1. Gene Therapy Research 徐国彤 MD， PhD Tongji University School of Medicine

2. Infectious Disease AIDS Severe Acute Respiratory Syndromes (SRAS)
Human immunodeficiency virus (HIV)
AIDS
Severe Acute Respiratory Syndromes (SRAS)
Influenza
Swine Flu (H1N1)
Avian influenza (bird flu)
Hand, foot and mouth disease (HFMD) ……
A young Bangladeshi girl infected with smallpox (1973).
smallpox

3. Drug-resistant bacteria
NMD-1 "Superbug" Poses a "Cause for Worldwide Concern"
Tuberculosis (TB): A fell plague

4. Cancer Lung cancer: Liver cancer: Stomach cancer:
Cross section of a human lung. The white area in the upper lobe is cancer; the black areas are discoloration due to smoking.
Liver cancer:
Left lobe liver tumor in a 50 year old male
Stomach cancer:
A suspicious stomach ulcer that was diagnosed as cancer on biopsy.

5. Hereditary diseases Rhematoid arthitis
Chondrodysplasia congenita (先天性软骨发育不良)

6. Hereditary diseases Leber’s congenital amaurosis (LCA)
Retinitis pigmentosa (RP)

7. Age-related macular degeneration Huntington disease Multiple sclerosis
Age-related diseases
Alzheimer’s disease
Normal Vision
AMD
Alzheimer’s disease
Parkinson disease
Age-related macular degeneration
Huntington disease
Multiple sclerosis ……
Age-related macular degeneration

8. Yes!
Current Treatments?
Other therapies?

9. What’s gene therapy?
Imagine that you accidentally broke one of your neighbor's windows.
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?
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!
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.

10. Content The definition of gene therapy strategies of gene therapy
Types and routes of gene therapy
Vectors of gene therapy
Gene therapy for clinical use
Problems and Perspective

11. What is gene therapy?
Gene therapy is the treatment of diseases based on the introduction of genetic material into target cells of the body.
Although the technology is still in its infancy, it has been used with some success. Scientific breakthroughs continue to move gene therapy toward mainstream medicine.
“ Gens” as medicine
The therapeutic drug is produced in small factory “cells”.

12. Content The definition of gene therapy strategies of gene therapy
Types and routes of gene therapy
Vectors of gene therapy
Gene therapy for clinical use
Problems and Perspective

13. The strategies of gene therapy
Gene replacement/correction
Replacing a mutated gene that causes disease with a healthy copy of the gene
Gene silencing/gene interference
Inactivating, or “knocking out,” a mutated gene that is functioning improperly
Gene augmentation/gene addition (modification)
The addition of a functional copy of a gene to the genome of an organism, or introducing a new gene into the body to help fight a disease
“Suicide gene”
Insert genes whose products metabolize normal drugs and ↑ their toxicity to proliferating–ie tumor cells ……

14. Gene replacement
Delivery of a gene whose function is absent due to loss-of-function mutations in the affected gene. This can be used in autosomal recessive diseases (RP or LCA) or in those that are autosomal dominant due to haploinsufficiency or dominant-negative mutations (RP).
Gene replacement by using gene homologous recombination (gene targeting).

15. Gene silencing
Delivery of a gene and/or nucleic acid to inhibit the expression of a gene or a gene product with abnormal function. This approach is useful in autosomal dominant diseases (RP) arising from gain-of-function mutations.
RNA interference (RNAi)
Triple helix-forming oligonucleotides (TFO)
Ribozyme ……

16. Gene addition/gene augmentation
Delivery of a gene whose product provides beneficial effects independently of the primary defective gene; without actually substituting that gene for the flawed or absent gene in the DNA (AAV2-RPE65 for LCA).
NATURE BIOTECHNOLOGY VOLUME 24 NUMBER 8 AUGUST 2006

17. Assisted killing of disease cells by immune system cells (gene augmentation)
Gene vaccine: IL-2, 4, TNFa, INFr into tumor cells
Adaptive immonotherapy: Cytokines into TIL
Immunoenhancement: MHC-I into tumor cell
In situ modification of immunogenity of : CLT

18. “Suicide gene”
Suicide gene: A gene whose expression in a cell is lethal for that cell under specific conditions.
Suicide genes form the basis of a strategy for making cancer cells more vulnerable, more sensitive to chemotherapy.
The approach has been to attach parts of genes expressed in cancer cells to other genes for enzymes not found in mammals that can convert a harmless substance (pro-drug) into one that is toxic to the tumor.

19. Samples of suicide gene
Pro-drug
Pro-drug gene
Toxic product
ganciclovir (gcv)
herpes simplex virus thymidine kinase (HSV-tk)
Gcv-ppp
5-fluorocytosine (5-Fc)
Cytosine deaminase (CD)
5-Fu /
Toxin
Diphtheria toxin

20. In vivo gene therapy for brain tumorsTK
gcv
gcv-ppp

21. Content The definition of gene therapy
Basic process (strategies) of gene therapy
Types and routes of gene therapy
Vectors of gene therapy
Gene therapy for clinical use
Problems and Perspective

22. Types of Gene Therapy Germ line gene therapy Somatic gene therapy
Target cell
ex vivo gene therapy
in vivo gene therapy
in situ
systematical
Delivery method

23. Germ line gene therapy Host cells: sperm or eggs Methods: Features:
Types of Gene Therapy
Germ line gene therapy
Host cells: sperm or eggs
Methods:
----To treat a pre-embryo before implantation in the mother, with the
use of IVF
----To treat the germ cells (sperm or egg cells) of afflicted adult
Features:
---functional genes are ordinarily integrated into their genomes
---effects would be heritable and would be passed on to later
generations.
---highly effective
---technical and ethical reasons

24. Somatic gene therapy Host cell: somatic cells of a patient Methods:
Types of Gene Therapy
Somatic gene therapy
Host cell: somatic cells of a patient
Methods:
----Inserting the gene into any location within the genome to replace a nonfunctional gene, which is the most commonly used
----Switching the abnormal gene  for a normal gene through homologous recombination. ----Fixing  through selective reverse mutation, which returns the gene to its normal function
Features
---Any modifications and effects will be restricted to the individual patient only
and will not be inherited by the patient's offspring or later generations
---widely used

25. Types of Gene Therapy Germ line gene therapy Somatic gene therapy
Target cell
ex vivo gene therapy
in vivo gene therapy
in situ
systematical
Delivery method

26. Types of Gene Therapy
ex vivo:
---incorporate gene into cells outside the body and then deliver altered cells to patient

27. in vivo: Types of Gene Therapy
---Systematically: vector carrying the gene is introduced directly into the body, often through a blood vessel
---in situ: vector carrying the gene is injected into localized and accessible body part

28. In-vivo In-vivo systematic delivery Ex-vivo
ex vivo or in situ delivery are currently preferred over systematic delivery
In-vivo
In situ delivery
In-vivo
systematic delivery
Ex-vivo
Examples:
- brain
- muscle
- eye
- joints
- tumors
Examples:
- intravenous
- intra-arterial
- intra-peritoneal V
Examples:
- bone marrow
- liver cells
- skin cells

29. Somatic Gene therapy: widely studied
Chronic treatment
'Use genes as drugs':
Correcting disorders by
somatic gene transfer
Acute treatment
Preventive treatment
Hereditary disorders
Acquired disorders
Loss-of-function
Gain-of-function

30. Target Sites for Gene Therapy

31. Target Sites for Gene Therapy
Cell /tissue
advantage
examples
Endothelium
Can form capillaries to secrete gene product into bloodstream
clotting factor for hemophilia
skin
Can take small piece of skin from patient to grow into large graft
Skin graphs can deliver therapeutic proteins
lung
cells lining the passageways are easily accessible
aerosol spray to treat Cystic Fibrosis
Nerve system
ability to treat common illnesses and injuries Challenge: neurons do not divide
gene therapy on fibroblasts to allow them to produce neurotransmitters
Muscle
Easily accessible, near bloodstream
Treatment for Duchenne Muscular Dystrophy
Liver
Multiple functions, capacity to regenerate
Treatment for familial hypercholesterolemia

32. Remember! Efficiency Specificity Persistence Toxicity
The four technical basic questions in somatic gene therapy
Remember!
Efficiency
Persistence
Specificity
Toxicity
Efficiency of gene transfer
Specificity of gene transfer
Persistence of gene transfer
Toxicity of gene transfer
The variables
which disease?
which gene?
which vector?
which target organ?
which type of delivery?

33. Content The definition of gene therapy
Basic process (strategies) of gene therapy
Types and routes of gene therapy
Vectors of gene therapy
Gene therapy for clinical use
Problems and Perspective

34. The Ideal Vector for Gene Transfer
High concentration of virus allowing many cells to be infected or transduced
Convenience and reproducibility of production
Ability to transduce dividing and non-dividing cells
Ability to integrate into a site-specific location in the host chromosome, or to be successfully maintained as stable episome
A transcriptional unit that can respond to manipulation of its regulatory elements
Ability to target the desired type of cell
No components that elicit an immune response

35. Virural vectors Non-virual vectors
Vector of gene therapy
Virural vectors
Non-virual vectors

36. Vectors for gene transfer
Transduction of somatic cells can be obtained both by both viral and non-viral nucleic acid transfer.
Viral vectors:
Gene delivery can be accomplished with high efficiency by using viruses modified as follows: the viral genome is partially or completely deleted of viral genes, which are generally substituted in the vector by an expression cassette containing the desired promoter–transgene combination.
Non-viral vectors:
Nucleic acids can be additionally delivered as naked DNA or as a complex with lipids or cationic polymers. These compounds usually improve the efficacy of DNA delivery to the target cells. Doublestranded short interfering RNA sequences (siRNAs), used to induce RNA interference of a target transcript, are usually delivered via nonviral methods.

37. Delivery System Viral Vector Non-viral Techniques Retrovirus Nake DNA
Lentivirus
Oligonucleotide
Adenovirus
Lipoplexes and polyplexes
Adeno-Associated Virus (AAV)
Ballistic DNA Injection
Herpes simplex virus (HSV)
Microinjection
Alphaviruses
Electroporation
Pox viruses (Vaccinia virus)
Calcium phosphate transfection ……

38. viral Vs non-viral vectors
(transfection) A
Why are viruses 'better'?
viral transfer is much more efficient
nonviral transfer must solve a number of hurdles - serum protection/stability - target docking - endosomal escape - nuclear trafficking - genomic integration - immunological camouflage B
Nuclear envelope barrier!
viral transfer
(Infection)
direct nuclear shuttling!

39. 1 Retroviral vectors Viral vector
Retrovirus virions contain a protein capsid that is lipid encapsulated. Virions range in diameter from 80 to 130 nm.
The viral genome is encased within the capsid along with the proteins integrase and reverse transcriptase.
The genome consists of two identical positive (sense) single-stranded RNA molecules ranging in size from 3.5 to 10kb.

40. Retrovirus vectors Advantages Disadvantages
High transduction efficiency
Requires dividing cells for infectivity
Insert size up to 8kB
Low titers ( )
Integrates into host genome resulting in sustained expression of vector
Integration is random, insertional mutagenesis
Extremely well studied system
In vivo delivery remains poor. Effective only when infecting helper cell lines
Vector proteins not expressed in host 　/

41. 2 lentiviral vectors Advantages : Disadvantages:
Belongs to retrovirus family, Lenti, in latin, means slow
Advantages :
High-efficiency infection of dividing and non-dividing cells;
Long-term stable expression of a transgene;
Low immunogenicity.
Disadvantages:
Random integration: insertional mutations; loss of function of important genes; cancer, etc

42. 3 adenoviral vector Advantages Disadvantages
High transduction efficiency
Expression is transient (viral DNA does not integrate)
Insert size up to 8kb
Cytopathogenicity (viral protiens can be expressed in host)
High viral titer ( )
Immunogenicity (In vivo delivery hampered by host immune response
Infects both dividing and non-dividing cells /

43. 4 recombinant AAV (rAAV) vectors
Recombinant vectors are generated by deleting the rep and cap sequences from the genome and by inserting the therapeutic gene of interest between the ITRs.
Hybrid vectors have been generated by including the same AAV vector genome (usually derived from AAV2) in external surface proteins (capsids) from other AAV serotypes; the resulting recombinant vectors (rAAVs) are indicated as ‘rAAV 2/1, 2/2, 2/3, 2/4, 2/ /n’, with the first number indicating the genome (i.e. AAV2 in this case) and the second the capsid.
Different rAAV serotypes have different capsids, tropism and transduction characteristics.

44. Producing recombinant AAV vectors

46. rAAV2/1-CMV-EGFP
rAAV2/5-CMV-EGFP
rAAV2/5-RHO-EGFP
Intravitreal AAV2-EGFP
Subretinal AAV2-EGFP

47. rAAV vector Advantages:
All virus genes removed, Lack of initiating an immune response;
Stable expression and safe;
Ability to infect a variety of dividing and non-dividing cells
Non-pathogenic
Disadvantages:
Small genome limits size of foreign DNA larger than 5 kb;
Must be closely screened for adenoviral or HSV contamination
Labor intensive production
except Hematopoietic cells

48. 5 Herpes simplex viruses I
Advantages:
Preferential neurons
Large insert size
Could provide long- term CNS gene expression
High titer
No integration
disadvantages:
Hard to prepare, cell toxicity
transient expression in non-neuron cells
Low transduction efficiency

49. Retrovirus
AAV
FDA approved

50. Cationic Lipids, Liposomes
Non-Viral Vectors
Non-Viral Vectors
Advantages:
Simple large scale production
Low host immunogenicity
Methods
disadvantages
Calcium Phosphate
Low Efficiency
DEAE Dextran
Cationic Lipids, Liposomes
Direct DNA Injections
Electroporation
Transient expression

51. DNA injection with
Electroporation
DNA injection alone
Electroporation

52. Limitations of current vectors
r-Adenovirus
- no persistence
- limited packaging
- toxicity, immunogenicity
Biolistic bombardment
or local direct injection
- limited area
r-AAV
- no integration in host g.
- very limited packaging
- autoimmunity?
Electroporation
- limited organ access
Liposomes, gene correction & Co.
- rather inefficient transfer
r-Retrovirus (incl. HIV)
- limited packaging
- random insertion
- unstable genome
General
- low transfer efficiency
- no or little genomic integration
General
- antibody response
- limited packaging
- gene silencing
- Manufacturing limitations
The future will probably see an increasing interest in viral-like, but artificial particles
Solutions:
- synthetic viruses
(“Virosomes”)

53. Content The definition of gene therapy
Basic process (strategies) of gene therapy
Types and routes of gene therapy
Vectors of gene therapy
Gene therapy for clinical use
Problems and perspective

54. Bubble boy disease
David Vetter, who was born with a genetic disorder leaving him no natural immunities against disease, became famous for living behind plastic barriers to protect him from germs. He died at age 12 of 1984

55. Why ADA deficiency is ideal target for the first gene therapy???
1.The pathological effects are reversible
2.The gene defect：the loss of function of a single gene
3.Tight control ：not important (ADA levels vary widely in the normal population)
4. Target gene：very small and easy to manipulate
5. Target cells: lymphocytes, which are accessible, easy to culture and easy to
put back into the body of the patient
6.The alternative treatments are expensive and/or hazardous

56. ADA deficiency: The First clinical Trial
September 14, NIH, French Anderson and R. Michael Blaese perform the first gene therapy 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.
Ashanti DaSilva
Andrew Gobea
Effective method: treatment of stem cells from
umbilical cord blood in infants
Lack of ADA blocks a biochemical pathway that normally breaks down a metabolic toxin into uric acid, which is then excreted. Without ADA, the substance that ADA normally acts upon (Deoxyadenosine) builds up and destroys T cells. Without helper T cells to stimulate them, B cells cannot mature into the plasma cells that produce antibodies Both branches of the adaptive immune system fail. The child becomes very prone to infections and cancer, and despite medical treatment, usually does not live beyond a year in the outside environment.
Culver, Anderson, and Blaese with gene therapy patients.

57. #2: Visual behavior in the children-as assessed by the ability to walk-showed substantial improvements after treatment (Y. 2007) (Video)

58. Before treatment (Video 1)
After treatment (Video 2)

59. Diseases were approved for gene therapy26 10 9 10 5 10 9

61. Died from a massive immune response against viral vector
Ornithine Transcarbamylase (OTC) deficiency
Jesse Gelsinger
OTC is an X-linked recessive disorder where one of five enzymes required to break down amino acids liberated from dietary proteins is absent. The nitrogen from the amino acids combines with hydrogens to form ammonia (NH3), which rapidly accumulates in the blood stream and travels to the brain, causing coma and death. Symptoms can be somewhat controlled by following a low-protein diet and taking drugs that bind ammonia.

62. Gene therapy in China Hemaphilla B:X-linked recessivie F IX
First disease with gene therapy
Retroviral vector and AAV (2 non-specific, 1 muscle, 5 liver)
Autologous skin fibrolast transplantation
Subcutanous graft
薛京伦教授

63. Content The definition of gene therapy
Basic process (strategies) of gene therapy
Types and routes of gene therapy
Vectors of gene therapy
Gene therapy for clinical use
Problems and Perspective

64. 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

65. Regulated target gene expression in gene therapy
Intrinsic regulatory elements of genes
Normal cells-specific :DAT—dopaminergic neuron
NSE---all neurons
Diseased cell-specific: AFP, CEA
Extrinsic regulatory elemmnts of gene
HSP response element
Diseased microenviroment
HIF and HRE
Inducible element
Tet on/tet off (tetracyclin-resisitance operon)
Inducible, regulated
expression

66. Inducible cell-specific expression
Ex vivo, somatic gene therapy
Disease mechanism
Suffered cell/tissue
Choice of vector
Inducible cell-specific expression

68. What impact is gene therapy likely to have on medicine in the future?
"Someday people will look back on the era before gene therapy in the same way we look back on the era before antibiotics and vaccines. It is now possible to think about treating a whole series of diseases with a one-shot therapy that would last a lifetime.“
Dr. Rochelle Hirschhorn (Professor of Medicine, New York University)

69. Thanks for your attention!