Name: Author Spotlight: Assessing Intrathecal Gene Therapy Efficacy in Juvenile Rats
Uploaded: 2024-03-29T14:00:00
Description: Watch this Scientific Journal Video about Author Spotlight: Assessing Intrathecal Gene Therapy Efficacy in Juvenile Rats at JoVE.com

The authors would like to thank Steven Gray, Matthew Rioux, Nanda Regmi, and Lacey Stearman of UT Southwestern for a productive discussion of the challenge posed by juvenile rats for intrathecal injection. This work was partly supported by funding from Jaguar Gene Therapy (to JLFK).

This study was approved by the Emory University Institutional Animal Care and Use Committee (IACUC). Sprague-Dawley rats (28-30 days of age, mass in the range of about 90-135 g, males and females) were used in the present study.
1. Preparation of the vector
<ol>
	<li>Thaw the AAV9 vector (see Table of Materials) on ice at the beginning of the procedure.</li>
	<li>Centrifuge the microcentrifuge tube containing the vector briefly in a table-top centrifuge to e...

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

<ol>
	<li>Wurster, C., Petri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+spinal+muscular+atrophy+research.">Progress in spinal muscular atrophy research.</a> Curr Opin Neurol. 35 (5), 693-698 (2022).</li><li>Wu, K. Y., 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=Retinitis+pigmentosa:+Novel+therapeutic+targets+and+drug+development.">Retinitis pigmentosa: Novel therapeutic targets and drug development.</a> Pharmaceutics. 15 (2), 685 (2023).</li><li>Larkin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+FDA-approved+gene+therapy+for+hemophilia.">First FDA-approved gene therapy for hemophilia.</a> JAMA. 329 (1), 14 (2023).</li><li>Lee, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nadofaragene+firadenovec:+First+approval.">Nadofaragene firadenovec: First approval.</a> Drugs. 83 (4), 353-357 (2023).</li><li>Taghian, T., 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=A+safe+and+reliable+technique+for+CNS+delivery+of+AAV+vectors+in+the+cisterna+magna.">A safe and reliable technique for CNS delivery of AAV vectors in the cisterna magna.</a> Mol Ther. 28 (2), 411-421 (2020).</li><li>Donsante, A., 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=Intracerebroventricular+delivery+of+self-complementary+adeno-associated+virus+serotype+9+to+the+adult+rat+brain.">Intracerebroventricular delivery of self-complementary adeno-associated virus serotype 9 to the adult rat brain.</a> Gene Ther. 23 (5), 401-407 (2016).</li><li>Pellot, J. E., Jesus, O. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suboccipital+puncture.+[Updated+2022+Jul+25].">Suboccipital puncture. [Updated 2022 Jul 25].</a> StatPearls [Internet]. , (2022).</li><li>Elliger, S. S., Elliger, C. A., Aguilar, C. P., Raju, N. R., Watson, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elimination+of+lysosomal+storage+in+brains+of+MPS+vii+mice+treated+by+intrathecal+administration+of+an+adeno-associated+virus+vector.">Elimination of lysosomal storage in brains of MPS vii mice treated by intrathecal administration of an adeno-associated virus vector.</a> Gene Ther. 6 (6), 1175-1178 (1999).</li><li>De La Calle, J. L., Paino, C. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV9:+A+potential+blood-brain+barrier+buster.">AAV9: A potential blood-brain barrier buster.</a> Mol Ther. 17 (3), 403-405 (2009).</li><li>Hudry, E., Vandenberghe, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+AAV+gene+transfer+to+the+nervous+system:+A+clinical+reality.">Therapeutic AAV gene transfer to the nervous system: A clinical reality.</a> Neuron. 101 (5), 839-862 (2019).</li><li>Georg-Fries, B., Biederlack, S., Wolf, J., Zur Hausen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+proteins,+helper+dependence,+and+seroepidemiology+of+a+new+human+parvovirus.">Analysis of proteins, helper dependence, and seroepidemiology of a new human parvovirus.</a> Virology. 134 (1), 64-71 (1984).</li><li>Schulz, M., 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=Binding+and+neutralizing+anti-aav+antibodies:+Detection+and+implications+for+rAAV-mediated+gene+therapy.">Binding and neutralizing anti-aav antibodies: Detection and implications for rAAV-mediated gene therapy.</a> Mol Ther. 31 (3), 616-630 (2023).</li><li>Gray, S. 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F., 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=Cerebrospinal+fluid+leakage+and+headache+after+lumbar+puncture:+A+prospective+non-invasive+imaging+study.">Cerebrospinal fluid leakage and headache after lumbar puncture: A prospective non-invasive imaging study.</a> Brain. 138, 1492-1498 (2015).</li><li>Hordeaux, J., 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=Adeno-associated+virus-induced+dorsal+root+ganglion+pathology).">Adeno-associated virus-induced dorsal root ganglion pathology).</a> Hum Gene Ther. 31 (15-16), 808-818 (2020).</li><li>Semple, B. D., Blomgren, K., Gimlin, K., Ferriero, D. M., Noble-Haeusslein, L. 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A wide variety of diseases affect the CNS. Providing a functional copy of the relevant gene via a viral vector is an attractive treatment strategy for those that are recessive and monogenic in nature, such as spinal muscular atrophy. However, the blood-brain barrier (BBB) excludes most gene therapy vectors given intravenously11. Those that can cross the BBB, such as AAV9, must be given in high doses to overcome the vector loss due to peripheral transduction12. The age is al...

The promise of viral-mediated gene therapies has finally been realized in recent years with the FDA approval of treatments for spinal muscular atrophy, retinal dystrophy, factor IX hemophilia, cancer, and more1,2,3,4. Countless other therapeutics are currently in development. Gene therapy aims to deliver a therapeutic gene to a patient&#39;s cells. The products of this new gene can replace the missing activity from a deficient endogenous gene, inhibit a toxic gene, kill cancerous cells, or provide some other beneficial function.
For diseases affecting the central nervous system (CNS), delivering the gene therapy vector directly to the target tissue is often desirable. Non-systemic approaches provide two benefits: they minimize off-target side effects that may be caused by peripheral transduction, and they greatly reduce the amount of vector needed to achieve adequate levels of transduction in the target tissue5.
There are a variety of approaches to delivering gene therapy vectors to the CNS. Intraparenchymal injection, the injection of a vector directly into the spinal cord or brain tissue, can be used for delivery to a defined region. However, for many diseases, broad transduction of the CNS is desired. This can be accomplished by delivering a vector to the cerebrospinal fluid (CSF)5, the fluid that flows in and around the brain and spinal cord. There are three primary ways to deliver vectors to the CSF. The most invasive approach is intracerebroventricular delivery, which involves drilling a burr hole through the skull and advancing a needle through the brain into the lateral ventricles. This yields transduction throughout the brain. However, the procedure may cause intracranial hemorrhage, and the approach generally produces only limited transduction of the spinal cord6. Injection into the cisterna magna at the base of the skull is less invasive, but carries the risk of damage to the brainstem. While often used in animal research5, injection into the cisterna magna is no longer used routinely in the clinic7. Lumbar puncture is the least invasive approach to access the CSF. This involves placing a needle between two lumbar vertebrae and into the lumbar cistern.
Lumbar puncture for vector delivery is routinely performed in adult rats and mice and in neonatal mice8,9. The authors of this study recently performed lumbar punctures in juvenile rats (28-30 days of age) to deliver adeno-associated virus serotype 9 (AAV9) vectors. In adult rats, a neonatal lumbar puncture needle was placed vertically between the L3 and L4 vertebrae9. Proper placement results in a tail flick and CSF flowing up into the needle reservoir. In juvenile rats, though, neither of these read-outs could be achieved. The authors then attempted to adapt an adult mouse procedure using a 27 G insulin syringe inserted at an angle between L5 and L610. In adult mice, which are typically smaller than P28 rats, this does not produce a tail flick, but incorrect needle placement is evident by the backflow of the injectate. In juvenile rats, however, this approach uniformly led to the injectate being delivered epidurally, likely resulting from different elasticity between adult mice and juvenile rats of the tissue layers surrounding the spinal cord. Catheter approaches were evaluated next. Specifically, a catheter was introduced through an incision in the dura of the lumbar cistern and up to the mid-thoracic spinal cord; however, this approach led to substantial reflux of the injectate back out of the incision site during delivery. Attempts to place the catheter into the intrathecal space percutaneously using a guide needle were also unsuccessful. Due to the narrowness of the interlaminar width, the catheter would likely hit the rostral lamina and fail to advance.
Here, a method is described to achieve successful and reproducible solution delivery via a lumbar puncture in the juvenile rat. This approach can be used for viral vectors, and likely also for cells, pharmaceuticals, and other therapeutics.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>200 &#181;L filtered pipette tips</td>
			<td>MidSci</td>
			<td>PR-200RK-FL</td>
			<td>Pipetting virus</td>
		</tr>
		<tr>
			<td>AAV9-GFP</td>
			<td>Vector Builder</td>
			<td>P200624-1005ynr</td>
			<td>AAV9 vector expressing GFP</td>
		</tr>
		<tr>
			<td>Absorbable Suture with Needle Coated Vicryl Polyglactin 910 FS-2 3/8 Circle Reverse Cutting Needle Size 4 - 0 Braided</td>
			<td>McKesson</td>
			<td>J422H</td>
			<td>Suture</td>
		</tr>
		<tr>
			<td>Bench pad</td>
			<td>VWR</td>
			<td>56616-031</td>
			<td>Surgery</td>
		</tr>
		<tr>
			<td>Braintree Scientific Isothermal Pads, 8&#39;&#39; x 8&#39;&#39;</td>
			<td>Fisher Scientific</td>
			<td>50-195-4664</td>
			<td>Maintains body temperature</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>McKesson</td>
			<td>1013922</td>
			<td>Analgesic</td>
		</tr>
		<tr>
			<td>Buprenorphine-ER (1 mg/mL)</td>
			<td>Zoopharma</td>
			<td></td>
			<td>Extended-release analgesic</td>
		</tr>
		<tr>
			<td>Cotton swabs</td>
			<td>Fisher Scientific</td>
			<td>19-365-409</td>
			<td>Blood removal</td>
		</tr>
		<tr>
			<td>Drape, Mouse, Clear Plastic, 12&#34; x 12&#34;, with Adhesive Fenestration</td>
			<td>Steris</td>
			<td>1212CPSTF</td>
			<td>Surgical drape</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td>Forceps</td>
		</tr>
		<tr>
			<td>Electric Blanket</td>
			<td>CVS Health</td>
			<td></td>
			<td>CVS Health Series 500 Extra Long Heating Pad</td>
		</tr>
		<tr>
			<td>Eppendorf Research plus, 1-channel pipette, variable, 20&#8211;200 &#181;L</td>
			<td>Eppendorf</td>
			<td>3123000055</td>
			<td>Pipetting virus</td>
		</tr>
		<tr>
			<td>Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>14059-11</td>
			<td>Curved surgical scissors</td>
		</tr>
		<tr>
			<td>Friedman-Pearson Rongeurs</td>
			<td>Fine Science Tools</td>
			<td>16121-14</td>
			<td>Laminectomy</td>
		</tr>
		<tr>
			<td>Halsey Needle Holders</td>
			<td>Fine Science Tools</td>
			<td>12001-13</td>
			<td>Needle driver</td>
		</tr>
		<tr>
			<td>Insulin Syringes with Ultra-Fine Needle 12.7 mm x 30 G 3/10 mL/cc</td>
			<td>BD</td>
			<td>328431</td>
			<td>Syringe</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>McKesson</td>
			<td>803250</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>Isopropanol wipes</td>
			<td>Fisher Scientific</td>
			<td>22-031-350</td>
			<td>Skin disinfection</td>
		</tr>
		<tr>
			<td>Lidocaine, 1%</td>
			<td>McKesson</td>
			<td>239935</td>
			<td>Local anesthesia</td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes: 1.5mL</td>
			<td>Fisher Scientific</td>
			<td>05-408-137</td>
			<td>Loading the syringe</td>
		</tr>
		<tr>
			<td>Povidone-iodine</td>
			<td>Fisher Scientific</td>
			<td>50-118-0481</td>
			<td>Skin disinfection</td>
		</tr>
		<tr>
			<td>Scalpel Handle - #4</td>
			<td>Fine Science Tools</td>
			<td>10004-13</td>
			<td>Scalpel blade holder</td>
		</tr>
		<tr>
			<td>Sure-Seal Induction Chamber</td>
			<td>Braintree Scientific</td>
			<td>EZ-17</td>
			<td>Anesthesia box</td>
		</tr>
		<tr>
			<td>Surgical Blade Miltex Carbon Steel No. 11 Sterile Disposable Individually Wrapped</td>
			<td>McKesson</td>
			<td>4-111</td>
			<td>#11 Scalpel blade</td>
		</tr>
		<tr>
			<td>SYSTANE NIGHTTIME Eye Ointment</td>
			<td>Alcon</td>
			<td></td>
			<td>Eye ointment</td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>VWR</td>
			<td>97063-702</td>
			<td>Injection</td>
		</tr>
	</tbody>
</table>

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

Intrathecal Vector Delivery in Juvenile Rats via Lumbar Cistern Injection

Our research focuses on the development of gene therapies for diseases that affect the central nervous system. As of December, 2023, the FDA has approved AAV-based gene therapies for a handful of genetic stores, including spinal muscular atrophy, Leber congenital amaurosis, Duchenne muscular dystrophy, and Hemophilia A and B.Many more treatments are currently being investigated in preclinical and clinical trials. Devising safe and effective interventions for genetic diseases requires leveraging a range of approaches from careful natural history studies in patients to preclinical work in tissue culture and animal models, and gene therapy is really only one of several technologies that are used for this purpose.Many gene therapy experiments use neonatal or adult animals. Few evaluate treatments in juveniles, even though this represents a significant patient population for genetic disorders. So, for older children, body size and the immune system pose significant barriers to intravenous delivery of gene therapies.Intrathecal delivery may overcome this, but we need appropriate models to test this hypothesis. This procedure that we have developed will allow us to evaluate the efficacy of this approach in juvenile rats.

To determine the accuracy of the injection technique, a dye, trypan blue, was used as a surrogate for the therapeutic. This dye readily binds to proteins, so it generally stays within the structure into which it was injected. This means the dye may not accurately predict the post-injection distribution of the therapeutic; it is simply used to reveal the accuracy of the injection. When successfully introduced into the lumbar cistern, trypan blue binds to the dura mater, staining the perimeter of the spinal cord blue. Howe...

Watch this Scientific Journal Video about Author Spotlight: Assessing Intrathecal Gene Therapy Efficacy in Juvenile Rats at JoVE.com

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 ...