Gene therapy aims to treat or prevent disease by introducing, removing, or modifying genetic material within a patient’s cells. The technology has advanced from theoretical concept to approved treatments for several genetic disorders and cancers.
Gene Addition and Gene Editing
Two broad strategies exist. Gene addition delivers a functional copy of a gene to compensate for a defective one, without modifying the endogenous genome. Gene editing uses engineered nucleases to directly modify the genome, correcting the underlying mutation. Gene addition is simpler and more advanced clinically, but gene editing offers the potential for permanent correction.
Viral Vectors
Viruses are naturally evolved gene delivery vehicles that have been adapted for therapeutic use. Adeno-associated virus vectors are the most commonly used in clinical trials. AAV is non-pathogenic, infects both dividing and non-dividing cells, and persists as an episome. AAV vectors have a limited cargo capacity of about 4.7 kilobases and can trigger immune responses at high doses. Different AAV serotypes have distinct tissue tropisms, allowing targeting of specific organs. AAV serotype 2 has natural tropism for the retina, while AAV9 crosses the blood-brain barrier.
Lentiviral vectors are derived from HIV and integrate their cargo into the host genome, providing long-term expression. These vectors are constructed using DNA ligation and cloning techniques. They can carry larger payloads of up to 8 kilobases and infect both dividing and non-dividing cells. Integration carries a risk of insertional mutagenesis, but self-inactivating vectors have improved safety. Lentiviral vectors are used in CAR-T cell therapy, where patient T cells are engineered ex vivo to express a chimeric antigen receptor targeting cancer cells.
Non-Viral Delivery
Non-viral methods avoid some limitations of viral vectors, particularly immunogenicity and cargo capacity. Lipid nanoparticles encapsulate nucleic acids for delivery, protecting them from degradation and facilitating cellular uptake. LNPs were successfully used in mRNA vaccines and are being developed for gene therapy. Electroporation uses electrical pulses to create transient pores in cell membranes, allowing DNA entry. Physical methods such as hydrodynamic injection, where DNA solution is rapidly injected into the bloodstream, can achieve gene expression in the liver of animal models.
Approved Gene Therapies
Several gene therapies have received regulatory approval. Luxturna treats inherited retinal dystrophy caused by RPE65 mutations, delivering a functional copy of the gene using AAV2. Zolgensma treats spinal muscular atrophy by delivering the SMN1 gene using AAV9. Strimvelis treats adenosine deaminase deficiency by ex vivo transduction of hematopoietic stem cells with a retroviral vector. CAR-T cell therapies including Kymriah and Yescarta are approved for certain B-cell malignancies. Recent approvals include hemophilia B and hemophilia A gene therapies using AAV vectors.
CRISPR-Based Gene Editing
CRISPR-Cas9 has revolutionized gene editing by enabling precise, efficient modification of specific genomic sequences. The system consists of a Cas9 nuclease guided by a single guide RNA that base-pairs with the target DNA sequence. The Cas9 nuclease creates a double-strand break at the target site, which is then repaired by either non-homologous end joining or homology-directed repair.
NHEJ can disrupt a gene by introducing small insertions or deletions, useful for knocking out disease-causing genes. HDR can introduce precise sequence changes when a repair template is provided, allowing correction of mutations. Base editing uses a modified Cas9 fused to a deaminase enzyme to directly convert one base to another without creating a double-strand break. Prime editing uses a Cas9 nickase fused to a reverse transcriptase to write new genetic information at a specific site.
Challenges and Risks
Gene therapy faces several challenges. Immune responses to viral vectors can limit efficacy and cause adverse reactions. The innate immune system recognizes viral capsids and nucleic acids, while adaptive immunity generates neutralizing antibodies that prevent re-administration and may preclude treatment in patients with pre-existing immunity. Off-target editing remains a concern for CRISPR-based approaches, potentially causing unintended mutations. Delivery to target tissues is challenging for many diseases, particularly those affecting the brain, muscle, or lung. Long-term durability of expression and the potential for delayed adverse effects require extended follow-up in clinical trials.
