By

Farshid Mokhberi

Shahid Behehsti University Of Medical Sciences and Health Service

What is Gene Therapy It is a technique for correcting defective genes that

are responsible for disease development There are four approaches:

1. A normal gene inserted to compensate for a nonfunctional gene.

2. An abnormal gene traded for a normal gene3. An abnormal gene repaired through selective reverse

mutation4. Change the regulation of gene pairs

The Beginning…In the 1980s, Scientists began to look into gene

therapy.They would insert human genes into a bacteria cell.Then the bacteria cell would transcribe and translate

the information into a proteinThen they would introduce the protein into human

cells

The First CaseThe first gene therapy was performed on September

14th, 1990Ashanti DeSilva was treated for SCID

Sever combined immunodeficiency Doctors removed her white blood cells, inserted the

missing gene into the WBC, and then put them back into her blood stream.

This strengthened her immune systemOnly worked for a few months

How It Works A vector delivers the therapeutic gene into a patient’s

target cell The target cells become infected with the vector The vector’s genetic material is inserted into the

target cell Functional proteins are created from the therapeutic

gene causing the cell to return to a normal state

Principle of gene therapy

An abnormal gene could be swapped for a normal gene through homologous recombination.

The abnormal gene could be repaired through selective reverse mutation, which returns the gene to its normal function.

The regulation (the degree to which a gene is turned on or off) of a particular gene could be altered.

Approaches of gene therapy 1. Gene modification

Replacement therapy Corrective Gene therapy

2. Gene transfer

Physical Chemical Biological

3. Gene transfer in specific cell line

Somatic gene therapy Germ line gene therapy

Vectors in gene therapy

Some of the different types of viruses used as gene therapy vectors:

Retroviruses Adenoviruses Adeno -associated viruses

Herpes simplex viruses

Viral methods for gene therapy

Non-viral methods

Injection of Naked DNA Physical & chemical Methods to Enhance

Delivery Electroporation Gene Gun Sonoporation Magnetofection Oligonucleotides

Electroporation Is a method that uses short pulses of high voltage to carry

DNA across the cell membrane.

This shock is thought to cause temporary formation of pores in the cell membrane, allowing DNA molecules to pass through.

Electroporation is generally efficient and works across a broad range of cell types.

However, a high rate of cell death following electroporation has limited its use, including clinical applications.

Gene Gun DNA is coated with gold particles and loaded into a

device which generates a force to achieve penetration of DNA/gold into the cells.

Example: If the DNA is integrated in the wrong place in the genome, for example in a tumor suppressor gene, it could induce a tumor.

This has occurred in clinical trials for X-linked severe combined immunodeficiency (X-SCID) patients.

Non-viral Options Direct introduction of therapeutic DNA

But only with certain tissue Requires a lot of DNA

Creation of artificial lipid sphere with aqueous core, liposome Carries therapeutic DNA through membrane

Chemically linking DNA to molecule that will bind to special cell receptors DNA is engulfed by cell membrane Less effective

Trying to introduce a 47th chromosome Exist alongside the 46 others Could carry a lot of information But how to get the big molecule through membranes?

Delivery of gene by direct and cell based methods

Advantages of gene therapy

In case of ‘silence’ a gene. In the case of someone with HIV, which had not yet developed into AIDS, scientists could save them the pain and suffering of the disease by using gene therapy to ‘silence’ the disease before its onset.

Gene therapy has the potential to eliminate and prevent hereditary diseases such as cystic fibrosis and is a possible cure for heart disease, AIDS and cancer.

These sceptics would almost certainly choose gene therapy, especially if it was the last hope for them or one of their loved ones – as is the case for many gene therapy patients.

Disadvantages of Gene Therapy

Short-lived nature of gene therapy.

Immune response - Genes injected with a virus may trigger an immune response against the virus. Problems with viral vectors (once inside the patient, the viral vector could recover its ability to cause disease).

Multigene disorders - The genetic material might not get into the right cell, or the right place in the cell’s DNA.

Ethical issues surrounding gene therapy

Who decides which traits are normal and which constitute a disability or disorder?

Will the high costs of gene therapy make it available only to the wealthy?

Could the widespread use of gene therapy make society less accepting of people who are different?

Should people be allowed to use gene therapy to enhance basic human traits such as height, intelligence, or athletic ability?

Application of gene therapy

Parkinson’s diseases Alzheimer’s disease Cyctic fibrosis Diabetic Neuropathy

Viral vectorsThe use of viral vectors as a tool for clinical gene

therapy did not emerge until the 1980s.

Mammals have equally evolved highly complex mechanisms to protect themselves against invading pathogens such as viral gene transfer vectors.

VirusesReplicate by inserting their DNA into a host cellGene therapy can use this to insert genes that encode

for a desired protein to create the desired traitFour different types

Remember!The success of in vivo gene therapy not only depends

on the ability to control the immune response toward the input vector, but also to the therapeutic transgene.

Retroviruses Created double stranded DNA copies from RNA genome

The retrovirus goes through reverse transcription using reverse transcriptase and RNA

the double stranded viral genome integrates into the human genome using integrase integrase inserts the gene anywhere because it has no

specific site May cause insertional mutagenesis

One gene disrupts another gene’s code (disrupted cell division causes cancer from uncontrolled cell division)

vectors used are derived from the human immunodeficiency virus (HIV) and are being evaluated for safety

Retroviruses

Retroviruses were the first type of vector ever used for gene therapy and are now the second most common vector used in clinical trials.

All retroviruses integrate their DNA into the host genome, leading to long-term expression of the target gene.

This useful trait is also the major problem with retrovirus vectors.

problem with retrovirus vectors Insertion of new DNA into the middle of an existing

open reading frame would disrupt function.

Insertion of new DNA with viral promoters could induce transcription of nearby protooncogenes.

Another problem is that some retroviruses can only infect dividing cells.

How to solve their problemPerforming the transduction in vitro and screening

for tumor cells before re-injection can help with these issues,

No one plasmid contains all the genes to produce the vector, as each one only produces a single component of the virus.

no plasmid contains viral replication genes, so the final vector is not capable of selfreplication.

LentivirusesThey are also capable of targeting non-dividing cells.

There is some concern that lentiviruses derived from HIV could undergo homologous recombination wild type virus.

Adenoviruses Are double stranded DNA genome that cause

respiratory, intestinal, and eye infections in humans The inserted DNA is not incorporate into genome Not replicated though

Has to be reinserted when more cells divide Ex. Common cold

AdenovirusesWild type adenoviruses are common, being responsible

for roughly 10% of upper respiratory infections.

their large DNA capacity has made them very popular vectors.

Accounting for almost a quarter of all clinical trials.

AdenovirusesAdenoviruses do not integrate their DNA,instead they become episomes.

This removes any possibility of inducingCancer.

the new DNA will eventually be degraded, requiring re-injection.

ProblemsMost people have antibodies for them.If the adenovirus dosage is too high this can cause a

severe immune reaction.Short-term immunosuppression can help, but can

lead to opportunistic infections

Tactic to solve the problemsSome promise is to use different adenovirus

serotypes, or replacing the virus antigens with those of a different serotype.

changing the viral promoters are retained in the vector can reduce the immune response.

adeno-associated virus (AAV)AAV is a small, nonenveloped single-stranded DNA virus.

This virus infects humans, and is from the Parvoviridae family.

AAV has a high safety profile (because it does not induce a large inflammatory response).

adeno-associated virus (AAV)Can transduce (transfer into a cell) a wide variety of

tissues and cells in vivo.

long-term expression can be achieved without integration.

adeno-associated virus (AAV)Is growing in popularity due to its lack of

pathogenicity and immune responseAAV does not cause any disease and cannot replicate

without coinfection from an adenovirus.Without this helper virus it integrates into the host

genome at a site that does not seem to be tumorigenic in humans.

adeno-associated virus

It provokes almost no immune response in most.

AAV vector problem

Its small size limits the length of the gene that can be inserted to under 5 kb.

This excludes some genes, but is enough for other useful ones.

herpes simplex virus (HSV)

HSV genome is large, measuring 152 kb.

It is possible to insert additional genes of ~10 kb in size into the intact viral genome.

There are three main classes of HSV-1 genes, namely immediate-early (IE or a) genes, the early (E or b) genes and the late (L or g) genes.

herpes simplex virus (HSV)

After various non-essential DNA sequences have been removed it is possible to insert or ‘package’ ~30 kb of foreign genetic material into the virion.

Can live in neurons in a latent state that does not appear to affect normal cellular physiology

This has sparked interest in this virus as a potential vector in the treatment of neurological disorders.

HSV vector problem

Damaging effects

Long-term gene expression in HSV vectors

HSV vector problemThe direct introduction of HSV into the brain as

would be required for testing genes or in gene therapy procedures will result in a lethal encephalitis due to viral replication.

During the onset of latent infection the virus shut down

HSV vector problemShut down of gene expression also occurs for any

exogenous genes, other virus promoters such as the immediate early promoter of cytomegalovirus or a variety of cellular promoters.

This results in expression of the foreign gene being observed for only a few days at the most.