To determine if the route of injection significantly impacts the degree to which the SMA phenotype is corrected in SMA mice following treatment with scAAV9-SMN, we performed a titration experiment to identify a relatively low concentration of vector that still resulted in significant survival (data not shown). This concentration (2×1010) as opposed to a maximal dose was used in subsequent experiments since a lower concentration was more likely to reveal differences regarding the ability to rescue the SMA phenotype. SMNΔ7 neonatal pups received IV or ICV injections with identical concentrations of the self-complementary AAV9 vector expressing full-length SMN cDNA. SMN protein levels were examined at PND7 and PND14 in disease relevant tissues, including brain and spinal cord, following scAAV9-SMN delivery (). As anticipated, following the ICV injection directly into the brain, SMN expression was dramatically increased in the brain and spinal cord to levels comparable to unaffected heterozygous animals (). IV injected animals expressed higher levels of SMN as well, although not to the same extent as ICV injected tissues. To monitor the gross phenotypic changes in treated mice, weight and survival were measured in each of the treatment groups (). The ICV and IV treated animals gained significantly more weight than untreated controls (), gaining nearly 2-fold more weight from PND3 to their peak weight. We also observed that IV treated animals experienced more early deaths compared to ICV treated mice ().
Fig. 1 Western blot showing protein expression is increased to near normal levels following ICV treatment with scAAV9-SMN, while IV treatment results in a more modest increase. Western blots of (A) PND 7 and (B) PND14 brain tissue. (C) PND7 and (D) PND14 spinal (more ...)
Fig. 2 scAAV-SMN ICV treated animals gain significantly more weight and experience fewer early deaths than IV treated SMA animals while both groups gain significantly more weight and live longer than untreated SMA controls (Smn−/−;SMN2+/+;SmnΔ7 (more ...)
Not all phenotypic parameters, however, were different between ICV and IV treatment groups. At early time points both treatment groups were visually indistinguishable from unaffected controls (). Later on despite the differences in body weight and survival, animals in both treatment groups developed mild ear and tail necrosis around 50–60 days of age (), similar to previous reports [9
]. However, the distal necrosis resolved shortly thereafter and did not progress further.
Time-to-right is an established means to monitor gross mobility for SMA mice [19
]. Therefore, SMA mice were subjected to a time-to-right assessment beginning on PND10. Interestingly, IV treated animals begin righting themselves at an earlier age than ICV treated animals and a greater percentage of the IV treated animals as compared to the ICV treated animals were able to right throughout PND10 to PND18 (). However, by PND17 ICV animals that did right, righted faster than IV treated animals (). Later in life, motor function and coordination in surviving animals was measured by rotarod and grip strength tests. Both ICV and IV treated animals performed as well as normal animals in the rotarod test () indicating that motor coordination, as measured by this assay, is fully rescued regardless of the route of injection. Grip strength analysis revealed that ICV treated animals have significantly better forearm strength when compared to IV treated animals (). Likewise, ICV treated animals also have slightly larger muscle fibers (Fig. 5E), perhaps accounting for the difference in strength observed in the grip strength assays. As expected based on their dramatic improvement in weight and survival, both IV and ICV treated animals have significantly improved tibalis anterior fiber size when compared to untreated SMA controls (Fig. 5A–D).
Fig. 4 Treatment with scAAV9-SMN results in increased muscle fiber size. (A–D) Representative muscle cross sections taken at 10X of a unaffected het control (Smn+/−;SMN2+/+;SmnΔ7+/+ ), (B) untreated SMA (Smn−/−;SMN2+/+ (more ...)
Recently, four reports have demonstrated that viral-mediated gene replacement in SMA can significantly rescue the SMNΔ7 mouse model [8
]. These reports differ in the usage of a variety of parameters in their therapeutic approach including: viral serotype, viral promoter, route of injection, and time of injection [9
]. Together these studies represent the most profound improvement in phenotype and survival seen in the SMNΔ7 mouse model to date. However, without a direct comparison, it is difficult to compare these studies in order to determine the most efficient and effective course of treatment and to assess serotype and promoter activity that can profoundly impact gene expression in AAV vectors [20
This study was performed to directly compare the influence of the injection route on the SMA phenotype. Here we chose to compare the two injection techniques that have been used thus far in viral gene therapy treatment of SMA: ICV and IV injections [8
]. In doing so, we have found that route of injection makes a dramatic difference in survival and mouse phenotype. ICV injections have proven to be more effective in rescuing the SMNΔ7 mouse. We propose that this is due to the direct introduction of virus into the central nervous system (CNS) [12
]. Because SMA is primarily a disease of the CNS, using ICV injections to physically overcome the blood-brain barrier is advantageous. Conversely, systemically injected viral particles must cross the blood-brain barrier before they are able to transduce motor neurons, reducing their efficiency and requiring more viral particles to achieve results similar to those of the ICV injection. However, it is known that there is a need for SMN protein in the peripheral organ systems, including, but not limited, to the heart [18
]. While IV injections are able to meet this need directly, it is likely that some of the ICV injected vector is able to escape the blood-brain barrier in the same fashion that the IV delivered vector is able to penetrate it, especially within the context of SMA compromised neonatal animals. Thus, ICV injections are able to meet the need for SMN protein in peripheral tissues, while first meeting the primary need for SMN protein in the CNS, resulting in a more complete phenotypic rescue.
When considering the clinical translation of these approaches, ICV injections, while easily executed in mice, are difficult to perform in humans. However, intrathecal injections, while still not as straightforward as IV administration, directly overcome the blood-brain barrier and can be performed in safely in humans [24
]. Additionally, intrathecal injections have recently been shown to result in efficient motor neuron transduction [20
] when using AAV9. Thus, intrathecal delivery may prove to be a desirable route of administration should AAV gene therapy for SMA reach clinical trials. While the results of this study emphasize the importance of route of delivery when examining gene therapy in the context of SMA, route of delivery is an important component of any therapeutic.