Transgenic or prednisolone-driven Klf15 upregulation improved SMA mouse phenotypes. A scAAV2/8-Spc5-12 vector expressing Klf15 was injected at birth into severe and intermediate SMA mice. Survival beat the GFP control only in Smn2B/− mice, and 1E11 vg/pup was lethal.
A self-complementary AAV2/8 vector carrying the synthetic muscle-specific Spc5-12 promoter driving eGFP (AAV8-GFP) expresses GFP in differentiated C2C12 myoblasts, confirmed by flow cytometry and live imaging 3 days after transduction. Injected intravenously via the facial vein at postnatal day 0 (1E11 vg/pup) into wild-type FVB/N mice, AAV8-GFP significantly raises GFP mRNA in quadriceps at both P2 and P7, with higher levels at P7 and variability between animals.
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A self-complementary AAV2/8 vector carrying the Spc5-12 promoter driving a codon-optimized Klf15 cDNA (AAV8-Klf15) significantly increases Klf15 mRNA in differentiated C2C12 myoblasts. At 2E10 vg/pup injected at postnatal day 0, AAV8-Klf15 raises quadriceps Klf15 mRNA in Smn+/−;SMN2 and Smn−/−;SMN2 mice at P2 and P7, with higher levels at P7 and variability between animals. P2 levels (~5–10 fold over control littermates) are similar to those of transgenic mice overexpressing muscle-specific Klf15, while P7 levels are significantly greater than in the transgenic mice.
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AAV8-Klf15 (scAAV2/8-Spc5-12-codon-optimized Klf15) at 1E11 vg/pup, injected at postnatal day 0, causes spontaneous death in both Smn−/−;SMN2 SMA mice and Smn+/−;SMN2 control littermates without affecting weight, whereas AAV8-GFP at the same dose is not harmful. The Klf15 vector is therefore toxic at this dose, and supraphysiological Klf15 is the likely cause, although only one AAV8-Klf15 preparation was used and batch-specific impurities are not excluded. The dose is reduced to 2E10 vg/pup for subsequent experiments.
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AAV8-Klf15 at 2E10 vg/pup, injected at postnatal day 0, does not improve weight or survival beyond the AAV8-GFP control in severe Taiwanese Smn−/−;SMN2 SMA mice. AAV8-Klf15-treated mice outlive untreated mice (log-rank p = 0.0048), but AAV8-GFP-treated mice do as well (p = 0.0034 versus untreated), and AAV8-GFP-treated mice weigh slightly more than AAV8-Klf15-treated and untreated mice. A lower AAV8-Klf15 dose of 1E10 vg/pup shows no significant survival benefit relative to 2E10 vg/pup.
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AAV8-Klf15 at 2E10 vg/pup, injected at postnatal day 0, has no significant effect on survival of Smn+/−;SMN2 control littermates relative to untreated or AAV8-GFP-treated mice, and AAV8-Klf15-treated mice show a small but significant weight increase only at 6 months of age.
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AAV8-Klf15 at 2E10 vg/pup, injected at postnatal day 0, modestly extends survival of intermediate Smn2B/− SMA mice relative to both untreated mice (p < 0.0001) and AAV8-GFP-treated mice (p = 0.0008). It does not change weight during the nursing period up to P21, and the later post-weaning weight gain does not prevent early death. The benefit is small and is seen in the intermediate Smn2B/− model but not in the severe Taiwanese Smn−/−;SMN2 model.
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The AAV8-GFP control vector (scAAV2/8-Spc5-12-eGFP, 2E10 vg/pup at postnatal day 0) itself modestly extends survival of Smn2B/− mice (p = 0.0021 versus untreated) and of Taiwanese Smn−/−;SMN2 mice (p = 0.0034 versus untreated) and increases preweaning weight in Smn−/−;SMN2, Smn+/−;SMN2 and Smn2B/− mice. The AAV8 vector alone therefore confounds attribution of SMA phenotype benefits to a transgene, and the cause is unresolved, with possible effects on the immune system proposed.
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The AAV8-Spc5-12 vector is not selective for skeletal muscle in newborn mice. After intravenous injection at postnatal day 0 (2E10 vg/pup), GFP and Klf15 mRNA from AAV8-GFP and AAV8-Klf15 are higher in liver (~15 fold for GFP and ~10 fold for Klf15 relative to skeletal muscle) and in heart (~40 fold GFP and ~50 fold Klf15 at P2, ~70 fold GFP and ~10 fold Klf15 at P7) than in skeletal muscle. AAV8 capsid-specific qPCR shows more vector genome content in heart and liver than in muscle, particularly at P2.
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Abstract
Spinal muscular atrophy (SMA) is a neuromuscular disease caused by loss of the survival motor neuron (SMN) gene. While there are currently two approved gene-based therapies for SMA, availability, high cost, and differences in patient response indicate that alternative treatment options are needed. Optimal therapeutic strategies will likely be a combination of SMN-dependent and -independent treatments aimed at alleviating symptoms in the central nervous system and peripheral muscles. Krüppel-like factor 15 (KLF15) is a transcription factor that regulates key metabolic and ergogenic pathways in muscle. We have recently reported significant downregulation of Klf15 in muscle of presymptomatic SMA mice. Importantly, perinatal upregulation of Klf15 via transgenic and pharmacological methods resulted in improved disease phenotypes in SMA mice, including weight and survival. In the current study, we designed an adeno-associated virus serotype 8 (AAV8) vector to overexpress a codon-optimized Klf15 cDNA under the muscle-specific Spc5-12 promoter (AAV8-Klf15). Administration of AAV8-Klf15 to severe Taiwanese Smn−/−;SMN2 or intermediate Smn2B/− SMA mice significantly increased Klf15 expression in muscle. We also observed significant activity of the AAV8-Klf15 vector in liver and heart. AAV8-mediated Klf15 overexpression moderately improved survival in the Smn2B/− model but not in the Taiwanese mice. An inability to specifically induce Klf15 expression at physiological levels in a time- and tissue-dependent manner may have contributed to this limited efficacy. Thus, our work demonstrates that an AAV8-Spc5-12 vector induces high gene expression as early as P2 in several tissues including muscle, heart, and liver, but highlights the challenges of achieving meaningful vector-mediated transgene expression of Klf15.
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