BIOTEKNOLOGI FARMASI

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

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 disease
are examples.
Chromosome disorders caused by an
excess or deficiency of the genes.
Down syndrome is caused by an extra
copy of a chromosome, but no
individual gene on the chromosome is
abnormal.
Multifactorial inheritance disorders
caused by a combination of small
variations in genes. Heart disease,
most cancers and Alzheimer's disease
are examples.

Sickle Cell
Disorder

Genetic screening can detect genetic disorders.

Genetic screening involves the testing of DNA.

determines risk of having

or passing on a genetic
disorder
used to detect specific
genes or proteins
can detect some genes
related to an increased
risk of cancer
can detect some genes
known to cause genetic
disorders

Disorder

GENE THERAPY

Gene therapy is the genetic alteration of

cells to correct the effects of a diseasecausing mutation.

GENE THERAPY

INCORPORATION OF CLONED DNA

INTO HUMANS AND OTHER ANIMALS
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
SCID (severe combined immunodeficiency)
Cystic fibrosis; CFRT gene.

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
- Design animal model for human
disease.

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 delivered

into cells or Nucleus (genes).

Effect on the cell

membrane or diffuse
into cells

Mechanisms

Usually cure the causes of the

diseases

Usually relieve the

symptoms or signs

Duration of
Effect

Can be permanent and also can

be passed down to next
generation in germline gene
therapy.

Usually stop the effect

once stop taking it.

Ethics

Major Issues

Usually Not

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:

1) Short-lived nature of gene therapy. Very hard to achieve any longterm benefits without integration and even with it.
2) Immune response. It reduces gene therapy effectiveness and
makes repetitive rounds of gene therapy useless
3) Problems with viral vectors . Toxicity, immune and inflammatory
responses, 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.

Gene Carrier Human Application

Considerations

Specificity
Effective
Safety

Only desired cells or

tissue Be Delivered.

The delivery efficiency

Dependent on the
disease requirements
Biocompatible
Non-cytotoxicity
Non-immunogenecity
Non-inflammation
Non-Tumor Generation

The success of gene therapy is based on:

efficient gene transfer into target cells

adequate level of transgene expression

persistence of gene expression
regulation of gene expression
tolerance to transgene product
safety

Barriers Of Gene Delivery

METHODS OF GENE DELIVERY

-- Injection of naked DNA into tumor by simple needle and syringe
-- DNA coated on the surface of gold pellets
which are air-propelled into the epidermis

(gene-gun), mainly non applicable to

cancer

-- DNA transfer by liposomes

(delivered by the intravascular, intratracheal,
intraperitoneal or intracolonic routes)

-- 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.

GENE TRANSFER SYSTEMS

Vectors under
study as gene
delivery vehicles

Retroviruses

Virotherapy

Liposomes

Adenoviruses

AdenoAssociated
Viruses

Naked DNA

Gene Carrier/Gene Vector

Retrovirus

Herpes
Simplex V

Adenovirus

AAV

Liposo
me

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

DNA

Yes or No

Polymer

No

Generally, viral vector system show higher gene transfer efficiency than nonviral gene carrier system, but viral systems have potential risk of wild type
virus regeneration, immunogenecity and cancer formation.

IDEAL CHARACTERISTICS
OF GENE DELIVERY VECTOR
1. High titer or concentrations (>108 particles/ml)
2. Easy and reproducible method of production
3. Precise and stable introduction of transgene
4. Vector should not elicit immune response
in the host
5. Transgene should be responsible
for its regulatory elements (on/off system)
6. Vector should be able to target specific cell types

NON-VIRAL VECTORS
Versatile design
No integration into host chromosome
Non-immunogenic and non-toxic
Possible multiple and repeated injections
Well-characterized and reproducible

pharmaceutical products
Simple and less expensive GMP production

NON-VIRAL VECTORS
Cationic liposomes: lipid molecules used
as vehicles for nucleic acid (e.g. DOTMA
and DOTAP)
Molecular conjugates: lipid molecules can
be conjugated to cancer cells specific
ligand

Cell

NON-VIRAL VECTORS
Cationic liposomes: lipid molecules used
as vehicles for nucleic acid
Molecular conjugates: lipid molecules can
be conjugated to cancer cells specific
ligand

Cell

Nucleus

LIPOSOMES
Liposomes resemble animal cell in that the

outer membrane consists of a double layer of

lipid molecules
These synthetic bubbles can be designed to
harbor a plasmid in which original genes have
been replaced by those intended to be
therapeutic
Liposomes loaded with some medicinal
substance might fuse with cells and deliver the
contents into the cellular interior

DNA delivery of genes by liposomes

Cheaper than viruses

No immune response
Especially good
for in-lung delivery (cystic fibrosis)
Low transfection efficiency
Transient expression
Inhibited by serum
Some cell toxicity

100-1000 times more plasmid DNA needed

for the same transfer efficiency as for viral vector

The efficiency of lipid-mediated

gene 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 and

into the cell nucleus where transcription occurs

Ideal Vector Life Cycle

VIRAL VECTORS
Retroviruses
Lentiviruses
Herpes simplex virus (HSV)
Adeno-associated viruses (AAV)
Adenoviruses (Ad)

GENE THERAPY
Retrovirus

Therapeutic human gene

Retroviral genes are replaced with therapeutic human

gene, making the retrovirus incapable of self-replication.

Packaging cell contains

machinery for duplicating
the replicationdeficient retrovirus

Unpackageable
helper provirus
allows
retrovital
replication

Multiple virions, carrying

the retrovirus, are produced
virions

Human target cell

RNA
Reverse
transcription

DNA

Integration of
replicationdefective retrovius
and therapeutic
gene into
host

Nucleus

Therapeutic
gene product

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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 killer
gene
Gene ablation targeted inhibition of gene expression

CANCER
VACCINE

Enhancement of immunological
responses to the tumour
Modification of anti-oncogenes
Manufacture of anti-cancer factors

GENETIC DEFECTS

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

Ashanti DaSilva

Andrew Gobea

Ornithine Transcarbamylase (OTC)

deficiency
Died from a massive
immune response against
viral vector

Jesse Gelsinger

Contributions of gene therapy

Good news: Promising advances during the
last two decades in recombinant DNA
technology.
Bad news: Efficacy in any gene therapy
protocol not definitive.
1. Shortcomings in all current gene
transfer vectors.
2. Inadequate understanding of
biological interactions of vector and
host.
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