delivering gene therapy vectors via lumbar puncture in the juvenile rat

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

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.

Author Spotlight: Assessing Intrathecal Gene Therapy Efficacy in Juvenile Rats

Administración intratecal de vectores en ratas juveniles mediante inyección en cisterna lumbar

Administración de vectores de terapia génica mediante punción lumbar en ratas juveniles

Comience colocando al animal anestesiado en la plataforma quirúrgica. Aplique ungüento lubricante para los ojos de cada animal para evitar que la córnea se seque durante la cirugía. Desinfecte el sitio quirúrgico afeitado con tres rondas alternas de povidona yodada e isopropanol.A continuación, coloque un rollo de papel toalla o un tubo de 1,5 centímetros de diámetro debajo del animal, justo rostral hasta las caderas. Cubra al animal con un paño fenestrado, centrando la fenestración sobre la columna lumbar para el acceso quirúrgico. Para verificar la profundidad de la anestesia en el animal, pellizque cada pata y observe la ausencia de cualquier respuesta de retirada.Luego, con una hoja de bisturí número 11, haga una incisión en la piel de tres centímetros de largo a lo largo de la línea media desde las vértebras L2 hasta L6. Ahora inserte un par de tijeras quirúrgicas curvas estériles entre el músculo y la piel, y abra suavemente las puntas para separar la piel del músculo subyacente. Retire la fascia que cubre las apófisis espinosas de las vértebras L2 a L5.Pipetee de 25 a 35 microlitros del vector AAV9 en la tapa de un tubo de microcentrífuga estéril. Extraiga todo el volumen del vector de la tapa en la jeringa de insulina. A continuación, identifique las apófisis espinosas L5 y L4 en el animal.Coloque la aguja de la jeringa a la izquierda de la apófisis espinosa L5, alineándola con el extremo caudal de la apófisis. Avance la aguja de la jeringa hacia adelante unos ocho milímetros sobre la lámina L5 y luego debajo de la lámina L4 hasta la cisterna lumbar hasta que se haga contacto con el hueso. Presione el émbolo de la jeringa lentamente durante aproximadamente cinco segundos.A continuación, retire lentamente la aguja del lugar de inserción. Usando una sutura 4-0 o grapas quirúrgicas, comience a cerrar la incisión en un extremo de la herida. Para evaluar el éxito de la inyección de tinte, después de sacrificar al animal, se realiza una incisión de cuatro centímetros de largo en el músculo paralelo a la columna vertebral a ambos lados de las apófisis espinosas.Use pinzas de columna vertebral o tijeras para eliminar delicadamente el músculo entre las apófisis espinosas. Con un rongeur, extraiga las apófisis espinosas desde la L6 hasta la columna torácica inferior. Inserte la punta inferior del rongeur debajo de la lámina L5 y retire metódicamente el hueso que recubre la médula espinal sacándole varios bocados.

Research

JoVE Journal

Neuroscience

Delivering Gene Therapy Vectors via Lumbar Puncture in the Juvenile Rat

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 ...

Investigación

Neurociencias

Preparing Reagents to Deliver an Adeno-Associated Serotype 9 Vector

Preparación de reactivos para administrar un vector adenoasociado al serotipo 9