GENE THERAPY Medical biotechnology

Maheen Alam 12-1011

Objectives To be able to learn and understand new techniques

being used in Gene therapy To see the basic differences between protien and gene

therapy To be able to learn the ethical issues related to gene

therapy To be able to understand the treatment HIV as an

example of gene therapy

1) New approaches to gene therapy

In conventional treatments of gene therapy viral and non-viral vectors are commonly used for the delivery of the gene.

These are used to deliver normal copies of a gene into a cell that tends to contain mutated copies of a gene.

However there are times that when you do add the good copy of the gene it might not work.

Dominant negative:

For example there are certain cases when a mutated gene

might produce a protein that prevents the normal protein from

doing its job and in this case if you simply add the normal

gene it won’t help. Mutated genes that work this way are

called dominant negative.

How do we then deal with a dominant negative? In this situation one could either repair the product of the

mutated gene or they could get rid of it altogether. Some new methods have been developed by scientists

which serve as potential approaches to gene therapy. Every technique being used for this purpose requires an

efficient and specific means of delivering the gene to the target cells.

Some of these are1. SMaRT2. Triple-helix forming oligonucleotides3. Antisense4. Ribozymes

A technique for repairing mutations: SMaRT: SMaRT stands for spliceosome-mediated RNA

Trans-splicing. This technique tends to target and repair the

messenger RNA transcripts that have been copied from the mutated gene.

Instead of replacing the entire gene this technique tends to repair a particular section of the mRNA that contains the mutation.

SMaRT involves three steps1) Delivery of a RNA strand that pairs specifically

with the intron next to the mutate segment of mRNA. Once bound, this RNA strand prevents spliceosomes from including the mutated segment in the final, spliced RNA product.

2) Simultaneous delivery of a correct version of the segment to replace the mutated piece in the final mRNA product

3) Translation of the repaired mRNA to produce the normal, functional protein

Techniques to prevent production of a mutated protein: Triple-helix forming oligonucleotides Triple-helix-forming oligonucleotide gene therapy

targets the DNA sequence of a mutated gene to prevent its transcription.

This technique involves the delivery of short, single-stranded pieces of DNA, called oligonucleotides, that bind specifically in the groove between the double strands of the mutated gene's DNA.

Binding produces a triple-helix structure that prevents that segment of DNA from being transcribed into mRNA.

Antisense Antisense gene therapy aims to turn off a

mutated gene in a cell by targeting the mRNA transcripts copied from the gene.

Antisense gene therapy involves the following steps:Delivery of an RNA strand containing the

antisense code of a mutated geneBinding of the antisense RNA strands to

the mutated sense mRNA strands, preventing the mRNA from being translated into a mutated protein

RibozymesLike antisense, ribozyme gene therapy aims to turn off a mutated gene in a cell by targeting the mRNA transcripts copied from the gene. This approach prevents the production of the mutated protein. Ribozyme gene therapy involves the

following steps:Delivery of RNA strands engineered to

function as ribozymes.Specific binding of the ribozyme RNA to

mRNA encoded by the mutated geneCleavage of the target mRNA, preventing it

from being translated into a protein

2) Protein Therapy vs. Gene TherapyProtein therapy Gene therapy Therapeutic proteins are used to

medically treat a disease. They are used for a wide array of

diseases In these cases the protein is either

lacking or deficient, or the therapeutic protein is used to inhibit a biological process.

Protein therapy uses well defined, precisely structured proteins

The optimal doses of individual protein for a particular treatment are already defined

Also the biological effects are well known in this case.

 

Gene therapy can actually be considered a form of pro tein therapy.

Instead of the therapeutic usage of the protein itself, genes are used.

Gene therapy works by placing into a cell a defined gene to either replace a defective gene or to increase the amount of a specific gene in a targeted cell/tissue

This is done in order to produce a higher amount of the desired protein.

To deliver the therapeutic gene either a carrier (vector DNA) must be used

Or the therapeutic DNA must be introduced as “naked” DNA, most often as plasmid DNA, into the target cells.

  

 There are still serious, unsolved problems related to gene therapy including: 1. Difficulty integrating the therapeutic DNA (gene) into the genome of target cells2. Risk of an undesired immune response 3 Potential toxicity, immu nogenicity, inflammatory responses and oncogenesis related to the viral vectors; and 4. The most commonly occurring disorders in humans such as heart disease, high blood pressure, diabetes, Alzheimer’s disease are most likely caused by the combined effects of variations in many genes, and thus injecting a single gene will not be beneficial in these diseases.

The benefits of protein therapy include:

Using a human protein with no immuno genic response

No need for viral vectors

Localized effect at the target tissue, and

Predictability of dose.

On the other hand, an obstacle of protein therapy is the

mode of delivery: oral, intrave nous, intra-arterial, or

intramuscular routes of the protein’s administration are

not always as effective as desired; the therapeutic protein

can be metabolized or cleared before it can enter the

target tissue.

 It seems that protein therapy will become the treatment

modality of choice for many disorders for at least the

next 10 years—at least until further research has

resolved the hurdles and risks related to gene therapy.

3) Ethical and Social Concerns in Germ-line Gene Therapy

Many unique technical and ethical considerations have

been raised by this new form of treatment

Several levels of regulatory committees have been

established to review each gene therapy clinical

trial prior to its initiation in human subjects. 

 

Ethical considerations include

a) deciding which cells should be used

b) how gene therapy can be safely tested and evaluated in

humans

c) what components are necessary for informed consent

d) and which diseases and/or traits are eligible for gene

therapy research.

Germ line gene therapy is difficult as stable integration and gene expression requires gene replacement or repair; however currently only gene addition can be done.

Gene addition could result in insertional mutations and productions of chimeras

Genetic enhancement is another issues which could be misused by totalitarian governments

Also as it tends to be expensive only a certain class can avail the treatment.

The treatment can cause unintended consequences and might affect evolution to a greater degree.

Germ line modifications tend to pose a risk to future generations.

4) Double whammy gene therapy clears HIV from body (Phase I study in 2011)

This study was conducted on 6 patients in California A person with HIV who didn't take antiretroviral drugs

for three months remained free of the virus, thanks to a groundbreaking gene therapy.

The success raises the prospect of keeping HIV in check permanently without antiretrovirals.

The gene therapy works by locking the virus out of the CD4 white blood cells it normally infects.

In this small phase I study they had one virus-free patient and 10-fold reductions in another two.

Zinc fingers: To deliver the treatment, doctors remove blood from the

patient and isolate CD4 and other white blood cells. Specialised molecular "scissors" called zinc finger

proteins enter the cells and sabotage a gene called CCR5, which makes a protein that helps HIV to enter cells.

It is unclear what role CCR5plays normally, although researchers know that cells can survive without it – and will remain uninfected by HIV.

These cells are then returned to the patient in the hope that they will multiply and provide a permanent source of cells immune to HIV, potentially locking out HIV completely.

Double sabotage The secret to making the treatment work best, according

to research, is therefore to eliminate both genes that make CCR5 in as many cells as possible. If only one is sabotaged, cells can still make enough CCR5 protein to allow the virus to invade. In doubly sabotaged or "bi-allelic" cells, there is no way in.