Gene Therapy Is a Process: Normal Genes Replace Abnormal Genes

Gene therapy addresses genetic disorders by replacing faulty genes with functional ones, offering potential treatments for previously incurable conditions like inherited diseases and certain cancers. Advances in gene delivery and genome editing have made this field increasingly viable for medical applications.

Key Components

Successful gene therapy begins with identifying and selecting the genetic material to replace the defective gene. This requires understanding the target gene’s function, its role in disease, and the specific mutations causing dysfunction. Advances in genomic sequencing, such as whole-exome and whole-genome sequencing, have enabled precise identification of disease-causing mutations. For example, in spinal muscular atrophy (SMA), mutations in the SMN1 gene lead to motor neuron degeneration, and gene therapy aims to restore function by delivering a healthy copy of this gene.

Once the therapeutic gene is identified, it must be packaged into a delivery system. Vectors serve as carriers that protect the genetic material and facilitate its entry into target cells. Viral vectors, such as adeno-associated viruses (AAV) and lentiviruses, are widely used due to their efficiency. AAV-based therapies, like Zolgensma for SMA, have demonstrated long-term efficacy. Nonviral vectors, including lipid nanoparticles and electroporation, offer alternatives that avoid risks like insertional mutagenesis.

Beyond delivery, gene expression must be regulated to ensure appropriate protein production. Promoters and enhancers, which influence gene activity, are carefully selected. In hemophilia gene therapy, liver-specific promoters drive expression of clotting factor genes in hepatocytes. Inducible promoters allow gene expression to be turned on or off in response to specific signals, improving safety and efficacy.

Core Steps In Replacing Abnormal Genes

Replacing defective genes begins with selecting target cells capable of integrating and expressing the introduced gene. For blood disorders like sickle cell disease, hematopoietic stem cells must self-renew and differentiate. In contrast, for conditions affecting non-dividing tissues like retinal cells in Leber congenital amaurosis, the therapeutic gene must persist long enough to provide lasting benefits.

Once the appropriate cells are identified, the therapeutic gene must be introduced in a way that ensures stable and effective expression. This involves designing the genetic construct with regulatory elements like promoters and enhancers to mimic natural gene regulation. For Duchenne muscular dystrophy (DMD), researchers use a shortened but functional version of the dystrophin gene—microdystrophin—to optimize packaging into delivery vectors while retaining therapeutic benefits.

After gene introduction, successful uptake and incorporation into target cells must be ensured. For disorders requiring long-term correction, such as adenosine deaminase severe combined immunodeficiency (ADA-SCID), the therapeutic gene must integrate into the host genome. Lentiviral vectors have been used in clinical trials to achieve stable integration in hematopoietic stem cells, leading to durable immune function restoration. In contrast, for conditions where temporary gene expression suffices, such as certain cancer immunotherapies, episomal vectors enable gene expression without altering the genome, reducing insertional mutagenesis risks.

Viral Versus Nonviral Vectors

Delivering therapeutic genes effectively depends on the vector used. Viral vectors, such as adeno-associated viruses (AAVs), are commonly used for diseases requiring long-term gene expression. AAVs persist in cells without integrating into the genome, making them suitable for conditions like SMA. Lentiviral vectors integrate into the host genome, providing a more permanent solution for disorders like beta-thalassemia, where continuous functional hemoglobin production is needed.

Despite their efficiency, viral vectors present challenges, including production complexity and size limitations. AAVs can only carry genetic payloads up to 4.7 kilobases, restricting their use for larger genes. Researchers have explored dual-vector systems, where a gene is split across two AAVs that recombine inside the cell, though this method introduces inefficiencies.

Nonviral vectors, such as lipid nanoparticles (LNPs) and electroporation techniques, offer alternatives without viral packaging limits. LNPs have gained prominence in RNA-based therapies, as seen in COVID-19 mRNA vaccines. Their ability to encapsulate and protect nucleic acids while facilitating cellular uptake makes them promising for gene therapy. Electroporation, which uses electrical pulses to create temporary pores in cell membranes, enhances direct gene transfer. This method has been successful in CAR-T cell therapies, where genetic modifications enhance immune cells’ tumor-targeting capabilities.

Ex Vivo And In Vivo Approaches

Gene therapy relies on two primary strategies: ex vivo and in vivo approaches. Ex vivo therapy involves extracting a patient’s cells, modifying them in a controlled environment, and reintroducing them. This method is effective for hematopoietic stem cell disorders like sickle cell disease and certain immunodeficiencies. By modifying cells outside the body, researchers can ensure precise genetic alterations and verify successful gene integration. The FDA-approved gene therapy Zynteglo treats beta-thalassemia using this approach, integrating a functional beta-globin gene into hematopoietic stem cells via a lentiviral vector.

In vivo gene therapy delivers genetic material directly into the patient’s body, targeting affected tissues without cell extraction. This approach is essential for disorders where cells cannot be easily harvested and reinfused, such as DMD and certain retinal diseases. AAV vectors are commonly used for in vivo delivery due to their ability to reach post-mitotic cells and sustain gene expression. Luxturna, a gene therapy for inherited blindness caused by RPE65 mutations, exemplifies this strategy by introducing functional genes into retinal cells via subretinal injection.

Mechanisms Of Integration In Host Cells

Once a therapeutic gene is delivered, its integration and expression determine treatment success. Some strategies rely on stable genomic integration, while others utilize episomal expression, allowing gene function without genome alteration. The choice depends on target tissue, duration of expression required, and risks associated with genome insertion.

Lentiviral vectors integrate their genetic payload into the host genome through reverse transcription, ensuring long-term expression but carrying insertional mutagenesis risks. Clinical trials for hematopoietic disorders have demonstrated their effectiveness, with patients achieving sustained therapeutic benefits. In contrast, AAV vectors primarily persist as episomal DNA, avoiding genomic disruption while still providing durable expression in non-dividing cells. This makes AAV-based therapies particularly advantageous for neurological and muscular disorders, where stable but non-integrating gene delivery achieves lasting therapeutic effects without altering the host genome.