Part VI Gene Therapy'ppt

1
??? ???? ?????? ??????
2
Application of Nanotechnology in Gene
Therapy???? ??? ????? ?????????? ???? ? ????
???? ? ??????? ?????oskueekr_at_mums.ac.ir
3
What is gene therapy? Why is it used?

• Gene therapy Introduction of normal genes into
  cells that contain defective genes to
  reconstitute a missing protein product
• GT is used to correct a deficient phenotype so
  that sufficient amounts of a normal gene product
  are synthesized ? to improve a genetic disorder

4
How is Gene Therapy Carried Out?

• Modification of somatic cells by transferring
  desired gene sequences into the genome.
• Somatic cells necessary to ensure that inserted
  genes are not carried over to the next generation.

5
What can gene therapy be applied to?

• Cancers
• Inherited disorders
• Infectious diseases (viral or bacterial)
• Immune system disorders
• Vaccination

6
(No Transcript)
7
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)

8
What is required forGene 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

9
Gene Therapy Strategies

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

10
Gene Augmentation

• For diseases caused by loss of gene function
• More copies of normal gene
• raise levels of gene product
• restore normal phenotype
• Apply to Monogenic recessive diseases eg.
  cystic fibrosis, haemophilia, muscular dystrophy

11
Gene Correction - Chimeraplasty
Targeting strand
DNA
Me-RNA
Me-RNA
X


X
3
5
Correcting strand
Mutator base
12
Targeted inhibition

• Ribozymes
• can cleave (or repair) mRNA
• Triple helix oligonucleotides
• block gene transcription
• Antisense oligos
• block mRNA translation
• SiRNA (Knockdown)

13

• All gene therapy strategies depend
• on getting the gene or genetic material
• into the target cells

Transduction
14
Different Delivery Systems are Available

• Two main routes
• In vivo versus ex vivo
• In vivo delivery of genes takes place in the
  body
• Ex vivo delivery takes place out of the body,
  and then cells are placed back into the body

15
Gene delivery approaches

• Physical methods
• Viral vectors
• Non-viral vectors

16
Physical methods

• Calcium phosphate
• Injection of naked DNA
• Electroporation in vivo or ex vivo
• Ballistic technology the gene gun

Advantage Disadvantage

• Not limited by size or number of genes

• Inefficient

17
Viral vectors

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

18
(No Transcript)
19

• 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

20
Non-Viral methods

• Cationic Lipids (Liposomes)
• Cationic Polymers
• Retrotransposons (jumping genes)
• Human artificial chromosomes (HACs)

21

• Advantages
• Low toxicity
• Nonimmunogenicity
• Easy synthesis
• Disadvantage
• The overall transfection pathways are inefficient

22
Problems with GT

• Gene delivery
• Safety
• Toxicity
• Immune response
• Integration
• Malignant transformation
• Germline integration
• Viral replication through recombination
• Efficacy
• Target cell uptake
• Control of gene expression
• Expense

23
Example Severe Combined Immunodeficiency
Disease (SCID)

• bubble boy syndrome
• SCID is caused by an Adenosine Deaminase
  Deficiency (ADA)
• Gene is located on chromosome 22 (32 Kbp, 12
  exons)
• Deficiency results in failure to develop
  functional T and B lymphocytes

24

• September 14, 1990 _at_ NIH, French Anderson and R.
  Michael Blaese perform the first GT Trial
• Ashanti (4 year old girl)
• Her lymphocytes were gene-altered (109) ex vivo
  ? used as a vehicle for gene introduction using
  a retrovirus vector to carry ADA gene (billions
  of retroviruses used)
• Cynthia (9 year old girl) treated in same year
• Problem WBC are short-lived, therefore
  treatment must be repeated regularly

25
The Biotech Death of Jesse Gelsinger

• Ornithine transcarbamylase (OTC) deficiency
• Case study Jesse Gelsinger
• GT began Sept. 13, 1999, Coma on Sept. 14, Brain
  dead and life support terminated on Sept. 17,
  1999
• Cause of death Respiratory Disease Syndrome

26
(No Transcript)
27

• Jaundice, kidney failure, lung failure and brain
  death
• Adenovirus triggered an overwhelming inflammatory
  reaction ? massive production of monokine IL-6
• ? multiple organ failure

28

• Every realm of medicine has its defining moment,
  often with a human face attached. Polio had
  Jonas Salk. In vitro fertilization had Louise
  Brown, the first test-tube baby. Transplant
  surgery had Barney Clark, the Seattle dentist
  with the artificial heart. AIDs had Magic
  Johnson. Now gene therapy has Jesse Gelsinger.

29
Another major blow came in January 2003

• FDA placed a temporary halt on all gene therapy
  trials using retroviral vectors in blood stem
  cells.
• FDA took this action after it learned that a
  second child treated in a French gene therapy
  trial had developed a leukemia-like condition.
• Both this child and another who had developed a
  similar condition in August 2002 had been
  successfully treated by gene therapy for X-linked
  severe combined immunodeficiency disease (X-SCID)

30
What factors have kept gene therapy from becoming
an effective treatment for genetic disease?

• Short-lived nature of gene therapy
• Immune response
• Problems with viral vectors
• Multigene disorders

31
Summary

• Gene Therapy is still in its infancy
• Early promise and hope not fulfilled
• Risks, including death, seen as a major issue
• But, recent trials look more promising

32
DNA delivery systems

• DNA delivery systems
• 1) viral vector-mediated systems
• 2) non-viral vector mediated systems

33
Limitations of viral vectors

• Toxic immunological reactions
• Restricted targeting of specific cell type
• Limited DNA carrying capacity
• Production packaging problems
• High cost
• Long term effect of the integrated gene

34
Non-viral methods

• Mechanical
• microinjection
• pressure
• particle bombardment
• electrical (electroporation)

35

• Chemical(easiest, most versatile, most
  effective, most desirable)
• Calcium phosphate
• Liposomal formulations
• Cationic polymers (polycations)

36
Comparison of efficiency toxicity
37
Steps of DNA delivery

• DNA condensation and complexation
• Endocytosis
• Nuclear targeting/entry

38
Major barriers to DNA delivery

• Low uptake across the plasma membrane
• Inadequate release of DNA with limited stability
• Lack of nuclear targeting

39
Schematic drawing of barriers
40
Mechanism

• Condensation of DNA
• Cellular uptake
• Release from endosome
• Nuclear transport
• Vector unpackaging

41
Non-viral vectors

• Cationic peptides
• Cationic lipids (liposomes)
• Cationic polymers
• Poly(ethylenimine) (PEI)
• Poly( L-lysine) (PLL)
• Other polymeric delivery systems

42
Cationic peptides

• amphiphilic peptides which can undergo
  conformational changes in acidic environments
  escaping the endosomal/lysosomal pathways.
• contain the positively charged amino acids (i.e.
  histidine, lysine and/or arginine)

43
Cationic lipids (liposomes)

• Since their introduction as gene carriers,
  liposomes have become one of the most studied
  non-viral vectors.
• amphiphilic molecules one or two fatty acid side
  chains (acyl) or alkyl, a linker and a
  hydrophilic amino group.

44
Poly (L-lysine) (PLL)

• one of the first cationic polymers
• lysine as the repeat unit thus, they posses a
  biodegradable nature.
• PLL has poor transfection ability when applied
  alone or without modifications.
• PEGylation One popular modification that can
  increase both the transfection ability and the
  circulation half-life

45
Other polymeric delivery systems

• Many other cationic polymers such as chitosans (a
  biodegradable linear aminopolysaccharides) were
  tested for gene transfer.

46
PEI
47
Cytotoxicity

• To be useful in gene therapy, polyplexes should
  be non-cytotoxic.
• In the case of PEIs, the gene delivery efficiency
  increases with molecular mass up to 25 kDa and
  then slumps, while the cytotoxicity rises
  linearly.
• damage to the plasma membrane, may be responsible
  for cytotoxicity.

48
In vivo gene delivery using polycations

• in vivo gene delivery faces a variety of
  obstacles
• anatomical size constraints
• interactions with biological fluids and
  extracellular matrix
• binding to a variety of non-target cell types

49
How to overcome barriers
50
(No Transcript)