1. Human Health and Disease
Lecture 5

2. Gene Therapy
Definition: the introduction of normal genes into cells in place of missing or defective ones in order to correct genetic disorders.
Approaches:
Replacing a mutated gene that causes disease with a healthy copy of the gene.
Inactivating, or “knocking out,” a mutated gene that is functioning improperly.
Introducing a new gene into the body to help fight a disease.

3. Objectives Successfull gene therapy should be:
Efficient (determines the efficacy of gene delivery)
Cell Specific
Safe
“One of the challenges of gene therapy is the efficient delivery of genes to target cells. Although the nucleic acids containing the genes can be generated in the laboratory with relative ease, the delivery of these materials into a specific set of cells in the body is far from simple”.

4. Types of Gene Therapy

5. Viral Vectors
Definition: Viral vectors are a tool to deliver genetic material into cells. This process can be performed inside a living organism (''in vivo'') or in cell culture (''in vitro'').

6. Key Properties of Viral Vectors
''Safety'': Although viral vectors are occasionally created from pathogenic viruses, they are modified in such a way as to minimize the risk of handling them. This usually involves the deletion of a part of the viral genome critical for viral replication. Such a virus can efficiently infect cells but, once the infection has taken place, requires a helper virus to provide the missing proteins for production of new virions.
''Low toxicity'': The viral vector should have a minimal effect on the physiology of the cell it infects.
''Stability'': Some viruses are genetically unstable and can rapidly rearrange their genomes. This is detrimental to predictability and reproducibility of the work conducted using a viral vector and is avoided in their design.
''Cell type specificity'': Most viral vectors are engineered to infect as wide a range of cell types as possible. However, sometimes the opposite is preferred. The viral receptor can be modified to target the virus to a specific kind of cell.
''Identification'': Viral vectors are often given certain genes that help identify which cells took up the viral genes. These genes are called Markers, a common marker is antibiotic resistance to a certain antibiotic. The cells can then be isolated easily as those that have not taken up the viral vector genes do not have antibiotic resistance and so cannot grow in a culture with antibiotics present.

7. Families of Viruses for Viral Vectors
retroviruses
adenoviruses
adeno-associated viruses
herpes simplex viruses
picornaviruses,
alphaviruses

8. Retroviruses
Retroviruses were the first viruses to be modified for gene delivery, and are used in the majority of all gene therapy clinical trials.

9. Replication Cycle of Retroviruses
Retroviral particles encapsidate two copies of the full-length viral RNA, each copy containing the complete genetic information needed for virus replication. Retroviruses possess a lipid envelope and use interactions between the virally encoded envelope protein that is embedded in the membrane and a cellular receptor to enter the host cells.
Using the virally encoded enzyme reverse transcriptase, which is present in the virion, viral RNA is reverse transcribed into a DNA copy. This DNA copy is integrated into the host genome by integrase, another virally encoded enzyme. The integrated viral DNA is referred to as a provirus and becomes a permanent part of the host genome. The cellular transcriptional and translational machinery carries out expression of the viral genes. The host RNA polymerase II transcribes the provirus to generate RNA, and other cellular processes modify and transport the RNA out of the nucleus. A fraction of viral RNAs are spliced to allow expression of some genes whereas other viral RNAs remain full-length. The host translational machinery synthesizes and modifies the viral proteins. The newly synthesized viral proteins and the newly synthesized full-length viral RNAs are assembled together to form new viruses that bud out of the host cells
A retrovirus binds to a receptor on the cell surface, enters the cell, and reverse transcribes the RNA into double-stranded DNA, viral DNA integrates into the cell chromosome to form a provirus. Cellular machinery transcribes and processes the RNA, and translates the viral proteins. Viral RNA and proteins assemble to form new viruses, which are released from the cell by budding.

10. Genome Structure of Retroviruses
Genome structures of an oncovirus, a lentivirus, and a spumavirus. A, proviral structure of murine leukemia virus with the genome size of 8.8 kb; B, proviral structure of human immunodeficiency virus type 1 with the genome size of 9.7 kb; C, proviral structure of human foamy virus with the genome size 12.3 kb.

11. Types of Retroviruses Simple Retroviruses: Oncoviruses
Complex Retroviruses: Lentiviruses, spumaviruses, and some oncoviruses
Simple and Complex retroviruses encode gag (group- specific antigen) (is the genetic material that codes for the core structural proteins of a retrovirus), 
pro (protease) (key enzymes in viral propagation and are initially synthesized with other viral protein that are subsequently cleaved by the viral protease activity at specific sites to produce mature, functional units) 
pol (polymerase), and
env(envelope) genes.

12. Basic concept of Retroviruses and Helper Cells
When a replication-competent retrovirus infects a natural host cell, it can form a provirus in the host genome, express viral genes, and release new infectious particles to infect other hosts. In most gene therapy applications, it is not desirable to deliver a replication-competent virus into a patient because the virus may spread beyond the targeted tissue and cause adverse pathogenic effects.
Therefore, in most retroviral systems designed for gene delivery, the viral components are divided into a vector and a helper construct to limit the ability of the virus to replicate freely.
The term vector generally refers to a modified virus that contains the gene(s) of interest and cis-acting elements needed for gene expression and replication. Most vectors contain a deletion(s) of some or all of the viral protein coding sequences so that they are not replication-competent. Helper constructs are designed to express viral genes lacking in the vectors and to support replication of the vectors. The helper function is most often provided in a helper cell format although it can also be provided as a helper virus or as cotransfected plasmids. Helper cells are engineered culture cells expressing viral proteins needed to propagate retroviral vectors; this is generally achieved by transfecting plasmids expressing viral proteins into culture cells. Most helper cell lines are derived from cell clones to ensure uniformity in supporting retroviral vector replication. Helper viruses are not used often because of the likelihood that a replication-competent virus could be generated through high frequency recombination. Helper functions can also be provided by transient transfection of helper constructs to achieve rapid propagation of the retroviral vectors.

13. Basic Concepts in Retrovirus Vectors and Helper Cells
A, propagation of retroviral vectors in helper cells. Helper cells produce the viral proteins that are used to assemble viral particles containing RNA transcribed from the viral vector. Target cells do not express viral proteins and cannot generate viral particles containing the vector RNA. B, cis-acting elements needed in a prototypical retroviral vector. The plasmid backbone contains a drug resistance gene and a bacterial origin of replication (ori).

14. Helper Cells
‘Helper cells are designed to support the propagation of retroviral vectors. The viral proteins in the helper cells are expressed from helper constructs that are transfected into mammalian cells. Helper constructs vary in their mode of expression and in the genes they encode. Most of the currently available helper cell lines are listed in a table in a recent review.’

15. Vectors Based on Different Retroviruses
Vector DNA is first introduced into the helper cells by transfection, electroporation, or lipofection.
After introduction of the DNA into the helper cells, the vector DNA integrates into the helper cell and is expressed.
The viral RNA is expressed from the 5′ LTR and consists of all the sequences between the two R regions.
This viral RNA contains the packaging signal and is packaged into the viral particles efficiently.
During retroviral replication, the plasmid backbone sequences outside the two LTRs are not transferred to the target cells.

17. Vectors Targeted to Specific Cells
One strategy is designed to control gene delivery at the point of virus entry into the host cell by using natural or genetically engineered envelope proteins that interact with cell-type-specific receptors.
Another strategy is designed to control expression of the therapeutic gene in specific cell types by using tissue-specific promoters.

18. Lecture prepared from the following review paper
52/4/493.full

19. Adenovirus Medium-sized (90-100nm)
Nonenveloped (without an outer lipid bilayer)
Double stranded DNA
Derived from Human Adenoids (tonsils) in 1953
They have a broad range of vertebrate hosts; in humans, 100 distinct adenoviral serotypes have been found to cause a wide range of illnesses, from mild respiratory infections in young children (known as the common cold) to life-threatening multi- organ disease in people with a weakened immune system.
Serotype or serovar are distinct variations within a species of bacteria or viruses or among immune cells of different individuals.
Different types/serotypes are associated with different conditions:
respiratory disease (mainly species HAdV-B and C)
conjunctivitis (HAdV-B and D)
gastroenteritis (HAdV-F types 40, 41, HAdV-G type 52)
obesity or adipogenesis (HAdV-A type 31, HAdV-C type 5, HAdV-D types 9, 36, 37) 

21. How Adenovirus enters the host cell?
Entry of adenoviruses into the host cell involves two sets of interactions between the virus and the host cell.
Entry into the host cell is initiated by the knob domain of the fiber protein binding to the cell receptor. The two currently established receptors are: CD46 for the group B human adenovirus serotypes and the coxsackievirus adenovirus receptor (CAR) for all other serotypes.
This is followed by a secondary interaction with an integrin molecule. It is the co-receptor interaction that stimulates entry of the adenovirus.
Binding to integrin results in endocytosis of the virus particle via clathrin-coated pits.
Attachment to integrin stimulates cell signaling and thus induces actin polymerization resulting in entry of the virion into the host cell within an endosome.

22. Entry in the nucleus
Once the virus has successfully gained entry into the host cell, the endosome acidifies, which alters virus topology.
These changes, as well as the toxic nature of the pentons, destroy the endosome, resulting in the movement of the virion into the cytoplasm.
With the help of cellular microtubules the virus is transported to the nuclear pore complex, whereby the adenovirus particle disassembles.
Viral DNA is subsequently released, which can enter the nucleus via the nuclear pore.
After this the DNA associateswith histone molecules. Thus, viral gene expression can occur and new virus particles can be generated.

23. Benefits Their basic biology has been studied extensively,
The viral genome can accommodate large heterologous transgene insertions,
They readily infect quiescent and dividing cells,
They can be amplified to high titers and they have previously been shown to be relatively safe for use in humans.
The family Adenoviridae consists of five genera, including genus Mastadenovirus and genus Aviadenovirus, which infect mammals and birds respectively.
The adenovirus vector most commonly used for clinical trials and experimental gene therapy applications is species C adenovirus, HAdV-C5.

24. Drawbacks
Adenovirus delivered genes can be lost due to genetic instability therefore repeated doses are necessary to maintain the expression of transgene.
They would not integrate into the host genome, their gene expression is too short term.
Immunologic responses against adenoviruses have made their clinical application limited to a few tissues, such as  liver, lung (especially for CF(Cystic Fibrosis) treatment), or localized cancer gene therapy.

25. Drawbacks
Although the risk of serious disease following natural adenovirus infection is rare and the viral genome would not integrate into the host genome, gene therapy by adenoviral vectors has caused serious bad side effects and even death of some patients.

26. Adeno-associated vectors
Adeno-associated vectors (AAV) are like adenoviral vectors in their features but because of having some deficiency in their replication and pathogenicity, are safer than adenoviral vectors.
In human, AAVs are not  associated with any disease.
Another special character of AAV is their ability to integrate into a specific site on chromosome 19 with no noticeable effects cause long-term expression in vivo. 
AAVs have been used in the treatment of some diseases, such as CF, hemophilia B, Leber congenital amaurosis, and AAT (Alpha-1 antitrypsine) deficiency.

27. The use of antisense oligodeoxynucleotides targeted to the renin-angiotensin system [hormone system that regulates blood pressure and water (fluid) system] and adeno-associated virus vector delivery of antisense DNA offers a new approach to prolonged hypertension therapy with a single administration.

28. Drawbacks
The major disadvantages of these vectors are complicated process of vector production and the limited transgene capacity of the particles (up to 4.8 kb). 

29. Comparison

30. Lecture prepared from
PMC /#!po=

31. Cystic Fibrosis
Average life span:25-30 years

32. Conventional Treatment
the use of physiotherapy, antibiotics and pancreatic supplements.
Many patients require treatment four times daily, including a considerable time spent on physiotherapy.
This combination of treatment has helped increase life expectancy considerably to the current median of approximately 30 years of age.
Despite small further improvements, more recently it has become apparent that there is a need for a more effective and convenient therapy.
The identification of the gene responsible for CF (the cystic fibrosis transmembrane conductance regulator or CFTR protein) in 1989, was responsible for the advent of potential new treatments for CF.
Gene therapy, the transfer of a normal copy of the CFTR gene into the lungs of CF patients, was proposed as an attractive new option.

33. Cystic Fibrosis, a case study
etherapy/casestudy/

34. News of Gene therapy in CF
environment
s/Gene-therapy-breakthrough-for-cystic- fibrosis.aspx
3 July 2015