fundamentals of gene therapy

What is gene therapy?

Gene therapy is the therapeutic modification of genetic material to prevent, treat, or potentially cure disease by correcting or altering the underlying genetic cause rather than only treating symptoms.

Why is gene therapy considered fundamentally different from traditional pharmacology?

Traditional pharmacology typically modifies protein activity temporarily, whereas gene therapy alters the underlying genetic blueprint itself, potentially producing long-term or permanent therapeutic effects.

List the major approaches used in gene therapy.

• Gene replacement

• Gene silencing

• Gene editing

• Cell-based gene therapy

What is gene replacement therapy?

A strategy in which a functional copy of a defective gene is introduced to restore normal protein production

What is gene silencing?

Suppression of harmful gene expression using approaches such as siRNA, antisense oligonucleotides, or CRISPR-mediated knockout

What is gene editing?

Direct modification of endogenous DNA sequences using targeted genome editing technologies such as CRISPR/Cas9, TALENs, or ZFNs

What is cell-based gene therapy?

A therapeutic approach in which cells are genetically modified outside the body and then reinfused into the patient.

What are the two defining properties of stem cells?

1. Self-renewal

2. Potency (ability to differentiate into specialised cell types)

Define self-renewal.

The ability of stem cells to divide repeatedly while remaining undifferentiated.

What is potency in stem cell biology?

The ability of a stem cell to differentiate into specialised cell types

Differentiate between totipotent, pluripotent, and multipotent stem cells

• Totipotent: can generate an entire organism

• Pluripotent: can generate almost all body cell types

• Multipotent: limited to specific lineages

What are embryonic stem cells (ESCs)?

Pluripotent stem cells derived from the inner cell mass of the blastocyst with the ability to generate nearly any cell type.

What are the major advantages of embryonic stem cells?

• Extensive differentiation potential

• High proliferative capacity

• Major regenerative potential

What are the major limitations of embryonic stem cells?

• Ethical concerns

• Tumour/teratoma risk

• Genomic instability

What are induced pluripotent stem cells (iPSCs)?

Adult somatic cells genetically reprogrammed into a pluripotent embryonic-like state

Which transcription factors are classically used to generate iPSCs?

• Oct4

• Sox2

• Klf4

• c-Myc

Why are iPSCs important in modern medicine?

They allow patient-specific therapies, disease modelling, reduced immune rejection, and personalised regenerative medicine.

What are the risks associated with iPSCs?

• Tumour formation

• Incomplete reprogramming

• Epigenetic abnormalities

What is a mutation?

A permanent alteration in DNA sequence that may affect protein structure, expression, or function

What is a frameshift mutation?

A mutation caused by insertion or deletion of nucleotides not in multiples of three, altering the downstream reading frame

Why are frameshift mutations usually severe?

Because they alter all downstream codons, often producing misfolded proteins or premature stop codons

What is a point mutation?

A single nucleotide substitution within DNA.

Differentiate between silent, missense, and nonsense mutations

• Silent: no amino acid change

• Missense: different amino acid inserted

• Nonsense: premature stop codon formed

What mutation causes sickle cell disease?

A missense mutation in the β-globin gene causing glutamic acid to be replaced by valine.

Explain the molecular mechanism underlying sickle cell disease.

The valine substitution creates hydrophobic interactions that promote HbS polymerisation under low oxygen conditions, deforming RBCs into sickle shapes

Why is sickle cell disease considered an excellent target for gene therapy?

Because it is a single-gene disorder with a well-characterised mutation and accessible haematopoietic stem cells

Why are DNA double-strand breaks (DSBs) important in genome editing?

Genome editing technologies intentionally induce DSBs to activate endogenous DNA repair pathways that perform the edit

Why are DNA double-strand breaks potentially dangerous?

Improper repair can cause chromosomal instability, mutations, or cancer

What is homology-directed repair (HDR)?

A high-fidelity DNA repair pathway that uses a homologous template to repair DNA accurately.

Outline the steps of HDR

• DSB formation

• DNA end resection

• Template alignment

• DNA synthesis

• DNA ligation

What are the major advantages of HDR?

Allows precise genome correction and targeted sequence insertion.

What are the major limitations of HDR?

• Low efficiency

• Mainly active in dividing cells

• Poor activity in neurons

What is non-homologous end joining (NHEJ)?

An error-prone DNA repair pathway that rejoins broken DNA ends without a repair template.

Why is NHEJ error-prone?

Because DNA ends are often processed before ligation, generating insertions or deletions (indels)

Why is NHEJ useful in gene therapy despite being error-prone?

Because it can intentionally disrupt harmful genes through frameshift mutations or gene knockout.

What are zinc finger nucleases (ZFNs)?

Engineered genome editing proteins combining zinc finger DNA-binding domains with the FokI nuclease domain

How do ZFNs generate DNA cleavage?

Two ZFNs bind opposite DNA strands, allowing FokI nuclease domains to dimerise and create a DSB

What are the limitations of ZFNs?

• Complex engineering

• Expensive development

• Difficult design process

What are TALENs?

Genome editing systems using transcription activator-like effector DNA-binding domains fused to FokI nucleases.

What advantages do TALENs have over ZFNs?

• Easier design

• Greater modularity

• Higher specificity

What is CRISPR/Cas9?

A programmable genome editing system derived from bacterial adaptive immunity that uses guide RNA-directed Cas9 nuclease to cut DNA at specific sites

What are the two major components of CRISPR

• Guide RNA (gRNA)

• Cas9 endonuclease

What is the function of guide RNA in CRISPR?

It directs Cas9 to complementary target DNA sequences via base pairing.

What is the role of Cas9?

Cas9 is an RNA-guided endonuclease that introduces site-specific DNA double-strand breaks

What is a PAM sequence?

A short DNA motif adjacent to the target sequence required for Cas9 binding and cleavage

Outline the CRISPR/Cas9 editing process.

1. gRNA designed

2. Cas9-gRNA complex formed

3. PAM recognised

4. Target DNA bound

5. Cas9 cleaves DNA

6. Repair occurs via HDR or NHEJ

What is base editing?

A CRISPR-derived technology allowing single nucleotide changes without creating full DNA double-strand breaks

What are the advantages of base editing?

• Lower toxicity

• Reduced chromosomal damage

• Greater precision for point mutations

What is prime editing?

A genome editing technology combining Cas9 nickase with reverse transcriptase to allow highly precise insertions, deletions, and substitutions

Why is prime editing considered potentially safer than conventional CRISPR?

Because it avoids full double-strand breaks and reduces off-target genomic damage

What are off-target effects in genome editing?

Unintended DNA modifications occurring at non-target genomic sites.

Why are off-target effects dangerous?

They may disrupt tumour suppressor genes, activate oncogenes, or create genomic instability

How can off-target effects be reduced?

• Improved gRNA design

• High-fidelity Cas9 variants

• Base editing

• Prime editing

Why are vectors needed in gene therapy?

Because DNA/RNA molecules are large, negatively charged, and cannot efficiently cross cell membranes unaided.

Why are viruses commonly used as gene delivery vectors?

Because viruses evolved naturally to deliver genetic material efficiently into host cells

What are adenoviral vectors?

Double-stranded DNA viral vectors capable of strong transient gene expression without genome integration

What are the major advantages of adenoviral vectors?

• High transduction efficiency

• Large cargo capacity

• Infect dividing and non-dividing cells

What are the major limitations of adenoviral vectors?

• Strong immune responses

• Inflammatory toxicity

• Temporary expression

What are adeno-associated viruses (AAVs)?

Small single-stranded DNA viruses widely used for relatively safe long-term gene delivery

Why are AAV vectors clinically important?

Because they have low immunogenicity, relatively good safety profiles, and enable long-term expression

What is the major limitation of AAV vectors?

Small cargo capacity

What are retroviral/lentiviral vectors?

RNA viral vectors that integrate therapeutic genes into the host genome

What is insertional mutagenesis?

Random viral genome integration that may activate oncogenes or disrupt essential genes.

What are liposomes?

Artificial lipid vesicles used to encapsulate and deliver nucleic acids into cells.

What are the advantages of non-viral delivery systems?

• Lower immunogenicity

• Improved safety

• Easier manufacturing

What are the limitations of non-viral delivery systems?

• Lower delivery efficiency

• Reduced cellular uptake

• Shorter expression duration

Why are lipid nanoparticles (LNPs) important?

They enable efficient RNA delivery and became especially important in mRNA vaccine technology

What is ADA-SCID?

A severe combined immunodeficiency caused by deficiency of adenosine deaminase (ADA), leading to toxic metabolite accumulation and lymphocyte destruction

Outline the gene therapy strategy used for ADA-SCID

• Remove patient haematopoietic stem cells

• Insert functional ADA gene using viral vector

• Reinfuse corrected cells

What are CAR-T cells?

Patient T cells genetically engineered to express chimeric antigen receptors targeting cancer cells.

How do CAR-T cells kill tumour cells?

Engineered receptors recognise tumour antigens, activating T cells to destroy cancer cells

Differentiate between somatic and germline gene editing.

• Somatic editing affects only the treated individual

• Germline editing affects reproductive cells and can be inherited by future generations

Why is germline editing ethically controversial?

Because genetic changes become heritable and may permanently alter future generations

List major ethical concerns surrounding gene therapy.

• Designer babies

• Equity of access

• Long-term safety

• Consent of future generations

• Eugenics concerns

What are the major current limitations of gene therapy?

• Delivery efficiency

• Immune responses

• Off-target effects

• Tumour risk

• High cost