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Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
Vector delivery
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Vector delivery

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  • NT-3 = neurotrophin 3BDNF= brain-derived neurotrophic factortrk = tyrosine kinase receptor
  • Doxycycline = tetracycline type antibioticTetracycline inducible systemTheoretically it should be possible to switch the expression on/off depending on the presence of doxycyclineMethod:Transfect cells (e.g. schwann cells) with gene of interest, add factor required for “on”
  • nACHR = nicotinic acetylcholine receptorNCAM = neuronal cell adhesion moleculep75NTR = p75 neurotrophin receptor
  • EYFP = Enhanced Yellow Fluorescent ProteinIpsilateral = on the same sideContralateral = on the opposite side
  • ~ 300- 400 neurons that correspond to the area and since 32- 43 neurons were found to be EYFP + ~10% of motor neurons can be transducedths way using the titer levels as outlined by thegroupUsing a higher concentration of virus may not be the best mechanism as the immune system may have a more pronounced effect
  • + EYFP staining only seen in a subset of neurons since these are the once that are innervating the muscle of the injected site!!!!
  • Transcript

    • 1.
    • 2. Gene Delivery to the Spinal Cord
      Pierre Zwiegers
      `
      Pierre Zwiegers28th-January-2009
    • 3. Presentation Outline:
      • Histological results of the up regulated progranulin experiment:
      • 4. Overview of axonal retrograde transport
      • 5. Types of Viral Vectors:
      - Retroviruses (incl. Lentiviruses)
      - Adenoviruses
      - Adeno-associated viruses
      • Viral Pseudotyping
      • 6. Mechanistic considerations for the intracellular up regulation of gene products
      • 7. Suggested experimental workflow
    • Progranulinupreglation in a murine model
      …the project from hell…
    • 8. 2007: In Vitro assessment of PGRN’s neuroprotective properties
      ii) 24 hr PGRN pre-incubation +
      24hr MPTP co-incubation
      i) 24 hr PGRN pre-incubation
      • PC-12 cells pre-incubated with the neurotrophic factor showed decreased cell death
      in response to a toxic MPTP insult
      • Possible explanation of apparent paradoxical result:
      - Increased proliferation results in increased MPTP susceptibility
      - Degradation of PGRN following 48 hrs in solution results in gain of
      toxic function
       Turned out that the contradictory results were abrogated when a “fresh” batch of
      PGRN was used
    • 9. 2008: Will upregulated PGRN mediate a neuroprotective effect
      against BSSG as mediated by lentiviral gene delivery?
      • Outline:
      - Experimental Male CD-1 animals were unilaterally injected in the right
      gastrocnemius muscle with a PGRN- expressing lentiviral vector
      - Feeding of 2mg BSSG /day for 15 weeks followed by a 5 week wash-out
      - 4 goups in total : PGRN-BSSG; PGRN-control; control-BSSG; control-control
      • Behavioral Assays:
      - Ethovision - Rotorod
      - DigiGait - Leg Extension
      Histochemical Assays:
      Neuronal Numbers:
      - Cresyl Violet
      - anti-ChAT
      Stress Markers:
      - HSP70
      - ATF3
      Kaspar B.K. et al
    • 10. Histological Results
    • 11. Prgn-BSSG
      Prgn-Chow
      Saline-BSSG
      Saline-Chow
      Histology: Neuronal Number
      Cresyl Violet
      • No statistically significant differences were found
    • Saline-Chow
      Saline-BSSG
      PGRN-Chow
      PGRN-BSSG
      Left
      14
      Right
      12
      10
      8
      6
      4
      2
      0
      Saline Chow
      Saline BSSG
      Prgn Chow
      Prgn BSSG
      Histology: Neuronal Number
      ChAT
      # of Motor Neurons
      ChATPositive Neurons in Lumbar Spinal Cord
      • No statistically significant differences were found even though the expected trend
      is evident
       Perhaps due to low levels of cells transduced?
    • 12. Histology: Stress Markers
      HSP70
      HSP70 Positive Cell Counts in Lumbar Spinal Cord
      Saline-Chow
      Saline-BSSG
      PGRN-Chow
      PGRN-BSSG
      Left
      Right
      • No statistically significant differences were found
    • Histology: Stress Markers
      ATF3
      ATF3 Positive Cell Counts in Lumbar Spinal Cord
      Saline-Chow
      Saline-BSSG
      PGRN-Chow
      PGRN-BSSG
      Left
      Right
      • No statistically significant differences were found
    • PITFALLS
      • Lack of significant BSSG-induced neuropathology
      • 13. PGRN did not mediate the expected neuroprotective effects
      • 14. Inability to demonstrate viral presence and/or PGRN upregulation:
      • 15. The majority of the animals were transfected with a vector
      that did NOT contain a GFP construct
      • Cannot conclusively deduce GFP expression in those that received the
      GFP-vector (autofluoresence)
      • No unfixed/unprocessed tissue samples on which to run
      Northern/Southern/Western blots or PCR
      • PGRN IHC will be problematic as the protein is not exclusively located in
      the CNS (there is evidence that the protein is localized in specified
      neuronal cell populations within the brain)
      - Would we really expect a DRASTIC difference compared to
      endogenous PGRN levels?
    • 16. Retrograde Axonal Transport
    • 17. Retrograde Transport:
      Spinal Cord
      Muscle
      Kaspar B.K. et al
      Wikipedia.
    • 18. Viral Vectors
      ADENOVIRUS
      ADEN|O-ASSOCIATED VIRUS
      RETROVIRUS
      LENTIVIRUS
    • 19. Adenovirus
      • Genetic information: dsDNA
      • 20. Expression: Transient
      • 21. Immunogenecity: High
      • 22. Host Range: Broad
      • 23. Since the DNA is not integrated into the genome of the host, these genes
      STOP replicating prior to cell division and subsequently are lost to the daughter
      cells
       Will require re-administration so that descendant cells will express
      gene of interest
      CELL
      VECTOR
      NUCLEUS
      http://www.virobathe.org/M055250-Adenovirus-SPL.jpg
    • 24. Adeno-associated virus
      • Genetic information: ssDNA
      • 25. Expression: Long Term
      • 26. Immunogenecity: Low/none
      • 27. Host Range: Broad
      • 28. AAV can infect non-dividing cells and thus can be useful in delivering genes to
      neurons
      • The recombinant AAV cannot integrate into the host genome since the
      virulence genes have been removed
      • Drawback: the small amount of DNA that can be carried
      CELL
      EPISOME
      VECTOR
      NUCLEUS
      http://junghokingdompropatel.com/images/virusadn.jpg
    • 29. Retrovirus
      • Genetic information: ssRNA
      • 30. Expression: Long Term
      • 31. Immunogenecity: Low/none
      • 32. Host Range: Limited
      • 33. Can be replication-competent/deficient depending on whether the genes for
      virion replication are present/absent
      • Random integration of viral genome can result in essential gene mutagenesis/
      tumourgenesis
      • Seems to only infect rapidly dividing cells, therefore transfecting neurons would be
      problematic
      CELL
      RT
      INTEGRASE
      VECTOR
      NUCLEUS
      http://www.memeticians.com/2007/11/28/retrovirus/retrovirus.jpg
    • 34. Lentivirus
      • Genetic information: ssRNA
      • 35. Expression: Long Term
      • 36. Immunogenecity: Low/none
      • 37. Host Range: Limited
      • 38. Integrate into the genome of non-dividing cells (e.g. neurons) at a random
      position
      • Random site of integration into host genome
      • 39. A murine cancer model did not show an increase in tumorogenic prevalence
      CELL
      RT
      VECTOR
      INTEGRASE
      NUCLEUS
      http://www.uiowa.edu/~gene/protocols.htm
    • 40. Viralpseudotyping
    • 41. Pseudotyping
      • Vector entry into the cell is mediated by the interaction between viral coat
      proteins and host cell surface receptors/molecules
      • Consequently, to increase the possible host range of the vector, one could
      engineer the vehicle so as remove the endogenous protein coat and express
      other viral/chimeric proteins
       Stay tuned, some of the papers that will shortly be discussed utilized
      pseudotyped vectors/chimeric proteins
      A
      B
      B’
      • B’ exhibits the tropism originally found in A
    • Mechanisms for gene upregulation
    • 42. Abdellatifet al.
      • Wished to elucidate the efficacy of viral-mediated gene delivery through an
      intraspinal injection
      • Gene of interest was D15A, a mutant NT-3 that binds to trkB and trkC
      • 43. D15A exhibits both NT3 and BDNF activities and thus have implications for SCI’s
    • Abdellatifet al.
      Generalized Experimental Overview:
      • Spinal cord (Sprague-Dawley rats) was
      exposed via laminectomy, with the dura
      incised to mediate access to the cord
      • 1ul was injected (viral stocks/PBS/Cell susp.)
      directly in to the spinal cord
      • Plethora of vectors tested:
       Lentivirus
       Adenovirus
       Retrovirus
      • Performed assays included:
      - ELISA
      - BCA Protein Assay
      - IHC (astrocytes, oligodendrocytes, neurons,
      microglia, immunomarkers)
      Exposed SC
      Visualized Laminectomy
      http://www.pcrm.org/resch/anexp/osu_about.html
    • 44. Abdellatifet al.
      Summary of Results :
      • Lentiviralvectors exhibit the most stable levels of gene expression and hence
      are more suitable for long-term transgene delivery
      • Ex-vivo retroviral delivery of transgenes are appropriate for transient delivery
      • 45. Protein levels are dependent upon viral titer
      • 46. Protein levels downregulate disproportionately in comparison to intracellular
      markers (e.g. GFP) following transfection and may subsequently not reflect actual
      levels of secreted transgene product
    • 47. Abdellatifet al.
      Results:
      • 1 wk after injection, expression of D15A was significantly higher in both goups
      • 48. Gene expression was dependent upon viral titer
      • 49. D15A expression was NOT significantly different between titer-matched vectors
    • Abdellatifet al.
      Results:
      • Lentiviral-mediated gene delivery resulted in stable expression up to 4 weeks
      following the injections
      • Note the adenoviral vector(w/o soluble Ab) showed marked downregulation
      over the study period
    • 50. Abdellatifet al.
      Results:
      • Ex-vivo vector delivery will be beneficial if one wishes for temporal control of
      transgene expression
      • After infected cells injected into spinal cord, water was
      supplemented with Doxycycline
      • See significant reduction in D15A levels if Dox
      administration ended after 1 week
      • No significant difference in D15A between the two time
      points in animals that received Dox for the entire period
      • Note: the “No Dox” group exhibited statistically significant D15A levels as
      compared to “Normal”  indicates the inherent leakiness of the system
    • 51. Mentis et al.
      • Wished to investigate a less neuroinvasive mechanism for vector delivery to the
      spinal cord utilizing a pseudotypedLentivirus (HIV-1)
      • HIV-1 was pseudotyped with glycoproteins derived from Rabies Virus (RV) in an
      attempt to confer RV tropism
    • 52. Mentis et al.
      Why Rabies Virus Glycoproteins?
      • The tropism of the virus causes transduction from the site of entry, via peripheral
      nerves, to the CNS where it results in encephalitis
      • “Receptors” for RV include nAChR at NMJ, NCAM, p75NTR
      http://medicineworld.org/images/blogs/9-2007/rabies-4120.jpg
    • 53. Mentis et al.
      Generalized Experimental Overview:
      • Two RV glycoprotein genes were derived from the mouse-brain adapted
      challenge virus (CVS-24) termed CVS-N2cand CVS-B2c
      • cDNA encoding CVS-N2c or CVS-B2c and a marker gene (EYFP) was incorporated
      into the HIV-1 genome
      • Intramusclar injections of pseudotyped HIV-1 vectors were performed on P12
      Swiss-Webster mice:
       Gastrocnemius
       Tibialisanteroir
       Soleus
      • Sacrificed two weeks after vector delivery
      Rosenberg P. et.al. PNAS 2004;101:9387-9392
    • 54. Mentis et al.
      Results:
      • EYFP expression was observed after transfection with both types of pseudotyped
      vectors
      • As expected, gastrocnemius-mediated delivery resulted with EYFP expression in
      Lumbar levels L4-L6
      • EYFP expression was restricted to the VENTRAL HORN on the ipsilateral side of
      the injection
      • ~ 10% of the motor neurons were transduced
      Kaspar B.K. et al
    • 55. Mentis et al.
      Results:
      • Motor neurons that activate a specific muscle define a motor nucleus
      • 56. Motor nuclei that serve hind limb muscles are found in the lumbar spinal cord
    • Mentis et al.
      Results:
      • Injection and retrograde transport of Fast Blue allows one to identify the
      neurons of a specific pool
    • 57. Mentis et al.
      Results:
      • All EYFP-positive neurons were co-labeled with ChAT, indicating transfected cells
      are motor neurons
    • 58. Mentis et al.
      Results:
      • Only Fast Blue motor neurons expressed EYFP  consequently viral transgene
      expression was localized to those neurons innervating the injected muscle
    • 59. Mentis et al.
      Results:
      • Depending on the muscle, transduction efficiencies on average, were between
      8% and 26%
      • No significance difference in infectivity between the pseudotyped vectors
    • 60. Putative experimental design
    • 61.
    • 62. General Outline:
      CD-1 male mice
      Vehicle
      Bilateral intra-gastrocnemiusinjection
      Progranulin-expressing lentivirus
      bilateral intra-gastrocnemius injection
      3 weeks
      3 weeks
      BSSG (2 mg /day)
      diet
      Normal
      chow diet
      BSSG (2 mg /day)
      diet
      Normal
      chow diet
      15 weeks of feeding + 5 weeks wash-out
      Behavioural Analysis
      Polymerase Chain Reaction
      Biochemical Analysis
      Histological Analysis
    • 63. Vector Design:
      PGRN
      PGRN
      PGRN
      PGRN
    • 64. Expression Assays:
      Following homogenization, run a gel and
      perform a WB
      OR
      PCR amplify using engineered primers
      Northern/ Western Blots
      * 25 L of 1 x 108 TU/mL
      into the right gastrocnemius
      Southern Blots
    • 65. Insert: Applause!
      I thank you for your pity

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