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

Intratekal vektorlevering hos unge rotter via lumbal cisterninjektion

Levering af genterapivektorer via lumbalpunktur i den unge rotte

Begynd med at placere det bedøvede dyr på operationsplatformen. Påfør smørende øjensalve på hvert dyrs øjne for at forhindre udtørring af hornhinden under operationen. Desinficer det barberede operationssted med tre skiftevis runder povidonjod og isopropanol.Placer derefter en rulle papirhåndklæde eller et rør med en diameter på 1,5 centimeter under dyret lige rostralt til hofterne. Draper dyret med et fenestreret draperet, centrer fenestrationen over lændehvirvelsøjlen for kirurgisk adgang. For at verificere dybden af anæstesi hos dyret skal du klemme hver pote og observere for fraværet af tilbagetrækningsrespons.Brug derefter et skalpelblad nummer 11 til at lave et tre centimeter langt hudsnit ned ad midterlinjen fra L2 til L6 ryghvirvler. Indsæt nu en steril buet kirurgisk saks mellem musklen og huden, og åbn forsigtigt spidserne for at adskille huden fra den underliggende muskel. Fjern fascien, der dækker de spinøse processer af L2 til L5 ryghvirvler.Der pipetteres 25 til 35 mikroliter af AAV9-vektoren i hætten på et sterilt mikrocentrifugerør. Træk hele vektorens volumen fra hætten ind i insulinsprøjten. Identificer derefter L5- og L4-spinøse processer på dyret.Placer sprøjtenålen til venstre for L5-spinøs proces, og juster den med den haleformede ende af processen. Før sprøjtenålen frem ca. otte millimeter over L5-laminaen og derefter under L4-laminaen ind i lændecisternen, indtil der er kontakt med knoglen. Tryk sprøjtestemplet langsomt ned i ca. fem sekunder.Træk derefter langsomt nålen ud af indføringsstedet. Brug en 4-0 sutur eller kirurgiske hæfteklammer til at begynde at lukke snittet i den ene ende af såret. For at evaluere succesen med farvestofinjektion, efter aflivning af dyret, skal du lave et fire centimeter langt snit i musklen parallelt med rygsøjlen på begge sider af de spinøse processer.Brug rygpincet eller saks til forsigtigt at fjerne musklen mellem de spinøse processer. ved hjælp af en rongeur udvindes de spinøse processer fra L6 til den nedre thorax rygsøjle. Indsæt den nederste spids af rongeur under L5-laminaen og fjern metodisk knoglen over rygmarven ved at tage flere bid ud af den.

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 ...

Preparing Reagents to Deliver an Adeno-Associated Serotype 9 Vector

Forberedelse af reagenser til at levere en adeno-associeret serotype 9 vektor