Gene Therapy and Pharmacogenomics

Spec Mapping — OCR H420 Module 6.1.3 — Manipulating genomes, content statements covering somatic and germline gene therapy, the use of viral and non-viral vectors for therapeutic gene delivery, named examples of gene therapy in clinical use, pharmacogenomics and personalised medicine, and the ethical implications of CRISPR-Cas9 gene editing in humans (refer to the official OCR H420 specification document for exact wording). This lesson closes Module 6.1.3 and synthesises the molecular tools (cloning, vectors, CRISPR) into their highest-stakes application — direct intervention in human genomes.

Gene therapy is the treatment of disease by introducing functional genes into a patient's cells. Pharmacogenomics is the use of an individual's genetic information to tailor drug treatment. Both are central to 21st-century personalised medicine. OCR A-Level Biology A specification 6.1.3 requires you to distinguish somatic from germline therapy, describe the vectors used, and give named examples of diseases treated.

This lesson also introduces the most consequential genome-editing technology to date: CRISPR-Cas9, the programmable nuclease system discovered by Jennifer Doudna at Berkeley and Emmanuelle Charpentier at Umeå and Berlin. Their 2012 paper showed that the bacterial CRISPR-Cas9 immune system could be reprogrammed with a synthetic single guide RNA to cut any chosen DNA target — a technical breakthrough that earned them the 2020 Nobel Prize in Chemistry (the first awarded to two women jointly). Paraphrased: their insight was that the natural Cas9 nuclease, normally guided by paired tracrRNA and crRNA molecules to invading phage DNA, could be redirected to any genomic sequence simply by changing the guide RNA's 20-nucleotide targeting region. CRISPR was not the first genome editor (zinc-finger nucleases and TALENs preceded it) but it was the first that any reasonably equipped lab could deploy within days at a reagent cost of a few pounds, transforming gene therapy from an esoteric speciality to a routine technique within a decade.

Key Definitions:

• Gene therapy — the introduction of a functional allele into cells to correct a genetic disorder.
• Somatic cell gene therapy — genetic modification of body cells; changes are not passed to offspring.
• Germline gene therapy — genetic modification of gametes or embryos; changes are heritable.
• Pharmacogenomics — the study of how genetic variation affects response to drugs.
• Personalised medicine — tailoring medical treatment to an individual's genotype.

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Why Gene Therapy?

Genetic disorders caused by a single faulty gene (monogenic diseases) are in principle curable by replacing the faulty allele with a functional one. Over 10,000 monogenic disorders are known, from cystic fibrosis (CF) and sickle cell anaemia to the lysosomal storage diseases and severe combined immunodeficiency (SCID). Many are recessive, so a single functional copy is enough to produce normal phenotype — ideal targets for gene therapy.

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Somatic vs Germline Gene Therapy

Somatic Cell Gene Therapy

Only body (somatic) cells are modified, so the change affects the individual but is not passed to offspring. All gene therapy trials in humans to date are somatic. The modified cells may be:

• Removed, modified, and returned (ex vivo) — e.g. bone marrow stem cells for SCID.
• Treated inside the body (in vivo) — e.g. delivering a vector directly into the lungs of a CF patient.

Somatic therapy typically needs repeated treatment because modified cells are eventually replaced by the body.

Germline Gene Therapy

Modification of gametes, fertilised eggs or early embryos means every cell of the resulting individual — and all their descendants — carries the change. This is currently illegal for humans in the UK, EU and most other countries because of ethical concerns: unknown long-term effects, the inability of unborn people to consent, and fears of "designer babies". The only reported case (He Jiankui, China, 2018) who edited two embryos to resist HIV sparked international condemnation and led to his imprisonment.

Feature	Somatic	Germline
Cells treated	Body cells	Gametes or embryos
Heritable	No	Yes
Consent from future generations	Not needed	Impossible
Currently permitted in humans	Yes (clinical trials)	No (banned)
Permanence	Lost as cells die	Permanent in lineage

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Vectors for Gene Therapy

Getting a gene into a patient's cells requires a vector. The ideal vector is safe, specific, efficient and persistent. Different vectors suit different tissues.

Viral Vectors

Viruses are natural gene delivery machines. Their virulence genes are removed and replaced with the therapeutic gene.

• Retroviruses — integrate into the host genome. Used for SCID therapy but caused leukaemia in some early trials by disrupting oncogenes.
• Lentiviruses (e.g. HIV-derived) — integrate into non-dividing cells; safer than other retroviruses.
• Adenoviruses — do not integrate, so expression is transient. Used for CF and some cancers.
• Adeno-associated viruses (AAV) — small, low immunogenicity, can give long-lasting expression. Used in the first FDA-approved gene therapy, Luxturna (RPE65 for inherited blindness, 2017).

Non-Viral Vectors

• Liposomes — lipid vesicles that fuse with target cell membranes. Used in aerosol form for CF lungs.
• Lipid nanoparticles (LNPs) — refined liposomal formulations that protect mRNA payloads and target hepatocytes; the delivery platform behind the Pfizer-BioNTech and Moderna COVID-19 mRNA vaccines and behind RNAi drugs such as patisiran.
• Naked DNA injected directly into tissue — inefficient but safe; used in some cancer DNA vaccines.
• Electroporation — electrical pulses open transient pores; routinely used ex vivo to transfect T cells for CAR-T manufacturing.
• Nanoparticles — gold, silica or polymer-based; increasingly used in research and clinical settings.

flowchart TD
    A[Therapeutic gene or gRNA/Cas9 cargo] --> B{Vector choice}
    B -->|Viral| C[AAV: low immunogenicity, episomal, long expression]
    B -->|Viral| D[Lentivirus: integrates, non-dividing cells, ex vivo CAR-T]
    B -->|Viral| E[Adenovirus: transient, high capacity, immunogenic]
    B -->|Non-viral| F[Lipid nanoparticle: mRNA, hepatocyte tropic]
    B -->|Non-viral| G[Liposome: aerosolised, CF lungs]
    B -->|Non-viral| H[Electroporation: ex vivo T cells]
    C --> I[Patient cell]
    D --> I
    E --> I
    F --> I
    G --> I
    H --> I

Vector	Genome capacity	Integration	Persistence	Best use
AAV	~4.7 kb	No (episomal)	Years (post-mitotic cells)	Luxturna, Zolgensma, haemophilia
Lentivirus	~8 kb	Yes	Permanent	CAR-T, Strimvelis SCID, sickle-cell ex vivo
Adenovirus	~36 kb	No	Weeks	CF aerosol, oncolytic, COVID vaccines
Retrovirus	~8 kb	Yes (random)	Permanent (but oncogene risk)	Older SCID trials, now largely replaced
Lipid NP	~unlimited mRNA	No	Days	mRNA vaccines, RNAi drugs

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CRISPR-Cas9 Gene Editing

Until ~2012, gene therapy meant gene addition: a working copy of a faulty gene was added to a cell via a vector, leaving the defective endogenous copy in place. Gene editing is fundamentally different — it modifies the patient's own genome at a precise location.

Mechanism

CRISPR-Cas9 has two components:

1. Guide RNA (gRNA) — a synthetic ~100-nucleotide RNA whose first 20 nucleotides ("spacer") are complementary to the target DNA sequence.
2. Cas9 endonuclease — a large protein from Streptococcus pyogenes that uses two nuclease domains (RuvC and HNH) to cleave double-stranded DNA.

The gRNA loads onto Cas9, the complex scans the genome for the gRNA's target sequence, and binding occurs only if the target is immediately followed by a protospacer adjacent motif (PAM) — the sequence 5'-NGG-3' for S. pyogenes Cas9. The PAM is essential: it is what distinguishes the Cas9-cleavable target from the CRISPR-array sequence in the bacterial genome itself. On binding, Cas9 makes a blunt double-strand break approximately 3 bp upstream of the PAM.

SVG — guide RNA + Cas9 at target

Cellular repair: NHEJ vs HDR

The cell repairs the double-strand break by one of two pathways:

• Non-homologous end joining (NHEJ) — fast, error-prone. The cell simply re-ligates the broken ends, often introducing small insertions or deletions (indels) at the junction. NHEJ is the default repair pathway in non-dividing cells. Used for gene knockout: a few inserted or deleted bases at an exon shift the reading frame and abolish protein function.
• Homology-directed repair (HDR) — slow, precise. Active only in S/G2 phases when a sister chromatid is available, HDR uses a homologous template to repair the break. By providing a synthetic donor template with the desired sequence change flanked by homology arms, the experimenter can introduce a precise edit (point mutation correction, knock-in of a small tag). HDR is much less efficient than NHEJ in mammalian cells, which is a major engineering challenge.

flowchart TD
    A[Cas9 + gRNA] --> B[Target DNA + PAM]
    B --> C[Blunt double-strand break ~3 bp from PAM]
    C --> D{Cell repair pathway}
    D -->|Default| E[NHEJ — error-prone re-ligation]
    E --> F[Indels disrupt reading frame → knockout]
    D -->|If donor template supplied + S/G2 phase| G[HDR — homology-directed repair]
    G --> H[Precise sequence edit / knock-in]

CRISPR vs traditional cloning

Feature	Traditional recombinant DNA	CRISPR-Cas9 editing
Target choice	Limited to restriction-enzyme sites	Any 20-nt sequence next to NGG PAM
Edits the host genome?	No — adds an extra copy via vector	Yes — modifies the patient's own DNA
Precision	Whole-gene insertion at random locus (viral) or extrachromosomal (plasmid)	Single-base precision possible (HDR)
Cost per experiment	High (custom restriction-enzyme strategy)	Low (synthesise new gRNA for ~£10)
Time to design + test	Weeks to months	Days
Repair pathway controlled by?	Researcher (ligation in vitro)	Cell's endogenous DSB repair (NHEJ default)

flowchart TD
    A[Design experiment: knockout gene X] --> B[Pick target sequence ending in NGG PAM]
    B --> C[Order 20-nt synthetic gRNA from supplier]
    C --> D[Combine with Cas9 RNP or transfect Cas9 + gRNA plasmid]
    D --> E[Deliver to cells: electroporation / LNP / virus]
    E --> F[Cas9 cuts; NHEJ introduces indels]
    F --> G[Sequence to confirm; clone single-cell knockouts]

CRISPR ethics — somatic vs germline

CRISPR can in principle be applied to somatic cells (single individual, like classical gene therapy) or to germline cells (heritable, all descendants affected). The 2018 He Jiankui CCR5 case is the historical inflection point: a researcher in China reported in 2018 the first reported germline-edited human births, having edited the CCR5 gene in embryos in an attempt to confer HIV resistance. The work was broadly condemned by the international scientific community for ethical violations of research protocols, regulatory frameworks, informed-consent practices, and the principle of clinical equipoise. He was subsequently imprisoned by Chinese authorities. The case is taught not because germline editing is intrinsically beyond debate, but because the procedural failures (consent, oversight, safety review, justification) were unambiguous.

The mainstream scientific consensus, articulated in the 2017 and 2020 NASEM reports and the 2019 WHO advisory committee, is that heritable germline genome editing in humans is not currently safe or appropriate, while accepting that the question of whether and when it might be is a legitimate subject for ongoing societal deliberation. OCR examiners want you to navigate these distinctions with academic care.

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Named Examples of Gene Therapy

Severe Combined Immunodeficiency (SCID)

Children born with SCID ("bubble boy disease") lack functioning T and B lymphocytes and die young without treatment. A common form is ADA-SCID, caused by mutation of the adenosine deaminase gene.

• In 1990 Ashanti DeSilva became the first human gene therapy patient, receiving her own T cells modified ex vivo with a retrovirus carrying the ADA gene.
• Later trials (France, UK) treated X-linked SCID with retroviruses but caused leukaemia in 5 of 20 children when the vector disrupted the LMO2 oncogene. This led to a pause in retroviral trials.
• Modern self-inactivating lentiviral vectors have largely overcome this problem, and gene therapy for SCID is now approved in the EU (Strimvelis, 2016).

Cystic Fibrosis (CF)

CF is caused by mutation of the CFTR gene, producing a faulty chloride channel. Thick mucus builds up in airways, causing recurrent infections. Gene therapy for CF has been pursued since the gene was identified in 1989, using adenoviruses and liposomes to deliver functional CFTR to airway epithelial cells by aerosol.

Results have been disappointing — gene expression is transient and the mucus layer blocks access to the cells. In 2019 the UK CF Gene Therapy Consortium reported modest improvements in lung function. Today, most CF patients benefit more from Kaftrio (a small-molecule CFTR modulator) than from gene therapy, but the two approaches are complementary.

Spinal Muscular Atrophy (SMA)