ONPS2431 — Molecular Biology & Genetics

Gene therapy is the introduction of genetic material into cells to compensate for faulty genes or to treat disease. This can involve adding a functional copy of a gene, silencing a faulty gene, or editing mutations. Option A describes germline therapy (not permitted in humans). Option C is not accurate as gene therapy doesn't involve surgical removal. Option D describes antibiotic treatment, not gene therapy.

The four main approaches to gene therapy are: (1) gene augmentation—adding a functional copy of a gene; (2) gene inhibition—silencing or reducing expression of a faulty gene; (3) gene editing—repairing or replacing the faulty sequence; and (4) killing specific cells—eliminating cells with genetic damage (used in cancer therapy). Protein replacement therapy is a different therapeutic approach, not one of the four primary gene therapy strategies.

Germline gene therapy is currently NOT permitted in humans due to ethical concerns about making heritable changes and not being able to obtain informed consent from future generations. Most human gene therapy is somatic, affecting only the treated patient. Germline therapy is only used experimentally in animals for research purposes.

Ex vivo gene therapy involves extraction of cells from the patient, modification outside the body in a laboratory environment (in vitro conditions), and then reimplantation of the modified cells back into the patient. This approach allows for better control and monitoring of the genetic modification process compared to in vivo delivery.

Retrovirus:
• Genome: Can carry 8kb RNA
• Target: Dividing cells only (require active transcription)
• Integration: Integrates randomly into host DNA
• Side Effects: Site of integration inappropriate, immune response

Adenovirus:
• Genome: 7.5kb dsDNA
• Target: Dividing and non-dividing cells
• Integration: Does not integrate, degrades quickly
• Side Effects: Problematic immune response only

Retroviruses carry RNA genomes that are reverse transcribed to DNA and integrate into the host genome at random sites. They can only infect dividing cells because they need the nuclear envelope to break down during mitosis. Adenoviruses carry double-stranded DNA that enters the nucleus but doesn't integrate—it remains episomal and degrades, requiring repeated doses. Adenoviruses can infect both dividing and non-dividing cells. The main risk with retroviruses is insertional mutagenesis (disruption at the site of integration) plus immune response; adenoviruses mainly trigger immune responses since they don't integrate.

AAV vectors have a small packaging capacity of approximately 4.7 kb of DNA, which limits the size of genes that can be delivered. This is a significant constraint compared to other viral vectors. However, AAV vectors have advantages: they can infect both dividing and non-dividing cells, have low immunogenicity, and can integrate site-specifically into chromosome 19 or remain episomal. The small size allows them to be administered at high doses.

The guide RNA directs Cas9 to the target DNA sequence first. Once at the target site, Cas9 checks for the presence of a PAM sequence immediately adjacent to the target site. If the PAM is present, Cas9 makes the double-strand break. The PAM recognition occurs after guide-directed targeting, not before. This two-step recognition (guide RNA + PAM) ensures high specificity.

CRISPR-induced double-strand breaks are repaired by two main pathways: NHEJ (Non-Homologous End Joining) is error-prone and quick, often introducing small insertions or deletions; HDR (Homology-Directed Repair) is precise but slower, using a homologous DNA template to accurately repair the break. For gene editing, HDR is preferred for precise corrections, while NHEJ can be used to disrupt genes. Options C and D describe other DNA repair pathways not involved in CRISPR repair.

Jesse Gelsinger, an 18-year-old with ornithine transcarbamylase (OTC) deficiency (a urea cycle disorder causing ammonia buildup), died in 1999 from a severe immune reaction to an adenovirus vector. His death was a major setback for gene therapy and highlighted the risks of immune responses to viral vectors. The case led to significant regulatory changes and increased safety protocols in gene therapy research.

Luxturna delivers a functional copy of the RPE65 gene via an AAV vector to photoreceptor cells in the retina. RPE65 encodes a protein essential for the visual cycle. Mutations in RPE65 cause inherited retinal dystrophy leading to progressive blindness. Luxturna is one of the first FDA-approved gene therapies and has shown significant success in improving vision in patients with biallelic mutations in RPE65.

• Gene Augmentation → A (Adding a functional copy of a normal gene)
• Gene Inhibition → B (Silencing or reducing expression of a mutated gene)
• Gene Editing → C (Repairing the DNA sequence of a faulty gene)
• Cell Death (Oncolytic) → D (Eliminating cells with genetic damage)

These four approaches represent the main strategies in gene therapy. Gene augmentation compensates for loss-of-function mutations by adding a normal copy. Gene inhibition reduces harmful effects of dominant mutations or upregulated genes. Gene editing directly fixes the faulty sequence, which is the most precise approach. Oncolytic therapy uses vectors or modified genes to kill cancer cells.

Liposomes are less immunogenic than viral vectors, reducing the risk of harmful immune reactions. They are also relatively simple to manufacture and can be produced at large scales. However, liposomes have lower transfection efficiency than viral vectors, cannot integrate into the genome, and typically carry smaller DNA fragments than some viral vectors. The main advantage is their safety profile and ease of production.

HDR (homology-directed repair) uses a homologous DNA sequence as a template to repair double-strand breaks created by CRISPR. This mechanism allows scientists to introduce specific genetic changes by providing a corrected template. In contrast, NHEJ (non-homologous end joining) simply rejoins the broken ends without a template, often introducing errors.

This is the key distinction between the two delivery approaches. In vivo therapy involves injecting a vector directly into the patient (e.g., Luxturna injected into the eye). Ex vivo therapy removes cells from the patient, modifies them in culture, and returns them (e.g., CAR-T cell therapy removes T cells, modifies them ex vivo, and reinfuses them). Each approach has advantages and disadvantages regarding efficiency, safety, and practicality.

Lentiviral vectors, derived from HIV, have a major advantage over simple retroviruses: they can infect both dividing and non-dividing cells because they can cross the nuclear envelope independently. Traditional retroviruses require the nuclear envelope to break down during mitosis, limiting them to dividing cells. Lentiviruses integrate into the genome (like retroviruses), trigger some immune response, and have moderate packaging capacity (~9 kb).

Gene therapy raises significant ethical questions, including: the line between treating disease and enhancement; potential misuse for non-therapeutic purposes ("designer babies"); equitable access since treatments are expensive; germline modification implications; and unintended off-target effects. These concerns require careful regulation and societal discussion. Gene therapy is not limited to elderly patients and does not prevent all cancers.

• SCID-X1 (severe combined immunodeficiency X-linked) patients had their bone marrow stem cells removed
• A retroviral vector was used to insert a functional copy of the IL2RG gene
• Modified cells were reinfused into patients (ex vivo gene therapy)
• Initially successful: immune function was restored in several patients
• However, retroviral integration caused insertional mutagenesis in some patients
• The vector integrated near the LMO2 proto-oncogene, triggering leukemia development
• This case demonstrated the dangers of insertional mutagenesis and led to regulatory changes and exploration of safer vectors (AAV, lentiviral with safety modifications)

The SCID-X1 trial showed that while gene therapy could be effective, the method of delivery was critical. The risk of random integration into the genome could activate oncogenes or disrupt tumor suppressors, causing secondary malignancies. This led researchers to develop self-inactivating (SIN) viral vectors and to favor integrase-deficient or non-integrating vectors for certain applications.

Zolgensma is a gene therapy that delivers a functional copy of the SMN (survival motor neuron) gene via an AAV vector to treat spinal muscular atrophy (SMA). SMA is caused by mutations in the SMN gene, leading to progressive muscle weakness and paralysis. Zolgensma is administered intravenously as a one-time treatment and has shown remarkable efficacy, particularly in infants and young children, preventing disease progression and promoting motor development.

• Luxturna → AAV (Adeno-Associated Virus)
• Zolgensma → AAV (Adeno-Associated Virus)
• SCID-X1 Trial → Retrovirus

AAV vectors have become the preferred choice for many approved gene therapies due to their low immunogenicity, ability to target non-dividing cells, and safety profile. Both Luxturna and Zolgensma use AAV vectors for in vivo delivery. The early SCID-X1 trial used retroviral vectors for ex vivo gene therapy in hematopoietic stem cells, which led to the insertional mutagenesis complications discussed earlier.

ADA-SCID is caused by mutations in the ADA (adenosine deaminase) gene, which encodes an enzyme critical for purine metabolism. Without functional ADA, toxic metabolites accumulate in lymphocytes, causing their death and leading to severe immunodeficiency. ADA-SCID was one of the first diseases successfully treated with gene therapy (early 1990s), using ex vivo delivery to modify T cells and hematopoietic stem cells. This landmark success helped establish gene therapy as a viable therapeutic approach.