GENE THERAPY

Gene therapy can be used to treat genetic diseases. The effectiveness of gene therapy depends on the
site of action. For example, in cystic fibrosis, the site of action is the cells lining the wind-pipe which are
replaced regularly so the effect is short-lived. On the other hand, patients suffering from hemophilia B
may only need one injection to completely cure them of the condition.

There are two types of gene therapy, somatic and germ-line but the process in both is effectively the
same. Gene therapy is the insertion of the normal gene which will replace the faulty gene.

Germ-line therapy aims to repair the faulty gene in the gametes, which means that the child should be
born free of the condition.

1. EXTRACTION or obtaining the required gene.

Identify the gene involved.
For example, the cystic fibrosis gene is found on chromosome number 7.

2. COPYING of the gene.

Make copies of the normal allele and insert into a vector (viruses/liposomes)

3. INSERTION of the gene into a vector.

Use the vector to insert the allele into target cells.

4. INSERTION of the gene into the genome of the organism.

STEP II - Use of REVERSE TRANSCRIPTION to make a copy of the gene

1. CFTR gene containing cells on chromosome number 7.

2. Extract mRNA which codes for CFTR.
3. mRNA for CFTR
4. mRNA acts as a template to synthesize a single strand of copy DNA from DNA nucleotides,
catalyzed by the enzyme reverse transcriptase.
5. Single stranded copy of DNA (cDNA) formed.
6. Now copy DNA (cDNA) acts as a template to synthesize the complementary DNA strand. This is
catalyzed by the enzyme DNA polymerase.
7. A copy of the human CFTR gene has been produced artificially.

STEP III – Inserting the required gene into the vector

1. Cut out the required gene with restriction endonuclease enzyme which produces sticky ends
(exposed bases)
2. Obtain plasmid from suitable bacteria (double stranded DNA)
3. Cut plasmid with same restriction enzyme which produces the same sticky ends.
4. Use reverse transcription to make a copy gene of desired gene or then add sticky ends to the
sticky ends of plasmid.
5. The sticky ends have exposed bases. These will attract the bases of the complementary sticky
ends. The bases will now join with hydrogen bonds by specific base pairing rule. Because the
sticky ends attract each other, the insertion of the gene into plasmid is more likely.
 6. The enzyme DNA ligase is used to join the cut ends of the DNA of the plasmid and gene. The
sugar-phosphate backbones are joined.

Recombinant DNA has now been produced because the gene DNA has been joined to the plasmid
DNA.

STEP IV – Insertion of gene into the genome of the organisms

There are two main methods for introducing healthy CFTR genes into lung epithelium cells.

1. Wrapping the gene in lipid molecules (liposomes) to enable it to pass through the membranes
of lung epithelium cells. Liposomes (made up of fats) are sprayed into nostrils of the patient
which fuses with the cell membrane passing the genetic material into the cell.
2. Using a harmless virus (adenovirus) to transfer the healthy gene into the host cells. The viruses
are treated to make them harmless. The gene is inserted and then the viruses are introduced
into the nostrils of the patients where we are infected with some viruses, the viral DNA
becomes incorporated into our cells own DNA. With other viruses the viral DNA remains
independent within the nucleus of our cells. In trials with CFTR, the second type of virus is used.
3. The use of virus is a potentially efficient form of gene transfer but it has been found to
produce an inflammatory response, with treated patients experiencing symptoms such as
headache, fatigue, fever and raised heart rate.

1. Copy of the normal allele is inserted into a loop of DNA (called a plasmid)
2. The plasmids are then combined with liposomes (spherical phospholipid bilayers)
3. The positively changed head groups combine with the DNA (a weak acid and so negatively
charged) to form a liposome DNA complex.
4. The cystic fibrosis patient breathes in an aerosol containing these complexes using a nebulizer.
The liposomes fuse with epithelial cell membrane and carry the DNA into the cells.

PROBLEMS WITH CYSTIC FIBROSIS GENE THERAPY –

1. Only about 25% of the normal chloride transport function is restored.

2. Effect is temporary (upto two weeks) as epithelial cells are replaced with faulty genes.
3. Use of viruses can cause side effects (fever, headache, higher heart rate)
4. Delivery is very inefficient, especially with liposomes (only about 1 in every 1000 genes gets into
epithelial cells)

REVERSE TRANSCRIPTION – It is the reverse of normal transcription. A particular sequence of DNA is

copied from a mRNA template.

REVERSE TRANSCRIPTASE – It is an enzyme which catalyzes reverse transcription.

RESTRICTION ENDONUCLEASE – These are enzymes which act as molecular scissors, cutting DNA at a
specific base sequence. Different restriction enzymes cut at different base sequences.

GENOME – The complete genetic information of an organism.