Direct central nervous system delivery provides enhanced protection following vector mediated gene replacement in a severe model of Spinal Muscular Atrophy

Abstract

Spinal Muscular Atrophy (SMA), an autosomal recessive neuromuscular disorder, is the leading genetic cause of infant mortality. SMA is caused by the homozygous loss of Survival Motor Neuron-1 (SMN1). SMA, however, is not due to complete absence of SMN, rather a low level of functional full-length SMN is produced by a nearly identical copy gene called SMN2. Despite SMN’s ubiquitous expression, motor neurons are preferentially affected by low SMN levels. Recently gene replacement strategies have shown tremendous promise in animal models of SMA. In this study, we used self-complementary Adeno Associated Virus (scAAV) expressing full-length SMN cDNA to compare two different routes of viral delivery in a severe SMA mouse model. This was accomplished by injecting scAAV9-SMN vector intravenously (IV) or intracerebroventricularly (ICV) into SMA mice. Both routes of delivery resulted in a significant increase in lifespan and weight compared to untreated mice with a subpopulation of mice surviving more than 200 days. However, the ICV injected mice gained significantly more weight than their IV treated counterparts. Likewise, survival analysis showed that ICV treated mice displayed fewer early deaths than IV treated animals. Collectively, this report demonstrates that route of delivery is a crucial component of gene therapy treatment for SMA.

Introduction

Spinal Muscular Atrophy (SMA) is caused by the homozygous loss of Survival Motor Neuron-1, SMN1 [1], [2]. The human genome contains two nearly identical SMN genes, SMN1 and SMN2, however, only SMN1 functions as the disease-determining gene [3], [4]. SMN1 and SMN2 differ by a silent C to T transition at the 5′ end of exon 7 [5], [6]. This difference alters the alternative pre-mRNA splicing ratios from the two genes, resulting in high levels of full-length product from SMN1, whereas SMN2 produces low levels of full-length SMN and an abundant alternatively spliced isoform, SMNΔ7. The truncated isoform is unstable and cannot compensate for the loss of SMN1 [3]. Despite the ubiquitous expression of SMN, preferential loss of motor neurons occurs in SMA. Because SMA is monogenic, vector-based gene replacement of SMN1 is an attractive option for the treatment of SMA. Encouraging reports have been published using a relatively severe model of SMA called SMNΔ7. These mice lack endogenous mouse Smn, but express the human SMN2 gene and the cDNA encoding the alternatively spliced isoform produced by SMN2, SMNΔ7 (Smn^−/−; SMN2^+/+; SMNΔ7^+/+) [7]. Untreated SMNΔ7 animals live approximately 14 days with disease symptoms becoming overtly apparent around day 7 [7]. Delivery of full-length SMN cDNA to SMNΔ7 neonates using scAAV8 or scAAV9 vectors resulted in significant extensions in survival ranging from an average of 60–200+ days [8], [9], [10], [11], with some treated mice displaying a full rescue in terms of lifespan and motor function. However, it remains unclear whether the different injection paradigms or the vector serotype was the primary cause for the differences in the degree of phenotypic rescue.

In this report we utilized a scAAV9-SMN vector and examined two routes of injection in neonatal SMNΔ7 mice [12]. Pups received injections of 2 × 10¹⁰ viral genomes via the facial vein (IV) or directly into the brain ventricles (ICV) on postnatal day 2 (PND2). We demonstrate that at this relatively low viral titer, animals receiving ICV injections gained significantly more weight and lived longer than animals receiving IV injections. As expected, animals receiving ICV injections also had higher SMN protein levels in the brain and lumbar spinal cord as compared to IV injected animals. From these results, we conclude that the route of injection for scAAV9-SMN has a significant impact upon the degree of phenotypic rescue and sheds light upon the development of disease and potential therapeutic implications.