delivering gene therapy vectors via lumbar puncture in the juvenile rat

CNS 유전자 치료를 위한 어린 쥐에서 AAV9의 요추 천자 전달

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.

저자 스포트라이트: Assessing Intrathecal Gene Therapy Effect in Juvenile Rats

Intrathecal vector delivery in juvenile rats via lumbar cistern injection(요추 수조 주입을 통한 어린 쥐의 척수강내 벡터 전달)

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.

Delivering Gene Therapy Vectors via Lumbar Penncture in the juvenile rat(어린 쥐에서 요추 천자를 통한 유전자 치료 벡터 전달)

마취된 동물을 수술 플랫폼에 놓는 것으로 시작합니다. 수술 중 각막 건조를 방지하기 위해 각 동물의 눈에 윤활 눈 연고를 바르십시오. 면도한 수술 부위를 포비돈, 요오드, 이소프로판올을 번갈아 3회 소독합니다.그런 다음 종이 타월 롤이나 직경 1.5cm 튜브를 엉덩이 바로 아래에 놓습니다. 동물을 창호 드레이프로 드레이프하고 수술 접근을 위해 요추 위의 창호 중앙에 놓습니다. 동물의 마취 깊이를 확인하려면 각 발을 꼬집고 금단 반응이 없는지 관찰하십시오.그런 다음 11번 메스 칼날을 사용하여 L2에서 L6 척추뼈까지 정중선을 따라 3cm 길이의 피부를 절개합니다. 이제 근육과 피부 사이에 멸균 된 곡선 수술 용 가위를 삽입하고 끝을 부드럽게 열어 아래 근육에서 피부를 분리합니다. L2에서 L5 척추의 가시돌기를 덮고 있는 근막을 제거합니다.AAV9 벡터 25-35 마이크로리터를 멸균 마이크로 원심분리기 튜브의 캡에 피펫팅합니다. 캡에서 벡터의 전체 부피를 인슐린 주사기로 그립니다. 그런 다음 동물의 L5 및 L4 가시 돌기를 식별합니다.주사기 바늘을 L5 가시돌기의 왼쪽에 놓고 돌기의 꼬리 끝부분에 맞춥니다. 주사 바늘을 L5 라미나 위로 약 8mm 앞으로 전진시킨 다음 뼈와 접촉할 때까지 L4 라미나 아래로 요추 수조로 이동합니다. 주사기 플런저를 약 5초 동안 천천히 누릅니다.그런 다음 삽입 부위에서 바늘을 천천히 빼냅니다. 4-0 봉합사 또는 수술용 스테이플을 사용하여 상처의 한쪽 끝 부분을 절개 부위를 봉합하기 시작합니다. 염료 주입의 성공 여부를 평가하기 위해, 동물을 안락사시킨 후, 가시돌기의 양쪽에서 척추와 평행한 근육을 4cm 길이로 절개한다.척추 집게나 가위를 사용하여 가시돌기 사이의 근육을 섬세하게 제거합니다. Rongeur를 사용하여 L6에서 하부 흉추까지의 가시 돌기를 추출합니다. 룽게르의 아래쪽 끝부분을 L5 라미나 아래에 삽입하고 척수 위에 있는 뼈를 여러 번 물어 체계적으로 제거합니다.

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 ...