This document discusses gene therapy, including its strategies, methods of delivery, history, and applications. It provides an overview of key concepts such as: 1. Gene therapy aims to treat genetic diseases by inserting normal genes into cells to compensate for abnormal genes. Strategies include gene replacement, gene augmentation, and gene inhibition. 2. Viruses are commonly used as vectors to deliver therapeutic genes. Retroviruses and adenoviruses integrate into the genome but can cause mutations, while adenoviruses are safer but less efficient. 3. The basic process involves isolating the normal gene, inserting it into a viral vector, infecting target cells, and having the cells produce functional proteins to return to

1. Gene Therapy
Dr.Nawfal H.Aldujaili

2. Genes
o The basic unit of heredity
o Are carried on a chromosome
o Encode how to make a protein DNARNA proteins
o Proteins carry out most of life’s function.
o When altered causes dysfunction of a protein
o 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

3. Various methods for treating
genetic disease

4. Various methods for treating genetic disease

5. What is Gene Therapy
• It is a technique for The correction of a genetic deficiency
in a cell by the addition of new DNA to the cell. This
definition has been expanded to include treatments of
acquired diseases by the addition of new DNA.
• There are four approaches:
1. A normal gene inserted to compensate for a
nonfunctional gene.
2. An abnormal gene traded for a normal gene
3. An abnormal gene repaired through selective reverse
mutation
4. Change the regulation of gene pairs

6. Gene Therapy Strategies
• Gene Augmentation Therapy (GAT)
• Gene inhibition therapy.
• Gene replacement therapy
• Gene Correction (Chimeraplasty)
• Targeted killing of specific cells
• Prodrug therapy

7. Gene Therapy Strategies

8. Gene Therapy Strategies

10. DNA Delivery in gene therapy
• (safety, efficacy, and specific maintenance )
• Viral vector : About 70% of human gene therapy trials
have used viral vectors. eg Adenoviruses , adeno-
associated virus (AAV), Retroviruses
 Nonviral delivery in gene therapy

12. Nonviral delivery in gene therapy
 Naked nucleic acid (DNA or less often RNA). Many animal
cells can be transformed directly with purified DNA. The
therapeutic gene may be inserted into a plasmid and the
plasmid DNA used directly. Some 10% to 20% of gene therapy
trials have used unprotected nucleic acid.
 Particle. DNA is fired through the cell walls and membranes
on metal particles. This method was originally developed to get
DNA into plants,However, it has also been used to make
transgenic animals and is occasionally used for humans.
 Receptor-mediated uptake. DNA is attached to a protein that is
bombardmentrecognized by a cell surface receptor. When the
protein enters the cell, the DNA is taken in with it.

13.  Polymer-complexed DNA. Binding to a positively charged
polymer, such as polyethyleneimine, protects the negatively
charged DNA. Such complexes are often taken up by cells in
culture and may in principle be used for ex vivo gene therapy.
 Encapsulated cells. Whole cells engineered to express and
secrete a needed protein may be encapsulated in a porous
polymeric coat and injected locally. Foreign cells excreting
nerve growth factor have been injected into the brains of aging
rats. The rats showed some improvement in cognitive ability,
suggesting that this approach may be of value in treating
conditions such as Alzheimer’s disease.
 Liposomes are spherical vesicles composed of phospholipid.
They have been used in around 10% of gene therapy trials

14. Risks of Nonviral Vectors for Gene Therapy
 insertion mutagenesis could activate oncogenes or
inhibit tumor suppressor genes if the plasmid
integrates
 the compounds that are used to facilitate the entry
of DNA into a cell might have some toxicity
Major advantage of using nonviral vectors is the lack of
risk of generating a wild-type virus via
recombination. In addition, episomal plasmids do
not pose the risk of insertional mutagenesis since
they do not integrate into the chromosome

15. Two Types of Gene Therapy
• Somatic gene therapy involves
introducing a “good “ gene into
targeted cells with the end results of
treating the patient-not the future
children
• Germline gene therapy involves
modifying the genes in egg or sperm
cells, which will then pass any genetic
changes to future generations as well

18. Application of gene therapy
• Cancers
• Inherited disorders
• Infectious diseases (viral or bacterial)
• Immune system disorders
• Vaccination
CancerCancerAIDSAIDSDiabetesDiabetes
Parkinson’Parkinson’
s Diseases Disease

20. Gene therapy and cancer
Most cancer result from
• Activation of an oncogene that leads to tumor
formation
• Inactivation of gene that normally suppresses
formation of a tumor

21. Cancer approaches using gene
therapy
• Delivery of genes encoding toxic molecules to cancer cells to kill
them
• Delivery of genes encoding chemokines to cancer cells to activate the
immune response to recognize and kill them
• Antibody therapies: DNA vaccines with genes that encode antibodies
to cancer specific proteins in tumor cells
• Insertion of normal tumor suppressor genes into cells
• Antisense therapy: DNA that blocks synthesis of proteins encoded by
deleterious genes

22. 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

23. What is required for
Gene Therapy to be possible?
• Understanding of the disease process
• Structure/function of gene to be introduced
• Efficient delivery of gene
• Control of gene expression
• Prevention/control of immune responses
• Animal model and assessment of function
• Clinical trial

24. General Steps of Human Gene Therapy
1. Identification and characterization of gene
2. Cloning of gene
3. Choice of vector (Insert normal allele into
vector)
4. Method of delivery

25. Thanks for
your attention

34. Brief History
• 1967- Nobelist Marshall Nirenberg wrote of programming cells.
• 1974- National Institute of Health took lead in recombinant DNA (rDNA)
research regulation.
• 1980-Dr. Martin Cline performs first DNA transfer into bone marrow cells.
• 1984- US Office of Technology Assessment stressed difference between
somatic and germ-line therapy
• 1990- NIH performed first approved gene therapy procedure
• 1999- Jesse Gelsinger becomes first fatality in gene therapy.
• 2003- FDA placed a temporary halt on all gene therapy trials using retroviral
vectors in blood stem cells
• 2005- 637 GT clinical trials (3464 patients)

38. Basic Strategies of Human Gene Therapy
1. Isolate and then clone the normal allele by PCR
2. Insert normal allele into a disabled virus
– Retroviruses and adenoviruses are the most common vectors
– Retroviruses are much more efficient at forming a provirus,
but have a greater chance of mutating to cause disease
– Adenoviruses are safer, but are relatively inefficient as a
vector
– Liposomes (lipid spheres) are also used as vectors
 e.g. Gene therapy for Cystic Fibrosis involves using an
inhaler to bring liposomes containing the CFTR gene to
the cells lining the lungs)

39. 3. Infect host cells with recombinant virus
a. Add recombinant virus directly to individual
e.g. Jesse Gelsinger—
 Had Ornithine Transcarbamylase Deficiency;
Causes build up of ammonia in liver cells
since they cannot convert the ammonia (toxic)
produced by amino acid metabolism to urea
(less toxic)
 Died in Sept.’99 due to a severe immune
response to the genetically modified
adenovirus containing the OTC gene
b. Isolate host cells from body and then add
recombinant virus (e.g. blood stem cells in gene
therapy for Gaucher disease)
• Inject genetically engineered cells back into the
body
Basic Strategies of Human Gene Therapy

42. How It Works
• A vector delivers the therapeutic gene into a
patient’s target cell
• The target cells become infected with the viral
vector
• The vector’s genetic material is inserted into the
target cell
• Functional proteins are created from the
therapeutic gene causing the cell to return to a
normal state

44. The first clear enunciation of the concept of exploiting
antisense compounds as therapeutic agents was in the
work of Zamecnik and Stephenson in 1978.
A-A-T-G-G-T-A-A-A-A-T-G-G
The revolution in the availability of viral and human
genomic sequences enhanced the development of the
antisense technology.
Over the past decade, substantial development in
antisense science and manufacturing led to the approval
of the first antisense drug fomivirsen (VitraveneTM
) for
the treatment of AIDS-related CMV retinitis.
History

45. Fomivirsen (VitraveneTM
)
In the meantime up to 50 new antisense compounds have
entered phase I/II, and in some cases phase III trials.

46. Gene Therapy Strategies
• Gene replacement
• Gene Augmentation Therapy (GAT)
• Gene Correction (Chimeraplasty)
• Targeted killing of specific cells
• Targeted inhibition of gene expression
(Gene ablation)

47. 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

48. Viral vectors
• Retroviruses
– eg Moloney murine leukaemia
virus
(Mo-MuLV)
– Lentiviruses (eg HIV, SIV)
• Adenoviruses
• Herpes simplex
• Adeno-associated
viruses (AAV)

49. 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

50. Adenoviruses
• 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

51. Adenovirus cont.
http://en.wikipedia.org/wiki/Gene_therapy

52. Adeno-associated Viruses
• 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

53. Herpes Simplex Viruses
• Double stranded DNA viruses that
infect neurons
• Ex. Herpes simplex virus type 1
http://www.ucmp.berkeley.edu/alllife/virus.html

55. Advantages
&Disadvantage
• Scientists are trying to
– Manipulate the viral genome to remove the disease-causing genes and
introduce therapeutic genes.
• Viruses introduce potential other problems in the body, such as:
– Toxicity
– Immune and inflammatory responses
– Gene control and targeting issues
– Capacity

56. Non-Viral methods
• Cationic Lipids (Liposomes)
• Cationic Polymers
• Retrotransposons (jumping genes)
• Human artificial chromosomes
(HACs)

57. Advantages
&Disadvantage
• Advantages
– Low toxicity
– Nonimmunogenicity
– Easy synthesis
• Disadvantage
– The overall transfection pathways are
inefficient

58. 58
Major barriers to DNA
delivery
• Low uptake across the plasma
membrane
• Inadequate release of DNA with
limited stability
• Lack of nuclear targeting

59. 59
Schematic drawing of barriers

60. Medicinal Chemistry of Antisense OligonucleotidesMedicinal Chemistry of Antisense Oligonucleotides
One of the major challenges for antisense
approaches is the stabilization of oligonucleotides,
as unmodified oligodeoxynucleotides are rapidly
degraded in biological fluids by nucleases.

61. 1.1.Functional Genomics and Target Validation:Functional Genomics and Target Validation:
Antisense oligonucleotides can be used to
selectively manipulate the expression of chosen
gene or genes. The process results in :
– A pharmacophore with a well-understood
mechanism of action.
– Well characterized distribution and a safe side
effect profile which could be used as a human
therapeutic.

62. Clinical Trials of Antisense OligonucleotidesClinical Trials of Antisense Oligonucleotides
– To date, one antisense oligonucleotide (fomivirsen) has been
approved by the FDA for local administration to treat CMV
retinitis.
– In 1996, only a handful of antisense molecules was in clinical
trials. However, the past few years has seen explosive growth
in the number of antisense- related clinical trials. Currently,
there are near to 50 antisense compounds in trials for various
diseases, up to 10 of which are in phase III, with an additional
20 in Phase II.

63. Unsuccessful Gene therapies
• Jesse Gelsinger, a gene therapy patient who lacked ornithine
transcarbamylase activity, died in 1999.
• Within hours after doctors shot the normal OTC gene attached
to a therapeutic virus into his liver, Jesse developed a high
fever. His immune system began raging out of control, his
blood began clotting, ammonia levels climbed, his liver
hemorrhaged and a flood of white blood cells shut down his
lungs.
• One problem with gene therapy is that one does not have
control over where the gene will be inserted into the genome.
The location of a gene in the genome is of importance for the
degree of expression of the gene and for the regulation of the
gene (the so-called "position effect"), and thus the gene
regulatory aspects are always uncertain after gene therapy

64. Successful Gene Therapy for Severe
Combine Immunodeficiency
• Infants with severe combined immunodeficiency are
unable to mount an adaptive immune response,
because they have a profound deficiency of
lymphocytes.
• severe combined immunodeficiency is inherited as
an X-linked recessive disease, which for all practical
purposes affects only boys. In the other half of the
patients with severe combined immunodeficiency,
the inheritance is autosomal recessive — and there
are several abnormalities in the immune system
when the defective gene is encoded on an autosome.

65. Severe Combine
Immunodeficiency Continued
• A previous attempt at gene therapy for
immunodeficiency was successful in children with
severe combined immunodeficiency due to a
deficiency of adenosine deaminase. In these
patients, peripheral T cells were transduced with a
vector bearing the gene for adenosine deaminase.
The experiment was extremely labor intensive,
because mature peripheral-blood T cells were
modified rather than stem cells, and the procedure
therefore had to be repeated many times to achieve
success.

66. Successful One Year Gene Therapy
Trial For Parkinson's Disease
• Neurologix a biotech company announced that they
have successfully completed its landmark Phase I
trial of gene therapy for Parkinson's Disease.
• This was a 12 patient study with four patients in
each of three dose escalating cohorts. All procedures
were performed under local anesthesia and all 12
patients were discharged from the hospital within 48
hours of the procedure, and followed for 12 months.
Primary outcomes of the study design, safety and
tolerability, were successfully met. There were no
adverse events reported relating to the treatment.

67. Parkinson's Disease Cont.
• The gene transfer procedure utilized the
AAV (adeno-associated virus) vector, a virus
that has been used safely in a variety of
clinical gene therapy trials, and the vehicle
that will be used in all of the company's first
generation products, including epilepsy and
Huntington's disease. In its Parkinson's
disease trial, Neurologix used its gene
transfer technology.

68. Recent Developments
• Genes get into brain using liposomes coated in
polymer call polyethylene glycol
– potential for treating Parkinson’s disease
• RNA interference or gene silencing to treat
Huntington’s
– siRNAs used to degrade RNA of particular sequence
– abnormal protein wont be produced
• Create tiny liposomes that can carry therapeutic
DNA through pores of nuclear membrane
• Sickle cell successfully treated in mice

69. 69
How to overcome barriers