Gene therapy is the process of introducing new genetic material into cells to compensate for abnormal genes or to repair damaged genes. This process has the potential to cure many monogenic diseasescurrently considered incurable, such as cancer, Huntington’s disease, HIV/AIDS, and cystic fibrosis.

How Gene Therapy Works

In order to modify or repair a mutated gene, during gene therapy, genetic material is delivered to cell nuclei via a viral vector such as recombinant Adeno-associated virus (AAV), which carries a therapeutic gene into targeted cells, or by direct injection of the therapeutic gene into the target cells.

Non-integrated AAV therapeutic DNA remain as extrachromosomal units, providing the benefit of recombinant protein expression without causing potential oncogenic mutations due to integration.

Once the therapeutic gene reaches the target cells, transcription and translation processes produce the desired protein, followed by posttranslational modifications. The recombinant protein then performs its normal function within the cell, correcting the genetic mutation that caused the disease.

Somatic and Germline Gene Therapy

Somatic gene therapy is the therapeutic delivery of nucleic acids into the cells of a patient's body to treat disease without germline transfer to future generations. The extrachromosomal location of recombinant genes in somatic cells is generally favored for therapeutic applications without risking mutations due to vector insertions. Germline gene therapy requires the recombinant therapeutic gene to be stably integrated into the host genome in cells destined for germline development.

Gene Therapy Vectors

There are many different types of vectors, including viruses, and nucleic acid carriers such as liposomes, and nanoparticles. Each type of vector and carrier has its own advantages and disadvantages.

• Adeno-associated virus (AAV) is the most used vector for gene therapy, especially since it lacks viral genes and is efficient with recombinant gene insertions. Vector construction using AAV involves splicing a new gene, including specific regulatory sequences, into restriction enzyme sites on an AAV vector backbone. An endosome coats the newly created AAV-new gene construct, which helps deliver the genetic payload through the cell’s outer surface membranes and into the cell nucleus for transcription and translation of recombinant protein.
• Lentiviral vectors are also useful for gene therapy, although integration into host genomes limits applications with recombinant therapeutic proteins. These viral vectors can infect both dividing and non-dividing cells and target specific cell types.
• Retroviruses include RNA viruses that insert a DNA copy into a host’s genome by utilizing viral-encoded reverse transcriptase. Retroviral vectors achieve high titers with recombinant insertions, can transfect a wide variety of host cells, and can result in stable insertions of novel gene constructs for gene therapy applications.

In addition to vectors that contain recombinant nucleic acid sequences, carriers are useful for delivering genetic payloads to the nuclei of host cells. These non-viral approaches have several advantages over viral delivery, including the lack of immunogenicity, the ability to target specific cell types, and greater safety. However, non-viral methods are often less efficient than viral delivery and can be more difficult to scale up for large-scale clinical use.

• Liposomes are small lipid (fat) molecules that can carry DNA or RNA into cells. They are less likely to provoke an immune response than viruses but are not as efficient at delivering genetic material to cells.
• Nanoparticles are very small particles that can carry DNA or RNA into cells. Similarly, nanoparticles do not elicit an immune response in comparison to viruses, and they have the advantage of targeting specific cell types. However, nanoparticles are not as efficient as AAV vectors.

Ex vivo and in vivo gene therapy

Ex vivo gene therapy is a type of gene therapy in which cells are removed from the body, genetically modified, and then returned to the patient. CAR-T (chimeric antigen receptor T cell therapy) is an example of ex vivo gene therapy combined with cell therapy. T-cells are genetically modified and are then reintroduced to a patient. FDA approvals include CAR-T applications for leukemia and lymphoma. This approach is often used when the targeted cells are not readily accessible or when a large number of cells need to be modified. Ex vivo gene therapy has been used to treat other diseases, including genetic disorders and viral infections.

In vivo gene therapy delivers therapeutic genes directly into the cells of a patient's body. This is typically done using a viral vector. The vector is usually injected into the patient's bloodstream via intravenous infusion (IV). However, success with in vivo gene therapy has also been achieved with injections to the heart, eye, and other tissues.

How Gene Therapy Can Cure or Treat Diseases

There are a number of different ways that gene therapy can be used for therapeutic applications. Most involve introducing a recombinant gene using a viral vector such as AAV and new gene-editing techniques that offer site-specific gene alterations.

For example, gene therapy can be used to:

• Replace a defective gene with a healthy copy
• Introduce a new genetic element (DNA or RNA) into cells to help fight infection or disease
• Inactivate a harmful gene
• Regulate the activity of genes
• Perform site-specific gene repair

By adding a fully functional recombinant gene, transcription and translation of a recombinant protein can correct a specific disease-causing genetic defect. For example,

• Pyruvate kinase (PK) deficiency is an inherited disorder that results in the inability to produce enough of the enzyme pyruvate kinase. PK deficiency can cause hemolytic anemia, a condition in which red blood cells are destroyed faster than they can be produced. Gene therapy is a promising treatment option for PK deficiency, where a normal copy of the gene that encodes for pyruvate kinase is inserted into cells using a viral vector. This allows the cells to produce the enzyme in enough quantity to function properly.
• mRNA constructed to elicit an immune response to SARS-CoV-2 spike proteins has effectively been used for vaccination against Covid-19.
• Downregulation of harmful gene activity can be accomplished by using anti-sense constructs and has applications with certain cancers where curtailing oncogenic activity is advantageous.
• CRISPR-Cas9 is a powerful tool for genome editing that can be used to make precise changes to specific DNA targets. AAV vectors incorporating CRISPR-Cas9 have been used as delivery vehicles intended for in situ gene modifications, which have been effective with various gene therapy applications. Gene editing tools like CRISPR are well suited for monogenic diseases where a single base change can result in a cure. Clinical trials with CRISPR-Cas9 have resulted in positive outcomes for sickle-cell disease and beta-thalassemia.

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