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

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

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

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

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

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

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

7 Age-related diseases Normal VisionAMD  Alzheimer’s disease  Parkinson disease  Age-related macular degeneration  Huntington disease  Multiple sclerosis  …… Age-related macular degeneration Alzheimer’s disease

8 Current Treatments? Other therapies? Yes!

9 What’s gene therapy? Imagine that you accidentally broke one of your neighbor's windows. I.Stay silent: no one will ever find out that you are guilty, but the window doesn't get fixed. II.Repair it with some tape: not the best long-term solution. III.Put in a new window: not only do you solve the problem, but also you do the honorable thing. 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? 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!

10  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 Content

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  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 Content

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 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. 1.RNA interference (RNAi) 2.Triple helix-forming oligonucleotides (TFO) 3.Ribozyme 4.……  Gene silencing

16 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  Gene addition/gene augmentation

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 Pro-drugPro-drug geneToxic product ganciclovir (gcv)herpes simplex virus thymidine kinase (HSV-tk) Gcv-ppp 5-fluorocytosine (5-Fc) Cytosine deaminase (CD) 5-Fu /ToxinDiphtheria toxin Samples of suicide gene

20 In vivo gene therapy for brain tumors gcvgcv-ppp TK

21  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 Content

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

23 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 Types of Gene Therapy

24 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 Types of Gene Therapy

25 Target cell Delivery method Germ line gene therapy Somatic gene therapy ex vivo gene therapy in vivo gene therapy in situ systematical

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

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

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

30 Target Sites for Gene Therapy

31 Cell /tissueadvantageexamples EndotheliumCan form capillaries to secrete gene product into bloodstream clotting factor for hemophilia skinCan take small piece of skin from patient to grow into large graft Skin graphs can deliver therapeutic proteins lungcells lining the passageways are easily accessible aerosol spray to treat Cystic Fibrosis Nerve systemability to treat common illnesses and injuries Challenge: neurons do not divide gene therapy on fibroblasts to allow them to produce neurotransmitters MuscleEasily accessible, near bloodstreamTreatment for Duchenne Muscular Dystrophy LiverMultiple functions, capacity to regenerateTreatment for familial hypercholesterolemia Target Sites for Gene Therapy

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

33  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 Content

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 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 VectorNon-viral Techniques RetrovirusNake DNA LentivirusOligonucleotide AdenovirusLipoplexes and polyplexes Adeno-Associated Virus (AAV)Ballistic DNA Injection Herpes simplex virus (HSV)Microinjection AlphavirusesElectroporation Pox viruses (Vaccinia virus)Calcium phosphate transfection ……

38 viral Vs non-viral vectors A B Non-viral vector (transfection) viral transfer (Infection) Nuclear envelope barrier! direct nuclear shuttling! 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 -...

39  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. 1 Retroviral vectors Viral vector

40 AdvantagesDisadvantages High transduction efficiency Requires dividing cells for infectivity Insert size up to 8kBLow titers (10 6 - 10 7 ) 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 / Retrovirus vectors

41 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 2 lentiviral vectors Belongs to retrovirus family, Lenti, in latin, means slow

42 AdvantagesDisadvantages 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 (10 10 -10 13 ) Immunogenicity (In vivo delivery hampered by host immune response Infects both dividing and non- dividing cells / 3 adenoviral vector

43 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, 2/5...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. 4 recombinant AAV (rAAV) vectors

44 Producing recombinant AAV vectors


46 rAAV2/1-CMV-EGFP rAAV2/5-CMV-EGFP rAAV2/5-RHO-EGFP Intravitreal AAV2-EGFPSubretinal AAV2-EGFP

47 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 rAAV vector

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 FDA approved RetrovirusAAV

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

51 Electroporation DNA injection alone DNA injection with Electroporation

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

53  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 Content

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 Ashanti DaSilva Andrew Gobea September 14, 1990 @ NIH, French Anderson and R. Michael Blaese perform the first gene therapy trial. –Ashanti (4 year old girl) Her lymphocytes were gene-altered (~10 9 ) 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. Effective method: treatment of stem cells from umbilical cord blood in infants 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 therapy 26 10 9 9 5


61 Ornithine Transcarbamylase (OTC) deficiency Jesse Gelsinger Died from a massive immune response against viral vector

62 Gene therapy in China Hemaphilla B:X-linked recessivie F IX First disease with gene therapy 1990.12 Retroviral vector and AAV (2 non-specific, 1 muscle, 5 liver) Autologous skin fibrolast transplantation Subcutanous graft 薛京伦教授

63  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 Content

64 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 Problems with Gene Therapy

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


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)


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