Gene Therapy. 2022 Dec
Yi A. Chen, Mark W. Kankel, Sam Hana, Shukkwan Kelly Lau, Maria I. Zavodszky, Olivia McKissick, Nicole Mastrangelo, Jessica Dion, Bin Wang, Daniel Ferretti, David Koske, Sydney Lehman, Kathryn Koszka, Helen McLaughlin, Mei Liu, Eric Marshall, Attila J. Fabian, Patrick Cullen, Galina Marsh, Stefan Hamann, Michael Craft, Jennifer Sebalusky, H. Moore Arnold, Rachelle Driscoll, Adam Sheehy, Yi Luo, Sonia Manca, Thomas Carlile, Chao Sun, Kirsten Sigrist, Alexander McCampbell, Christopher E. Henderson, and Shih-Ching Lo
Products used in the paper Details Operation
Plasmid construction and AAV vector packaging The sgRNAs were synthesized and cloned into the expression constructs by PackGene (Worcester, MA, USA). Each AAV construct contains a U6 promoter that drives expression of a sgRNA, and either a CBA promoter that drives expression of a green fluorescence protein (eGFP) fused with a KASH domain or a CAG promoter that drives expression of a mCherry fluorescence protein. All AAV vectors, including AAV-PHP.B and AAV-PHP.eB, were purchased from PackGene (Worcester, MA, USA). Request Quote

Research Field: CNS

Keywords: Gene therapy, Gene delivery

AAV Serotype: AAV-PHP.B and AAV-PHP.eB,

Dose: 100 μl of AAV buffered with PBS (1E12 vg per mouse)

Routes of Administration: Neonatal intracerebroventricular (ICV) injection, intrathecal injection (IT) and intravenous injection (IV) of AAV

Targeted organ: brain

Animal or cell line strain: H11Cas9 mice [B6J.129-Igs2tm1.1(CAG-cas9*) Mmw/J; and transgenic SOD1.G93A mice (B6.Cg-Tg(huSOD1*G93A)1Gur/J; stock #: 004435) were purchased from the Jackson Laboratory. All mice were backcrossed for at least eight generations to C57BL/6 mice. Homozygous H11Cas9 female mice and heterozygous SOD1-G93A male mice were crossed to generate H11Cas9−/+; SOD1G93A−/+ mice and age-matched H11Cas9−/+; huSOD1.G93A−/− littermates.


CRISPR-based gene editing technology represents a promising approach to deliver therapies for inherited disorders, including amyotrophic lateral sclerosis (ALS). Toxic gain-of-function superoxide dismutase 1 (SOD1) mutations are responsible for ~20% of familial ALS cases. Thus, current clinical strategies to treat SOD1-ALS are designed to lower SOD1 levels. Here, we utilized AAV-PHP.B variants to deliver CRISPR-Cas9 guide RNAs designed to disrupt the human SOD1 (huSOD1) transgene in SOD1G93A mice. A one-time intracerebroventricular injection of AAV.PHP.B-huSOD1-sgRNA into neonatal H11Cas9 SOD1G93A mice caused robust and sustained mutant huSOD1 protein reduction in the cortex and spinal cord, and restored motor function. Neonatal treatment also reduced spinal motor neuron loss, denervation at neuromuscular junction (NMJ) and muscle atrophy, diminished axonal damage and preserved compound muscle action potential throughout the lifespan of treated mice. SOD1G93A treated mice achieved significant disease-free survival, extending lifespan by more than 110 days. Importantly, a one-time intrathecal or intravenous injection of AAV.PHP.eB-huSOD1-sgRNA in adult H11Cas9 SOD1G93A mice, immediately before symptom onset, also extended lifespan by at least 170 days. We observed substantial protection against disease progression, demonstrating the utility of our CRISPR editing preclinical approach for target evaluation. Our approach uncovered key parameters (e.g., AAV capsid, Cas9 expression) that resulted in improved efficacy compared to similar approaches and can also serve to accelerate drug target validation.

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