Cloned gene inserted into DNA of

selected somatic cells

- Gene not passed to offspring

- Vector used to introduce cloned gene

into host DNA/nuclei• Retrovirus

• Adenovirus

•

liposome

- Examples


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Gene Therapy vs Conventional Therapy

Gene Therapy Conventional Therapy

Materials Nucleotide Acid, DNA, RNA;etc.

Cells, Tissues, Or Organs.

Small molecules,Peptide, Proteins.

Delivery Usually required to be deliveredinto cells or Nucleus (genes).

Effect on the cellmembrane or diffuse

into cells

Mechanisms Usually cure the causes of thediseases

Usually relieve thesymptoms or signs

Duration of

Effect

Can be permanent and also can

be passed down to nextgeneration in germline genetherapy.

Usually stop the effect

once stop taking it.

Ethics Major Issues Usually Not


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GENERAL CONCERNS

The Food and Drug Administration (FDA) has not yet approved

any human gene therapy product for sale.

Four major problems with gene therapy:

2) Immune response. It reduces gene therapy effectiveness and

makes repetitive rounds of gene therapy useless

3) Problems with viral vectors . Toxicity, immune and inflammatoryresponses, also fears that viral vector may recover disease-causing ability

4) Multigene disorders. Most commonly occurring disorders,

such as heart disease, Alzheimer's disease, arthritis, and diabetes,

are caused by the combined effects of variations in many genes.

1) Short-lived nature of gene therapy. Very hard to achieve any long-

term benefits without integration and even with it.


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Gene Carrier Human Application

Considerations

Safety

EffectiveSpecificity

BiocompatibleNon-cytotoxicity

Non-immunogenecityNon-inflammationNon-Tumor Generation

The delivery efficiencyDependent on thedisease requirements

Only desired cells or

tissue Be Delivered.


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efficient gene transfer into target cells

adequate level of transgene expression

persistence of gene expression

regulation of gene expression

tolerance to transgene product

safety

The success of gene therapy is based on:


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Barriers Of Gene Delivery


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METHODS OF GENE DELIVERY

-- Injection of naked DNA into tumor by simple needle and syringe

-- DNA transfer by liposomes (delivered by the intravascular, intratracheal,

intraperitoneal or intracolonic routes)

-- DNA coated on the surface of gold pellets which are air-propelled into the epidermis

(gene-gun), mainly non applicable to cancer

-- Biological vehicles (vectors) such as viruses and bacteria.

Viruses are genetically engineered

so as not to replicate once inside the host.

They are currently the most efficient means of gene transfer .


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GENE TR NSFER SYSTEMS

Vectors under

study as gene

delivery vehicles

Virotherapy Liposomes “Naked” DNA

Retroviruses Adenoviruses

Adeno-

Associated

Viruses


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Gene Carrier/Gene Vector

Retrovirus Herpes

Simplex V

Adenovirus AAV Liposo

me

DNA Polymer

Integration Yes Non Non Yes Non

Expression Stable Transient Transient Stable Transient

Transfection Efficient Efficient Efficient Low Low

Immune Response No Yes High No Yes Yes or No No

Generally, viral vector system show higher gene transfer efficiency than non-

viral gene carrier system, but viral systems have potential risk of wild type

virus regeneration, immunogenecity and cancer formation.


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IDEAL CHARACTERISTICSOF GENE DELIVERY VECTOR

1. High titer or concentrations (>108 particles/ml)

3. Precise and stable introduction of transgene

2. Easy and reproducible method of production

4. Vector should not elicit immune response

in the host

6. Vector should be able to target specific cell types

5. Transgene should be responsible

for its regulatory elements (on/off system)


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NON-VIRAL VECTORS

Versatile design

No integration into host chromosome

Non-immunogenic and non-toxic Possible multiple and repeated injections

Well-characterized and reproduciblepharmaceutical products

Simple and less expensive GMP production


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NON-VIRAL VECTORS

Cationic liposomes: lipid molecules usedas vehicles for nucleic acid (e.g. DOTMAand DOTAP)

Molecular conjugates: lipid molecules canbe conjugated to cancer cells specificligand

Cell


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NON-VIRAL VECTORS

Cationic liposomes: lipid molecules usedas vehicles for nucleic acid

Molecular conjugates: lipid molecules canbe conjugated to cancer cells specificligand

CellNucleus


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LIPOSOMES

Liposomes resemble animal cell in that theouter membrane consists of a double layer oflipid molecules

These synthetic bubbles can be designed toharbor a plasmid in which original genes have been replaced by those intended to betherapeutic

Liposomes loaded with some medicinalsubstance might fuse with cells and deliver thecontents into the cellular interior


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DNA delivery of genes by liposomes

Cheaper than virusesNo immune response

Especially good

for in-lung delivery (cystic fibrosis)

100-1000 times more plasmid DNA needed

for the same transfer efficiency as for viral vector

Low transfection efficiency

Transient expression

Inhibited by serum

Some cell toxicity

http://www.pharmj.com/Editorial/19990828/pictures/parenteralFig4.gif


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The efficiency of lipid-mediatedgene transfection is dependent on

several steps: Adsorption of the transfection complex to the

cellular surface

Escape from the endosome/lysosome

Translocation across the nuclear membrane andinto the cell nucleus where transcription occurs


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Ideal Vector Life ycle


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VIRAL VECTORS

Retroviruses

LentivirusesHerpes simplex virus (HSV)

Adeno-associated viruses (AAV)

Adenoviruses (Ad)


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Retrovirus

Retroviral genes are replaced with therapeutic human

gene, making the retrovirus incapable of self-replication.

Therapeutic human gene

GENE THERAPY


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Integration of

replication-

defective retrovius

and therapeutic

gene into

host

Therapeutic

gene productNucleus

Human target cell

RNA

Reverse

transcription

DNA


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Gene therapy could be very different for different diseases

• Gene transplantation

(to patient with gene deletion)

• Gene correction

(To revert specific mutation in the gene of interest)

• Gene augmentation

(to enhance expression of gene of interest)

• Targeted killing of specific cells by introducing killergene

• Gene ablation – targeted inhibition of gene expression


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Enhancement of immunological


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CANCER“VACCINE”

Enhancement of immunological

responses to the tumour

Modification of anti-oncogenes

Manufacture of anti-cancer factors

GENETIC DEFECTS


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GENETIC DEFECTS

I i i l G Th T


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Ashanti DaSilva

Initial Gene Therapy Treatments

ADA deficiency periodic treatment of white blood cells in

older children treatment of stem cells from umbilical

cord blood in infants

Andrew Gobea


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Ornithine Transcarbamylase (OTC)deficiency

Jesse Gelsinger

Died from a massiveimmune response against

viral vector


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Contributions of gene therapy

Good news: Promising advances during the

last two decades in recombinant DNAtechnology.

Bad news: Efficacy in any gene therapyprotocol not definitive.

1. Shortcomings in all current gene

transfer vectors.

2. Inadequate understanding of

biological interactions of vector and

host.


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Bioteknologi, minggu 14, terapi gen.pdf