outline how genetic diseases can be treated with gene therapy, using the examples severe combined immunodeficiency (SCID) and inherited eye diseases

Genetic Technology Applied to Medicine – A‑Level Biology 9700

Objective: Outline how genetic diseases can be treated with gene therapy, using severe combined immunodeficiency (SCID) and inherited eye diseases as examples, and relate the techniques to the full Cambridge “Genetic Technology” (Topic 19) syllabus.

1. Foundations of Genetic Technology

1.1 Recombinant DNA (rDNA) Technology

• Restriction enzymes cut DNA at specific palindromic sites, producing sticky or blunt ends.
• DNA ligase joins compatible ends, allowing insertion of a gene of interest into a plasmid (vector).
• Typical plasmid features for therapeutic use:

  • Origin of replication (for bacterial amplification)
  • Selectable marker (e.g., ampR)
  • Multiple cloning site (MCS)
  • Eukaryotic promoter, poly‑A signal and, if required, an intron for efficient expression

• The recombinant plasmid is amplified in E. coli, purified and then packaged into a delivery vehicle (viral or non‑viral).

1.2 Core Molecular‑Biology Techniques (AO1)

• Polymerase Chain Reaction (PCR) – exponential amplification of a specific DNA fragment using primers, thermostable DNA polymerase and thermal cycling.
• Gel electrophoresis – separates DNA fragments by size; visualised with ethidium bromide or safe‑alternatives.
• DNA sequencing (Sanger or next‑generation) – determines the exact nucleotide order, confirming mutations or vector integrity.
• Cloning & transformation – ligated plasmid introduced into competent bacteria (e.g., CaCl₂‑treated E. coli); colonies screened for correct inserts.

1.3 Gene‑Editing Technologies

• CRISPR‑Cas9 – a guide RNA (gRNA) directs Cas9 to a 20‑bp target adjacent to a PAM (NGG). Cas9 creates a double‑strand break (DSB) repaired by:

  • Non‑homologous end joining (NHEJ) – error‑prone, creates insertions/deletions (knock‑out).
  • Homology‑directed repair (HDR) – uses a supplied DNA template for precise correction (knock‑in).

• Advantages over older tools (ZFNs, TALENs): simplicity, multiplexing, lower cost.
• Clinical illustration: ex‑vivo CRISPR correction of the HBB gene in hematopoietic stem cells for sickle‑cell disease (Phase I/II, 2023‑2024).

1.4 RNA‑Based Therapeutic Approaches

• RNA interference (RNA‑i) – siRNA or shRNA guide the RISC complex to degrade complementary mRNA, silencing a disease‑causing gene.
• Antisense oligonucleotides (ASOs) – bind pre‑mRNA to modify splicing or block translation (e.g., nusinersen for SMA).
• mRNA therapy – synthetic mRNA encodes a functional protein; delivered in lipid nanoparticles (platforms now adapted for enzyme replacement).
• Example: siRNA targeting mutant huntingtin mRNA (clinical trials for Huntington’s disease).

1.5 DNA Fingerprinting & Forensic Biotechnology (Topic 19.3)

• Highly variable short‑tandem repeat (STR) loci are amplified by PCR.
• Products are separated by capillary electrophoresis; the resulting profile is unique to an individual.
• Techniques (PCR, electrophoresis, sequencing) are identical to those used for confirming vector integration or detecting off‑target edits in the laboratory.

1.6 Transgenic Organisms & Agricultural Biotechnology (Link to Topic 19.4)

• Same rDNA principles are used to create GM crops and animals.

  • Bt‑cotton – bacterial cry gene confers insect resistance.
  • Golden Rice – genes for β‑carotene synthesis increase vitamin A content.
  • Marker‑assisted selection and gene‑stacking rely on selectable markers and promoters introduced via transformation.

• Understanding vector design, promoter choice and biosafety is essential for both medical and agricultural applications.

2. Gene‑Therapy Strategies (AO2)

1. Gene addition – delivery of a functional copy of a defective gene (most common for monogenic disorders).
2. Gene editing – precise correction of the mutation in situ (CRISPR‑Cas9, TALENs, ZFNs).
3. RNA‑based silencing – reduction of harmful allele expression (RNA‑i, ASOs).

3. Case Study 1 – Severe Combined Immunodeficiency (SCID)

3.1 Molecular Basis

• SCID includes several inherited forms; the most frequent is X‑linked SCID caused by mutations in IL2RG (common γ‑chain of interleukin receptors).
• Loss of the γ‑chain blocks signalling required for T‑cell development, resulting in absent functional T‑cells (and often B‑cells).

3.2 Ex‑vivo Gene‑Addition Therapy (Current Standard)

1. Cell collection: Harvest hematopoietic stem cells (HSCs) from bone‑marrow or cord blood.
2. Vector design: Self‑inactivating (SIN) lentiviral vector carrying a functional IL2RG cDNA under an internal promoter. Lentiviruses integrate into the host genome, giving long‑term expression in dividing HSCs.
3. Transduction (ex‑vivo): HSCs are cultured with cytokines and exposed to the viral vector; integration is verified by PCR and flow cytometry.
4. Conditioning regimen: Low‑dose busulfan or cyclophosphamide creates niche space in the patient’s marrow.
5. Re‑infusion: Gene‑corrected HSCs are returned intravenously; they home to the marrow, differentiate into T‑cells and re‑establish immunity.

3.3 Clinical Outcomes & Safety

• Long‑term immune reconstitution reported in > 80 % of treated children (median follow‑up > 10 years).
• Key safety issue: insertional mutagenesis. SIN lentiviral vectors reduce enhancer‑driven activation of oncogenes compared with earlier γ‑retroviral vectors.
• Regulatory status (2024): Approved in the EU (Strimvelis – a γ‑retroviral product) and FDA‑approved trials for lentiviral IL2RG therapy.

3.4 Emerging Gene‑Editing Alternative

• CRISPR‑Cas9 delivered as ribonucleoprotein (RNP) complexes to HSCs can correct the IL2RG mutation via HDR.
• Advantages: precise correction without random integration.
• Challenges: low HDR efficiency in HSCs, potential off‑target cleavage, need for efficient delivery.

4. Case Study 2 – Inherited Eye Diseases (Inherited Retinal Dystrophies)

4.1 Molecular Basis

• Mutations in genes required for photoreceptor or retinal pigment epithelium (RPE) function cause diseases such as Leber congenital amaurosis (LCA) and retinitis pigmentosa.
• Example: Biallelic mutations in RPE65 disrupt the visual cycle, leading to early‑onset blindness.

4.2 In‑vivo Gene‑Addition Using AAV (Current Standard)

1. Vector choice: Adeno‑associated virus (AAV) serotype 2, 8 or 9 – non‑integrating, low immunogenicity, natural tropism for RPE cells.
2. Transgene cassette: Human RPE65 cDNA driven by a ubiquitous or RPE‑specific promoter, flanked by inverted terminal repeats (ITRs) for packaging.
3. Delivery: Sub‑retinal injection creates a bleb between the RPE and photoreceptor layer; 100–200 µL of vector (~10¹¹ vector genomes) is administered under local anaesthesia.
4. Expression & outcome: Transduced RPE cells produce functional RPE65, restoring the visual cycle. Clinical trials (e.g., Luxturna) report improved light sensitivity and visual acuity within weeks, with durability up to five years.

4.3 Other In‑vivo Approaches (Emerging)

• Gene editing: AAV‑CRISPR systems aim to correct RPE65 or ABCA4 mutations directly in the retina (Phase I trials ongoing).
• RNA‑based therapy: Antisense oligonucleotides for exon skipping in specific RP mutations (e.g., QR‑110 for CEP290‑related LCA).

5. Comparison of Gene‑Therapy Approaches

6. Clinical Development Pathway (AO3)

1. Pre‑clinical stage: In‑vitro efficacy, animal models, vector biodistribution, toxicology.
2. Phase I: Small cohort – safety, dose‑finding.
3. Phase II: Expanded cohort – preliminary efficacy.
4. Phase III: Large, randomised, confirmatory efficacy & safety.
5. Regulatory review: EMA (EU) / FDA (US) – assessment of CMC, clinical data and risk‑management plan.
6. Post‑marketing surveillance (Phase IV): Long‑term follow‑up for insertional events, immune reactions, durability of benefit.

7. Ethical, Legal & Social Issues (ELSI) – Dedicated Sub‑section

• Somatic vs. germ‑line therapy: Current clinical work is limited to somatic cells; germ‑line editing would affect future generations and is prohibited in most jurisdictions.
• Informed consent: Special considerations for paediatric patients – assent from the child plus parental/guardian consent.
• Equity & cost: Luxturna ≈ US $425 000 per eye; Strimvelis ≈ €200 000 per patient. Raises questions about NHS/insurance coverage and global accessibility.
• Regulatory frameworks: EU Clinical Trials Regulation (CTR 536/2014), US FDA Gene Therapy Guidance (2022), WHO recommendations on human genome editing.
• Public perception: Media hype vs. realistic expectations; importance of transparent communication about risks, benefits and uncertainties.

8. Suggested Practical Investigation (AO2 & AO3)

Title: Detecting Integration of a Lentiviral Vector in Cultured Human Hematopoietic Stem Cells

1. Isolate CD34⁺ HSCs from peripheral blood (ethical approval required).
2. Transduce cells with a SIN‑lentiviral vector carrying a GFP reporter under an EF1α promoter.
3. After 48 h, extract genomic DNA from a sample of the cells.
4. Perform PCR using primers flanking the 5′ LTR–genome junction; run products on a 1 % agarose gel.
5. Analyse GFP expression by flow cytometry to assess functional transduction.
6. Variables to investigate: multiplicity of infection (MOI), presence of polybrene, exposure time.
7. Safety considerations: Biosafety Level‑2 containment, proper disposal of viral waste, use of PPE.
8. Data analysis: calculate transduction efficiency (% GFP⁺ cells) and correlate with PCR band intensity (integration frequency).

9. Summary

Gene therapy offers a route to correct the underlying genetic defect rather than merely treating symptoms. Ex‑vivo strategies, exemplified by X‑linked SCID, modify stem cells outside the body and rely on integrating vectors for durable expression. In‑vivo approaches, such as AAV‑mediated delivery for RPE65‑related blindness, exploit the immune‑privileged eye to achieve long‑term correction without genomic integration. Mastery of recombinant DNA methods, basic molecular‑biology techniques, vector biology, gene‑editing tools and RNA‑based therapies underpins both medical and agricultural biotechnology. While clinical successes are increasing, careful attention to safety, ethical and legal considerations, regulatory pathways and equitable access remains essential for responsible translation of these powerful technologies.