This work was funded by the ALS Association, Karyopharm Therapeutics, Inc., Meira GTx, and a charitable donation for ALS research from Thomas Lawson, for which we are grateful. We thank Elysse Orchard and Donna Burney for advice and training. JAS was supported by the Lied Foundation and National Institutes of Health grant GM103418.

All procedures followed the appropriate NIH guidelines. All of the procedures followed animal protocols that were approved by the Animal Care and Use Committee at LSU Health Sciences Center in Shreveport. Female Sprague-Dawley rats at 6 weeks of age were used for this procedure.
1. Determine the doses/volumes of vector needed
<ol>
	<li>Weigh the rats to be injected and determine the amount of viral vector needed for each animal based on the titer (concentration) of the vector preparation. Use vector doses that have been successful for efficient expression in the rat CNS previously (see Jackson et al.6). 
	NOTE: For AAV9 TDP-43 or AAV9 GFP, a typical dose is between 3 x 1013 - 1 x 1014 vector genomes/kg. The young adult rats at 6 weeks of age weigh about 150 g.</li>
	<li>Make equivalent doses per kg in all animals for systematic comparisons. Ensure the volume does not exceed standard advised injection volumes (250 - 500 &#181;L for a 250 g rat). A typical injection volume is 200 &#956;l to a young adult rat.</li>
</ol>
2. Prepare work station
<ol>
	<li>Gather all required supplies: one paraffin film square (about 5 x 5 cm) per animal, one alcohol preparation pad per animal, gauze pads (2 - 3 per animal), pipet tips, and a micropipette.</li>
	<li>Prepare the working surface by cleaning the surface with 70% ethanol. Place a heating pad down with the temperature set to low (~37 &#176;C). Place a bench pad on top of the heating pad.</li>
	<li>Prepare the gas anesthesia (isoflurane). Ensure adequate oxygen and gas anesthesia for the procedure. Clean the induction chamber and anesthesia mask with 70% ethanol, and place the anesthesia mask on the bench pad.</li>
</ol>
3. Prepare the vector
<ol>
	<li>Thaw the vector at room temperature, and label one sterile microcentrifuge tube for each animal.</li>
	<li>Pipet the volume of AAV determined in step 1.1 into the appropriate tube and bring the volume up to the desired injection volume (here, 200 &#956;l) with lactated Ringer&#39;s solution or saline.</li>
	<li>Pulse vortex the sample for 1 - 2 s and then pulse centrifuge for 5 s to bring the sample to the bottom of the tube.</li>
	<li>Pipet the total injection volume for the rat out of the tube and onto the square of paraffin film.</li>
	<li>Place a 30 gauge needle on a 1 mL syringe.</li>
	<li>Place the needle with the bevel facing down into the vector on the paraffin film. Pull back on the syringe plunger until all the virus is in the needle and syringe. Be careful to avoid drawing air bubbles into the needle, which becomes easier with practice.</li>
</ol>
4. Prepare the rat
<ol>
	<li>Turn the oxygen up to 1 L/min and set the isoflurane anesthesia to 5%. Ensure that the gas anesthesia is flowing to the induction chamber by checking all connecting hoses and stopcock orientations.</li>
	<li>Place the rat in the gas anesthesia induction chamber about 3 min, until the rat is unresponsive.</li>
	<li>Ensure adequate depth of anesthesia by pinching the toe. There should be no movement in the foot or leg after the toe pinch.</li>
	<li>Reduce the isoflurane anesthesia to 2% for maintenance of anesthesia and adjust the stopcock so that the anesthesia is flowing to the anesthesia mask.</li>
	<li>Place the rat&#39;s nose in the anesthesia mask and adjust the rat so that it is lying on its side.</li>
	<li>Identify the lateral tail vein. 
	NOTE: The lateral tail veins lie at approximately 10 and 2 o&#39;clock with the dorsal top of the tail as 12 o&#39;clock. With the rat lying on its side, the lateral tail vein should be facing up.</li>
	<li>Wipe the injection area with the alcohol preparation pad. The injection site is two-thirds down the length of the tail.</li>
</ol>
5. Inject the vector
<ol>
	<li>Firmly hold the tail slightly above the injection area with one finger directly over the lateral tail vein; this will dilate the vein.</li>
	<li>With the bevel facing up, align the needle with the visible tail vein in the same direction.</li>
	<li>Pierce the skin over the tail vein while taking great care to avoid the other hand holding the tail and pressing on the tail vein.</li>
	<li>Release pressure from the tail vein to allow for normal blood flow.</li>
	<li>Move the needle into the vein. To ensure that the needle has punctured the tail vein, pull back slightly on the plunger. If the needle is in the vein, blood will flow into the needle.
	<ol>
		<li>Reposition the needle as needed. Then, to stabilize the needle, move the hand above the injection site to below the injection site and hold the end of the syringe against the tail.</li>
	</ol>
	</li>
	<li>Slowly inject the vector into the tail (approximately 20 &#181;L/s). 
	NOTE: The vein may lose color as the vector flows through it. Signs that the solution was injected outside of the vein include blebbing, blanching of the skin, or resistance when injecting the vector. With practice, extra-vascular exposure can be minimized.</li>
	<li>After the vector has been administered, remove the needle from the rat and press a gauze pad against the injection site to help stem the bleeding for 30 - 60 s.</li>
</ol>
6. Clean-up
<ol>
	<li>Turn off the isoflurane anesthesia and oxygen. Monitor the rat until it regains consciousness, and then return it to its cage.</li>
	<li>Carefully place all needles in the sharps container. Dispose of all other disposable materials that are potentially contaminated with AAV into an appropriate biohazard container. Clean the working stations with 70% ethanol.</li>
</ol>
7. Evaluate hindlimb escape reflex
NOTE: Hindlimb deficits usually arise 2 - 6 weeks after TDP-43 gene transfer, depending on the vector dose.
<ol>
	<li>On a flat surface clear of debris, with two fingers grab the rat&#39;s tail approximately 2 cm from the body. Lift the rat gently until the forelimbs are hanging while viewing the ventral underside of the rat.
	<ol>
		<li>Perform the procedure weekly throughout the experimental time-course after gene transfer (e.g., up to 12 weeks after gene transfer). 
		NOTE: Also score forelimb deficits in this manner, but hindlimb deficits are more likely to occur in this model, depending on the vector dose used.</li>
	</ol>
	</li>
	<li>Observe the hindlimbs for 10 s. Note if one or both hindlimbs clench towards the midline during the 10 s.</li>
	<li>Repeat this test for a total of three times with 30 s between trials. If one or both hindlimbs are clenched for each of three trials, the rat is showing motor dysfunction. Record the data (manually in notes) as either unilateral or bilateral hindlimb deficit present if there is consistent clenching across all three trials.</li>
</ol>
8. Evaluate motor function on the rotarod
<ol>
	<li>To set up the rotarod, place a bench pad beneath the rotarod and set acceleration to 4 - 40 revolutions per minute (rpm) over 2 min (the rate increases by ~0.3 rpm/s). For a 12 week time-course, run the subjects at 2, 4, 8, and 12 weeks after gene transfer.</li>
	<li>Allow the rat to acclimate to the testing room for 20 min.</li>
	<li>To train the rat to use the rotarod for the first time, first start the rotarod at a set speed of 4 rpm, then place the rat on the rotarod. Allow the rat to walk for one full revolution at 4 rpm, then start the acceleration and allow the rat to walk on the rotarod until it falls off. Continue training until the rat can consistently walk for at least 40 s. A healthy, young adult rat will be readily trained in this manner.
	<ol>
		<li>In the case of a rat with a noticeable motor impairment, which may prevent the rat from achieving a baseline latency to fall of 40 s, provide the rat 3 - 5 training attempts before beginning rotarod scoring.</li>
	</ol>
	</li>
	<li>To begin rotarod testing, place the rat on the rotarod and start acceleration of the rotarod. Note the time at which the rat falls from the rotarod; this is the latency to fall. If the rat remains on the rotarod after 120 s, cap the session at this point, remove the rat from the rotarod and record a fall latency of 120 s.</li>
	<li>Repeat the testing two more times for a total of three trials. To minimize the effect of fatigue, space the trials at least 5 min apart. Average the scores of the three trials. An unimpaired adult rat typically has an average latency to fall of &#62; 40 s.</li>
	<li>Place the rat back in the cage, and clean the rotarod and surrounding areas with 70% ethanol.</li>
</ol>
9. Quantification of GFP expression in the cerebellum
<ol>
	<li>Anesthetize the rats and perfuse with phosphate buffered saline (PBS) and then 4% paraformaldehyde. Dissect the brain and spinal cord and soak the tissues in the fixative overnight and then move them to a 30% sucrose solution. After equilibration, cut 50 &#956;m coronal sections on a sliding microtome with freezing stage4,5,6.</li>
	<li>Choose 3-6 sections of the cerebellum that are evenly spaced in the vermis, the central lobe of the cerebellum. Fluorescently label the GFP to maximize the signal. Use the primary antibody for GFP at 1:500 and use the Alexa-488 conjugated secondary antibody at a dilution of 1:3004,5,6. 
	NOTE: See references 4,5,6 for more detail on the sampling, sectioning, and staining procedures.</li>
	<li>Photomicrograph each section in the defined region of interest with a low magnification lens using a microscope. Use a 2.5X objective (N.A. 0.12) and filter set 10, 450-490/515-565 excitation/emission). Keep the camera settings (exposure times, e.g., 2 s) the same across samples to be analyzed. Save as a .TIF file.</li>
	<li>Open the photomicrograph in the widely available Scion image program; two windows will open. Close the window indicated as &#34;Indexed Color&#34;.</li>
	<li>Choose &#34;Options&#34;, then &#34;Density Slice&#34;; a range of shades will be indicated in red on the Look-Up Table (LUT) window. In the window, use the cursor to fill in the specific GFP labelling only and stop when the non-specific background begins to be picked up in the image. 
	NOTE: This will highlight the fluorescent area on the photomicrograph to be quantified. The settings should be optimized to specifically capture all of the visibly fluorescent cells. With practice, this method can precisely highlight the specific fluorescence.</li>
	<li>Before measuring fluorescent area, first ensure that the measurements made are in the correct units (pixels). To do this, click &#34;Analyze&#34;, then &#34;Set Scale&#34;; the fourth line should say &#34;Units&#34;. From the drop-down list, choose pixels to measure in pixels, then click &#34;OK&#34;. Toggle the analysis options to &#34;Include Interior Holes&#34; to ensure the inclusion of the entire cell body in the analysis.</li>
	<li>To measure the fluorescent area, click &#34;Analyze&#34; and then click &#34;Measure&#34;. To see this value, click &#34;Analyze&#34; then click &#34;Show Results&#34;; a results window will appear. The fluorescent area measurement will be under the heading labeled &#34;Area&#34;.</li>
	<li>To ensure standardization across the measurements, do not change the highlighted values in the LUT window.</li>
	<li>Once all measurements have been recorded, average the values for each animal (from 3 - 6 sections per animal). Use an appropriate statistical test to compare the transduced areas between the groups.6</li>
</ol>

<ol>
	<li>Foust, K., Nurre, E., Montgomery, C., Hernandez, A., Chan, C., Kaspar, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+AAV9+preferentially+targets+neonatal+neurons+and+adult+astrocytes.">Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes.</a> Nat. Biotechnol. 27 (1), 59-65 (2009).</li><li>Duque, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+administration+of+self-complementary+AAV9+enables+transgene+delivery+to+adult+motor+neurons.">Intravenous administration of self-complementary AAV9 enables transgene delivery to adult motor neurons.</a> Mol. Ther. 17 (7), 1187-1196 (2009).</li><li>Gombash Lampe, S., Kaspar, B., Foust, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+injections+in+neonatal+mice.">Intravenous injections in neonatal mice.</a> J. Vis. Exp. (93), e52037 (2014).</li><li>Wang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansive+gene+transfer+in+the+rat+CNS+rapidly+produces+amyotrophic+lateral+sclerosis+relevant+sequelae+when+TDP-43+is+overexpressed.">Expansive gene transfer in the rat CNS rapidly produces amyotrophic lateral sclerosis relevant sequelae when TDP-43 is overexpressed.</a> Mol. Ther. 18 (12), 2064-2074 (2010).</li><li>Dayton, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+forelimb+impairment+in+rats+expressing+a+pathological+TDP-43+25+kDa+C-terminal+fragment+to+mimic+amyotrophic+lateral+sclerosis.">Selective forelimb impairment in rats expressing a pathological TDP-43 25 kDa C-terminal fragment to mimic amyotrophic lateral sclerosis.</a> Mol. Ther. 21 (7), 1324-1334 (2013).</li><li>Jackson, K., Dayton, R., Klein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV9+supports+wide-scale+transduction+of+the+CNS+and+TDP-43+disease+modeling+in+adult+rats.">AAV9 supports wide-scale transduction of the CNS and TDP-43 disease modeling in adult rats.</a> Mol. Ther. Methods Clin. Dev. 2, 15036 (2015).</li><li>Jackson, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+of+forelimb+function+by+UPF1+gene+therapy+in+a+rat+model+of+TDP-43-induced+paralysis.">Preservation of forelimb function by UPF1 gene therapy in a rat model of TDP-43-induced paralysis.</a> Gene Therapy. 22 (1), 20-28 (2015).</li><li>Jackson, K., Dayton, R., Deverman, B., Klein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Better+targeting,+better+efficiency+for+wide-scale+neuronal+transduction+with+the+synapsin+promoter+and+AAV-PHP.B.">Better targeting, better efficiency for wide-scale neuronal transduction with the synapsin promoter and AAV-PHP.B.</a> Front. Mol. Neurosci. 9, 116 (2016).</li><li>Jackson, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Severe+respiratory+changes+at+end+stage+in+a+FUS-induced+disease+state+in+adult+rats.">Severe respiratory changes at end stage in a FUS-induced disease state in adult rats.</a> BMC Neuroscience. 17 (1), 69 (2016).</li><li>Xu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CMV-beta-actin+promoter+directs+higher+expression+from+an+adeno-associated+viral+vector+in+the+liver+than+the+cytomegalovirus+or+elongation+factor+1+alpha+promoter+and+results+in+therapeutic+levels+of+human+factor+X+in+mice.">CMV-beta-actin promoter directs higher expression from an adeno-associated viral vector in the liver than the cytomegalovirus or elongation factor 1 alpha promoter and results in therapeutic levels of human factor X in mice.</a> Hum Gene Ther. 12 (5), 563-573 (2001).</li><li>Jackson, K., Dayton, R., Klein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+vector+&amp;#34;magic+bullet&amp;#34;:+targeted+expression+in+the+central+nervous+system+after+peripheral+delivery+using+the+synapsin+promoter.">Gene vector &amp;#34;magic bullet&amp;#34;: targeted expression in the central nervous system after peripheral delivery using the synapsin promoter.</a> Expert Opin Ther Targets. 20 (10), 1153-1154 (2016).</li><li>van Hulst, R., Klein, J., Lachmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+embolism:+pathophysiology+and+treatment.">Gas embolism: pathophysiology and treatment.</a> Clin. Physiol. Funct. Imaging. 23 (5), 237-246 (2003).</li></ol>

The authors have nothing to disclose.

A variety of delivery routes have been tested for AAV vectors: intra-parenchymal, intra-cerebroventricular, intra-thecal, intravenous, intranasal, intra-muscular, etc. Each route may have specific advantages as well as drawbacks. Tail vein administration of AAV to adult rats is a reliable method for achieving consistent transduction of the CNS. The peripheral injection method avoids the introduction of an injection cannula into the CNS tissues and is therefore minimally invasive. The efficient CNS expression that can be ...

Recombinant adeno-associated virus (AAV) vectors are indispensable tools for CNS research because they are so efficient for transducing neurons in vivo. AAV vectors are very versatile for studying different transgenes and protein isoforms, different tissues, different host species, and different routes of administration. For instance, AAV can be administered to mice by a peripheral, relatively non-invasive, intravenous injection to transduce neurons throughout the CNS as first described in Foust et al. and Duque et al. (see JOVE paper by Gombash et al. (2014)).1,2,3 This gene delivery approach is used in rats to efficiently express either green fluorescent protein (GFP) or the ALS related protein, transactive response DNA-binding protein of 43 kDa (TDP-43) in the CNS.4,5,6,7,8,9 Working in rats is significant because the rat&#39;s physiologic and metabolic parameters are closer to humans as compared to mice and there are behavioral and toxicological assays designed specifically for rats. Furthermore, more transgenic rat lines are becoming available that can be utilized in AAV gene transfer studies.
Methods are detailed for expansive CNS gene transfer in the rat, and rapid, reliable quantification of the outcomes. Wide-scale CNS transduction is used to mimic the symptomatology of ALS in rats by expressing TDP-43 throughout the spinal cord. The method is tail vein injections of the TDP-43 vector to young adult rats as used in Jackson et al.6,8,9 After several weeks, TDP-43-induced motor deficits are scored by two methods: escape reflex and rotarod as used in Dayton et al. and Jackson et al.5,6,7,9 For the control GFP vector, in post-mortem analysis, the fluorescent area of the cerebellum is calculated as an index of transduction efficiency as used in Jackson et al.6,8 The analysis of cerebellum has proven to be a rapid and reliable index of the degree of CNS transduction after peripheral gene delivery and should be applicable to a variety of approaches attempting peripheral-to-central gene transfer.

<table><tbody><tr><td>Scale</td><td>Pelouze</td><td>SP5</td><td></td></tr><tr><td>Nalgene bucket</td><td>Thermo Scientific</td><td>12014000</td><td>4000 mL</td></tr><tr><td>Microcentrifuge tube</td><td>USA Scientific</td><td>1615-5500</td><td></td></tr><tr><td>Lactated Ringer&#039;s Solution</td><td>Baxter Healthcare</td><td>0338-0114-04</td><td></td></tr><tr><td>Mini vortexer</td><td>Fisher Scientific</td><td>128101</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>22624415</td><td></td></tr><tr><td>Laboratory film</td><td>Bemis</td><td>PM996</td><td></td></tr><tr><td>1-mL syringe</td><td>BD</td><td>309626</td><td>Comes with a 25g needle attached.&lt;br/&gt; Replace with 27-30g needle for injection</td></tr><tr><td>30g needle</td><td>BD</td><td>305106</td><td></td></tr><tr><td>Isoflurane</td><td>Piramal Healthcare</td><td>66794-013-25</td><td>Isoflurane USP 250 mL</td></tr><tr><td>Anesthesia mask</td><td>Kopf Instruments</td><td>906</td><td></td></tr><tr><td>Gauze</td><td>Henry Schein</td><td>100-2524</td><td>2&quot; x 2&quot; (5cm x 5cm)</td></tr><tr><td>Pipet tips</td><td>USA Scientific</td><td>1111-0816</td><td></td></tr><tr><td>Micropipet</td><td>Gilson</td><td>4642080</td><td></td></tr><tr><td>Gas anesthesia vaporizer</td><td>SurgiVet</td><td>4214322</td><td>Classic T3</td></tr><tr><td>Gas anesthesia scavenger</td><td>SurgiVet</td><td>32373B10</td><td>Enviro-PURE</td></tr><tr><td>Heating pad</td><td>Sears</td><td>17280</td><td></td></tr><tr><td>Bench pad</td><td>VWR</td><td>56617-006</td><td></td></tr><tr><td>Rotarod</td><td>Panlab/Harvard Apparatus</td><td>76-0772</td><td>4 rats/ 4 mice</td></tr><tr><td>70% Ethanol</td><td>Decon Laboratories, Inc.</td><td>2401</td><td>140 proof</td></tr><tr><td>70% Isopropanol</td><td>VWR</td><td>89108-160</td><td></td></tr><tr><td>Scion Image</td><td>Scion Corporation</td><td></td><td></td></tr><tr><td>GFP Tag Polyclonal Antibody</td><td>Invitrogen</td><td>A11122</td><td>Primary Antibody - 1:500 dilution</td></tr><tr><td>AlexaFluor 488</td><td>Invitrogen</td><td>A11070</td><td>Secondary Antibody - 1:300 dilution</td></tr><tr><td>Axioskop 40 microscope</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

methods and tips for intravenous administration of adeno-associated virus to rats and evaluation of central nervous system transduction

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.

TDP-43 induced motor impairments should arise within 2 - 6 weeks depending on the dose of the AAV TDP-43 used (Figure 1). Unsuccessful tail vein injections will result in partial or no impairments; the AAV must reach the bloodstream to produce the effect. A successful injection of AAV GFP will result in GFP expression throughout the brain and spinal cord in a vector-dose dependent manner. When using the strong recombinant promoter to drive expression, the cyt...

Watch this Scientific Journal Video about Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction at JoVE.com

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

Methods for a wide-scale central nervous system gene delivery in the rat are covered. In this example, the purpose is to mimic a disease that affects the entire spinal cord. The widespread transduction can be used to deliver a therapeutic protein to the CNS from a one-time, peripheral administration.

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), ...

methods-tips-for-intravenous-administration-adeno-associated-virus-to

Research

JoVE Journal

Neuroscience

11.4K Views.  Louisiana State University Health Sciences Center. Methods for a wide-scale central nervous system gene delivery in the rat are covered. In this example, the purpose is to mimic a disease that affects the entire spinal cord. The widespread transduction can be used to deliver a therapeutic protein to the CNS from a one-time, peripheral administration. 

Video: Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

Production and Titering of Recombinant Adeno-associated Viral Vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

Immunology and Infection

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Medicine

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

Direct Intrathecal Injection of Recombinant Adeno-associated Viruses in Adult Mice

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, and excellent transduction efficacy in the CNS.&#160;Previous studies have demonstrated the therapeutic potency of AAV-delivered gene therapy in neurodegenerative disorders by IT administration. However, high rates of unpredictable failure due to the technical limitation of IT administration in small animals have been reported. Here, we established a scoring system to indicate the success extent of lumbar puncture in small animals by adding 1% lidocaine hydrochloride into the injection solution. We further show that the extent of transient weakness following injection can predict the transduction efficiency of AAV. Thus, this IT injection method can be used to optimize therapeutic trials in mouse models of CNS diseases that afflict wide regions of the CNS.

Here we present a direct intrathecal injection technique using 1% lidocaine hydrochloride in a viral solution to ensure efficient adeno-associated virus delivery to small animals and establish a scoring system to predict transduction efficiency in the central nervous system according to the degree of transient weakness induced by lidocaine.

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, ...

Purification of AAV Vectors by Single-Step Heparin Affinity Chromatography

Isolation of Adeno-Associated Viral Vectors Through a Single-Step and Semi-Automated Heparin Affinity Chromatography Protocol

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. However, the commonly used methods to purify AAVs make use of cesium chloride or iodixanol density gradient ultracentrifugation. Despite their advantages, these methods are time-consuming, have limited scalability, and often result in vectors with low purity. To overcome these constraints, researchers are turning their attention to chromatography techniques. Here, we present an optimized heparin-based affinity chromatography protocol that serves as a universal capture step for the purification of AAVs.
This method relies on the intrinsic affinity of AAV serotype 2 (AAV2) for heparan sulfate proteoglycans. Specifically, the protocol entails the co-transfection of plasmids encoding the desired AAV capsid proteins with those of AAV2, yielding mosaic AAV vectors that combine the properties of both parental serotypes. Briefly, after the lysis of producer cells, a mixture containing AAV particles is directly purified following an optimized single-step heparin affinity chromatography protocol using a standard fast protein liquid chromatography (FPLC) system. Purified AAV particles are subsequently concentrated and subjected to comprehensive characterization in terms of purity and biological activity. This protocol offers a simplified and scalable approach that can be performed without the need for ultracentrifugation and gradients, yielding clean and high viral titers.

Author Spotlight: Improved Method for Production and Purification of Adeno-Associated Viral Vectors

This manuscript describes a detailed protocol for the generation and purification of adeno-associated viral vectors using an optimized heparin-based affinity chromatography method. It presents a simple, scalable, and cost-effective approach, eliminating the need for ultracentrifugation. The resulting vectors exhibit high purity and biological activity, proving their value in preclinical studies.

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. ...

Bioengineering

Lumbar Puncture Delivery of AAV9 in Juvenile Rats for CNS Gene Therapy

Intrathecal Vector Delivery in Juvenile Rats via Lumbar Cistern Injection

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a toxic gene, or provide a gene whose product can modulate the biology of the disease. The delivery method for the therapeutic vector can take many forms, ranging from intravenous infusion for systemic delivery to direct injection into the target tissue. For neurodegenerative disorders, it is often desirable to skew transduction towards the brain and/or spinal cord. The least invasive approach to target the entire central nervous system involves injection into the cerebrospinal fluid (CSF), allowing the therapeutic to reach a large fraction of the central nervous system. The safest approach to deliver a vector into the CSF is the lumbar intrathecal injection, where a needle is introduced into the lumbar cistern of the spinal cord. This technique, also known as a lumbar puncture, has been widely used in neonatal and adult rodents and in large animal models. While the technique is similar across species and developmental stages, subtle differences in size, structure, and elasticity of tissues surrounding the intrathecal space require accommodations in the approach. This article describes a method for performing lumbar puncture in juvenile rats to deliver an adeno-associated serotype 9 vector. Here, 25-35 &#181;L of vector were injected into the lumbar cistern, and a green fluorescent protein (GFP) reporter was used to evaluate the transduction profile resulting from each injection. The benefits and challenges of this approach are discussed.

Author Spotlight: Assessing Intrathecal Gene Therapy Efficacy in Juvenile Rats

A surgical procedure is described to perform injections into the lumbar cistern of the juvenile rat. This approach has been used for the intrathecal delivery of gene therapy vectors, but it is anticipated that this approach can be used for a variety of therapeutics, including cells and drugs.

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a ...

Direct Vagus Nerve Injection Protocol For Rats

There is a relative abundance of strategies and methodologies to facilitate drug delivery to the central nervous system. However, drug delivery directly to the peripheral nervous system is less common, with fewer detailed methods publications available to aid researchers. Here, we describe a direct nerve injection method for peripheral nervous system drug delivery, using the vagus nerve as a model nerve. This method can be used in the treatment of autonomic nervous system disorders through targeting of the left vagus nerve, although this general injection method can be extrapolated to injection of other nerves with minor modification. This method explains all critical steps involved in the procedure involving microsurgery in anesthetized adult rats under a dissecting microscope. The use of a tracking dye is described to facilitate the monitoring of injection fidelity in real time. Illustrations of successful and failed injections are provided. If carried out properly, direct vagus nerve injections can be conducted in a safe manner that is well-tolerated by the rat without post-delivery complications. For example, once the surgeons were trained in this method, six out of six rats were successfully injected without any complications. This method of direct nerve injection for preclinical rat studies is capable of delivering agents (including but not limited to gene therapy) to peripheral nerves.

We present a protocol for direct vagus nerve injection in rats, enabling drug delivery directly into the nerve without post-injection complications. This method applies to preclinical neurological studies involving autonomic nervous system manipulation. It can be used for direct nerve injection for other nerves in rats and other species, with necessary modifications.

There is a relative abundance of strategies and methodologies to facilitate drug delivery to the central nervous system. However, drug delivery directly ...

AAV-Based Therapies via Intravenous Injection in Preclinical Mouse Models

Consistent Delivery of Adeno-Associated Virus via Lateral Tail-Vein Injection in Adult Mice

Many disorders affect multiple organs or involve different regions of the body, so it is critical to deliver therapeutics systemically to target the affected cells located in different sites. Intravenous injection is a widely used systemic delivery route in preclinical studies that assess treatments intended for body-wide administration. In adult mice, it involves the intravenous administration of the therapeutic agent into the mouse&#39;s lateral tail veins. When mastered, tail-vein injections are safe and fast, and only require simple and commonly available tools. However, tail-vein injections are technically challenging and require extensive training and continuous practice to ensure the accurate delivery of the intended dose.
Here we describe a detailed, optimized, lateral tail-vein injection protocol that we have developed based on our experience and on recommendations that had been previously reported by other groups. Other than the mouse restrainers and insulin syringes, this protocol requires only reagents and equipment that are readily available in most labs. We found that following this protocol results in consistently successful intravenous delivery of adeno-associated virus (AAV) into the tail veins of unsedated 7-9 week-old mice. Additionally, we describe the optimized protocols for the histological detection of fluorescent reporter proteins and vector genome per diploid genome (vg/dg) quantification used to assess AAV transduction and biodistribution. The goal of this protocol is to aid experimenters in easily performing tail-vein injections successfully and consistently, which can reduce the practice time needed to master the technique.

Author Spotlight: Developing Gene Therapies for Muscular Dystrophies and Neuromuscular Disorders

Here we detail an optimized protocol for mouse lateral tail-vein injection to systemically administer adeno-associated virus (AAV) in adult mice. Additionally, we describe protocols of commonly used assays to assess AAV transduction.

Many disorders affect multiple organs or involve different regions of the body, so it is critical to deliver therapeutics systemically to target the ...