gene thereby methods of delivering gen clinical trials success and failure

2. Gene
A gene is the unit of heredity
and carries inherited information

3. Amazing Facts of Human
Genome
3 billion (3,000,000,000) letters in the DNA code in every cell in your body.
There is 6 feet of DNA in each of our cells packed into a structure only 0.0004
inches across (it would easily fit on the head of a pin).
There are 100 trillion (100,000,000,000,000) cells in the body
If all the DNA in the human body was put end to end it would reach to the sun
and back over 600 times (100 trillion x 6 feet divided by 93 million miles = 1200).
If we recited the genome at one letter per second for 24 hours a day
it would take a century to recite the book of life.
The vast majority of DNA in the human genome - 97% - has no known function.
Our DNA is 98% identical to that of chimpanzees.
Between humans, our DNA differs by only 0.2%, or 1 in 500 bases (letters).
(This takes into account that human cells have two copies of the genome.)
The estimated number of genes in both humans and mice is 30,000-45,000
; in the round worm (C. elegans), the number is approximately 19,000.

4. Chromosome to Gene

5. Genes per chromosome

6. Gene structure

7. exon 2
exon 1 exon n
promotor
5‘UTR
3‘UTR
Protein coding sequence
exon n-1
Gene structure

8. How Function is done

10. Our life is maintained
by molecular network systems
Molecular network
system in a cell

11. Proteins play key roles in a living system
⚫
Three examples of protein
functions
–
Catalysis:
Almost all chemical reactions in
a living cell are catalyzed by
protein enzymes.
–
Transport:
Some proteins transports
various substances, such as
oxygen, ions, and so on.
–
Information transfer:
For example, hormones.
Alcohol
dehydrogenate
oxidizes alcohols
to aldehydes or
ketones
Hemoglobin
carries oxygen
Insulin controls the
amount of sugar in
the blood

12. TERTIARY STRUCTURE (fold)
TERTIARY STRUCTURE (fold)
Genome
Expressome
Proteome
Metabolite
Functional Genomics
From gene to function

14. Picture of a Chromosome

15. Gene structure

16. 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

17. Dilemma
⚫
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.

18. Imagine
–
Imagine that you
–
accidentally broke one of your
neighbor's windows.

19. Behavior
⚫
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.

20. What is similar in gene therapy
.I
Stay silent: ignore the genetic disorder and nothing gets
fixed.
.II
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.
.III
Put in a normal, functioning copy of the gene: if you can do
this, it may solve the problem!

21. How to fix it
A
B C a beneficial gene
A
virus modified virus
⚫
A virus is found which replicates by inserting its genes into the host cell's genome.
This virus has three genes - A, B and C.
⚫
Gene A encodes a protein which allows this virus to insert itself into the host's
genome.
⚫
Genes B and C actually cause the disease this virus is associated with.
⚫
Replace B and C with a beneficial gene. Thus, the modified virus could introduce your
'good gene' into the host cell's genome without causing any disease.
⚫
So we use the modified virus to fix the “broken window”

22. Gene therapy using an Adenovirus vector.
A new gene is inserted into an adenovirus vector,
which is used to introduce the modified DNA into
a human cell. If the treatment is successful, ]
the new gene will make a functional protein.

23. Gene Therapy

25. Diseases
Which of the following diseases are current or potential targets of
gene therapy?
a. Cancer b. AIDS c. Diabetes d. Parkinson’s
Disease

26. Both
⚫
Acquired
Inherited

27. Genetic disorders
–
1-one single gene
–
Cystic fibrosis
–
Severe combined immunodeficiency
–
Adenosin deaminase and purine nucleoside
phosphorylase
–
2- gene therapy for cancer
–
3-gene therapy for cardiovascular diseases

28. Diseases
⚫
Acquired Inherited

29. Diseases
⚫
Genetic diseases:
⚫
Type 1: Single locus (gene) is defective and responsible for
⚫
the disease, 100% heritable.
⚫
:
Sickle cell anemia,
⚫
Hypercholesterolemia
⚫
Cystic fibrosis
⚫
Type 2: Polygenic traits, <100% heritable, may be dependent
⚫
on environmental factors and lifestyle.
⚫
Heart disease
⚫
Cancer
⚫
Diabetes
⚫
Alcoholism
⚫
Schizophrenia
⚫
Criminal behavior

30. QUESTIONS

31. What is Gene Therapy
⚫
It is a technique for correcting
defective genes that are responsible
for disease development
⚫
There are four approaches:
1
.
1-A normal gene inserted to
compensate for a nonfunctional gene.

32. techniques
1
.
2-An abnormal gene traded for a
normal gene
2
.
3- An abnormal gene repaired through
selective reverse mutation
3
.
-Change the regulation of gene pairs
4
-
-
3

33. ⚫
Insertion of genes into individual’s cell
⚫
Or
Tissue
To treat a disease
Hereditary disease in particular

34. Gene Therapy mainly aim to
⚫
Supplement a defective mutant allele
⚫
With a functional one

35. ⚫
Can be divided by two categories
Diseases like parkinsonism
AIDS
Diabetes

36. Gene therapy for AIDS
⚫
Gene therapy for infectious diseases
⚫
Hepatitis Papiloma herpes
⚫
Gene therapy for rheumatoid arthritis
⚫
Gene therapy neurological diseases

37. vectors in gene therapy

38. Ideal vector characteristics:
1. Insert size: one or more genes.
2. Targeted: limited to a cell type.
3. No immune response.
4. Stable: not mutated.
5. Production: easy to produce high
concentrations (titer).
6. Regulatable: produce enough protein to
cause an effect.

39. How
–
Doctors removed her white blood cells, inserted the
missing gene into the WBC, and then put them back into
her blood stream.
–
This strengthened her immune system
–
Only worked for a few months 

40. Tell The story
–
1990s - 1st successful gene therapy protocol
–
(William Anderson, Michael Blaise, and Ken Culver)
–
Treatment of ADA (adenosine deaminase) deficiency-
–
causes severe immune deficiency. Recessive disease
that
–
results in the buildup of waste products that kill T-
cells.
–
*Used retrovirus to infect T-cells and produce ADA.

41. Choices of Vectors
⚫
The ideal vector system would have the following characteristics:
⚫
(1) an adequate carrying capacity;
⚫
(2) to be undetectable by the immune system;
⚫
(3) to be non-inflammatory;
⚫
(4) to be safe to the patients with pre-existing lung inflammation;
⚫
(5) to have an efficiency sufficient to correct the cystic fibrosis
phenotype;
⚫
(6) to have long duration of expression and/or the ability to be
safely re-administered.
⚫
Viral vectors:
⚫
Vetrovirus
⚫
Adenovirus
⚫
Adeno-associated virus
⚫
Herpes Simplex Virus
Non-viral vectors:
Liposome
DNA–polymer conjugates
Naked DNA

42. Carrier molecules designed specifically
to enter cells & deposit therapeutic genes
Vectors can be viral or non-viral
Vectors

43. METHODS OF VECTOR DELIVERY

44. Germ line gene therapy
Somatic cell gene therapy
Gene augmentation
Gene replacement
Specific inhibition of gene expression
Targeted cell death
Gene therapy targets

45. Gene augmentation
most therapies simply add a useful gene into a selected cell type to
compensate for the missing or flawed version. Useful in treating loss of
function mutations such as Tumour Genes

46. Specific inhibition of gene expression
Involves silencing of specific genes like activated
oncogenes, by using molecules that degrade
RNA transcripts.
Strategies include
Antisense therapy
siRNA (small interfering RNA)
Ribozymes etc

47. Gene replacement
This strategy replaces the mutant copy with a correctly
functioning copy in situ. Useful for gain of function
mutations such as oncogenes

48. Antisense therapy
short stretches of
synthetic ssDNA
that target the
mRNA transcripts
of abnormal
proteins
preventing its
translation

49. siRNA therapy
Small interfering RNAs
short stretches (21-23nt) of
synthetic ds RNA
Has 3’ overhangs of 2 nt
Incorporates into RISC (RNA
induced silencing complex)
Target mRNA cleaved in the
middle

50. Targeted cell death

51. Targeted cell death

52. Catalytic RNAs that cleave target
mRNAs in a sequence-specific
manner
e.g. hammerhead ribozymes are
engineered to recognise specific
sequences and made resistant to
nucleases
Ribozymes

53. Viral vector strategy
Replication & virulence genes can
be substituted with therapeutic genes

54. designed to enter cell and deposit genes
Special vectors are constructed by
deleting or altering native sequence in
retroviral and lentiviral vectors, to
prevent the generation of replication
competent retroviruses (RCR)
typically caused by homologous
recombination
Retroviral vectors

55. Minimal HIV vector plasmid

56. Consist of
(1) consisting of the CMV/HIV LTR hybrid promoter
followed by the packaging signal (), the rev-binding
element RRE for cytoplasmic export of the RNA, the
transgene expression cassette consisting of internal
promoter(s) and transgenes'), and the 3' self-
inactivating (SIN) LTR. All genes coding for
enzymatic or structural HIV proteins have been
removed. Together with the HIV vector plasmid (1),
the HIV packaging plasmid (2), HIV rev (3), and an
envelope expressing plasmid (4) are needed for HIV
vector production.

57. Packaging retroviral vectors
Gag, pol and env genes on physically separate fragments without sequence
Recombinant viral proteins are infective but replication-deficient

58. Retroviral vectors
Advantages
1) long-term expression
2) low toxicity
3) high capacity
4) low antivectorial immunity allowing repeat administration
Problems
Lack of cell specificity:
Promiscuous: depositing genes into several cell types
resulting in reduced target efficiency and unwanted physiological effects
Random splicing into host DNA resulting
in normal gene disruption and/or alteration in gene function

59. Retroviruses
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

60. Retrovirus life cycle

61. Retrovirus life cycle
From: Alberts et al. Molecular Biology of the Cell

62. Retrovirus genome
5’ LTR Packaging Gene X Neor 3’ LTR
Encapsulation
(packaging)
Retrovirus vector construction for gene therapy

63. Gene therapy by injection of retrovirally transduced
autologous CD34+ hematopoietic stem cells (HSCs).

64. Disadvantage
insertional mutagenesis near the proto-oncogene LMO2
promoter (Science, 302:415-419, October 17, 2003)
2/11 X-SCID patients developed leukemia

65. Adenoviral vectors
do not insert into genome
temporary
lack of specificity
strong immune response

66. Adeno-associated viral vectors
Nature Reviews Genetics
1; 91-99 (2000);
Integrate into
genome but
small in size

67. Other virus used
Herpes simplex Delta hepatitis virus Pox virus

68. 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.
Aden-associated Viruses

69. Advantages
non-toxic
no immune response
Non-viral Vectors

70. Liposome delivery

71. liposome

72. liposome

73. Tumour-suppressor gene delivery

74. Conditional replicating virus

75. Non-viral Vectors
Receptor-mediated endocytosis

76. naked DNA
artificial human chromosomes
Non-viral Vectors Gene gun

77. Gene therapy in cancer
Based on
http://www.
wiley.co.uk/genetherapy
/clinical/

78. Conditionally replicating viruses
Replication of
a conditionally
replicating
virus, ONYX-
015, in a
cancer cell
from a patient
with head and
neck cancer
during Phase
II clinical
testing.

79. Current status
Food and Drug Administration
(FDA) has not yet approved
any human gene therapy product for sale

80. ⚫

81. Trial result
⚫
the procedure is not a cure. The
genetically altered white blood cells
work only temporarily. Ashanti gets
annual routine checkups at the NIH
and the procedure has been repeated
about a half-dozen times
.

82. Culver says
–
Genetics can be more than we probably can
even imagine today.
Genetics is basically the new microscope
for looking at human disease

83. The First Case
The first gene therapy was performed on
September 14th, 1990
Ashanti DeSilva was treated for SCID
Sever combined immunodeficiency

84. Time
14 September The Hero of the story
Genetics is basically the
new microscope for looking
at human disease
Ken Culver

85. Severe Combined Immunodeficiency
(SCID)
Rare condition caused by the lack
or reduction in the immune system
(‘bubble baby syndrome’)
Patients cannot make T lymphocytes and their
B lymphocytes fail to make essential antibodies for fighting infections.
Gene therapy in X-SCID patients
X-SCID caused by mutations in the X-linked gene
IL2RG, which encodes the common gamma chain
(gc) of the lymphocyte receptors for interleukin-2
(IL-2) and many other cytokines

86. Viral vector

87. –
On Sept. 14, 1990
–
Dr. Ken Culver,
–
a pediatrician and Presbyterian elder
–
joined a team at the NIH to conduct the first approved
"gene therapy" procedure on 4-year-old Ashanti.
.

88. trial
–
In Ashanti's procedure Culver and his team
–
removed white blood cells from her body,
–
let the cells grow in the lab
–
, inserted the missing gene in them
–
and then infused the genetically modified cells back into
Ashanti's bloodstream.
–
The treatment strengthened Ashanti's immune system.

89. DeSilva
–
She takes
–
singing
–
and piano lessons
–
and
–
lives a relatively normal life

90. The hero this time

91. ⚫
Ornithine transcarbomaylase deficiency
often becomes evident in the first few
days of life

92. Ornithin transcarbamylase
defeciency
⚫
Mutations in the
OTC
mutated gene
cause ornithine transcarbamylase
deficiency
Ornithine transcarbamylase
deficiency belongs to a class of genetic
the enzyme that starts a specific reaction within the urea cycle
is damaged or missing

93. What is the matter
–
The urea cycle cannot proceed normally, and nitrogen
accumulates in the bloodstream
–
in the form of ammonia.
–
Ammonia is especially damaging to
–
Nervous system

94. treatment
⚫
.
a low-protein formula called keto-acid
a low-protein formula called keto-acid and Na
Ornithine transcarbamylase deficiency is
believed to occur in approximately 1 in every 80,000 people.

95. cause
⚫
Mutations in the genes cause
OTC
gene cause
ornithine transcarbamylase deficiency

96. –
Ornithine transcarbamylase deficiency is an X-linked disorder.
–
A condition is considered X-linked if the mutated gene that causes
the disorder is located on the X chromosome
–
one of the two sex chromosomes.
–
A striking characteristic of X-linked inheritance is that fathers
cannot pass X-linked traits to their sons
.
–
.

97. male
–
In males (who have only one X
chromosome), one altered copy of the
gene in each cell is sufficient to cause the
condition.
.

98. female
–
In females (who have two X chromosomes),
mutations in both copies of the gene will cause the
disorder. Some females with only one altered copy
of the OTC gene also show signs and symptoms
of ornithine transcarbamylase deficiency

99. Not inherited
⚫
Gelsinger did not inherit the disease;
it was the result of a genetic
mutation and as such was not as
severe

100. trial
⚫
On Monday, September 13 1999,
Gelsinger was injected with
adenoviruses
carrying a corrected gene in the hope that it would manufacture
the needed enzyme

101. He is dead
⚫
He died four days later, apparently
having suffered a massive immune
response
September 17th at 2:30

102. ⚫
Food and Drug Administration
FDA
investigation concluded that the
scientists involved in the trial,
including the lead researcher Dr.
James M. Wilson (U Penn), broke
several rules of conduct
:

103. Number 1
⚫
Inclusion of Gelsinger as a substitute
for another volunteer who dropped
out, despite having high ammonia
levels that should have led to his
exclusion from the trial

104. 2-
⚫
Failure by the university to report
that two patients had experienced
serious side effects from the gene
therapy

105. 3-
⚫
Failure to mention the deaths of
monkeys given a similar treatment in
the informed consent
documentation
.

106. Jesse Gelsinger
⚫
The Gel singer case was a severe
setback for scientists working in the
field
.

107. What is in The future
⚫
We need more knowledge about
⚫
Gene function
⚫
Enteron
⚫
Exon

108. We need to advance our weapon
⚫
Safe vehicle
⚫
Sequence specific vehicle
⚫
Reducing immune response
⚫
Improve risk assesment