What are genes?

• Genes are present inside the chromosome, which are the basic physical and 
  functional units of hereditary.
  

• Genes are made up of specific sequences of bases and these gene bases, code 
  for specific protein production as needed by the human body for normal 
  metabolism.
  

• Proteins are required for normal cellular function, proper functioning of proteins 
  is required for production and maintaining of cellular structures.
  

• When alterations in gene takes place the proteins which are coded by these 
  gene are not able to function and causes genetic disorders.
  

• Gene therapy is done to repair the defective genes responsible for disease. 
  

     Gene therapy is done by: 

         1. Inserting the normal gene into the genome to replace the non functional 
             gene.
  
         2. By recombination: The abnormal gene is replaced by normal gene.
  
         3. By selective reverse mutation: The abnormal gene is repaired to become 
             normal and perform its normal function. 
  
How does gene therapy work?

• Genes are composed of Deoxyribonucleic acid – DNA. DNA is composed of 
  genetic information, by which they produce the specific proteins which are the 
  building blocks of the body. 
  

• Gene damage or missing gene may lead to inadequate or wrong protein 
  production leading to disease condition.
  

• In gene therapy, a normal gene is inserted into the genome to replace an 
  abnormal disease causing gene. 
  

• A carrier molecule “vector” can be used to deliver the therapeutic gene into the 
  patients target cells. 
  

• Viruses are microorganisms which infect humans and multiply in the genome of 
  the human body. 
  

• Scientists have tried to take advantage of this multiplication capacity of the 
  viruses to transfer the required genes into the human body for treatment 
  purposes. 
  

• The virus is manipulated by the scientists and the pathogenic disease causing 
  genes in the virus are removed and the therapeutic genes are inserted.
  

• The virus vector then unloads its genetic material containing the therapeutic 
  human gene into the target cell (human host). 
  

• The treated human produce the normal proteins due to the transfer of the 
  therapeutic genes. 

Types of gene therapy 

• There are two forms of gene therapy – Somatic gene therapy and Germ line 
  gene therapy. 
  

• Somatic gene therapy involves the manipulation of gene expression in cells that 
  will be corrective to the patient but not inherited to the next generation.
  

• Somatic cell gene therapy is at an early stage of development.
  

• Germ line gene therapy involves the genetic modification of germ cells (sperms 
  and eggs) in order to prevent a genetic defect from being transmitted to future 
  generations. 
  

Various strategies involved in somatic cell gene therapy are emerging and can be grouped under two broad categories as ex vivo and in vivo gene therapy:

Ex vivo gene therapy:

• Ex vivo gene therapy involves following procedure:
  
       1. Cells are collected from the affected individual.
       2. The genetic defect is corrected by transferring the genes into the isolated 
           cells. 
       3. The genetically corrected cells are selected and grown. 
       4. The genetically modified cells are transferred into the patient. 
       5. The use of patients’ owns cells (autologous cells) have no adverse 
           immunological response after transplantation.
       6. Vectors derived from mouse retroviruses are mostly used.
       7. Intact particles deliver the complete vector RNA to a host cell at a high 
           frequency.
       8. Retroviruses readily infect replicating cells, so actively growing target cells 
           are either treated with purified packaged retroviral vector particles directly 
           or co cultivated with packaging cell line.
  

• The transferred target cells are tested to ensure that:
  
         1. The desired gene product is produced in the gene transferred humans.
         2. It is ensured that the virus does not produce its pathogenic viral cellular 
             structures and causes infection in the human host. 
         3. It is ensured that the Retroviral vector DNA has not been inserted into a
             size that either alters the growth properties of the cells or interferes with
             normal cellular functions.
  

• The transduced cells are grown in culture, collected in large amounts, and then 
  introduced into the patient at various intervals; with the hope that the cells 
  will be maintained and the disease that is being treated will be corrected.
  

• Patients with genetic diseases that respond to bone marrow transplantation are 
  treated by ex vivo gene therapy.
  

• Bone marrow transplantation acts as a therapeutic procedure for different 
  genetic diseases, bone marrow contains totipotent embryonic stem cells at a 
  frequency of 10-4 to 10-5. These embryonic cells can divide and differentiate 
  into various important cell types, including B and T lymphocytes, macrophages, 
  Red blood cells, platelets and bone cells. 
  

• Ex vivo gene therapy is a procedure in which genetically engineered totipotent 
  stem cells are transplanted into a patient, where they would provide the 
  missing cell type or gene product.
  

In-vivo Gene therapy:

• Gene therapy involves direct delivery of a corrected gene into the cells of a 
  particular tissue of patient.

Viral gene delivery systems:

Retroviral vectors:

• Retroviruses are enveloped viruses, which contains a single stranded RNA 
  genome. 
  

• Most commonly used retroviral vector is Monkey murine leukaemia virus vector 
  system which is used to treat ADA-SCID.
  

• A recombinant retro virus vector genome that is packaged in the envelope 
  protein of another virus will have the binding specificity and infection spectrum 
  that are determined by the envelope protein called pseudo type formation.
  

• Retroviral vectors have the ability to integrate their genome into a host cell 
  genome.
  

• The viral genes “gag”, “pol” and “env” are present in retroviruses and these 
  genes are responsible for genomic and protein assembly.
   

• These vectors have potential for permanent expression in somatic cell.
   

• After transfection, the ssRNA is reverse transcribed into double stranded DNA, 
  by which the inserted genes gets integrated into host genome and translated 
  into proteins. 
   

• The retrovirus genome is approximately 10 kb and contains three genes:
       1.  gag (coding for core proteins),
       2.  pol (coding for reverse transcriptase)
       3.  env (coding for viral envelope proteins)
       4.  Each end of the genome has long terminal repeats (LTRs) which has the 
            promoter/enhancer regions.
   

• Some retroviruses contain proto-oncogenes which when mutated can cause 
  cancers, during packaging of vectors, these oncogenes are removed. 
   

• The retroviral vectors are manipulated to increase the yield of virus particles, 
  increase the efficiency of transduction, engineering them to infect non-dividing 
  cells and specifying cell type.

Lentivirus Vectors:
 

• Lentiviruses are a subclass of retroviruses which are able to infect both 
  proliferating & non-proliferating cells. 
  

• They are considerably more complicated than simple retroviruses, containing an 
  additional six proteins - tat, rev, vpr, vpu, nef & vif.
  

• Lentiviruses are internally engineered by a cytomegalovirus promoter.
  

• Lentiviral vectors used are derived from the Human Immunodeficiency Virus 
  (HIV) & are being evaluated for safety, with a view to removing some of the 
  non-essential regulatory genes. 

Adenoviral vectors:

• Adenoviruses are non-enveloped viruses containing a linear double stranded 
  DNA genome. 
   

• There are over 40 serotype strains of adenovirus, most of which cause benign 
  respiratory tract infections in humans. 
   

• Adeno virus infect a wide range of non dividing human cells and have been used 
  extensively as live vaccines against respiratory infections and gastro enteritis 
  without side effects. 
   

• Serotypes 2 or 5 are predominantly used as vectors. 
   

• The wild type adenovirus genome is approximately 35 kb of which up to 30 kb 
  can be replaced with foreign DNA. 
   

• After infection of target cell with recombinant adenovirus, the DNA is passed 
  into cell nucleus, where therapeutic gene is expressed. 
   

• Adenoviral vectors are very efficient at transducing target cells. 
   

• The recombinant DNA construct does not integrate into a chromosome and so 
  does not persist long. 
   

• Immune responses to the inserted virus can be avoided by giving 
  immunosuppressive therapy. 

Adeno-Associated Viruses:

• Adeno-associated viruses (AAV) are non-pathogenic human Parvo viruses. 
  

• Adeno associated virus is a small, single stranded human DNA virus that can 
  integrate into a specific site on chromosome 19. 
  

• Absence of pathogenicity makes AAV a good candidate as an ideal vector to 
  deliver therapeutic genes. 
   

• Recombinant AAV is generated by co transfection of two plasmids into a host 
  cell that has been infected with Adeno virus (helper virus). 
   

• AAV wild type genome is a single stranded DNA molecule, consisting of two 
  genes: 
         1.  rep: coding for proteins which control viral replication, structural gene 
             ex-pression & integration into the host genome. 
         2.  cap: codes for capsid structural proteins. 
   

• The total length of the insert is 4.7 kb. 
   

• It is possibly due to the simplicity of the viral capsid that the immune response 
  does not occur. 

Herpes Simplex Virus:

• Herpes simplex virus type 1 (HSV-1) is a human neurotropic virus. 
  

• HSV-1 virus is able to infect neurons. 
  

• HSV -1 genome is a linear double stranded DNA molecule of 152 kb.
  

• Three main classes of HSV-1 genes have been identified:
         1. Immediate-early (IE or alpha) genes, 
         2. Early (E or beta) genes 
         3. Late (L or gamma) genes. 
  

• The early genes encode genes for nucleotide metabolism & DNA replication. 
  

• Late genes code for structural proteins. 
  

• The entire cycle takes less than 10 hours & invariably results in cell death. 
  

• Two basic approaches have been used for production of HSV-1 vectors -    
  amplicons & recombinant HSV-1 viruses. 
  

• Amplicons are bacterially produced plasmids containing col E1 ori (an 
  Escherichia coli origin of replication), OriS (the HSV-1 origin of replication), 
  HSV-1 packaging sequence & a selectable marker.
  

• Both the helper & amplicon containing viral particles are delivered to the 
  recipient. 
  

• Deletion of a number of immediate-early genes reduces cytotoxicity

Non-viral Gene delivery systems:

• All viral vectors which are used to transfer genes induce an immunological 
  response to some degree & may have safety risks (such as insertional 
  mutagenesis & toxicity problems). 
  

• Furthermore their capacity is limited & large scale production may be difficult to 
  achieve. 
  

• Non-viral methods of DNA transfer require only a small number of proteins, have 
  a virtually infinite capacity, have no infectious or mutagenic capability & large 
  scale production is possible using pharmaceutical techniques. 
  

• Viral vectors are costly to maintain 
  

• Has limited cloning capacity 
  

• Viral proteins may induce inflammatory response.
  

• Limitations:
       1.  Frequency of transfection is often too low to create a therapeutic effect.
       2.  Duration of therapeutic gene ex-pression is too brief to provide an effective 
            treatment. 
  

• There are three methods of non-viral DNA transfer. namely: 
  

• Naked DNA 
  

• Plasmid liposomes complex
  

• Molecular conjugates 

Naked DNA:

• Naked DNA is directly injected inside the target tissue.
  

• In this procedure the pure DNA constructs are directly inserted into cells of 
  target tissues. 
  

• Therapeutic genes delivered are expressed in targeted tissues and gene 
  products are released into circulatory system.
  

• Secretion of therapeutic protein into circulatory system should facilitate into 
  target tissue. 
  

• DNA construct (genetic DNA) is surrounded by artificial lipid layers to form a 
  lipid sphere with an aqueous core which facilitates passage of DNA through the 
  cell membrane. 
  

• This method is a simple procedure and has shown good expression in target 
  genes. 
  

• This technique is used for DNA vaccine productions. 
  

• This technique does not produce immunity against the agents; it is relatively a 
  cheap technique and can be used for multiple deliver. 
   

• By transferring DNA, it is able to immunize with two serologically distinct 
  strains. 
   

• Naked DNA technique is used in cancer immunity, pancreatic insulin function, 
  and stimulation of blood vessels. 
   

• By administration of collagenase, papaverine the expression of gene product in 
  muscle tissue is improved. 
   

• Muscle specific promoter & intragene regulators also improve transgene 
  expression. 

Plasmid liposome complexes:

• Liposomes are lipid bilayers entrapping a fraction of aqueous fluid.
  

• Cationic lipid DNA complexes are used for transferring genes into lungs.
  

• They do not produce immunological response. 
  

• It can be used for transferring large piece of DNA. 
  

• Lipids consist of positively charged head group which binds to DNA and a 
  hydrophobic anchor for cell membranes. 
  

• DNA will be transported to cell nucleus where it is expressed. 
  

• It can be used as an aerosol directly induced into the lung tissues.

Molecular conjugates:

• Molecular conjugates are made up of protein or synthetic ligands to which DNA 
  is bound.
  

• Targeting proteins of Molecular conjugates are asialoglycoproteins, transferrin, 
  polymeric IgA and adenovirus.

Pro-drug Activation Therapy:

• Combination of HSV-thymidine kinase gene (HSV tk) and ganciclovir [GCV; 9-(1, 
  3-dihydroxy-2-propoxymethyl) guanine] has been used to eradicate 
  proliferating tumor cells. 
  

• Transfecting tumor cells in vivo with HSV tk gene under the control of an active 
  promoter and after few days ganciclovir is administered. 
  

• HSV tk phosphorylates ganciclovir to form monophosphate GCV. 
  Triphosphate-GCV inhibits DNA polymerase and terminates DNA synthesis, 
  causing death of tumor cell. 
  

• Triphosphate-GCV can pass unmodified cells by cell to cell contact and kill 
  these cells as well. 1 HSV tk expressing tumor cell can kill up to 10 unmodified 
  cells. This phenomenon is called “bystander effect”. 
  

• When gene under certain conditions causes death of its own cell – suicide 
  gene. 
  

• Prodrug- Inactive form of therapeutic agent that is activated biologically after 
  it is administered as part of a treatment. 

Two-gene therapy:

• Cancer therapy is given by using two different gene systems.
  

• GCG-HSVtk suicide gene therapy and gene based immunotherapy have been 
  combined to treat cancers. 
  

• Tumor cells transduced separately with HSV tk gene and cloned cytokine cDNA 
  or gene.
  

• Cytokines such as Interleukin-2, Interleukin-12, and others act as signals that 
  mobilize immune cells and stimulate immune response against tumor cells.
  

• Tumor cell peptides that are released, after cell death caused by suicide gene 
  therapy, will interact with immune cells and cause immunological reaction 
  against tumor cells. An added benefit is circulating tumor cell antibodies which 
  prevent initiation of cancer at other sites in the body.

Nucleic acid Therapeutic agents:

• Therapeutic systems using nucleotide sequences are devised to treat human 
  disorders.
  

• Small, single strand nucleotide sequences (oligonucleotides) could hybridize to a 
  specific gene or mRNA and reduce transcription or translation, lowering the    
  amount of protein synthesized.
  

• An oligonucleotides designed to bind to a gene and block transcription is called 
  antigen oligonucleotides and one that base pairs with specific mRNA is an  
  antisense oligonucleotides.
  

• Binding of oligonucleotides to transcription factor responsible for expression of 
  specific gene. Double stranded oligonucleotides that attach to DNA binding 
  proteins could prevent activation of transcription of specific genes.
  

• Synthetic DNA molecules that bind to proteins and prevent them from 
  functioning can be created.
  

• Ribozymes are natural RNA sequences that bind and cleave specific RNA 
  molecules. These could be engineered to target an mRNA and decrease the 
  amount of proteon.

Antisense RNA production:

• Antisense oligonucleotides must bind to a specified mRNA and prevent 
  translation of the protein.
  

• Studies are undergoing to use ex-pression vector to produce an antisense RNA 
  that suppresses pathogenic condition.

Antisense Oligonucleotides:

• Sequence specific effectiveness of chemically synthesized antisense 
  oligodeoxynucleotides on hybridization to an accessible site on target mRNA, 
  resistance to degradation by cellular nucleases, and are delivevered into cells.
  

• Oligonucleotides with about 15-20 nucleotides have sufficient specificity to 
  hybridize unique mRNA. 
  

• The most commonly used antisense oligonucleotides has a sulfur group in place 
  of free oxygen of phosphodiester bond the RNA-DNA duplex activates 
  endogenous enzyme ribonuclease (RNase) H, which cleaves the mRNA 
  component of hybrid molecule.
  

• Clinical trials are undergoing using phosphorothioate antisense oligonucleotides, 
  against CMV, HIV.

Nucleic acid pharmaceuticals:

• Administration of oligonucleotides that attaches to transcription or translation 
  factor could prevent expression of target gene.
   

• Nucleic acid molecules are capable of binding to proteins, and by devising an 
  oligonucleotide that binds to protein that does not normally bind to nucleic acid 
  molecule and as a consequence of this attachment, inhibit functioning of this 
  protein. 
   

• This type of oligonucleotide is called ‘Aptamer’. Potential anti-thrombin aptamer 
  inhibits thrombin.

Ribozymes – as therapeutic agents:

• Ribozymes are naturally occurring catalytic RNA molecules that have separate 
  catalytic and substrate binding domains. 
  

• The substrate domain of a ribozyme can be engineered to direct it to a specific 
  mRNA sequence. 
  

• The substrate binding sequence combines by nucleotide complementary and 
  catalytic portion cleaves the target RNA at specific site.

Oligonucleotide correction of Genetic conditions:

• Ability to convert a mutant base pair of a gene to the wild type (normal) 
  version would reverse the consequences of many different genetic conditions.
  

• Strategy using modified RNA-DNA oligonucleotides with 68 nucleotides has been 
  devised for this purpose.

History of events - Gene therapy: