delivering gene therapy vectors via lumbar puncture in the juvenile rat

Lumbalpunktionstillförsel av AAV9 hos unga råttor för CNS-genterapi

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.

Författare i fokus: Bedömning av intratekal genterapieffekt hos unga råttor

Intratekal vektorleverans hos unga råttor via injektion av ländryggscistern

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.

Tillförsel av genterapivektorer via lumbalpunktion hos den juvenila råttan

Börja med att placera det bedövade djuret på den kirurgiska plattformen. Applicera smörjande ögonsalva på varje djurs ögon för att förhindra att hornhinnan torkar ut under operationen. Desinficera det rakade operationsområdet med tre omväxlande omgångar povidonjod och isopropanol.Placera sedan en rulle hushållspapper eller ett rör med en diameter på 1,5 centimeter under djuret precis vid höfterna. Drapera djuret med ett fenestrerat draperi och centrera fenestrationen över ländryggen för kirurgisk åtkomst. För att verifiera djupet av anestesi hos djuret, nyp i varje tass och observera att det inte finns någon abstinensreaktion.Använd sedan ett skalpellblad nummer 11 och gör ett tre centimeter långt hudsnitt längs mittlinjen från L2 till L6 kotor. Sätt nu in en steril böjd kirurgisk sax mellan muskeln och huden och öppna försiktigt spetsarna för att separera huden från den underliggande muskeln. Ta bort fascian som täcker ryggradsutskotten i L2 till L5 kotorna.Pipettera 25 till 35 mikroliter av AAV9-vektorn i locket på ett sterilt mikrocentrifugrör. Dra ut hela vektorns volym från locket till insulinsprutan. Identifiera sedan L5- och L4-ryggradsprocesserna på djuret.Placera sprutnålen till vänster om L5 spinous process och rikta in den mot den kaudala änden av processen. För fram sprutkanylen cirka åtta millimeter framåt över L5-laminan och sedan under L4-lamina in i ländryggscisternen tills benet kommer i kontakt med benet. Tryck långsamt ned sprutkolven i cirka fem sekunder.Dra sedan långsamt ut nålen från insättningsstället. Använd en 4-0 sutur eller kirurgiska häftklamrar och börja stänga snittet i ena änden av såret. För att utvärdera framgången med färginjektionen, efter att ha avlivat djuret, gör ett fyra centimeter långt snitt i muskeln parallellt med ryggraden på båda sidor av ryggradsutskotten.Använd en pincett eller en sax för att försiktigt ta bort muskeln mellan ryggradsutskotten. med hjälp av en rongeur, extrahera de spinösa utskotten från L6 till nedre bröstryggen. Sätt in den nedre spetsen av rongeur under L5-lamina och ta metodiskt bort benet som ligger över ryggmärgen genom att ta flera tuggor ur det.

delivering-gene-therapy-vectors-via-lumbar-puncture-juvenile

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 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.

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 ...