Gene therapy was first introduced by Joshua Lederberg in 1963². Gene therapies frequently work by introducing new copies of an impaired gene or by replacing a non-functional or absent gene in a patient’s cells with a healthy copy of that gene. This concept was first proposed by Theodore Friedmann and Richard Roblin in 1972³.

In 1990, the U.S. FDA approved the first human gene therapy trial². The initial case involved treating adenosine deaminase deficiency-severe combined immunodeficiency (ADA-SCID). This early form of gene therapy employed the gene-addition approach, where a new gene was introduced into cells to either help fight a disease or replace a faulty gene.

The gene therapy process comprises three important facets:

• Silencing of genes using microRNA (miRNA), small interfering RNA (siRNA), and short hairpin RNA (shRNA). RNA interference is a natural cellular process that can be harnessed for therapeutic purposes by degrading mRNA transcripts associated with disease-causing genes.
• Replacement of genes, where the desired genes are administered in the form of viral vectors and plasmids. Viral vectors are predominantly used due to their efficiency in delivering genetic material into human cells.
• Modification of mutation by specific types of nucleases such as transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs) and proteins (Cas) associated with clustered regularly interspaced short palindromic repeats (CRISPR). Among these, CRISPR-Cas9 is particularly noted for its high efficiency and precision in making targeted DNA modifications.

Types of gene therapy

Gene therapy can be classified into two main types based on the target cells: somatic cell gene therapy (SCGT) and germline gene therapy^{2, 4}.

Somatic cell gene therapy

SCGT uses non-reproducing somatic cells that require gene modification to prevent disease. A correct or functional gene is inserted into a non-reproducing somatic cell. The method is used to treat or cure diseases in individual patients without changing the genetic material handed on to descendants. This type of therapy focuses on changing the genes of specific tissues or organs to treat disorders such as sickle cell anemia and cystic fibrosis. SCGT is the benchmark for fundamental gene therapy research. It has diverse applications, including the prevention and management of various genetic diseases and cancers, vaccine development, and wound healing.

SCGT in spinal muscular atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive disorder that causes progressive loss of the alpha motor neurons and motor nuclei, resulting in hypotonia, muscle weakness and atrophy⁵. The most common form of SMA is caused by changes in the survival motor neuron gene 1 (SMN1). Onasemnogene abeparvovec is an SCGT drug that is used to treat SMA by delivering a functional copy of SMN1 directly into the motor neurons via an adeno-associated virus vector⁶. The goal is to replace the missing or faulty gene responsible for the disease. Onasemnogene abeparvovec SCGT has led to significant improvements in survival rates for SMA patients.

Germline gene therapy

Germline gene therapy, or transgenesis, is the process that involves the insertion of modified genes into reproductive cells, such as sperms and eggs, or into pre-embryos, enabling genetic modifications to be passed down to subsequent generations⁴. This approach has the potential to eradicate inherited genetic diseases from a family lineage. However, it is a highly controversial area of research owing to ethical concerns surrounding the possibility of creating genetically modified humans.

Although germline gene therapy has not yet been widely adopted, it holds promise for eliminating genetic disorders such as Huntington’s disease and certain inherited cancers.

Gene therapy methods

Gene therapy involves strategies for modifying genetic material to treat or prevent diseases. These methods range from adding new genes and silencing harmful ones to editing genes for precise modifications.

Gene addition

Gene addition is a popular gene therapy strategy being investigated for single-gene diseases, which are conditions caused by mutations in one or both sets of human genes⁷. This procedure includes inserting functional (or healthy) copies of a gene into a person’s cells using a vector. This vector transports the functional gene to the patient’s cells. Once within the cell, the transported gene (called the transgene) instructs the cell to produce functional proteins. Because there is only one faulty gene to correct, adding a functional gene helps correct the malfunctioning one. For example, hemophilia B is treated by introducing a functional copy of the clotting factor IX gene into liver cells using viral vectors⁸. This approach aims to restore normal blood clotting function.

Gene addition is commonly used to treat hemoglobinopathies, including sickle cell disease and thalassemia. Ongoing research is also exploring its potential for treating lung disorders such as cystic fibrosis.

Gene silencing

Gene silencing, or knockdown, is a technique used to reduce or suppress the expression of specific disease-causing genes at either the transcriptional level or the translational level⁹. Antisense and RNA interference (RNAi) are the most well-known methods for achieving gene knockdown.

RNAi, also known as post-transcriptional gene silencing (PTGS), is a natural cellular process that regulates gene expression by degrading messenger RNA (mRNA) and preventing the production of harmful proteins. This method has significant therapeutic potential, allowing for the targeted suppression of genes responsible for conditions such as cancer, viral infections, and genetic disorders such as Huntington’s disease. In the case of Huntington’s disease, RNAi can target the responsible mutant huntingtin gene by degrading its mRNA transcript, thereby reducing toxic protein production¹⁰.

Gene editing

Gene editing is the process of creating specific breaks in the DNA by using various techniques, either with or without signals to repair them¹¹. The two primary methods of gene editing are disruption/inactivation and correction/insertion; in the former, a gene is turned off, while in the latter, a gene is either repaired or a new sequence is added.

The CRISPR-Cas9 system has emerged as the most widely used tool for gene editing owing to its high efficiency and precision¹². It is a system naturally found in bacteria as a defense mechanism against viruses and has two main components: the Cas enzyme and the guide ribonucleic acid (gRNA). The Cas enzyme, particularly Cas-9, acts as molecular scissors to cut the DNA. The gRNA contains a specific sequence that directs the Cas enzyme to the desired location in the DNA, ensuring precise targeting of the genetic material. Once the DNA is cut, the break can be repaired in different ways, either with or without specific repair instructions. This allows scientists to modify the DNA, correcting or deactivating genes as needed.

The CRISPR-Cas9 system has been used in research to correct genetic mutations associated with diseases such as sickle cell anemia (where hemoglobin subunit beta [HBB] genes are modified), muscular dystrophy, and certain types of cancer. This technology offers promising potential for developing new treatments for a wide range of genetic disorders.

Knockout cell lines are created by inactivating or deleting specific genes in cells. These cell lines are used extensively in research to study the function of genes and their role in disease mechanisms. By knocking out a gene, researchers can observe how its absence affects cellular processes, which helps in understanding disease pathogenesis and identifying potential drug targets.

Knockout cell lines and gene therapy are interconnected tools in biomedical research. Knockout cell lines provide valuable insights into gene function and disease mechanisms, which inform the development of gene therapies aimed at treating various cancers.

Gene replacement

Gene replacement is a technique that involves replacing the faulty gene with a functional copy to recover normal cell function¹³. This method is beneficial for treating genetic abnormalities caused by mutations that induce the absence or dysfunction of a specific protein. One famous example involves the treatment of SCID, also known as “bubble boy” sickness.

Gene therapy approaches

The study of gene therapy has evolved into two distinct strategies: ex vivo and in vivo gene therapy².

Ex vivo gene therapy

Ex vivo gene therapy is the process where genetic modifications of the cells can be achieved outside the body to produce therapeutic factors, which, in turn, are transplanted back into the patients. The genome of cells is modified, and genomic sequencing is employed to ensure that the desired alteration is performed and there are no adverse off-target effects. Finally, the gene-edited cells are reintroduced into the patient with the hope that the transplanted cells will replace the disease-causing cells.

Ex vivo gene therapy techniques are utilized to treat disorders affecting tissues that are easily accessible and transportable into and out of the body, such as the blood and skin. One such example is CAR-T cell therapy for blood cancer. In CAR-T cell therapy, T cells are extracted from a patient’s blood, genetically engineered to recognize cancer cells (eg, by targeting CD19), and then infused back into the patient¹⁴. This approach has shown significant success in treating refractory B cell malignancies.

The absence of an immune response is an obvious advantage of ex vivo gene therapy.

In vivo gene therapy

In vivo gene therapy is a technique that modifies somatic cells by promoting endogenous regeneration through the delivery of therapeutic factors. It is used to treat genetic diseases by adding functional genes or replacing dysfunctional ones. Additionally, it targets cancer with the help of oncolytic vectors, which are genetically engineered viruses that can selectively infect and destroy tumor cells, either directly or indirectly.

In vivo gene therapy is particularly suited for organs that are difficult to access or extract, such as the eye, brain, or liver. It has been successfully utilized in clinical trials to treat conditions such as inherited retinal disorders, hemophilia, and muscular dystrophy. Voretigene neparvovec-rzyl is an in vivo gene therapy approved for treating Leber congenital amaurosis type II, an inherited retinal disorder causing blindness¹⁵. It involves injecting a viral vector carrying a functional copy of the RPE65 gene directly into each retina during surgery.

Delivery methods for gene therapy

One of the key challenges of gene therapy is ensuring the delivery of the precise amount of genetic material to the correct cells in the body¹⁶. This is achieved using a vector, a vehicle designed to transport the gene of interest. The ideal vector should deliver the genetic material accurately to each target cell, enabling effective gene expression without causing harm.

An ideal gene therapy vector must possess a few features:

• Specific targeting: The vector must target the correct cell type, ensuring the gene reaches the intended cells without affecting healthy, non-target cells.
• Capacity for large genes: The vector must be able to carry the gene of interest, which may vary in size. For example, the dystrophin gene used in Duchenne muscular dystrophy therapy is over 2.4 million base pairs long, requiring a vector capable of carrying large genetic payloads.
• Sustained gene expression: The vector must facilitate long-lasting expression of the gene in the target cells to treat or cure the disease effectively.
• Non-immunogenicity: The vector should ideally be non-immunogenic or have minimal immunogenic properties to prevent immune reactions that could compromise therapy and patient safety.

Gene delivery methods can be categorized into two main approaches: viral vectors and non-viral delivery methods². Gene transfer using viral vectors is called transduction, whereas transfer via non-viral vectors is called transfection.

Viral vectors

The viral method is associated with greater technical demands and a higher risk of virus-related harm than non-viral delivery. Viral vectors are generally designed to be replication inadequate to ensure their safety. The ability of viruses to efficiently infect cells and transmit DNA to the host without eliciting an immune response makes them desirable as vectors.

The viral vectors are classified into two types:

• Integrating viral vectors
• Non-integrating viral vectors

Retroviral, lentiviral, and adeno-associated viral vectors can integrate into the human genome. In contrast, non-integrating vectors (eg, adenoviral vector) are maintained in the nucleus but do not integrate into the chromosomal DNA, resulting in transgene loss during cell division and transient expression of the foreign gene.

Adeno-associated virus (AAV) gene therapy

AAV vectors are one of the most widely utilized viral vectors in gene therapy. AAVs have no known pathogenicity, are low in immunogenicity, target non-proliferating cells, and may include discrete genome insertion sites. As a result, AAV can deliver a short length of genetic material (<5 kb) to a patient’s cells without causing harm¹⁷.

AAV vectors are employed in several gene therapies, including Duchenne muscular dystrophy (DMD) gene therapy. In DMD, the faulty dystrophin gene is replaced with a functioning form, which is then delivered to muscle cells via AAV vectors. Another application of AAV vectors is the “suicide” gene therapy for oral cancer cell lines. This approach involves introducing a gene into cancer cells to make them susceptible to a prodrug, which is an inactive precursor compound. The enzyme coded by the transgene converts the prodrug into a cytotoxic compound, selectively killing the cancer cells while sparing normal cells.

Retroviral and adenoviral vectors

Retroviral vectors contain RNA viruses that carry a reverse transcriptase gene, which transcribes viral genetic information into a double-stranded DNA intermediate. This DNA intermediate is subsequently incorporated into the host DNA, allowing the host cell machinery to generate all the required viral components.

Lentiviral vectors, a subtype of retroviral vectors, are widely used for their ability to infect both dividing and non-dividing cells. Lentiviruses are particularly useful for gene therapies targeting blood cells (eg, sickle cell disease, beta-thalassemia), as they can deliver genes to stem cells, which can then differentiate into various cell types.

Adenoviral vectors consist of DNA viruses that infect cells, shed their protein coats, and transport DNA to the nucleus, where it is transcribed. Because this DNA does not integrate into the host genome, its effects are temporary.

Non-viral delivery methods

Non-viral vectors include liposomes and naked DNA that act based on a plasmid (a closed, circular DNA strand). Therapeutic genes can be directly incorporated into the plasmid, and the resulting recombinant plasmid can be injected into cells in a variety of methods. For example, naked DNA can be administered directly into targeted tissues. Other alternatives for non-viral delivery methods include nanoparticle transfer and electroporation².

Nanoparticles are tiny particles that can encapsulate genetic material and be taken up by cells. Nanoparticles can be made from lipids, polymers, or even metals. These particles are particularly useful in gene therapies aimed at tissues that are difficult to target, such as tumors or the liver. A significant advantage of nanoparticles is that they can deliver a wide range of genetic materials, including large genes and CRISPR-based systems.

Electroporation uses electrical pulses to open cell membranes, allowing genetic material to enter the cells. This technique has been used to enhance gene delivery into both mammalian cells and bacteria. It is often used in the laboratory for research purposes and has been explored for genetic modification of muscle cells and cancer therapy.

Ultrasound is usually used to deliver ultrasound energy to an object to increase the delivery of therapeutic genes or drugs. This approach enhances gene delivery by altering vascular permeability via sonoporation. It helps in treating prostate tumors.

Hydrodynamic-based approach achieves efficient gene delivery and expression, for example, by rapidly injecting a large volume of DNA solution through an animal’s tail vein. However, this procedure may be hazardous to the experimental animal.

Gene gun immunization through the skin is a reliable and reproducible method of DNA vaccine delivery. In animal models, this technique can stimulate an immune response to both infectious disease-causing agents and cancer. Furthermore, gene gun immunization is a highly efficient way to achieve antigen presentation.

Non-viral vectors are still in the early stages of development. They are generally less efficient than viral vectors. Nevertheless, they remain a promising area of research in gene therapy due to their notable advantages, particularly their safety of administration and lack of immunogenicity.

Applications of gene therapy

The current applications of gene therapy are provided in the table below.

Additional potential applications

• Cancer research: Over 65% of global clinical trials focus on cancer-related therapies owing to the genetic mutations involved in many cancers. Gene therapy strategies, such as gene editing and immunotherapy, are being developed to target specific cancer-causing genes and improve treatment outcomes.
• Genetic conditions beyond rare diseases: Gene therapy holds great potential for treating a variety of genetic conditions once the challenges of delivery, safety, and efficiency are overcome. Conditions such as sickle cell anemia and beta-thalassemia have already seen promising results with gene editing approaches.
• Neurological disorders: Gene therapy is being explored for treating neurological conditions such as Alzheimer’s disease and Parkinson’s disease, where anomalous genes contribute to disease progression. By delivering therapeutic genes to the brain, researchers aim to slow or reverse damage caused by these conditions.
• Inherited eye diseases: Gene therapy has shown promise in treating inherited eye diseases, such as Leber congenital amaurosis and retinitis pigmentosa, by delivering functional copies of faulty genes directly to retinal cells. This has the potential to restore vision or prevent further deterioration in some cases.

Challenges and limitations of gene therapy

The delivery technique is one of the most challenging aspects of gene therapy¹⁸. Some viral vectors, such as AVV, have a limited capacity to deliver big genes, and non-viral approaches still need to become more efficient. Ensuring that genetic material is given to the appropriate cells without eliciting an immunological response is another key challenge.

Safety concerns

Gene therapy raises concerns regarding the possibility of immunological responses, off-target consequences, and long-term dangers. For example, the CRISPR gene editing technology might cause unexpected modifications to the genome, resulting in dangerous mutations. Furthermore, the use of viral vectors may cause detrimental immunological reactions.

Ethical and regulatory considerations

Ethical debates surrounding gene therapy, particularly germline gene therapy, remain a prominent concern¹⁹. Modifying the human germline raises concerns about ethical implications, such as proposals for the production of designer babies. Therefore, before approving gene treatments for clinical use, regulatory agencies such as the U.S. FDA must weigh the potential advantages against the necessity for stringent safety criteria.

Cost and accessibility

Gene therapy, although promising, is expensive to develop and administer. The complexity of creating customized treatments, the need for specialized infrastructure, and the long development timelines contribute to high costs. This can limit access to treatment, especially in low-resource settings, and raise concerns about the fairness of healthcare distribution. Making gene therapy affordable and accessible to a broader population is a significant challenge.

Technical limitations of gene editing technologies

CRISPR-Cas9 and other gene-editing tools have revolutionized the field; however, these technologies are not without their limitations. Issues such as the precision of edits, difficulty in editing certain genes, and limitations in targeting specific tissues need to be addressed. Additionally, the complexity of human genetics means that a “one-size-fits-all” approach will not be effective for all individuals, and treatments may need to be personalized, which complicates the development process.

Long-term efficacy

One of the key limitations of gene therapy is the uncertainty about its long-term efficacy. While short-term results can be promising, ensuring that the delivered genes remain functional and continue to produce the desired effect over time is a challenge. In some cases, the body may eventually reject the introduced genes, or the initial therapeutic effects may diminish as the body adapts. Continuous monitoring and adjustments to the treatment are necessary to maintain its effectiveness.

Lack of standardization

Gene therapy techniques and protocols are still evolving, and there is no universal standard for the best practices in administering these therapies. Variability in manufacturing processes, delivery mechanisms, and patient populations means that each therapy may require tailored approaches, leading to inconsistencies in treatment outcomes. Standardizing protocols to ensure reproducibility and consistency is an ongoing challenge for the field.

Public perception and acceptance

Public perception of gene therapy remains a significant barrier to its widespread acceptance. Engaging with the public, addressing misconceptions, and ensuring transparency in the research and approval processes is vital for widespread acceptance.

FAQs

What techniques are used in gene therapy?

Gene therapy employs techniques such as using viral vectors to transfer genes, CRISPR-Cas9 for precise gene editing, and non-viral methods such as electroporation or liposomes for gene delivery. It also comprises gene replacement to replace faulty genes, RNA-based medicines to alter gene expression, and stem cell therapy for focused treatment.

What are the potential benefits of gene therapy?

Gene therapy offers promising benefits as an innovative treatment, particularly for conditions with limited or no existing alternatives. Many diseases that can cause disability or death if untreated may be effectively managed with this approach. Research has shown that gene therapy can significantly slow or even stop the progression of several serious illnesses.

Gene therapy makes it possible to develop treatments that can specifically target any of the genes in the body. It can eliminate a person’s ailments permanently. Gene therapy has the potential to improve many people’s lives by contributing to cell lineage.

Why are bioactive proteins important in gene therapy research?

Bioactive proteins are important in gene therapy research because they can directly affect cellular functions and processes, resulting in the desired therapeutic effects. These proteins can be utilized to change gene expressions, repair or replace damaged genes and elicit specific biological responses. By adding bioactive proteins into gene therapy, researchers can improve treatment precision and effectiveness, target specific diseases, and reduce adverse effects. Furthermore, these proteins can function as biomarkers, allowing for more accurate monitoring and evaluation of therapy outcomes.

What role do antibodies play in gene therapy research?

Antibodies play an essential role in gene therapy research as they help target and deliver therapeutic genes to specific cells or tissues. They can be designed to identify specific cell receptors, increasing the precision and efficacy of gene delivery systems. Antibodies are also employed to monitor immunological responses to gene treatments, ensuring that the body does not build an inappropriate immune response. They can also help detect and neutralize possible viral vectors used in gene therapy, increasing safety and efficacy.

How is gene therapy regulated?

Gene therapy is primarily regulated by the U.S. Food and Drug Administration (FDA) and the National Institutes of Health (NIH). The FDA’s Center for Biologics Evaluation and Research (CBER) oversees gene therapy products, requiring an investigational new drug application before clinical trials and a biologics license application for market approval. The NIH’s Recombinant DNA Advisory Committee reviews research protocols to ensure safety standards are met. In Europe, gene therapies are regulated as advanced therapeutic medicinal products by the European Medicines Agency. These regulatory frameworks aim to balance innovation with safety concerns.

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