1. PHR 403 Pharmaceutical Biotechnology Gene Therapy
Course Instructor
Md. Samiul Alam Rajib
Senior Lecturer,
Department of Pharmacy
BRAC University

2. Overview of the chapter
Concept of Gene and genetic disorders
Ex vivo versus in vivo gene therapy
Potential target diseases for gene therapy
Gene transfer methods
Viral gene delivery systems
Non viral gene delivery systems
Anti-sense technology

3. Genes Are carried on a chromosome The basic unit of heredity
Encode how to make a protein
DNARNA proteins
Proteins carry out most of life’s function.
When altered causes dysfunction of a protein
When there is a mutation in the gene, then it will change the codon, which will change which amino acid is called for which will change the conformation of the protein which will change the function of the protein. Genetic disorders result from mutations in the genome.

4. Genetic disorders Chromosomal disorders Single Gene disorders
Gametes have abnormal chromosome numbers and mutations
Offspring inherit extra chromosome or are missing a chromosome
Caused by problems with meiosis

5. Genetic disorders Chromosomal disorders:
Nondisjunction of chromosomes during meiosis
Based on: Brooks/Cole – Thomson Learning

6. Genetic disorders Chromosomal disorders: Example: Down Syndrome
Down Syndrome Characteristics:
wide, rounded face
enlarged tongue
Lower cognitive ability
equal length fingers
webbed neck
Normal female karyotype with 46 chromosomes
Down syndrome karyotype with extra chromosome 21

7. Concept of Inheritance
Important points of inheritance:
Genes have different forms, called alleles
Each trait is controlled by effects of two alleles
Some alleles are:
Dominant/recessive
Other alleles are co-dominant

8. Concept of Inheritance
Case: Hairline trait
Allele for Widow’s Peak is dominant
Allele for Straight Hairline is recessive
A person with WW or Ww will have Widow’s Peak
A person with ww will have a Straight Hairline

9. Concept of Inheritance
Genotype Vs Phenotype
Genotype refers to the alleles
Phenotype refers to the appearance
Genotype
Phenotype WW
person has a widow’s peak Ww ww
person has a straight hair line

10. Genetic disorders Single gene disorders:
About 4,000 human diseases are thought to be inherited.
Scientists are making good progress figuring out where genes are located on chromosomes.
Genetic diseases are caused by
Mutations
incorrect sequences in the normal form of the gene.

11. Genetic disorders and human diseases
Huntington’s disease:
Results in a loss of muscle control and mental function.
The symptoms usually do not appear until after 30 years old.
1 in 10,000 people.
Caused by a dominant allele

12. Genetic disorders and human diseases
Cystic Fibrosis:
Mucus in bronchi is thick,
interfering with lung function
1 in 25 are carriers
One of the first disorders to be actively studied for gene therapy.
Most lethal autosomal recessive disorder in U.S.

13. Genetic disorders and human diseases

14. Genetic disorders and human diseases
Sickle Cell anemia:
Red blood cells are sickle shaped, issues with circulation causing anemia and pain

15. Genetic disorders and human diseases
Sickle cell anemia:
Co-dominant disorder found in African Americans.
1 in 400 African Americans
Can be fatal.
Possible cure: bone-marrow transplants
The sickle cell trait can prevent Malaria

16. Genetic disorders and human diseases
Hemophilia:
A disorder in which a person’s blood does not clot properly.
It is a recessive sex-linked, X-chromosome disorder.
1 in 10,000 males born are afflicted.
“Royalty Disease”

17. Genetic disorders and human diseases
Adenoside Deaminase (ADA) Deficiency:
ADA-deficient persons are affected by severe immunodeficiency, with recurrent infections that might be life-threatening.
First disease approved for gene therapy.
Autosomal recessive disorder.
The drug exists but is very expensive, needs to be injected in vein for life.

18. What is gene therapy?
Many medical conditions result from flaws, or mutations, in one or more of a person's genes. So, if a flawed gene caused our "broken window," can you "fix" it? What are your options?
Imagine that you accidentally broke one of your neighbor's windows.
Stay silent: no one will ever find out that you are guilty, but the window doesn't get fixed.
Repair it with some tape: not the best long-term solution.
Put in a new window: not only do you solve the problem, but also you do the honorable thing.
Stay silent: ignore the genetic disorder and nothing gets fixed.
Try to treat the disorder with drugs or other approaches: depending on the disorder, treatment may or may not be a good long-term solution.
Put in a normal, functioning copy of the gene: if you can do this, it may solve the problem!

19. What is gene therapy?
It is a technique for correcting defective genes that are responsible for disease development
There are four approaches:
A normal gene inserted to compensate for a nonfunctional gene (Gene transplantation).
An abnormal gene traded for a normal gene (Gene transplantation)
An abnormal gene repaired through selective reverse mutation (Gene correction)
Change the regulation of gene pairs (Gene augmentation)

20. Diseases for applying gene therapy
Target Cells
Severe Combined Immunodeficiency (SCID)
Bone marrow Cells or
T-Lymphocytes
Hemophilia
Liver, Muscle
Cystic fibrosis
Lung cells
Cancer
Many cell types
Neurological diseases (Parkinson’s, Alzheimer's)
Nerve Cells
Infectious diseases (AIDS, Hepatitis B)
White Blood Cells

21. in vivo Versus ex vivo Gene Therapy

22. In vivo Gene Therapy
1. The genetic material is transferred directly into the body of the patient
2. More or less random process;
small ability to control; less manipulations
3. Only available option for tissues
that can not be grown in vitro;
or if grown cells can not be transferred back

23. ex vivo Gene Therapy The genetic material is first transferred
into the cells grown in vitro
2. Controlled process;
Genetically altered cells are selected and expanded; more manipulations
3. Cells are then returned back to the patient

24. Gene delivery systems Non viral gene delivery systems
Direct introduction of therapeutic DNA
But only with certain tissue
Requires a lot of DNA
Creation of artificial lipid sphere with aqueous core, liposome Carries therapeutic DNA through membrane
Chemically linking DNA to molecule that will bind to special cell receptors
DNA is engulfed by cell membrane
Less effective 
Trying to introduce a 47th chromosome
Exist alongside the 46 others
Could carry a lot of information
But how to get the big molecule through membranes?

25. Gene delivery systems Viral gene delivery systems. Retro virus
Adenovirus
Adeno associated viruses (AAV)
Herpes simplex virus (HSV)

26. Non viral gene delivery systems
Direct introduction of therapeutic DNA:

27. Non viral gene delivery systems
Current attempts with naked DNA vaccination in infectious diseases:
HIV
Hepatitis
Influenza
Tuberculosis
Lyme disease
Malaria

28. Non viral gene delivery systems
Direct introduction therapeutic DNA: Ballistic DNA injections:
Inventyed for gene transfer in plant cells
Fully applicable to eukayotic cells
plasmid DNA shown here

29. Non viral gene delivery systems
Gene delivery using Liposomes:
Next level idea – why naked DNA?
Lets’ wrap it in something safe
to increase transfection rate
Lipids – are an obvious idea !
Therapeutic DNA

30. Non viral gene delivery systems
Gene delivery using Liposomes:
Cheaper than viruses
No immune response
Especially good
for in-lung delivery (cystic fibrosis)
times more plasmid DNA needed
for the same transfer efficiency as for viral vector

31. Viral gene delivery systems
Viruses:
Replicate by inserting their DNA into a host cell
Gene therapy can use this to insert genes that encode for a desired protein to create the desired trait
Four different types
Retro virus
Adenovirus
Adeno associated viruses (AAV)
Herpes simplex virus (HSV)

32. Viral gene delivery systems
By Retrovirus:
Created double stranded DNA copies from RNA genome
The retrovirus goes through reverse transcription using reverse transcriptase and RNA
the double stranded viral genome integrates into the human genome using integrase
integrase inserts the gene anywhere because it has no specific site
May cause insertional mutagenesis
One gene disrupts another gene’s code (disrupted cell division causes cancer from uncontrolled cell division)
vectors used are derived from the human immunodeficiency virus (HIV) and are being evaluated for safety

33. Viral gene delivery systems
By Adenovirus:
Are double stranded DNA genome that cause respiratory, intestinal, and eye infections in humans
The inserted DNA is not incorporate into genome
Not replicated though 
Has to be reinserted when more cells divide
Ex. Common cold

34. Viral gene delivery systems (Using Adenovirus)

35. Viral gene delivery systems
By Adeno associated virus:
Adeno-associated Virus- small, single stranded DNA that insert genetic material at a specific point on chromosome 19
From parvovirus family- causes no known disease and doesn't trigger patient immune response.
Low information capacity
gene is always "on" so the protein is always being expressed, possibly even in instances when it isn't needed.
hemophilia treatments, for example, a gene-carrying vector could be injected into a muscle, prompting the muscle cells to produce Factor IX and thus prevent bleeding.
Study by Wilson and Kathy High (University of Pennsylvania), patients have not needed Factor IX injections for more than a year

36. Viral gene delivery systems
By Herpes Simplex virus Type 1:
Double stranded DNA viruses that infect neurons
Ex. Herpes simplex virus type 1

37. Anti sense therapy Theory:
By introducing an antisense gene (or asRNA) into cells, the asRNA would “zip up” the complementary mRNA into a dsRNA that would not be translated
The “antisense effect” was highly variable, and in light of the discovery of RNAi, asRNA probably inhibited its target by inducing RNAi rather than inhibiting translation.
Used from ~1980 on, to repress specific genes
Alternative to gene knock-outs, which were/are very difficult to do in higher plants and animals

38. Anti sense therapy Mechanism:
An antisense oligonucleotide (ASO) is a single-stranded, chemically modified DNA-like molecule that is 17–22 nucleotides in length and designed to be complementary to a selected gene’s mRNA and thereby specifically inhibit expression of that gene
Therefore, the level of the target protein is reduced by blocking translation and by altering the subsequent cascades regulating cellular proliferation, differentiation, homeostasis and apoptosis

39. Anti sense therapy
Several mechanisms that explain how ASO inhibit translation have been proposed- 1. The most accepted involves the formation of an mRNA–ASO duplex formed through Watson–Crick binding, leading to RNase-H-mediated cleavage of the target mRNA. 2. Other proposed mechanisms include prevention of mRNA transport, modulation or inhibition of splicing, translational arrest, and formation of a triple helix through ASO binding to duplex genomic DNA resulting in inhibition of transcription. 3. Recent advancements: siRNA

40. Anti sense therapy

41. Current scenario
FDA hasn’t approved any human gene therapy product for sale
Reasons:
In 1999, 18-year-old Jesse Gelsinger died from multiple organ failure 4 days after treatment for omithine transcarboxylase deficiency.
Death was triggered by severe immune response to adenovirus carrier
January 2003, halt to using retrovirus vectors in blood stem cells because children developed leukemia-like condition after successful treatment for X-linked severe combined immunodeficiency disease

42. Problems with gene therapy
Short Lived
Hard to rapidly integrate therapeutic DNA into genome and rapidly dividing nature of cells prevent gene therapy from long time
Would have to have multiple rounds of therapy
Immune Response
new things introduced leads to immune response
increased response when a repeat offender enters
Viral Vectors
patient could have toxic, immune, inflammatory response
also may cause disease once inside
Multigene Disorders
Heart disease, high blood pressure, Alzheimer’s, arthritis and diabetes are hard to treat because you need to introduce more than one gene
May induce a tumor if integrated in a tumor suppressor gene because insertional mutagenesis

43. For the Curious minds!!
There is a very recent advancement in Genome editing (in 2014) The technology is called CRISPR/ Cas9 technology A video documentary is uploaded in TSR in case you are interested