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!!!!
  • Vector delivery

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

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