Gene Therapy Dr. Aws Alshamsan & Dr. Nermin Hassan Department of Pharmaceutics...

Gene TherapyDr. Aws Alshamsan & Dr. Nermin Hassan

Department of Pharmaceuticsaalshamsan@ksu.edu.sa

nhassan@ksu.edu.sa

Objectives of this lecture

By the end of this lecture you will be able to:1. Define the term “Gene Therapy”2. Use the correct terminology for gene transfer3. Realize the significance of gene therapy research

Objectives of this lecture

By the end of this lecture you will be able to:1. Describe the different strategies for gene therapy2. Select the suitable strategy based on the clinical case3. Understand the complexity of clinical application of gene therapy4. Evaluate proposed strategies according to the therapeutic need

What is gene therapy?

What is gene therapy?

Introduction of new genetic material into a cell for therapeutic purposes

Genetic Materials:

DNA

RNA

Problem Example Solution

Defect in protein expression G6PD deficiency Introduce a correct

version of the gene

Expression of harmful proteins Cancer Inhibit or block the

harmful proteins

Advantages of gene therapy• Specificity• Can be either temporary or permanent• Localization• Low immunogenicity

Gene Therapy

• Germ-Line Gene Therapy:• Introduction of genes into germ cells (sperm and ovum)

• Somatic Gene Therapy:• Introduction of genes into somatic cells

Germ-Line Gene Therapy

• Hypothetically, germ-line therapy prevents transfer of defective genes to subsequent generation

• Due to ethical and safety reasons, it is not presently accepted for human application

• However, it is used for the transgenic laboratory animals production

Gene therapy

In vivo Ex vivo

Advantage DisadvantageSpecificity Time and labor consuming

High transfection efficiency Invasive

Contamination

Not every cell type is growable

Ex vivo gene therapy• Most widely used in clinical trials

• Some attempts in practice

Advantage Disadvantage

Low cost None specificity

None invasive Low transfection efficiency

Less contamination

Ballistic DNA Injection (gene guns)

Invented for DNA transfer to plant cells

Fully applicable to eukaryotic cells

plasmid DNA shown here

LiposomesNext level idea – why naked DNA?

Lets’ wrap it in something safe to increase transfection rate

Therapeutic drugs

Lipids – are an obvious idea !

DNA delivery of genes by liposomes

Cheaper than viruses

No 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

In vivo gene therapy• Some organs are less suited for ex vivo e.g. (brain,

heart and lungs)

• More practical approach

Gene Therapy Considerations• What gene will be delivered or targeted?• What method will be used?

• Genetic material either for gene expression or downregulation

• Vector to carry the genetic material inside the cells

Elements for gene transfer

Expression Vector (Plasmid)

Viral VectorNon-Viral Vector

Gene Transfer• Transformation: introduction of genetic materials into

bacteria

• Transfection: introduction of genetic materials into eukaryotic cells (e.g. fungi, plant, or animal cells)

• Transduction: introduction of genetic materials using viruses

• Lipofection: introduction of genetic materials using liposomes

Stable vs. Transient Gene Transfer• Stable Gene Transfer: achieved by plasmid integration in the host

genome or episomal replication of the transferred plasmid.

• Transient Gene Transfer: the foreign DNA is usually not integrated into the nuclear genome and will be degraded or diluted through mitosis

Gene Therapy Strategies

•Replacement of a missing or defective gene

•Introduction of gene(s) to influence cellular process

•Interference with gene products

Replacement strategy

Applies to diseases caused by single gene defects

Transfer of a functional copy of the defective or missing gene

Examples: enzyme deficiencies

Replacement strategy

To apply this strategy, three requirements must be met:

1. The specific gene defect must be known

2. A functional copy of the gene must be available

3. Target cells must be available and amenable to transfection methods resulting in long-term expression

Replacement strategy

Gene with defect Disease/Disorder

Adenosine deaminase (ADA) SCID

a-1-antitrypsin Emphysema

CF transmembrane regulator Cystic fibrosis

Clotting factor VIII Hemophilia A

Clotting factor IX Hemophilia B

b-chain of hemoglobin Sickle cell anemia

Mucus in bronchi is thick, interfering with lung function

1 in 25 are carriers

One of the first disorders to be actively studied for gene therapy.

Most lethal autosomal recessive disorder in U.S.

Cystic Fibrosis

Based on: Harvard Family Health Guide, 1999

Sickle Cell Disease- recessive allele•Red blood cells are sickle shaped, issues with circulation causing anemia and pain

Hemophilia

• A disorder in which a person’s blood does not clot properly.

• It is a recessive sex-linked, X-chromosome disorder.

• 1 in 10,000 males born are afflicted.

“Royalty Disease”

ADA-deficient persons are affected by severe immunodeficiency, with recurrent infections that might be life-threatening.

First disease approved for gene therapy.

Autosomal recessive disorder.

The drug exists but is very expensive, needs to be injected in vein for life.

Adenoside Deaminase (ADA) Deficiency

Ashanti Disilva

Bubble Boy

David Phillip Vetter (September 21, 1971 – February 22, 1984)

Gene therapy trial

First successful clinical trial in gene therapy was initiated in September 14, 1990

Hematopoietic stem cells were isolated from the patient (4 y/o girl) and transduced with retroviral vector containing ADA gene

25% recovery of normal ADA in patient T cells

Why was ADA suitable?

Single gene defect

Gene was isolated and cloned in 1983

HSC are easy to obtain and maintain in vitro

Influence strategy

Applies to complex disorders were more than one gene is involved

Based on in vitro cloning of human genes that were derived from human tissue

Examples: cancer

Areas of investigation

Enhancement of anti-tumor response

Introduction of drug-resistance genes

Introduction of drug-sensitivity genes

Replacement of tumor suppressor genes

Introduction of drug-sensitivity genes Suicide gene therapy

Gene that converts non-toxic prodrug into a toxic metabolite

Bystander effect

Gancyclovir triphosphate

Problem: it can transfect normal cells too

Gene Therapy Strategies

•Replacement of a missing or defective gene

•Introduction of gene(s) to influence cellular process

•Interference with gene products

Interference strategy

Downregulation of gene expression at the mRNA level

Inhibition of mRNA translation

Interference nucleic acids

DNAAntisense oligodeoxynucleotide (ODN)DNAzyme

RNAAntisense RNARibozymeSmall interfering RNA (siRNA)Short hairpin RNA (shRNA)microRNA (miRNA)

RNA interference

(RNAi)

DNAzyme

DNAzyme binds target mRNA

mRNA cleavage and degradation

DNAzyme

AAAAAA….ATarget mRNA

protein

X

© American Society for Investigative Pathology

Ribozyme

Antisense ODN

• Sequence-selective oligonucleotide that can bind to a target mRNA to inhibit gene expression i.e. to inhibit translation

Antisense ODN

Antisense ODNAntisense ODN

mRNA

RNase H

We don’t have antisense ODN for every disease

• The main barrier to antisense strategy is optimal delivery in sufficient quantities to the correct target and for the desired time frame to achieve the desired level of gene inhibition

• ODNs are polyanionic macromolecule (large and charge)

• Stability issues in vivo

Designing Biologically Stable ODNs

Designing Biologically Stable ODNs

Fomivirsen Sodium (Vitravene)®

• FDA-approved for the local treatment of CMV retinitis in AIDS patients

Fomivirsen Sodium (Vitravene)®• Dose 150-330 μg intravitreal injection

• Every other week for 2 doses

• Cleared locally by exonucleases 1-2 hr after injection

Antisense RNA

What is RNAi?

• Post-transcriptional phenomenon that was initially discovered in plants

• Mediated by double-stranded RNA

siRNA

RISC

siRNA is bound by RISC and unwound by RNA helicase

RISC

Sense RNA strand degraded

© American Society for Investigative Pathology

RISC

AAAAAA….A

Target mRNA

mRNA cleavage and degradation

RISC-bound antisense strand directed to target mRNA

AAAAAA….A

Target mRNARISC

© American Society for Investigative Pathology

siRNA

www.nature.com/focus/rnai/animations/rnai_revised_320x180.mov

Antisense ODN v.s. siRNA

Antisense ODN siRNA

Nucleotide sugar Deoxyribose Ribose

Structure Single stranded Double stranded

Length 16-30 bp 19-21 bp

Molecular weight ~ 6-9 kDa ~ 13-14 kDa

Precursor vailability No Yes

Site of action Cytoplasm / Nucleus Cytoplasm

mRNA cleavage RNase H RISC

Degradation upon activity Yes No

Effective concentration 50-400 nM 5-100 nM

shRNA

Plasmid DNA

miRNA

cytoplasmic processing

Dicer (RNase III)

pre-miRNA

miRNA

Interact with target mRNA

RISCribosome

mRNA A(n)

RISC RISC

RISC RNA-Induced Silencing Complex

© American Society for Investigative Pathology

miRNA

RNA-induced silencing complex

General mechanisms of siRNA and miRNA

siRNA v.s. miRNA

siRNA v.s. miRNA

Production and Regulation

Strict adherence to GLP and GMP principles

Effectiveness in an appropriate animal model

Safety in rodents and larger animals such as monkeys

Clinical Trials

Good Laboratory Practices

Good Manufacturing Processes

Gene Therapy Successes

approved by the FDA for sale, some diseases have been experimentally successful:

• Melanoma (skin cancer)• Severe Combined Immunodeficiencies• Hereditary Blindness• Sickle Cell Anemia

• 2006: Scientists at the National Institutes of Health (Bethesda, Maryland) have successfully treated metastatic melanoma in two patients. This study constitutes one of the first demonstrations that gene therapy can be effective in treating cancer.

• 2007- 2011: Research is still ongoing and the number of diseases that has been treated successfully by gene therapy increases.

Retinal disease Colour blindnessAdrenoleukodystrophy

• 2011: Medical community accepted that it can cure HIV as in 2008, Gero Hutter has cured a man from HIV using gene therapy

Gene Therapy Disappointments

• In 1999 a boy died due to an immune response to an adenovirus gene therapy vector.

•Four children have developed cancer due to a retrovirus gene therapy vector

You are now able to: Define the term “Gene Therapy” Use the correct terminology for gene transfer Realize the significance of gene therapy research

Now you are able to: Describe the different strategies for gene therapy Select the suitable strategy based on the clinical case Understand the complexity of clinical application of gene therapy Evaluate proposed strategies according to the therapeutic need

http://www.wellesley.edu/Biology/Courses/219/Gen_news/i3_Gene_Therapy.jpg