Feb 11, Gene Therapy. Sam K.P. Kung Immunology Rm 417 Apotex Center

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1 Gene Therapy Sam K.P. Kung Immunology Rm 417 Apotex Center

2 Objectives: The concept of gene therapy, and an introduction of some of the currently used gene therapy vector Undesirable immune responses to gene therapy approach Potential gene therapy approach in modulating immune responses Examples of applications

3 It is an approach to treating disease by either modifying the expressions of an individual's genes or correction of abnormal genes. By administration of DNA rather than a drug, many different diseases are currently being investigated as candidates for gene therapy. These include cystic fibrosis, cardiovascular disease, infectious diseases such as AIDS and cancer. Cell Therapy can be defined as the infusion or transplantation of whole cells into a patient for the treatment of an inherited or acquired disease

4 Why do we need it? Feb 11, 2013

5 What do we need to do this? Administration Delivery system Efficiency and efficacy Target Gene X Therapeutic gene Safe

6 Gene Therapy Choice of vector in gene therapy application is important = Pathogens?

7 Limitations that hampered the progress gene delivery Inefficient Specific targeting of expression to the cells or tissues of interest only. Duration of expression Safety Immune responses

8 An ideal gene therapy vector Allow efficient and selective transduction of the target cells The vector is maintained inside the cells Expresses gene of interest at levels necessary for achieving therapeutic effects Safe

9 Gene Transfer Technology Non-viral (chemical, liposome, electroporation of DNA) Viral (gene transfer vector derived from virus)

10 Currently used gene therapy viral vectors Adenovirus Adeno-associated virus Alphaviruses Herpes Simplex Virus Retrovirus Lentivirus

11 Viral vectors used for gene delivery Feb 11, 2013

12 Viral vectors used for gene delivery Feb 11, 2013

13 Adenovirus Linear double-stranded DNA Genome size is ~ 35 kb Nonenveloped Transcription proceeds according to a carefully orchestrated program, with viral genes being transcribed early after infection, and others being transcribed at later times

14 Adenovirus Pros High titer virus stock is possible High level of gene expression Cons Preexisting immunity to the virus Strong immunogenicity Transient expression, no genome integration

15 Retrovirus The viral genome is a dimer of linear, positive-sense, single-stranded RNA Genome size is ~7-13 kb Require reverse transcription for replication and integration The genome contains three large genes, gag (group-specific antigen), Pol (polymerase) and env (envelope) Eg. Moloney Murine leukemia virus

16 Retrovirus Pros Long-term expression possible, genome integration Ability to pseudotype with other viral envelope proteins to Increase Target range Cons Inability to infect non-dividing cells Sites of Integration

17 Lentivirus The viral genome is a dimer of linear, positive-sense, single-stranded RNA Genome size is ~10 kb Require reverse transcription for replication and integration The genome contains three large genes, gag, Pol (polymerase) and env (envelope). In addition, six additional viral proteins Are the primary translation products of spliced mrna Eg. HIV-1

18 Lentivirus Human Immunodeficiency virus Pathogenesis/Clinical Significance Acquired Immunodeficiency Syndrome HIV envelope binds CD4 molecule (T helper cells) Transmission routes: sexual contact, blood, breast feeding So far, non-oncogenic

19 Lentivirus Pros Ability to infect both dividing and non-dividing cells Long-term expression possible, genome integration Ability to pseudotype with other viral envelope proteins to increase Target range Cons Safety concerns Sites of Integration

20 Major Success Story Retrovirus-based treatment of infants suffering from the X- chromosome-linked severe combined immunodeficiency disease (SCID) (bubble children). Following this treatment, these children have been able to live in the open air.

21 Figure 2 Longitudinal study of lymphocyte subsets from patient 1 (P1) and patient 2 (P2). M Cavazzana-Calvo et al. Science 2000;288: Published by AAAS

22 Updates (as of 2012) Feb 11, 2013

23 Discussion Feb 11, 2013

24 Choice of vectors in gene therapy applications is important Feb 11, 2013

25 Adenovirus Experience Feb 11, 2013

26 Adenoviruses Double-stranded DNA virus genome ~34 43 kb Species specific and of different serotypes 51 serotypes in humans 27 serotypes in simian 7 chimpanzees

27 Adenoviral vectors attractive candidate gene replacement vehicles Genome is well characterized and relatively easy to manipulate Adenoviruses cause mild diseases in immunocompetent human adults. Can be made replication-defective by deletions of crucial viral proteins Broad tropism infecting a variety of dividing and non-dividing cells High titers in culture

28 Adenoviral vectors attractive candidate gene replacement vehicles An example: Ornithine transcarbamylase deficiency

29 Ornithine Transcarbamylase Deficiency X-linked disorder resulting in a deficiency of the OTC enzyme, that leads to marked elevations of ammonia. Liver is the main target organ Animal models available in mice Current treatment: diet, sodium benzoate

30 2x10 11 vectors Tail vein injections into OTC deficient mice (spf, ash) Blood sampling

31 Urinary orotate excretion in OTC-deficient mice infused with adenoviral vectors carrying mouse OTC cdna Feb 11, 2013

Summary: Molecular Genetics and Metabolis 80 (2003), 148-158 An 18-year-old male with partial ornithine transcarbmaylase (OTC) deficiency participated in a pilot (safety) study of gene

The subject had high serum levels of IL-6 and IL-10 but normal TNF immediately after infusion of the vector.

32 Summary: Molecular Genetics and Metabolis 80 (2003), An 18-year-old male with partial ornithine transcarbmaylase (OTC) deficiency participated in a pilot (safety) study of gene therapy. The vectors were infused into the right hepatic artery at a dose of 6x1011 particles/kg. 98 h following gene transfer, he was dead. The subject had high serum levels of IL-6 and IL-10 but normal TNF immediately after infusion of the vector. This experience points to the limitations of animal studies in predicting human responses, the steep toxicity curve for replication defective adenovirus vectors, substantial subject-to-subject variation in host responses to systemically administered vectors, and the need for further study of the immune response to these vectors.

33 Molecular Therapy, 3:697 (2001) C57BL6 mice were injected intravenously with Ad-lacZ sacrificed at the indicated time points. Heart blood was taken for cytokine assays Spleen was harvested for culture of DCs and macrophages and used for immunohistochemical and X-gal staining.

34 Intravenous injection of mice with Ad-lacZ significantly induces elevation of serum inflammatory cytokines. Feb 11, 2013

35 Bangari and Mittal, Curr Gene Ther : Feb 11, 2013

36 Bangari and Mittal, Curr Gene Ther : Feb 11, 2013

37 Summary of the Adenovirus Experience Immediate hypersensitivity to adenovirus, which results in increased release of TNF-, IL-6 and IL-8 can have lethal consequences (toxicity). Cellular responses may develop and destroy successfully transduced cells or reduce transgene expression (limit transduction efficiency) Preexisting humoral immunity to vectors or development of antibodies against transgene product or vector components (limit transduction efficiency)

38 Approaches to avoid immune system Further Deletion of viral genes Ehrhardt et al., A gene-deleted adenoviral vector results in phenotypic correction of canine hemophilia B without liver toxicity or thrombocytopenia. Blood 102: (2003). Immunosuppression Ziller et al., Transient blocking of both B7.1 (CD80) and B7.2 (CD86) in addition to CD40- CD40L interaction fully abrogates the immune response following systemic injection of adenovirus vector. Gene Therapy 9: (2002). Switching serotypes Kass-Eisler et al., Circumventing the immune response to adenovirus-mediated gene therapy. Gene Ther. 3(2): (1996)

39 Approach to take advantage of the immune system Adenoviral vectors attractive vaccine vehicles

40 Replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity Shiver et al., Nature 415:331 (2002) Non-human primate SHIV-89.6P challenge CD4 counts and viral load

41 Figure 2 Post-challenge viraemia and CD4+ T-cell counts. Feb 11, 2013

42 Figure 1 Quantification of CD8+ T-cell responses by tetramer analysis during immunization. Feb 11, 2013

43 CD8 Feb 11, 2013 IFN-

44 HIV-1 based lentiviral vectors HIV-1 LTR gag vif vpr vpu tat rev LTR pol env nef HR CMVEGFP LTR CMV EGFP LTR

45 Key differences between retroviral and lentiviral vectors Cell-cycle requirements for successful transduction Preference in the Integration sites Vector silencing?

46 Manufacture of lentiviral vectors LTR CMV CMV CMV EGFP env + gag pol + LTR Gene Therapy Vector Packaging Vector Envelope Vector CaPO 4 transfection 293T cells Harvest Virus Supernatant

47 CD4 Feb 11, 2013 CS-RhMLV-E in vitro U3 R U5 Rh-MLV LTR EGFP U3 R U5 gag MoMLV CS-RhMLV-E CS-RhMLV-E CSCG Activated Human T cells EGFP Kung et al., J. Virol. 74:

48 Transplantation of transduced CD34+ cells in rhesus macaques GCSF SCF Peripheral Blood CD34+ cells 10 Gy FACS PBMC In vitro Lentiviral Transduction

49 Lentiviral vector marking of rhesus peripheral blood cells Granulocytes Monocytes Lymphocytes RBC Platelet CS-RhMLV-E HR CMVEGFP EGFP An and Kung et al., J. Virol. 75:

50 CD34 Feb 11, 2013 EGFP markings in rhesus bone marrow aspirates CS-RhMLV-E 95E132 HR CMVEGFP RC505 EGFP

57 New Designs Cell-specific targeting Non-integrating Lentiviral vectors mirna-regulated Lentiviral vectors

58 Morizono et al., J Virol Sep;75(17): Feb 11, 2013

59 Morizono et al., Nat Med Mar;11(3): Feb 11, 2013

60 Morizono et al., Nat Med Mar;11(3): Feb 11, 2013

61 Genetic engineering of T cells for adoptive immunotherapy Feb 11, 2013

68 HSC-based gene therapy approaches to treat HIV infection Kitchen S.G., et al., Virology 2011

69 Inhibitory agents used in HIV hematopoietic cell gene therapy trials Feb 11, 2013

72 Take Home Messages Gene therapy as a modern therapeutic for both hereditary and acquired diseases. The progress has been slower than anticipated because of the Setbacks and the major limitations on vector developments No universally applicable viral vector system is ideal for all clinical applications. Because of the various properties of each viral vector, the choice of a vector depends on factors such as packaging capacity, host range, cellor tissue-specific targeting, genome integration and duration of transgene expression.

73 Take home message SCIDs provide conditions that favor successful outcomes following gene therapy -stable integration of vector -selection pressure -lack of immune responses -in SCID-X1, no concern about unwanted ectopic expression, and de-regulated cell activation SCID-X1 treatments presented proof of principle for gene therapy clinically The use of retrovirus (and lentivirus) raises concern of insertational mutagenesis Limitations in animal models Need to improve vector designs and site-specific integration

74 Take home message Things to consider in designing a gene therapy protocol Host immunity presents a challenge to the simple rationale behind metabolic gene replacement therapy: supply the missing gene and cure the disease. Need to seriously consider making immunomodulation as important a part as are vectors and transgenes. Host immunity against certain viral vectors can be useful in vaccine designs. Ability to manipulate primary cells of hematopoietic lineages also allow direct genetic manipulation of immune responses.

VIROLOGY. Engineering Viral Genomes: Retrovirus Vectors

VIROLOGY. Engineering Viral Genomes: Retrovirus Vectors VIROLOGY Engineering Viral Genomes: Retrovirus Vectors Viral vectors Retrovirus replicative cycle Most mammalian retroviruses use trna PRO, trna Lys3, trna Lys1,2 The partially unfolded trna is annealed