Gene therapy

What is gene therapy and what are the two types?

Gene therapy is the introduction of nucleic acids (DNA or RNA) into cells to alter gene expression with the goal of preventing, stopping, or reversing diseases caused by genetic defects. The two main types are germ line gene therapy, and somatic gene therapy.

Outline and evaluate germ line and somatic gene therapy.

Germ line gene therapy involves modifying reproductive cells (sperm or egg), the introduced gene becomes part of the genome and can be inherited by future generations.

+ has potential for permanent cure, as every cell in offspring carries corrected gene

- technically very difficult, unpredictable effects for future generations, ethically controversial and illegal in most countries.

Somatic gene therapy invoices transferring therapeutic genes into body cells. Common in current research.

- the change only affects the treated individual and is not passed on to offspring.

Outline and explain the 4 key considerations for developing gene therapy

1. delivery, how will gene reach target?

• ex vivo: cells removed, modified in lab, then reinserted

• in situ: gene delivered directly into affected tissue

• in vivo: gene delivered through blood stream to target tissue

2. efficiency: what percentage of cells bust be altered to acheive a therapeutic effect?

3. expression control: should the gene be expressed continuously (constitutively) or regulated (controlled promoter)?

4. Safety: what happens if the gene is over-expressed? how long will the gene persist and stay active?

What are the three strategies for delivering the transgene?

1. Ex Vivo

• Cells removed from the patient → gene introduced in culture → modified cells returned to the body.
  ✅ Example: CAR-T cell therapy for cancer.

2. In Situ

• Gene is injected directly into the target tissue (e.g. muscle or eye).
  ✅ Example: Retinal gene therapy for inherited blindness.

3. In Vivo

• Gene delivered via bloodstream to reach internal organs.
  ✅ Example: Systemic delivery using viral vectors.

What are the main gene therapy approaches?

Gene addition e.g cystic fibrosis gene replacement, gene correction/alteration r.g genome editing using CRISPR, and gene knowckdown e.g gene interference.

Explain how gene addition is done as an approach in gene therapy.

Gene addition adds a therapeutic or corrective gene to provide a missing or faulty protein, the corrective gene can integrate into the chromosome or exist episomally (outside chromosome but still expressed). This is the most common approach used in pre-clinical and clinical trials. One example is cystic fibrosis gene replacement.

Explain how gene correction/alteration is done as an approach in gene therapy.

This appraoch uses engineered DNA binding proteins (e.g zinc finger nucleases, CRISPR-Cas9, TALENs) to introduce targeted double stranded breaks in DNA and stimulate homologous recombination. Its goal is to correct or induce mutations in the exact genomic location. Challenges of this approach include; 

• natural homologous recombination is very rare in mammalian cells

• random integration is often too frequent

How do ZNFs allow gene correction/alteration?

Zinc finger nucleases contain engineered DNA binding domains made of ‘zinc fingers’, each ZF binds to a 3-base DNA sequence, by combining several ZFs, scientist can design highly specific binding sites. The nuclease then cuts DNA, repair machinery fixes it using correct donor DNA, allowing correction.

Explain how gene knockdown is done as an approach in gene therapy.

The aim of this approach is to reduce or block the production of a harmful protein, achieved using RNA interference - a natural cellular mechanism. 

1. the siRNA or shRNA binds to target mRNA via complementary base pairing.

2. the mRNA is cut and degraded by the RISC (RNA induced silencing complex)

3. this prevents the synthesis of the target protein

What are vectors and what are the two main types?

In gene therapy, a vector is a vehicle used to deliver nucleic acids into patient cells. Vectors are essential for getting the therapeutic gene into the right cells, expressed at the right level, and for the right amount of time. The two main categories are viral vectors (most common) and non-viral vectors (simpler but less efficient).

What are the ideal properties of a gene therapy vector?

An effective vector should be:

1. target-cell selective - must deliver gene only to desired cells/tissues

2. transcriptionally competent - gene must be expressed for the required time

3. highly concentrated and active

4. immunologically neutral - minimal immune reaction in the patient.

In realuty each vector type has trade offs between safety, efficiency and duration of expression.

What are the main barriers to successful gene therapy?

Four key technical barriers limit the success of gene therapy. First, vector uptake, transport, and uncoating depend on factors such as blood supply, vector size, and receptor–ligand interactions, which affect delivery efficiency. Second, vector genome persistence is an issue because episomal DNA (non-integrated) is lost during cell division—making it better for non-dividing tissues—while integrating vectors can persist in dividing cells but risk disrupting host genes. Third, sustained transcriptional expression may be reduced due to epigenetic silencing of vector DNA, requiring expression duration to match therapeutic needs. Finally, the host immune response can target either the transgene product or the vector itself, reducing treatment efficacy.

Give an example of when vector integration lead to uncontrolled cell growth.

Integration-based vectors can trigger insertional mutagenesis — risk depends on the transgene and where it integrates.

X-linked severe combined immunodeficienct (SCID) is a lack of functional B and T cells due to defective IL2RG gene. A retroviral vector is used to insert functional IL2RG into bone marrow stem cells ex vivo, sucessfully restored immune finction in most patients however 5 out of 20 patients developed leukemia. This was because the vector integration activated LMO2 proto-oncogene, leading to uncontrolled cell growth. In contrast ADA-SCID gene therapy (using ADA gene rather than IL2RG) was sucessful as ADA gene does not have independent growth promoting activity.

What are the features, advantages, and drawbacks of retroviruses and lentiviruses in gene therapy?

Retroviruses integrate their genetic material into the host genome, allowing long-term and stable expression. They are efficient at transducing dividing cells, while lentiviruses can also target non-dividing cells. However, integration carries the risk of insertional mutagenesis, which can activate oncogenes, and their gene capacity is limited to about 7 kb. These vectors are mainly used for ex vivo modification of cells, such as in treating SCID and in CAR-T cell therapies

What are the main characteristics, benefits, and limitations of adenoviral vectors in gene therapy?

Adenoviruses do not integrate into the host genome, resulting in transient expression that typically lasts a few weeks. They can infect both dividing and non-dividing cells and have a large DNA capacity, allowing high levels of gene expression. However, they are highly immunogenic, can trigger strong immune and inflammatory responses, and cannot be reused with the same serotype due to immune memory. These features limit their repeated or systemic therapeutic use.

What are the features and clinical applications of AAV vectors in gene therapy?

AAV is a small, non-pathogenic virus capable of infecting both dividing and non-dividing cells with very low immunogenicity, making it extremely safe. It cannot replicate independently and provides stable expression in tissues like muscle, heart, and retina. Its main limitations are a small transgene capacity (~4 kb) and high production cost. Clinically, AAV has been used for Leber’s Congenital Amaurosis, where AAV carrying the RPE65 gene restores vision, and it shows promise for conditions like muscular dystrophy and heart failure.

What are non-viral vectors, and what are their advantages and limitations?

Non-viral vectors deliver DNA or plasmids using physical or chemical methods such as liposomes, electroporation, or nanoparticles. They are safe, non-immunogenic, inexpensive, and easy to produce, with no risk of insertional mutagenesis. However, they have poor in vivo efficiency, limited nuclear delivery, and short-lived gene expression since plasmids are lost over time. Due to these limitations, they are primarily used for research and ex vivo applications rather than systemic therapy.

What is cystic fibrosis and what happens in CFTR loss?

Cystic fibrosis is caused by mutations in the CFTR gene. CTFR encodes a Cl- channel located on the luminal sirface of epithelial cells in the airway. Normally CFTR allows Cl- to move out of cells, inhibiting Na+ absorption through the epiuthelial sodium channel (ENaC), helping maintain proper hydration of airway mucus. In the mutation there is a loss of Cl- secretion, ENaC is hyperactive leading to Na+ absorption, this causes water loss leading to thick and sticky mucus = blocked airwars, infections and inflammation.

Outline gene therapy approaches for CF

Since the discovery of the CFTR gene in 1989 over 20 clinical trials have been conducted, both viral and non-viral vectors were tested. There was some success; CFTR mRNA detected in airway cells, and partial restoration of Cl- secretion. However, Na+ hyper-absorption was not corrected, so clinical benefit was limited.

What are the problems associated with CFTR gene transfer?

1. Mucociliary clearance:

   • Airway defenses quickly remove vectors from the surface.

2. Thick mucus barrier:

   • In CF, mucus is dense and sticky, preventing vectors from reaching cells.

3. Airway inflammation:

   • Causes toxicity and immune responses to the vector.

4. Immune response:

   • Antiviral immunity reduces the success of repeat dosing.

5. Lack of receptors:

   • Few viral receptors on the apical (outer) surface of airway cells.

6. Lung structure complexity:

   • Airways vary in gene expression and cell type from proximal → distal regions,
     making it difficult to replicate natural CFTR expression patterns.

7. Unclear target cell type:

   • Should the therapy target surface epithelial cells or submucosal glands (where CFTR is normally high)?

   • Nebulised therapy mainly reaches surface cells, not deeper glands.

How is gene therapy used in treating cancer and what are the main strategies?

Cancer gene therapy aims to kill tumour cells or stop their growth by manipulating genes. The four main strategies include;

• immungene therapy which aims to boost immune response by expressing tumour antigens or cytokines.

• suicide gene therapy which delivers gene encoding enzymes that convert non-toxic prodrug into toxic compound in cancer cells or introduce pro-apoptotic genes to induce cell death, tumour suppressor gene replacement.

• tumour supressor gene replacement, this restores missing or mutated tumour supressors such as p53.

• anti-angiogenic therapy, this blocks blood vessel formation feeding the tumour.

• RNA based therapies, using miRNA or RNAi to silence oncogenes or block cancer promoting RNA molecules

Explain the use of oncolytic viruses in cancer gene therapy and give an example.

Some cancer gene therapies use replication competent viruses that specifically infect and destroy tumour cells, these are called oncolytic viruses.

Conditionally replicating adenoviruses (CRAds) are engineered adenoviruses that replicate only in tumour cells, killing them (oncolysis) while sparing normal cells.

1. virus infects tumour cell, replicates and causes lysis.

2. new viral particles spread to nearby cancer cells and repeats infection.

3. tumour mass is gradually destroyed

What are CRAds and how are they designed?

Conditionally replicating adenoviruses are engineered adenoviruses that replicate only in tumour cells, killing them while sparing normal cells. There are two main engineering strategies:

• deletion mutants: delete essential viral genes, because of this deletion, the virus can’t grow or replicate in normal healthy cells — only in tumour cells that can replace (or “make up for”) the missing function. e.g ONYC-015 lacks E1B 55 kDa gene, so it replicates only in p53-deficient tumour cell (not in healthy cells).

• promoter targeting: place viral genes under controll of tumour specific promoters e.g human telomerase promoter hTERT which is active in 90% of cancers and inactive in normal tissue, ensuring tumour specific replication.

Describe ONYX-015 as a deletion mutant conditionally replicating adenovirus (CRAds)

It is a mutant adenovirus (E1B-deleted) that replicates in p53 defective cancer cells, it has been tested in head and neck, brain, liver, colorectal, sarcoma, ovarian, and pancreatic cancers. Results show as a single agent it has modest effects, and when combined with cheotherapy it is a strong anti-tumour synergy.

Explain how combining CRAds with RNAi works in cancer gene therapy?

CRAds can be engineered to deliver RNAi molecules (shRNA/siRNA) into tumour cells, combining oncolysis and gene silencing. Examples include CRAd + siRNA against mutant Ras - this results in enhanced tumour cell killing with minimal toxicity to normal cells. Another example is CRAd expressing shRNA against VEGF, results in long lasting silencing of VEGF, resulting in anti-angiogenic and tumour suppressing effects.