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. 2010 Jan:Chapter 4:Unit 4.24.
doi: 10.1002/0471142301.ns0424s50.

Gene transfer into rat brain using adenoviral vectors

Mariana Puntel  1 , Kurt M Kroeger, Nicholas S R Sanderson, Clare E Thomas, Maria G Castro, Pedro R Lowenstein
Affiliations

Gene transfer into rat brain using adenoviral vectors

Mariana Puntel et al. Curr Protoc Neurosci. 2010 Jan.

Abstract

Viral vector-mediated gene delivery is an attractive procedure for introducing genes into the brain, both for purposes of basic neuroscience research and to develop gene therapy for neurological diseases. Replication-defective adenoviruses possess many features which make them ideal vectors for this purpose-efficiently transducing terminally differentiated cells such as neurons and glial cells, resulting in high levels of transgene expression in vivo. Also, in the absence of anti-adenovirus immunity, these vectors can sustain very long-term transgene expression within the brain parenchyma. This unit provides protocols for the stereotactic injection of adenoviral vectors into the brain, followed by protocols to detect transgene expression or infiltrates of immune cells by immunocytochemistry or immunofluorescence. ELISPOT and neutralizing antibody assay methodologies are provided to quantitate the levels of cellular and humoral immune responses against adenoviruses. Quantitation of adenoviral vector genomes within the rat brain using qPCR is also described.

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Figures

Figure 4.24.1
Figure 4.24.1
Optimal position of stereotactic frame, fiber-optic light source, and surgical microscope.
Figure 4.24.2
Figure 4.24.2
Drill and sterilized surgical tools required for stereotactic injection of adenoviral vectors into the rat brain. From left to right: drill, scissors, forceps with suture, syringe with bent needle, retractor, scalpel, forceps.
Figure 4.24.3
Figure 4.24.3
Correct placement of anesthetized rat in the stereotactic apparatus. (A) The ear bars are gently inserted into each ear canal so the head of the animal is firmly in place and does not wobble. (B) It is critical to ensure the animal’s tongue is extended outside of the mouth to prevent choking during surgery.
Figure 4.24.4
Figure 4.24.4
The syringe is positioned over the future injection site and lowered to the skull. A marker pen is used to mark the position of the needle on the skull. The skin overlying the skull is held back with a retractor.
Figure 4.24.5
Figure 4.24.5
The Hamilton syringe is slowly depressed to inject adenoviral vector into the brain. (A) 2 to 3 minutes after the needle is inserted into the brain, the syringe plunger is slowly depressed to inject 0.5 µl over the course of 1 min. (B) Precise finger position is used to control the speed of the injection.
Figure 4.24.6
Figure 4.24.6
Staining reaction for immunohistochemistry, using glucose oxidase (GOD), horseradish peroxidase (HRP), and 3′3′-diaminobenzidine (DAB).
Figure 4.24.7
Figure 4.24.7
Transgene expression at 3 and 30 days post-infection with different doses of vector and different vector backbones. Panels (A–D) show the immunohistochemical detection of the transgene product β-galactosidase (β-Gal) after administration of either 1 × 107 infectious units (panels A and B) or 1 × 109 infectious units (panels C and D) of a first-generation adenovirus vector expressing β-Gal from the hCMV promoter. Transgene expression remains stable over the 30-day period after injection of 107 infectious units, but is almost eliminated after injection of 109 infectious units. Panels E and F show transgene expression from 1 × 107 infectious units of a high-capacity adenovirus vector, expressing β-galactosidase from the hCMV promoter. Levels of expression from the high-capacity vector are similar to those from the equivalent dose of first-generation vector (from Thomas et al., 2000).
Figure 4.24.8
Figure 4.24.8
The area of the brain surrounding the injection site is dissected. (A) The brain is placed in the rat brain matrix and a razor blade is used to make a dorso-ventral cut separating the forebrain from the midbrain. (B) A second razor blade is used to make a dorso-ventral cut to remove the olfactory bulb. (C) A third razor blade is used to separate the right and left forebrain hemispheres.
Figure 4.24.9
Figure 4.24.9
Detection of blood-brain barrier permeability 2 days after administration of (A) saline, (B) 1 × 107 infectious units of adenovirus vector, or (C) 1 × 109 infectious units of adenovirus vector. Animals were injected with HRP via the tail vein 20 min before perfusion-fixation, and HRP within the brain was detected according to the Hanker-Yates method (E. Abordo-Adesida, M. Castro, and PR. Lowenstein, unpub. observ.).
Figure 4.24.10
Figure 4.24.10
Apparatus for perfusion-fixation by gravity.
Figure 4.24.11
Figure 4.24.11
Transgene expression and inflammation in brains injected with 1 × 104 infectious units (panels A,B,E, and F) or 1 × 107 infectious units (panels C,D,G, and H) of a first-generation adenovirus vector expressing β-galactosidase from the major intermediate early human CMV promoter (hCMV; panels B,F,D, and H) or the major intermediate early murine CMV promoter (mCMV; panels A,E,C and G). Expression from the mCMV promoter is approximately 3 log units higher than from the hCMV promoter, and thus substantial levels of transgene expression can be achieved from low doses of vector which elicit minimal levels of inflammation—evidenced here by ED1 staining of activated microglial cells and macrophages (from Gerdes et al., 2000).
Figure 4.24.12
Figure 4.24.12
Immunohistochemical detection of GFAP (A,C; activated astrocytes) or NeuN (B,D; neuronal nuclei) 3 days after injection of 1 × 107 or 1 × 109 infectious units of a first-generation adenovirus vector expressing β-galactosidase. Low-power images are shown on the left of each panel. The small box in the low-power images delineates the region which is shown at higher power on the right of each panel. Absence of GFAP or NeuN immunoreactivity within the injection site indicates astrocyte/neuronal death due to direct vector-mediated cytotoxicity or damage caused by the needle injection. Note the lesion in brains injected with 1 × 109 infectious units of vector extends throughout the region corresponding with the area of vector spread (Thomas et al., 2000).
Figure 4.24.13
Figure 4.24.13
Effect of injecting an RCA-contaminated stock of adenovirus vector into the brain. Panels A–D show the immunohistochemical detection of activated astrocytes (GFAP; panels A and B) and activated microglial cells and macrophages (ED1; panels C and D), 14 days after injection of 1 × 109 infectious units of adenovirus vector, either RCA-free (panels B and D), or heavily contaminated with RCA (panels A and C). Although the high dose of RCA-free vector has caused considerable inflammation (i.e., astrocyte activation and macrophage infiltration), injection of the same dose of RCA-contaminated vector has caused massive inflammation and tissue necrosis (E. Abordo-Adesida, M. Castro, and PR. Lowenstein, unpub. observ.).
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References

Literature Cited

    1. Akli S, Caillaud C, Vigne E, Stratford-Perricaudet ED, Poenaru E, Perricaudet M, Kahn A, Peschanski MR. Transfer of a foreign gene into the brain using adenovirus vectors. Nature Genetics. 1993;3:224–228. - PubMed
    1. Amalfitano A, Parks RJ. Separating fact from fiction: Assessing the potential of modified adenovirus vectors for use in human gene therapy. Curr. Gene Ther. 2002;2:111–133. - PubMed
    1. Barcia C, Gerdes C, Xiong W, Thomas CE, Liu C, Kroeger KM, Castro MG, Lowenstein PR. Immunological thresholds in neurological gene therapy: Highly efficient elimination of transduced cells may be related to the specific formation of immunological synapses between T cells and virus-infected brain cells. Neuron Glia Biol. 2006a;2:309–327. - PMC - PubMed
    1. Barcia C, Thomas CE, Curtin JF, King GD, Wawrowsky K, Candolfi M, Xiong WD, Liu C, Kroeger K, Boyer O, Kupiec-Weglinski J, Klatzmann D, Castro MG, Lowenstein PR. In vivo mature immunological synapses forming SMACs mediate clearance of virally infected astrocytes from the brain. J. Exp. Med. 2006b;203:2095–2107. - PMC - PubMed
    1. Barcia C, Jimenez-Dalmaroni M, Kroeger KM, Puntel M, Rapaport AJ, Larocque D, King GD, Johnson SA, Liu C, Xiong W, Candolfi M, Mondkar S, Ng P, Palmer D, Castro MG. Sustained, one year expression from high-capacity helper-dependent adenoviral vectors delivered to the brain of animals with a pre-existing systemic anti-adenoviral immune response: implications for clinical trials. Mol. Ther. 2007;15:2154–2163. - PMC - PubMed

Key References

    1. Dewey, et al. 1999 See above. A description of previously unforeseen long-term toxic side-effects of administering a potentially therapeutic adenovirus vector. This paper provides an excellent illustration of the importance of investigating long-term side effects of vector administration to the brain

    1. Easton RM, Johnson EM, Creedon DJ. Analysis of events leading to neuronal death after infection with E1-deficient adenovirus vectors. Mol. Cell. Neurosci. 1998;11:334–347. A detailed molecular study of the consequences of infecting peripheral neurons in culture

    1. Gerdes, et al. 2000 See above. Illustrates the different levels of expression achievable from two different adenovirus vectors with the same virus backbone, expressing the same transgene under the control of two different promoters

    1. Thomas, et al. 2000 See above. An in-depth investigation, in the rat brain, of the longevity of transgene expression, and inflammatory and cytotoxic side-effects from different doses of vector from 106 to 109 infectious units

    1. Wood, et al. 1996 See above.A review of the immune responses to adenovirus vectors injected into the brains of naive or primed animals

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