Gene Theraphy | Zoology Optional Notes for UPSC PDF Download

Introduction

Gene therapy is a groundbreaking treatment method diverging from conventional drug compounds. Instead, it utilizes genes or short oligonucleotide sequences as therapeutic agents. This innovative technique targets defective genes contributing to disease development. The core principle involves introducing one or more foreign genes into an organism, aiming to address hereditary or acquired genetic defects. This is achieved by packaging DNA encoding a therapeutic protein within a "vector," facilitating the transportation of DNA into cells. The expression of the inserted DNA by cell machinery treats the disease with minimal toxicity. The first FDA-approved gene therapy experiment occurred in 1990 on ADA-SCID, following the treatment of Ashanti DeSilva. Subsequently, around 1700 clinical trials employing various techniques and genes for numerous diseases have been conducted. Although successful trials have been reported for diseases like ADA-SCID, X-linked SCID, Leber's congenital amaurosis, Parkinson's disease, multiple myeloma, chronic and acute lymphocytic leukemia, and adrenoleukodystrophy, FDA approval is pending. Ongoing research explores gene therapy for diseases like Haemophilia, Tyrosinemia, Hyperbilirubinemia (Criglar-Nijjar Syndrome), Cystic Fibrosis, and various cancers. Despite three decades of research, only one product, Glybera, received FDA approval in November 2012 and might be available in late 2013. Glybera has the potential to cure lipoprotein lipase deficiency (LPLD), a rare disease.

Types of Gene Therapy

Gene therapy encompasses several approaches for rectifying faulty genes, with the most common involving the insertion of a normal gene into a specific genome location. It is classified into two types:

Somatic Gene Therapy

Somatic gene therapy targets the somatic cells of a patient for foreign gene transfer. The effects of the foreign gene are limited to the individual patient, not inherited by offspring or later generations.

Germ Line Gene Therapy

In germ line gene therapy, functional genes to be integrated into genomes are inserted into germ cells, such as sperm or eggs. This approach makes the therapy heritable.

Gene Therapy Strategies

Gene Augmentation Therapy (GAT)

Gene Augmentation Therapy (GAT) involves adding functional alleles to treat inherited disorders resulting from a genetic deficiency. GAT is applicable to autosomal recessive disorders, while dominantly inherited disorders pose challenges for this approach.

Targeted Killing of Specific Cells

This strategy employs genes encoding toxic compounds (suicide genes) or prodrugs to eliminate transfected or transformed cells. Particularly prevalent in cancer gene therapies, it utilizes compounds like thymidine kinase (TK) and prodrug ganciclovir. TK phosphorylates ganciclovir, leading to chain termination upon its incorporation into DNA.

Targeted Inhibition of Gene Expression

Targeted inhibition focuses on blocking the expression of diseased genes or new genes producing harmful proteins. Particularly effective for treating infectious diseases and certain cancers.

Targeted Gene Mutation Correction

This approach corrects defective genes to restore function either at the genetic level through homologous recombination or at the mRNA level using therapeutic ribozymes or RNA editing.

Gene Therapy Approaches

Classical gene therapy involves delivering therapeutic genes and optimizing their expression within the target cell. Foreign genes carry out functions such as producing lacking proteins, generating toxins to eliminate diseased cells, or activating immune cells to aid in diseased cell elimination.

Non-classical Gene Therapy

Non-classical gene therapy inhibits the expression of genes associated with pathogenesis or corrects genetic defects to restore normal gene expression.

Methods of Gene Therapy

There are two main gene therapy approaches:

Ex Vivo Gene Therapy

In ex vivo gene therapy, genes are transferred to cultured cells, selected, multiplied, and reintroduced into the patient. Autologous cells are used to avoid immune rejection. This approach is applicable to tissues like hematopoietic and skin cells. Figure 8-1.5.1 illustrates the schematic diagram for ex vivo gene transfer.

• Autologous cells are sourced from the patient, grown in culture, genetically corrected, and reintroduced.
• Applied to tissues that can be removed, corrected outside the body, and reintroduced for long-term engraftment.

In Vivo Gene Therapy

In in vivo gene therapy, cloned genes are directly transferred into patient tissues, particularly suitable for tissues where individual cells cannot be cultured in vitro efficiently (e.g., brain cells). Liposomes and viral vectors, especially recombinant retrovirus-infected cells (vector-producing cells or VPCs), are used for gene transfer to surrounding disease cells.

• Efficiency of gene transfer and expression determines success due to the lack of selection and amplification of expressing cells.

Difference Between In Vivo and Ex Vivo Gene Delivery Systems:

In Vivo

• Less invasive
• Technically simple
• Vectors introduced directly
• Safety check not possible
• Decreased control over target cells

Ex Vivo

• More invasive
• Technically complex
• No vectors introduced directly
• Safety check possible
• Close control possible

Target Sites for Gene Therapy

Therapeutic genes necessitate specific delivery to target sites for different diseases. The table below outlines diseases and their corresponding target sites for gene therapy:

Target Cells for Gene Transfer:

• Disease: Cancer

  • Target Cells: Tumor cells, antigen-presenting cells (APCs), blood progenitor cells, T cells, fibroblasts, muscle cells

• Disease: Inherited Monogenic Disease

  • Target Cells: Lung epithelial cells, macrophages, T cells, blood progenitor cells, hepatocytes, muscle cells

• Disease: Infectious Disease

  • Target Cells: T cells, blood progenitor cells, antigen-presenting cells (APCs), muscle cells

• Disease: Cardiovascular Disease

  • Target Cells: Endothelial cells, muscle cells

• Disease: Rheumatoid Arthritis

  • Target Cells: Synovial lining cells

• Disease: Cubital Tunnel Syndrome

  • Target Cells: Nerve cells

Vectors for Gene Therapy

Gene therapy utilizes vectors for gene delivery, classified into two types:

1. Viral Vectors

   • Adenovirus
   • Retrovirus
   • Adeno-Associated Virus
   • Lentivirus
   • Vaccinia virus
   • Herpes simplex virus

2. Non-viral Vectors

   • Lipid complex
   • Liposomes
   • Peptide/protein
   • Polymers

Note: Table 2 presents vectors employed in gene therapy, adapted from AR Prabhakar in Gene Therapy and its Implications in Dentistry, International Journal of Clinical Pediatric Dentistry, 2011; 4(2):85-92.

Direct Gene Transfer Methods:

• Mechanical methods
• Electroporation
• Gene gun

These methods are also employed to transfer genes directly into target cells.

Viral Vectors

Retroviruses, Adenoviruses, and Adeno-Associated Viruses (AAV)

Viral vectors play a crucial role in gene therapy, with some commonly used viral vectors being retroviruses, adenoviruses, and adeno-associated viruses (AAV). Less commonly used viral vectors include those derived from the Herpes simplex virus (HSV-1) and the baculovirus.

Adenoviral Vectors

Adenoviruses, large linear double-stranded DNA viruses, are popular gene transfer vectors. They are the second most commonly used vector for gene therapy in diseases like cystic fibrosis and certain cancers.

• Adenovirus Entry Mechanism:

  • Receptor-mediated endocytosis involves primary receptors (Coxsackie and Adenovirus Receptor, Heparan sulphate glycosaminoglycans, sialic acid, CD46, CD80, CD86, alpha domain of MHC I) and secondary receptors (integrins).
  • Some adenoviruses directly interact with integrins, such as fiber-deficient Ad2 virions.

• Adenoviral DNA Structure:

  • Inverted terminal repeats (ITRs) and a terminal protein (TP) covalently attached to 5' termini.
  • Genome classified into early (E1, E2, E3, E4) and late (L1, L2, L3, L4, L5) regions based on expressed proteins.

• Classes of Adenoviral Vectors:

  • First Generation Adenoviral Vectors:

    • Constructed by replacing the E1/E3 expression cassette with a candidate gene (3-4kb).
    • Requires cell lines providing E1 proteins for replication.
    • Advantages: High titers, infect various human cell types, high transduction efficiency.
    • Disadvantages: Short foreign gene expression, potential immune response causing inflammation.

  • Second Generation Adenoviral Vectors:

    • Developed to address issues of the first generation.
    • E1/E2 or E3/E4 expression cassettes deleted and replaced.
    • Requires cell lines to complement both E1 and E2 or E3 and E4.
    • Can carry DNA insert up to 10.5kb.
    • Advantages: Improved safety, increased transgene expression.
    • Disadvantages: Associated with immunological problems, challenging construction.

  • Third Generation Adenoviral Vectors (Gutless Adenovirus):

    • Also known as helper-dependent adenovirus.
    • Lacks all coding sequences, requires a helper virus (AAV, artificially disabled viruses).
    • Can carry DNA insert up to 36kb (high-capacity adenoviruses).
    • Advantages: Non-integrative, high-capacity vectors, easy construction, longer stability, reduced immune response.
    • Disadvantages: Potential helper virus contamination causing diseases like conjunctivitis, pharyngitis, cold, and respiratory disease.

Adeno-Associated Virus (AAV)

• A group of small, single-stranded DNA viruses requiring co-infection with a helper virus, like an adenovirus, for productive infection.
• AAVs have an insert size of 4.5 kb, ensuring long-term gene expression as they integrate into chromosomal DNA.
• Recombinant AAV vectors contain only the gene of interest, with 96% of viral genes deleted, ensuring high safety.
• Further details on Adeno-Associated Viruses are explained in Module 5-Lecture 1.

Retroviral Vectors

• Retroviruses, RNA viruses with reverse transcriptase activity, synthesize complementary DNA after infection.
• Retroviruses deliver a nucleoprotein complex (pre-integration complex) into the cytoplasm, and the viral RNA genome is reverse transcribed and integrated into the chromosome.

Tumor Retroviruses (e.g., Moloney's Murine Leukemia Virus):

• Widely used for recombinant vectors; produced at low titers due to deletion of all viral genes.
• Limited target cells, particularly those dividing shortly after infection.

Recombinant Lentiviruses:

• Non-pathogenic to humans, capable of transducing stationary cells.

Other Viral Vectors

• Herpes Simplex Virus Vectors (HSV-1):

  • 150 kb double-stranded DNA virus with broad host range.
  • Larger insert size (>20kb) but limited by short-term expression due to the inability to integrate into host chromosomes.

• Baculovirus:

  • Efficiently expresses very large genes.
  • Used for recombinant protein expression in insect cells, with the ability to infect hepatocytes.

• Simian Virus 40 Vectors (SV40):

  • Icosahedral papovavirus with a circular double-stranded DNA of 5.2kb.
  • Recombinant SV40 vectors allow expression of transduced genes.

Non-Viral Vectors

Chemical and Physical Methods:

1. Direct Injection/Particle Bombardment:

• DNA injection parenterally considered for diseases like Duchenne muscular dystrophy (DMD).
• Particle bombardment or "gene gun" technique coats DNA on metal micro-particles fired into cells/tissues.
  • Advantages: Simple and comparatively safe.
  • Disadvantages: Poor efficiency, low stable integration, potential damage in proliferating cells with repeated injection.

2. Microinjection:

• Delivers foreign DNA into a living cell using a glass micropipette under a powerful microscope.
• Tip diameter: 0.5 to 5 micrometers.

3. Particle Bombardment Method:

• Tungsten or gold particles coated with foreign DNA.
• Micro-projectile bombardment uses high-velocity metal particles to deliver DNA into target cells.
• Used for gene transfer in mammalian cells with cell viability dependent on helium pressure.

4. Liposomes Mediated:

• Liposomes: Spherical vesicles with synthetic lipid bilayers resembling biological membranes.
• DNA packaged into liposomes in vitro and transferred to targeted tissue; survives in vivo and enters cells by endocytosis.
  • Cationic liposomes, with a positive charge stabilized by binding negatively charged DNA, are popular for in vivo gene transfer.
  • Advantages: Easy preparation of liposomes with foreign DNA, no size restriction for transferred DNA.
  • Disadvantages: Low efficiency of gene transfer, transient expression as they do not integrate into chromosomal DNA.
    

Electroporation

• Electroporation involves applying an external electric field to protoplasts, altering cell membrane electrical conductivity and permeability.
• Exogenous molecules in the medium are taken up into the cytoplasm (transient transfection) or the nucleus (stable transfection).
• Efficiency can be increased by applying heat shock or using a small quantity of PEG during electroporation.

Advantages:

• Large numbers of cells can be processed simultaneously, reducing processing time.

Disadvantages:

• Improperly calculated voltage can damage cells.
• Lack of control over the environment may allow harmful substances or impurities to enter the cell membrane.

Sleeping Beauty Transposition

• Non-viral method offering stable DNA integration into vertebrate chromosomes.
• Consists of a sleeping beauty transposon and a sleeping beauty transposase.
• Transposon has terminal inverted repeats at the ends of the gene of interest, and transposase mediates excision and integration into a chromosome site with a TA dinucleotide dimer.

Gene Therapy Application:

• Transposase gene replaced by the gene of interest, transposase provided in trans.

RNA-DNA Chimera

• Introduction:
  • Also known as chimeroplast, corrects point mutations by mismatch repair.
  • 68-nucleotide double-stranded nucleic acid molecule with one DNA strand and another strand with two 10-nucleotide 2’-O methyl RNA stretches separated by a 5-nucleotide DNA stretch.
  • Contains two hairpin loops and a GC clamp.

Receptor-Mediated Endocytosis

• Can be viral or non-viral mediated gene transfer.
• Viral vectors attach to surface receptors and are internalized.
• Non-viral mode involves coupling DNA to a ligand binding specifically to a cell surface receptor, causing DNA transfer into cells by endocytosis.
• Transferrin receptor and transferrin used as a ligand, targeting proliferating and hematopoietic cells.

• Advantages:

  • High gene transfer efficiency.

• Disadvantages:

  • Does not allow integration of transferred genes.
  • Protein-DNA complexes are not stable in serum.

• Enhancement:

  • Coupling inactivated adenovirus to the DNA-transferrin complex can increase gene transfer efficiency and aid in receptor-mediated endocytosis and lysosomal escape.

Endosomal/Lysosomal Escape

Introduction:

• In gene therapy, genes can get entrapped in the endocytic pathway and face degradation by hydrolytic enzymes, a major limitation.
• Various approaches are developed for both viral and non-viral gene delivery systems to escape the endocytic pathway.

For Viral Gene Delivery System:

• Enveloped viruses penetrate endosome membranes, and non-enveloped viruses escape by forming pores or lysing vesicular membranes.

For Non-viral Gene Delivery System:

1. Proton Sponge Hypothesis:

   • Cationic polymers with protonable amine groups having pKa close to endosomal/lysosomal pH.
   • During endosome acidification, polymers become protonated, increasing H+ influx and Cl- concentration, leading to endosome swelling and rupture.

2. Flip-Flop Mechanism:

   • Electrostatic interaction of cationic lipoplexes and anionic lipids causes lateral diffusion of lipids.
   • Results in displacement of nucleic acids from lipoplexes to the cytoplasm.

3. Pore Formation:

   • Peptide-based gene delivery system, e.g., GALA, undergoes conformation change at low pH, forming a pore in the vesicular membrane when incorporated.

4. Photochemical Internalization:

   • Uses photosensitizers that bind to and localize in the plasma membrane.
   • Photosensitizers remain inactive in endosomal membrane during endocytosis.
   • When irradiated with light of a specific wavelength, they produce reactive oxygen species, leading to endosome lysis.

Advantages of Gene Therapy

• Cure of Genetic Diseases: Addition, removal, or replacement of genes can cure genetic diseases.
• Cancer Treatment: Gene therapy can be used to kill cancerous cells.
• Controlled Gene Expression: Gene expression can be controlled.
• Continuous Production of Therapeutic Protein: Therapeutic proteins are continuously produced inside the body, reducing long-term treatment costs.