Anti-SOD1 Nanobodies That Stabilize Misfolded SOD1 Proteins Also Promote Neurite Outgrowth in Mutant SOD1 Human
Meenakshi Sundaram Kumar1,2, Megan E Fowler-Magaw1,3, Daniel Kulick4
1Department of Neurology, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
Abstract:
ALS-linked mutations induce aberrant conformations within the SOD1 protein that are thought to underlie the pathogenic mechanism of SOD1-mediated ALS. Although clinical trials are underway for gene silencing of SOD1, these approaches reduce both wild-type and mutated forms of SOD1. Here, we sought to develop anti-SOD1 nanobodies with selectivity for mutant and misfolded forms of human SOD1 over wild-type SOD1. Characterization of two anti-SOD1 nanobodies revealed that these biologics stabilize mutant SOD1 in vitro. Further, SOD1 expression levels were enhanced and the physiological subcellular localization of mutant SOD1 was restored upon co-expression of anti-SOD1 nanobodies in immortalized cells. In human motor neurons harboring the SOD1 A4V mutation, anti-SOD1 nanobody expression promoted neurite outgrowth, demonstrating a protective effect of anti-SOD1 nanobodies in otherwise unhealthy cells. In vitro assays revealed that an anti-SOD1 nanobody exhibited selectivity for human mutant SOD1 over endogenous murine SOD1, thus supporting the preclinical utility of anti-SOD1 nanobodies for testing in animal models of ALS. In sum, the anti-SOD1 nanobodies developed and presented herein represent viable biologics for further preclinical testing in human and mouse models of ALS.
Insights
Researchers developed novel nanobodies targeting misfolded SOD1 protein in ALS. These biologics show promise in stabilizing mutant SOD1, restoring cellular function, and protecting motor neurons, advancing potential ALS therapies.
Area of Science:
- Neuroscience
- Biochemistry
- Protein Engineering
Background:
- Amyotrophic lateral sclerosis (ALS) is often linked to mutations in the SOD1 gene, leading to misfolded SOD1 proteins.
- Current gene silencing therapies for SOD1-ALS reduce both normal and mutated SOD1, lacking specificity.
Purpose of the Study:
- To develop highly selective anti-SOD1 nanobodies that target mutant and misfolded SOD1 forms over wild-type SOD1.
- To evaluate the therapeutic potential of these nanobodies in cellular and preclinical models of ALS.
Main Methods:
- Development and characterization of anti-SOD1 nanobodies.
- In vitro stabilization assays of mutant SOD1.
- Co-expression studies in immortalized cells and human motor neurons with SOD1 A4V mutation.
- In vitro selectivity assays comparing human mutant SOD1 and murine SOD1.
Main Results:
- Developed nanobodies that stabilize mutant SOD1 in vitro.
- Restored physiological subcellular localization and enhanced expression of mutant SOD1.
- Demonstrated protective effects in human motor neurons, promoting neurite outgrowth.
- Confirmed selectivity for human mutant SOD1 over endogenous murine SOD1.
Conclusions:
- Anti-SOD1 nanobodies are viable biologics for targeting misfolded SOD1 in ALS.
- These nanobodies offer a promising, selective therapeutic strategy for SOD1-ALS.
- Further preclinical testing in human and mouse models is warranted.
