Gene Therapy: Types, Strategies, Vectors, and Clinical Applications

Table of Content

• Introduction to Gene Therapy
• Types of Gene Therapy
• Modification of Somatic Cells in Gene Therapy
• Strategies for Somatic Cell Gene Therapy
• Vectors for Somatic Cell Gene Therapy
• Gene Therapy for Inherited Immunodeficiency Syndromes

Introduction to Gene Therapy

• Gene therapy involves the direct genetic modification of a patient’s cells in order to achieve a specific therapeutic goal.
• It is an experimental technique that uses genes to treat or prevent diseases, rather than relying solely on conventional drugs or surgeries.
• By administering DNA instead of a pharmaceutical drug, gene therapy allows targeted intervention at the genetic level, making it a promising approach for many disorders.
• A wide range of diseases are currently being investigated as potential candidates for gene therapy, including:
• Cystic fibrosis
• Cardiovascular diseases
• Acquired Immunodeficiency Syndrome (AIDS)
• Cancer
• Researchers are actively testing several different approaches to gene therapy, which include:
• Replacing a mutated gene that causes disease with a healthy, functional copy of the same gene.
• Inactivating or “knocking out” a mutated gene that is malfunctioning or expressed abnormally, thereby preventing its harmful effects.
• Introducing a new gene into the body to help the cells fight or resist disease, or to provide a missing biological function.

Types of Gene Therapy

Gene therapy is broadly classified into two main types:

1. Germ-line Gene Therapy

2. Somatic Gene Therapy

1. Germ-line Gene Therapy

• Germ-line gene therapy involves the genetic modification of germ cells, such as gametes (sperm or eggs), zygotes, or early-stage embryos.
• The genetic changes introduced through this approach are permanent and are passed on to future generations, affecting not only the treated individual but also their offspring.
• This type of therapy raises significant ethical, social, and moral concerns, which is why it has not been attempted in humans to date.
• Germ-line gene therapy is still considered an emerging and experimental technique and requires extensive refinement and validation before any potential application in humans.
• Compared to somatic gene therapy, germ-line therapy is far more complex and challenging, both technically and ethically.

2. Somatic Gene Therapy

• Somatic gene therapy is based on the principle that a defective or malfunctioning gene in a patient’s somatic (body) cells can be replaced or functionally compensated by introducing a normally functioning gene.
• Since the genetic modification is limited only to somatic cells, the changes are not heritable and do not affect future generations.
• The therapeutic effect of somatic gene therapy is therefore restricted to the individual patient receiving the treatment.
• All currently approved and ongoing gene therapy trials and clinical protocols are focused exclusively on somatic gene therapy.
• As gene therapy continues to show successful outcomes and the associated technologies become more advanced, precise, and safer, there is the possibility that germ-line applications may be explored in the future.
• Such advancements could lead to the concept of “designer babies,” which brings with it serious ethical and societal concerns.

Modification of Somatic Cells in Gene Therapy

Somatic cells can be genetically modified using several different therapeutic strategies, depending on the nature of the disease and the intended outcome.

The major approaches include:

1. Gene Augmentation (or Gene Addition)

2. Elimination of Pathogenic Mutations

3. Targeted Inhibition of Gene Expression

4. Targeted Killing of Specific Cells

1. Gene Augmentation (or Gene Addition)

• The primary objective of gene augmentation therapy is to introduce a functional copy of a gene into the patient’s cells.
• This functional gene supplements or compensates for a defective or non-functional gene already present in the genome.
• Gene augmentation is mainly used to treat diseases caused by loss of gene function, where the original gene product is absent or insufficient.
• Instead of repairing the defective gene, a normal copy is added to restore the required biological function.
• Cystic fibrosis is a classic and well-known candidate disease for gene augmentation therapy.

2. Elimination of Pathogenic Mutations

• This approach aims to restore the normal function of a mutated gene rather than adding an extra copy.
• The defective gene is repaired directly at the DNA level by replacing the DNA sequence that contains the pathogenic mutation with a normal, healthy equivalent sequence.
• An alternative strategy involves modifying gene splicing, where an exon containing the harmful mutation is skipped during mRNA processing.
• This exon skipping allows the production of a shortened but functional protein, thereby reducing or eliminating the harmful effects of the mutation.

3. Targeted Inhibition of Gene Expression

• This method is used to silence or suppress the expression of specific genes that contribute to disease.
• It is particularly important in cancer therapy, where it is used to inactivate activated oncogenes that drive uncontrolled cell division.
• Another major application is in autoimmune diseases, where gene inhibition is used to reduce unwanted or overactive immune responses.
• In inherited genetic disorders, this strategy can be used to silence a mutant allele while allowing the normal allele to function properly.

4. Targeted Killing of Specific Cells

• This strategy is especially useful in the treatment of cancer, where selective elimination of diseased cells is required.
• It can involve the direct killing of target cells through the introduction of genes that encode toxic proteins.
• Another important variation of this approach is immunotherapy, where gene transfection is used to stimulate a very strong immune response against cancer cells.
• This strategy works because the immune system is naturally designed to recognize non-self antigens.
• Although cancer cells share many antigens with normal body cells, they may:
• Express tumor-specific antigens, or
• Overexpress normal antigens, making them recognizable to the immune system.
• These antigen differences can provoke a weak natural immune response, which is normally insufficient to eliminate the cancer.
• Immunotherapy amplifies this weak immune response, enabling the immune system to more effectively recognize and destroy cancer cells.

Strategies for Somatic Cell Gene Therapy

Somatic cell gene therapy can be carried out using two main strategies:

1. Ex vivo Gene Therapy

2. In vivo Gene Therapy

1. Ex vivo Gene Therapy

• In ex vivo gene therapy, target cells are first removed from the patient and grown in culture outside the body.
• These cells are then genetically modified by introducing the desired nucleic acid or oligonucleotide into them.
• After modification, selected cells are amplified in culture to obtain sufficient numbers and then reintroduced into the patient.
• Since the genetic modification occurs outside the patient’s body, this approach is referred to as ex vivo gene therapy.
• This strategy is most suitable for disorders in which the target cells are:
• Removable from the patient
• Returnable after modification
• Long-lived and hardy, able to survive for extended periods
• Capable of engrafting successfully after replacement
• Common examples of cells used in ex vivo gene therapy include:
• Hematopoietic stem cells (blood-forming cells)
• Skin cells

2. In vivo Gene Therapy

• In vivo gene therapy involves direct delivery of the therapeutic gene into the patient, where the gene transfer occurs within the body.
• This strategy is the only option for disorders where:
• Target cells cannot be cultured in sufficient numbers in vitro (e.g., neurons in the brain)
• Cultured cells cannot be efficiently re-implanted into the patient
• Tissue targeting is a critical consideration to ensure the gene reaches the intended cells.
• Gene delivery can be performed either by:
• Direct injection of the gene construct into the target tissue, or
• Systemic administration, where the gene is delivered through circulation to reach the target tissue (e.g., portal vein injection for liver targeting)
• Some viral vectors naturally infect specific cell types, providing inherent tissue specificity.
• The success of in vivo gene therapy depends heavily on the efficiency of gene transfer and expression in the correct tissue.

Vectors for Somatic Cell Gene Therapy

In gene therapy, cloned genes, RNA, or oligonucleotides are delivered into a patient’s cells using vectors. Vectors can be broadly classified into:

1. Viral Vectors

2. Non-Viral Vectors

1. Viral Vectors

• Viral vectors are the most commonly used delivery method in gene therapy due to their high efficiency in infecting cells and delivering genetic material.
• Transduction refers to the process of gene transfer into cells using viruses.
• Some viral vectors can integrate into the host genome, allowing long-term expression of the therapeutic gene.
• Viruses vary in their tropism:
• Broad tropism: infect a wide range of cell types
• Narrow tropism: infect specific cells expressing certain receptors
• Large-genome viruses can accommodate larger DNA inserts, while non-essential viral genes are removed to make room for therapeutic genes.
• Viral vectors are typically replication-defective for safety, though replication-competent viruses like oncolytic viruses are sometimes used for cancer therapy.
• Common viral vectors include:

1. Retroviruses
2. Lentiviruses
3. Adenoviruses
4. Adeno-associated viruses (AAV)
5. Herpes simplex viruses (HSV)

1. Retroviral Vectors

• Account for ~34% of current gene therapy trials.
• Integrate stably into host chromosomes.
• Limitations: small insert size; cannot accommodate all regulatory elements of a gene.
• Integration is not completely random, with a preference for inserting into genes.
• Infect actively dividing cells only.

2 Lentiviral Vectors

• A subclass of retroviruses that can infect both dividing and non-dividing cells.
• Most are based on HIV, with unnecessary genes removed for safety.
• Exhibit a safer integration profile compared to standard retroviruses.

3. Adenovirus Vectors

• Large non-enveloped, double-stranded DNA viruses that infect vertebrates.
• Used in ~27% of gene therapy trials.
• Advantages: can infect a wide range of dividing and non-dividing cells; large capacity for foreign genes.
• Disadvantages: DNA does not integrate, so repeated administration may be required; repeated exposure may trigger fatal immune responses.
• Known for causing the first fatality in a gene therapy trial.

4. Adeno-Associated Virus (AAV) Vectors

• Nonpathogenic, single-stranded DNA viruses.
• Safe: do not cause disease in humans and remain inactive without a helper virus.
• Rep protein mediates integration into a specific site on chromosome 19.
• Most rAAV vectors lack rep for safety, functioning mainly episomally; occasional integration into host genome occurs.

5. Herpes Simplex Virus (HSV) Vectors

• Complex viruses with ≥80 genes; tropic for CNS neurons.
• Can establish lifelong latent, non-integrated infections in sensory ganglia.
• Useful for delivering genes to neurons in Parkinson’s disease or CNS tumors.

2. Non-Viral Vectors

• Non-viral gene delivery, known as transfection, transfers DNA, RNA, or oligonucleotides without viral involvement.
• Advantages: high safety, no integration into host genome.
• Disadvantages: low transfection efficiency, poor gene transfer, and low expression levels.

Challenges & Solutions:

• Plasmid DNA transport to the nucleus of non-dividing cells is inefficient.
• Strategies to improve nuclear entry:
• Conjugation with DNA- or protein-sequences that facilitate nuclear import.
• Compacting DNA to small sizes for passage through nuclear pores.
• Stable integration is usually not achieved, but tissues like muscle, which are non-dividing, can sustain expression for months.

1. Liposome Vectors

• DNA is encapsulated in lipid spheres (liposomes).
• Advantages: nontoxic, non-immunogenic, easy to prepare, and no DNA size limit.
• Disadvantages: low gene transfer efficiency, transient expression.
• Some liposomes are designed to target specific cells using antibodies, peptides, or carbohydrate ligands.
• DNA often ends up in endosomes rather than the nucleus; only some reaches the nucleus without integrating.

2. Microinjection and Particle Bombardment

• Microinjection: naked DNA is injected directly into target tissues, e.g., muscle.
• Particle bombardment (gene gun): DNA-coated metal particles are shot into cells.
• Advantages: safe and simple, successful gene transfer to multiple tissues.
• Disadvantages: low transfer rates and weak transgene expression.

Gene Therapy for Inherited Immunodeficiency Syndromes

• The first clinical trial for an inherited disorder began in September 1990.
• The initial protocol aimed to treat a 4-year-old girl with adenosine deaminase (ADA) deficient severe combined immunodeficiency (SCID).
• Gene therapy is also being investigated for other inherited diseases, including:
• SCID-X1
• Cystic fibrosis
• HIV

1. ADA-SCID

• ADA (Adenosine Deaminase) is essential for metabolism of adenine and guanine.
• Lack of ADA destroys T lymphocytes, leading to recurrent and persistent infections.
• Traditional treatments: bone marrow transplant and weekly ADA enzyme injections.
• Gene therapy was explored because:
• Transplants often fail
• Enzyme therapy is ineffective in many patients
• Clinical trials timeline:
• 1990: first trial started
• 1991: second trial initiated
• 1996: additional 11 children enrolled after no side effects observed
• Ex vivo approach used:
• ADA cDNA introduced into T lymphocytes via retroviral-mediated gene transfer
• Gene-corrected T cells infused back into patients
• Outcomes:
• At age 17, the first girl led a normal lifestyle with a strong immune system
• She experienced average infections, no side effects, and an increased number of T cells
• T cells do not have the longevity of stem cells, but ADA gene expression persisted for ≥13 years
• She still received ADA injections but showed marked improvement compared to enzyme therapy alone

2. SCID-X1

• SCID-X1 is an X chromosome-linked form of severe combined immunodeficiency.
• Caused by mutations in the γc-cytokine receptor gene, which responds to interleukins.
• Leads to T lymphocyte and natural killer (NK) cell deficiencies due to a block in differentiation.
• April 2000 trial in France:
• Two infants (8 and 11 months old) were treated
• Both were very ill with pneumonia, diarrhea, and skin lesions
• Bone marrow hematopoietic stem cells were treated ex vivo with retroviral γc-receptor cDNA vector
• Gene-corrected cells transfused back into patients
• Initial results were encouraging; clinical trials expanded.
• Complications:
• Both infants later developed leukemia
• Cause: retroviral vector insertion near LMO2 proto-oncogene
• LMO2 encodes a transcription factor needed for hematopoiesis; overexpression promotes leukemia

3. Cystic Fibrosis Gene Therapy

• Cystic fibrosis is a recessive genetic disease.
• Symptoms include:
• Excessive salt loss in sweat
• Thick, sticky mucus in airways
• Recurrent lung infections and irreversible lung damage
• Obstruction of pancreatic ducts
• Caused by mutations in CFTR (cystic fibrosis transmembrane conductance regulator) gene:
• CFTR protein is a cAMP-regulated chloride channel, keeping airways mucus-free
• ~70% of cases involve deletion of 3 base pairs, causing loss of phenylalanine at position 508
• Initial approaches:
• Adenovirus and liposome vectors delivering CFTR cDNA via nasal spray
• Results were disappointing due to immune response to adenovirus
• Current approaches:
• Adeno-associated virus (AAV), especially rAAV2, used for gene delivery
• rAAV2-CFTR therapy showed:
• Improved lung function for 30 days post-treatment
• Excellent safety profile in Phase II trials
• CFTR mRNA expression and cAMP-activated chloride channel function in nasal cells

4. HIV-1 Gene Therapy

• HIV-1 causes Acquired Immunodeficiency Syndrome (AIDS).
• Challenges:
• No completely effective drug treatments
• High viral genetic diversity complicates vaccine development
• Rapid mutation generates billions of unique virions daily in an infected person
• Gene therapy strategies:
• Protein-based approaches: toxins, antibodies
• RNA-based approaches: antisense, ribozymes, RNA aptamers, RNA interference
• Anti-HIV-1 Ribozymes:
• Target specific HIV-1 genes
• Strategy: combine ribozymes with different specificities to overcome viral variability
• Catalytic antisense RNAs:
• Target the LTR region, essential for viral replication
• Contain:
• Stem-loop antisense motif (binds RNA substrate)
• Hairpin/hammerhead motif (catalyzes cleavage)
• Achieve up to 90% inhibition of HIV-1 replication in eukaryotic cells (p24 reduction)
• Despite promising results, clinical application is still under development

5. Gene Therapy for Tumors

• Brain tumors are major targets for novel gene transfer.
• Basis: malignant tumor cells divide rapidly, unlike mostly quiescent mature brain cells, allowing selective targeting.
• Current therapeutic strategies include:
• Direct killing of tumor cells
• Expression of new tumor antigens on cancer cells to induce immune rejection
• Transfer of drug sensitivity genes to tumor cells to enhance chemotherapy efficacy