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 200days. 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.

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 cord. All tissues were collected on the respective days from animals injected on PND2 with 2×10¹⁰ viral particles. Controls were untreated SMA (Smn^−/−;SMN2^+/+;SmnΔ7^+/+ ) and unaffected, heterozygous (het) animals (Smn^+/−;SMN2^+/+;SmnΔ7^+/+ ).

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^+/+ ). (A) Average weight, in grams, of animals at each day of life: untreated (n=9), IV treated (n=6), ICV (n=4), and Het (n=24). Each treatment group is significantly different from the others by 1way ANOVA (p<0.0001). Tukey’s multiple comparison test: Untreated vs. ICV p<0.001, Untreated vs. IV p<0.001 and IV vs. ICV p<0.001 (B) Percent body weight gained from PND3 to peak weight of untreated, IV treated, and ICV treated SMA animals: untreated=130.6%, IV treated=435.9%, and ICV treated=1011.7%. Unpaired two-tailed student’s t test: untreated vs. ICV p<0.0001, untreated vs. IV p=0.0157, and IV vs. ICV p=0.0085. (C) Kaplan-Meier survival curve of untreated, IV treated, and ICV treated SMA animals. Log-Rank (Mantel-Cox) test: untreated vs. IV p=0.0004, untreated vs. ICV p=0.0019, and IV vs. ICV p=0.3251. (D–G) Representative images of treated SMA animals with both untreated SMA (Smn^−/−;SMN2^+/+;SmnΔ7^+/+ ) and unaffected controls (Smn^+/−;SMN2^+/+;SmnΔ7^+/+ ). (D) PND14 IV treated with controls, (E) PND14 IV treated with controls, (F) PND43 IV treated with unaffected littermate, and (G) PND63 ICV treated.

Fig. 3

ICV and IV scAAV9-SMN treatment improves motor function in SMA animals (Smn^−/−;SMN2^+/+;SmnΔ7^+/+ ). (A) Percent of pups able to right themselves on PND10-PND18. Time it took for each individual pup assessed to right on (B) PND14 and (C) PND17. (D) Rotorod performance and (E) Grip strength assessed for 10 consecutive days. (F) Mean grip strength quantified from the 10 day course of assessment. (D,E,F) Unaffected het controls (Smn^+/−;SMN2^+/+;SmnΔ7^+/+ ) n=10, ICV treated n=2, and IV treated n=2. All tested mice >80 days of age.

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^+/+;SmnΔ7^+/+ ), (C) ICV treated SMA animal, and (D) IV treated SMA animal. (E) Average tibialis anterior muscle fiber size (in um²) at P14 following treatment with 2×10¹⁰ viral particles. Untreated SMA n=5, Unaffected het n=8, IV treated n=8, and ICV treated n=5.