The treatment of many diseases has been improved by the use of genetic technology in medicine.
(a) Explain what is meant by genetic engineering.
(b)(i) Leber Congenital Amaurosis (LCA) is an inherited eye disease. In LCA, the photoreceptor cells in the retina die at an early age. This causes impaired vision (reduced eyesight) in children, which can progress to blindness.
Mutations in different genes cause different forms of LCA. One form of this disease, LCA2, is caused by a mutation in the RPE65 gene. Gene therapy has been used to treat LCA2.
Outline how an inherited eye disease, such as LCA2, is treated with gene therapy.
(ii) Suggest why the eye is a suitable organ for gene therapy.
(c) LCA10 is a different form of LCA caused by a recessive mutation in the CEP290 gene. This gene codes for the protein CEP290, which is involved in the correct functioning of photoreceptor cells in the retina.
The mutation in CEP290 causes an error to be made when the primary transcript is spliced to form messenger RNA (mRNA). The abnormal mRNA that is formed has an extra sequence of RNA nucleotides, known as exon X, between exon 26 and exon 27. Exon X contains a STOP codon.
Fig. 3.1 compares the effect of the mutation in CEP290 with the normal gene expression.
In 2022, research was carried out into possible treatment of LCA10 using genetic technology.
(i) A human clinical trial investigated a treatment of LCA10 using a short RNA nucleotide sequence known as Sepofarsen.
Sepofarsen binds to the section of the primary transcript containing the CEP290 mutation so that normal splicing occurs and functional CEP290 protein is synthesised.
- People in the clinical trial received regular treatment with Sepofarsen to the eye with the greatest loss of vision (treated eye) for a period of 12 months.
- Changes in the light perception (visual acuity) of both eyes were measured using a vision chart.
- A negative change in the visual acuity score shows an improvement in visual acuity.
Fig. 3.2 shows the results of the clinical trial over 12 months.
Describe the results of the clinical trial data shown in Fig. 3.2.
(ii) Another method being investigated to treat LCA10 is to use a gene editing tool known as the CRISPR/Cas9 system.
The CRISPR/Cas9 system uses a short length of RNA called guide RNA. Guide RNA is complementary to the target DNA and is linked to a nuclease enzyme called Cas9. Cas9 breaks phosphodiester bonds in DNA.
The cell repair mechanisms repair the cut in DNA after the modification has taken place.
- A vector delivers Cas9 and two specific guide RNAs to the photoreceptor cells.
- They act on the section of DNA which contains the mutation.
- Exon X is no longer added to the mRNA.
Explain how this method used to treat LCA10 is an example of gene editing.
▶️ Answer/Explanation
(a) Genetic engineering refers to the direct manipulation of an organism’s genes using biotechnology. It involves:
- Isolating and modifying specific genes from one organism
- Inserting these genes into another organism’s genome
- Making the modified genes functional in the new host
- Resulting in the expression of new traits or characteristics
This technology allows scientists to alter the genetic makeup of organisms to achieve desired outcomes, such as producing medicines, improving crops, or treating genetic disorders.
(b)(i) Gene therapy for LCA2 involves:
- Identifying and isolating the healthy, functional version of the RPE65 gene
- Inserting this gene into a viral vector (typically an adeno-associated virus)
- Injecting the viral vector directly into the retina of the affected eye
- The virus delivers the healthy gene to retinal cells
- The healthy gene integrates into the cells’ DNA
- The cells then produce the functional RPE65 protein
- This restores the normal function of photoreceptor cells and improves vision
(b)(ii) The eye is particularly suitable for gene therapy because:
- It’s relatively isolated from the rest of the body, reducing systemic effects
- It’s small in size, requiring only small amounts of therapeutic agents
- It has immune privilege, meaning it’s less likely to mount an immune response against the therapy
- The retina is easily accessible for direct injection
- Changes in vision provide clear indicators of treatment effectiveness
(c)(i) The clinical trial results show:
(c)(ii) This is gene editing because:
- CRISPR/Cas9 directly modifies the patient’s DNA at the precise location of the mutation
- The guide RNA targets the specific sequence between exons 26 and 27 where the mutation occurs
- Cas9 makes a precise cut in the DNA at this location
- The cell’s repair mechanisms then fix the DNA, removing the mutation
- This results in normal mRNA splicing without the inclusion of exon X
- The repaired gene can now produce functional CEP290 protein
- Unlike gene therapy which adds new genes, this method repairs the existing faulty gene