This document discusses various immunosuppressant drugs and their mechanisms of action. It begins by describing the immune system and cytokines. It then discusses several classes of immunosuppressant drugs including inhibitors of cytokine production/action like cyclosporine and tacrolimus, inhibitors of cytokine gene expression like corticosteroids, cytotoxic drugs like azathioprine and mycophenolate mofetil, antibodies that block T cell surface molecules, and others like sirolimus. It provides details on the mechanisms, pharmacokinetics, uses, and adverse effects of these immunosuppressant drugs.

1. IMMUNOSUPPRESSANT DRUGS AND
GENE THERAPY
Dr. Rupendra Bharti
Assist. Professor
Department of Pharmacology

2. Immune system
• Is designed to protect the host from harmful foreign
molecules.
• Allograft introduction can elicit a damaging
immune response.
• Immune system include two main arms
1) Cell –mediated immunity.
2) Humoral (antibody –mediated immunity).

4. Cytokines
• Cytokines are soluble, antigen-nonspecific
signaling proteins that bind to cell surface
receptors on a variety of cells.
• Cytokines include
– Interleukins,
– Interferons (IFNs),
– Tumor Necrosis Factors (TNFs),
– Transforming Growth Factors (TGFs)
– Colony-stimulating factors (CSFs).

5. • IL-2 stimulates the proliferation of antigen-primed
(helper) T cells.
Cell-mediated Immunity
• TH1 produce more IL-2, TNF-β and IFN-γ.
• Activate
– NK cells (kill tumor & virus-infected cells).
– Cytotoxic T cells (kill tumor & virus-infected cells).
– Macrophages (kill bacteria).

6. Cell-mediated Immunity

7. Humoral Immunity
B-lymphocytes TH2 produces (interleukins)
IL-4 & IL-5 which in turn causes:
• B cells proliferates & differentiates into
– memory B cells
– Antibody secreting plasma cells

8. Humoral Immunity

9. Mutual regulation of T helper lymphocytes
• TH1 interferon-γ:
inhibits TH2 cell proliferation
• TH2 IL-10:
inhibits TH1 cytokine production

10. IMMUNOSUPPRESSANT DRUGS
I. Inhibitors of cytokine (IL-2) production or
action:
1) Calcineurin inhibitors
• Cyclosporine
• Tacrolimus (FK506)
2) Sirolimus (rapamycin).
II. Inhibitors of cytokine gene expression
– Corticosteroids

11. IMMUNOSUPPRESSANT DRUGS CONT..
III. Cytotoxic drugs
 Inhibitors of purine or pyrimidine synthesis
(Antimetabolites):
– Azathioprine
– Myclophenolate Mofetil
– Leflunomide
– Methotrexate
 Alkylating agents
Cyclophosphamide

12. IMMUNOSUPPRESSANT DRUGS CONTD…
IV. Immunosuppressive antibodies
that block T cell surface molecules involved
in signaling immunoglobulins
– antilymphocyte globulins (ALG).
– antithymocyte globulins (ATG).
– Rho (D) immunoglobulin.
– Basiliximab
– Daclizumab
– Muromonab-CD3
V. Interferon
VI. Thalidomide

13. CYCLOSPORINE
Chemistry
Cyclosporine is a fungal polypeptide composed of 11
amino acids.
Mechanism of action
– Acts by blocking activation of T cells by inhibiting
interleukin-2 production (IL-2).
– Decreases proliferation and differentiation of T-
cells.

14. – Cyclosporine binds to cyclophilin (immunophilin)
intracellular protein receptors .
– Cyclosporine- immunophilin complex inhibits
calcineurin, a phosphatase necessary for
dephosphorylation of transcription factor (NFATc)
required for interleukins synthesis (IL-2).
– NFATc (Nuclear Factor of Activated Tcells).
– Suppresses cell-mediated immunity.
CYCLOSPORINE CONTD..

16. Pharmacokinetics of cyclosporine:
– Can be given orally or i.v. infusion
– Orally (25 or 100 mg) soft gelatin capsules, microemulsion.
– Orally, it is slowly and incompletely absorbed.
– Peak levels is reached after 1– 4 hours, elimination half life
24 h.
– Oral absorption is delayed by fatty meal (gelatin capsule
formulation)
– Microemulsion
( has higher bioavailability-is not affected by food).

17. • 50 – 60% of cyclosporine accumulates in blood
(erythrocytes – lymphocytes).
– Metabolized by CYT-P450 system (CYP3A4).
– Excreted mainly through bile into faeces, about
6% is excreted in urine.

18. Therapeutic Uses of Cyclosporine
– Organ transplantation (kidney, liver, heart)
either alone or with other immunosuppressive
agents (Corticosteroids).
– Autoimmune disorders (low dose 7.5 mg/kg/d).
e.g. endogenous uveitis, rheumatoid arthritis,
active Crohn’s disease, psoriasis, psoriasis,
nephrotic syndrome, severe corticosteroid-
dependent asthma, early type I diabetes.
– Graft-versus-host disease after stem cell
transplants

19. Adverse Effects of cyclosporine (Dose-
dependent)
Therapeutic monitoring is essential
– Nephrotoxicity (increased by NSAIDs and
aminoglycosides).
– Liver dysfunction.
– Hypertension, hyperkalemia.
(K-sparing diuretics should not be used).
– Hyperglycemia.
– Viral infections (Herpes - cytomegalovirus).

20. – Lymphoma (Predispose recipients to cancer).
– Hirsutism
– Neurotoxicity (tremor).
– Gum hyperplasia.
– Anaphylaxis after I.V.
Adverse Effects of Cyclosporine (Dose-dependent) cont..

21. Cyclosporine Drug Interactions
• Clearance of cyclosporine is enhanced by co-
administration of CYT p450 inducers
(Phenobarbitone, Phenytoin & Rifampin ) → rejection
of transplant.
• Clearance of cyclosporine is decreased when it is co-
administered with erythromycin or Ketoconazole,
Grapefruit juice → cyclosporine toxicity.

22. TACROLIMUS (FK506)
• A fungal macrolide antibiotic.
• Chemically not related to cyclosporine
• Both drugs have similar mechanism of action.
• The internal receptor for tacrolimus is immunophilin
( FK-binding protein, FK-BP).
• Tacrolimus-FKBP complex inhibits calcineurin.

24. Kinetics of Tacrolimus
• Given orally or i.v or topically (ointment).
• Oral absorption is variable and incomplete, reduced
by fat and carbohydrate meals.
• Half-life after I.V. form is 9-12 hours.
• Highly bound with serum proteins and concentrated
in erythrocytes.
• metabolized by P450 in liver.
• Excreted mainly in bile and minimally in urine.

25. USES as cyclosporine
• Organ and stem cell transplantation
• Prevention of rejection of liver and kidney
transplants (with glucocorticoids).
• Atopic dermatitis and psoriasis (topically).

26. Toxic effects
• Nephrotoxicity (more than CsA)
• Neurotoxicity (more than CsA)
• Hyperglycemia ( require insulin).
• GIT disturbances
• Hperkalemia
• Hypertension
• Anaphylaxis
NO hirsutism or gum hyperplasia
• Drug interactions as cyclosporine.

27. What are the differences between CsA and
TAC ?
TAC is more favorable than CsA due to:
• TAC is 10 – 100 times more potent than CsA in
inhibiting immune responses.
• TAC has decreased episodes of rejection.
• TAC is combined with lower doses of glucocorticoids.
But
• TAC is more nephrotoxic and neurotoxic.

28. Sirolimus (Rapamycin)
• SRL is macrolide antibiotic.
• SRL is derived from fungus.
• It binds to FKBP and the formed complex binds to
mTOR (mammalian Target Of Rapamycin).
• mTOR is serine-threonine kinase essential for cell
cycle progression, DNA repairs, protein translation.

29. • SRL blocks the prog Sirolimus (Rapamycin)
ression of activated T cells from G1 to S phase of cell
cycle (Antiproliferative action).
• It Does not block IL-2 production but blocks T cell
response to cytokines.
• Inhibits B-cell proliferation & immunoglobulin
production.
Sirolimus (Rapamycin) Contd..

31. Pharmacokinetics of Sirolimus
• Given orally and topically, reduced by fat meal.
• Extensively bound to plasma proteins
• metabolized by CYP3A4 in liver.
• Excreted in feces.
Pharmacodynamics
• Immunosuppressive effects
• Anti- proliferative action.
• Equipotent to CsA.

32. USES OF SIROLIMUS
• Solid organ allograft
• Renal transplantation alone or combined with (CSA,
tacrolimus, steroids, mycophenolate).
• Heart allografts
• In halting graft vascular disease.
• Hematopoietic stem cell transplant recipients.
• Topically with cyclosporine in uveoretinitis.
• Synergistic action with CsA

33. Toxic effects Sirolimus
• Hyperlipidaemia (cholesterol, triglycerides).
• Thrombocytopenia
• Leukopenia
• Hepatotoxicity
• Hypertension
• GIT dysfunction

34. Inhibitors of cytokine gene expression
Corticosteroids
– Prednisone
– Prednisolone
– Methylprednisolone
– Dexamethasone
They have both anti-inflammatory action
and immunosuppressant effects.

35. Mechanism of action
– Bind to glucocorticoid receptors and the complex interacts
with DNA to inhibit gene transcription of inflammatory
genes.
– Decrease production of inflammatory mediators as
prostaglandins, leukotrienes, histamine, PAF, bradykinin.
– Decrease production of cytokines IL-1, IL-2, interferon,
TNF.
– Stabilize lysosomal membranes.

36. – Decrease generation of IgG, nitric oxide and histamine.
– Inhibit antigen processing by macrophages.
– Suppress T-cell helper function.
– decrease T lymphocyte proliferation.
Mechanism of action

37. Kinetics
Can be given orally or parenterally.
Dynamics
1. Suppression of response to infection
2. Anti-inflammatory and immunosuppressant.
3. Metabolic effects.

38. Indications
– First line therapy for solid organ allografts &
haematopoietic stem cell transplantation.
– Autoimmune diseases as refractory rheumatoid
arthritis, systemic lupus erythematosus (SLE),
asthma.
– Acute or chronic rejection of solid organ
allografts.

39. Adverse Effects
– Adrenal suppression
– Osteoporosis, osteonecrosis
– Hypercholesterolemia
– Hyperglycemia
– Hypertension
– Cataract
– Glaucoma
– Infection
– HPA suppression

40. Cytotoxic drugs
 Inhibitors of purine or pyrimidine synthesis
(Antimetabolites):
– Azathioprine
– Myclophenolate Mofetil
– Leflunomide
– Methotrexate
 Alkylating agents
Cyclophosphamide

41. AZATHIOPRINE
CHEMISTRY:
– Derivative of mercaptopurine.
– Prodrug.
– Cleaved to 6-mercaptopurine then to 6-
mercaptopurine nucleotide, thioinosinic acid
(nucleotide analog).
– Inhibits de novo synthesis of purines required for
lymphocytes proliferation.
– Prevents clonal expansion of both B and T
lymphocytes.

43. Pharmacokinetics
– Orally or intravenously.
– Widely distributed but does not cross BBB.
– Metabolized in the liver to 6-mercaptopurine or
to thiouric acid (inactive metabolite) by
xanthine oxidase.
– excreted primarily in urine.
Drug Interactions:
– Co-administration of allopurinol with
azathioprine may lead to toxicity due to
inhibition of xanthine oxidase by allopurinol.

44. USES OF AZATHIOPRINE
• Acute glomerulonephritis
• Systemic lupus erythematosus
• Rheumatoid arthritis
• Crohn’ s disease.

45. Adverse Effects of Azathioprine
• Bone marrow depression: leukopenia,
• Thrombocytopenia.
• Gastrointestinal toxicity.
• Hepatotoxicity.
• Increased risk of infections.

46. MYCOPHENOLATE MOFETIL
– Is a semisynthetic derivative of mycophenolic
acid from fungus source.
– Prodrug; is hydrolyzed to mycophenolic acid.
Mechanism of action:
– Inhibits de novo synthesis of purines.
– mycophenolic acid is a potent inhibitor of inosine
monophosphate dehydrogenase (IMP), crucial for
purine synthesis →deprivation of proliferating
T- and B-cells of nucleic acids.

48. Pharmacokinetics:
– Given orally, i.v. or i.m.
– rapidly and completely absorbed after oral
administration.
– It undergoes first-pass metabolism to give the
active moiety, mycophenolic acid (MPA).
– MPA is extensively bound to plasma protein.
– metabolized in the liver by glucuronidation.
– Excreted in urine as glucuronide conjugate
– Dose : 2-3 g/d

49. CLINICAL USE OF MYCOPHENOLATE MOFETIL
– Solid organ transplants for refractory rejection.
– Steroid-refractory hematopoietic stem cell
transplant patients.
– Combined with prednisone as alternative to CSA
or tacrolimus.
– Rheumatoid arthritis, & dermatologic disorders.

50. ADVERSE EFFECTS MYCOPHENOLATE MOFETIL
– GIT toxicity: Nausea, Vomiting, diarrhea,
abdominal pain.
– Leukopenia, neutropenia.
– Lymphoma
Contraindicated during pregnancy

51. LEFLUNOMIDE
 A prodrug
 Active metabolite undergoes enterohepatic
circulation.
 Has long duration of action.
 Can be given orally
 Antimetabolite immunosuppressant.
 Pyrimidine synthesis inhibitor
 Approved only for rheumatoid arthritis

52. LEFLUNOMIDE
Adverse effects
1. Elevation of liver enzymes
2. Renal impairment
3. Teratogenicity
4. Cardiovascular effects (tachycardia).

53. Methotrexate
– Folic acid antagonist
– Orally, parenterally (I.V., I.M).
– Excreted in urine.
– Inhibits dihydrofolate reductase required for folic
acid activation (tetrahydrofolic)
– Inhibition of DNA, RNA &protein synthesis
– Interferes with T-cell replication.
– Rheumatoid arthritis & psoriasis and Crohn
disease
– Graft versus host disease

54. Adverse effects Methotrexate
– Nausea-vomiting-diarrhea
– Alopecia
– Bone marrow depression
– Pulmonary fibrosis
– Renal & hepatic disorders

56. Cyclophosphamide
– Alkylating agent to DNA.
– Prodrug, activated into phosphamide.
– Is given orally & intravenously
– Destroy proliferating lymphoid cells.
– Anticancer & immunosuppressant
– Effective in autoimmune diseases e.g rheumatoid
arthritis & systemic lupus erythrematosus.
– Autoimmune hemolytic anemia

57. Side Effects of Cyclophosphamide
– Alopecia
– Hemorraghic cystitis.
– Bone marrow suppression
– GIT disorders (Nausea -vomiting-diarrhea)
– Sterility (testicular atrophy & amenorrhea)
– Cardiac toxicity

58. Antibodies
block T-cell surface molecules involved in
signaling immunoglobulins
– Antilymphocyte globulins (ALG).
– Antithymocyte globulins (ATG).
– Rho (D) immunoglobulin.
– Basiliximab
– Daclizumab
– Infliximab

59. Antibodies contd…
Preparation
1. By immunization of either horses or rabbits
with human lymphoid cells producing
mixtures of polyclonal antibodies directed
against a number of lymphocyte antigens
(variable, less specific).

60. 2. Hybridoma technology
• produce antigen-specific, monoclonal antibody
(homogenous, specific).
• produced by fusing mouse antibody-producing cells
with immortal, malignant plasma cells.
• Hybrid cells are selected, cloned and selectivity of the
clone can be determined.
Antibodies preparation contd…

61. Recombinant DNA Technology
• Recombinant DNA technology can be used to replace
part of the mouse gene sequence with human genetic
material (less antigenicity-longer half life).
• Antibodies from mouse contain Muro in their names.
• Humanized antibodies contain ZU or XI in their
names.

62. Antibodies

63. Antibodies

64. Antilymphocyte globulins (ALG)
&Antithymocyte globulins (ATG)
• Polyclonal antibodies obtained from plasma or
serum of horses hyper-immunized with human
lymphocytes.
• Binds to the surface of circulating T-lymphocytes,
which are phagocytosed in the liver and spleen giving
lymphopenia and impaired T-cell responses &
cellular immunity.

65. Antilymphocyte globulins (ALG)
&Antithymocyte globulins (ATG) contd…
Kinetics
• Given i.m. or slowly infused intravenously.
• Half life extends from 3-9 days.
Uses
• Combined with cyclosporine for bone marrow
transplantation.
• To treat acute allograft rejection.
• Steroid-resistant rejection.

66. Adverse Effects
– Antigenicity.
– Leukopenia, thrombocytopenia.
– Risk of viral infection.
– Anaphylactic and serum sickness reactions (Fever,
Chills, Flu-like syndrome).

67. Muromonab-CD3
• Is a murine monoclonal antibody
• Prepared by hybridoma technology
• Directed against glycoprotein CD3 antigen of
human T-cells.
• Given I.V.
• Metabolized and excreted in the bile.

68. Mechanism of action of Muromonab-CD3
• The drug binds to CD3 proteins on T-lymphocytes
(antigen recognition site) leading to transient
activation and cytokine release followed by
disruption of T-lymphocyte function, their
depletion and decreased immune response.
• Prednisolone, diphenhydramine are given to
reduce cytokine release syndrome.

69. Uses Muromonab-CD3
• Used for treatment of acute renal allograft
rejection & steroid-resistant acute allograft
• To deplete T cells from bone marrow donor prior
to transplantation.
Adverse effects
• Anaphylactic reactions.
• Fever
• CNS effects (seizures)
• Infection
• Cytokine release syndrome (Flu-like illness to shock
like reaction).

70. Cytokine Release Syndrome
•It is a major side effect of anti-CD3 therapy, which typically
begins within 30 minutes after infusion (but can occur later also)
and may persist for hours.
•This is due to release of TNFα, ILs and interferons.
•The most common presenting symptoms are fever, chills/rigor,
nausea, vomiting, diarrhoea, headache, tremors, myalgia and
arthralgia.
•These symptoms usually are worst with the first dose. Tapering
the dose causes remission of symptoms.
•Pretreatment administration of corticosteroids prevents the
release of cytokines, hence reduces the first dose reaction. This
procedure is used a standard protocol for antiCD3 therapy.

71. Rho (D) immune globulin
• Rho (D) is a concentrated solution of human IgG1
containing higher titer of antibodies against Rho (D)
antigen of red cells.
• Given to Rh-negative mother within 24-72 hours
after delivery of Rh positive baby (2 ml, I.M.) to
prevent hemolytic disease of the next Rh positive
babies (erythroblastosis fetalis).

72. • The Treatment is advocated to Rh-negative
mothers at 26–28 weeks’s gestation with
history of:
– Miscarriages
– Previous ectopic pregnancies, or
– Previous abortions,
– When the blood type of the previous fetus is
unknown.

73. • The doses schedule as follows:
– Prophylaxis after delivery/abortion: 300mcg IM
within 72 hours of the event.
– Antepartum prophylaxis before 20 weeks of
gestation: 250IU, IM.
– Antepartum prophylaxis after 20 weeks of
gestation: 500 IU, IM.
– In case of transplacental hemorrhage: 1200mcg,
IM.

74. • It is well tolerated with mild discomforts
like pain at injection site and slight rise in
body temperature.
• Rho(D) immune globulin is injected to the
mother and not to infant.

75. Monoclonal antibodies

80. Monoclonal antibodies
Basiliximab and Daclizumab
 Obtained by replacing murine amino acid sequences
with human ones.
 Basiliximab is a chimeric human-mouse IgG (25%
murine, 75% human protein).
 Daclizumab is a humanized IgG (90% human
protein).
 Have less antigenicity & longer half lives than murine
antibodies

81. Mechanism of action
• IL-2 receptor antagonists
• Are Anti-CD25
• Bind to CD25 (α-subunit chain of IL-2 receptor on
activated lymphocytes)
• Block IL-2 stimulated T cells replication & T-cell
response system
• Basiliximab is more potent than Daclizumab.
Basiliximab and Daclizumab

82. Basiliximab and Daclizumab
• Given I.V.
• Half life Basiliximab (7 days )
• Daclizumab (20 days)
• are well tolerated - only GIT disorders
USES
• Given with CsA and corticosteroids for Prophylaxis
of acute rejection in renal transplantation.

83. Monoclonal antibodies
Infliximab
 A chimeric human-mouse IgG
 Directed against TNF-α
 Is approved for ulcerative colitis, Crohn’s disease
&rheumatoid arthritis
Omalizumab
 A humanized monoclonal IgE
 Directed against Fc receptor on mast &basophils
 Is approved for asthma in steroid-refractory patient

84. INTERFERONS
Three families:
• Type I IFNs ( IFN-α, β ):
• Acid-stable proteins; act on same target cell receptor
• Induced by viral infections
• Leukocyte produces IFN-α
• Fibroblasts & endothelial cells produce IFN-β
• Type II IFN (IFN-γ):
• Acid-labile; acts on separate target cell receptors
• Produced by Activated T-lymphocytes.

85. Interferon Effects
IFN- γ : Immune Enhancing
– Increased antigen presentations with macrophage,
natural killer cell, cytotoxic T-lymphocyte
activation
IFN- α, β :
– effective in inhibiting cellular proliferation
(more effective than IFN- γ in this regard)

86. USES OF INTERFERON
– Treatment of certain infections e.g. Hepatitis C
(IFN- α ).
– Autoimmune diseases e.g. Rheumatoid arthritis.
– Certain forms of cancer e.g. melanoma, renal cell
carcinoma.
– Multiple sclerosis (IFN- β): reduced rate of
exacerbation.
– Fever, chills, myelosuppression.

87. THALIDOMIDE
• A sedative drug.
• Teratogenic (Class-X).
• Can be given orally.
• Has immunomodulatory actions
• Inhibits TNF-α
• Reduces phagocytosis by neutrophils
• Increases IL-10 production

88. USES OF THALIDOMIDE
 Myeloma
 Rheumatoid arthritis
 Graft versus host disease.
 Leprosy reactions
 Treatment of skin manifestations of lupus
erythematosus

90. CLINICAL USES OF IMMUNOSUPPRESSIVE
AGENTS
DISEASE AGENT USED
Autoimmune Disease:
Acute glomerulonephritis
Autoimmune haemolytic anaemia.
Prednisone*,
mercaptopurine.
Cyclophosphamide.
Prednisone*,
cyclophosphamide,
mercaptopurine,
azathioprine, high dose δ-
globulin.

91. Organ transplant:
• Renal
• Heart
Cyclosporine, Azathioprine,
Prednisone, ALG, Tacrolimus.
• Liver Cyclosporine, Prednisone,
Azathioprine, Tacrolimus.
• Bone marrow Cyclosporine,
Cyclophosphamide,
Prednisone, Methotrexate,
ALG, total body radiation.

92. Gene Therapy

93. 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

94. Picture of a Chromosome

95. What is Gene Therapy
• It is a technique for correcting defective
genes that are responsible for disease
development
• There are four approaches:
a) A normal gene inserted to compensate for a
nonfunctional gene.
b) An abnormal gene traded for a normal gene
c) An abnormal gene repaired through selective
reverse mutation
d) Change the regulation of gene pairs

96. The Beginning…
• In the 1980s, Scientists began to look into
gene therapy.
– They would insert human genes into a bacteria
cell.
– Then the bacteria cell would transcribe and
translate the information into a protein
– Then they would introduce the protein into
human cells

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

98. 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

99. Picture

100. 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

101. 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

102. 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

103. Adenovirus cont.

104. 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

105. Herpes Simplex Viruses
• Double stranded DNA viruses that infect neurons
• Ex. Herpes simplex virus type 1

106. Non-viral Options
• 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?

107. Current Status
• 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

108. 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

109. 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

110. 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.

111. 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.

112. 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.

113. 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.

114. 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