Researchers develop the first polymer-based therapeutic for Huntington’s disease

In a longitudinal cross-sectional study published in Science Advances, researchers developed a protein-like polymer (PLP) and investigated its ability to inhibit the binding of valosin-containing protein (VCP) to mutant huntingtin (mtHtt) in models of Huntington's Disease (HD).

They found that the PLP prevented mitochondrial autophagy in HD models, displayed high stability and a long circulation half-life, and showed superior bioactivity and therapeutic potential in HD transgenic mice, outperforming traditional peptides.

Background

HD is a severe, inherited neurodegenerative disorder marked by motor dysfunction, cognitive decline, and high suicide rates. It results from a genetic mutation leading to the production of mtHtt, which disrupts cellular function by binding to VCP, causing excessive mitophagy and neuronal death.

Current treatments are mostly symptomatic and do not prevent disease progression. While peptide-based drugs show potential for targeting HD's molecular mechanisms, they often suffer from poor pharmacokinetics, rapid degradation, and limited cell penetration.

To address these issues, researchers, in the present study developed PLPs to prevent VCP-mtHtt binding. Further, they investigated the efficacy and properties of these peptides, highlighting their potential as a sustainable and effective HD treatment.

About the study

The HV3 peptide was modified to prevent disulfide bonding and enhance cellular uptake by adding charged residues. Four peptide sequences were synthesized, attached to a norbornene derivative, and polymerized. PLPs (P1-P4) were characterized using nuclear magnetic resonance and size exclusion chromatography with a multi-angle light scattering. HdhQ111 and HEK293T cells treated with HV3-TAT or PLPs were assessed for cell viability, protein binding, mitochondrial localization, and morphology. VCP binding affinity was measured using Bio-layer Interferometry.

Proteolytic stability of P1 was tested against pronase, chymotrypsin, elastase, and pepsin using gel electrophoresis and high-performance liquid chromatography (HPLC). Serum stability was evaluated. A fluorogenic EDANS-DABCYL assay was used to assess enzyme resistance, and liver microsome assays were conducted to examine degradation by HPLC. Post-treatment bioactivity was tested in HdhQ111 cells.

Hemocompatibility was assessed by activated clotting time (ACT) and hemolytic activity to evaluate effects on blood clotting and red blood cell stability for P1 and HV3-TAT. A C3a ezyme-linked immuno-sorbent assay tested complement activation at both therapeutic and higher doses. For in vivo pharmacokinetics and biodistribution, gadolinium-labeled P1 enabled quantification was done in blood and tissue following intravenous injection in mice. Toxicity was evaluated in healthy wild-type mice over two months. Mice models were treated with P1, HV3-TAT, or saline for efficacy testing, with assessments for motor coordination, body weight, and neuropathology. Western blot and immunohistochemistry were used to analyze neuronal markers, mtHtt aggregation, and VCP mitochondrial translocation.

Results and discussion

PLPs improved cell viability and effectively blocked VCP/mtHtt interactions, with P1 chosen for further study due to optimal uptake and charge properties. Confocal microscopy and flow cytometry confirmed P1's efficient cellular uptake and mitochondrial localization, along with the prevention of mitochondrial fragmentation. P1's dissociation constant was found to be 150-fold lower than HV3-TAT, attributed to slower off-rates and enhanced binding stability due to multivalency.

P1 demonstrated strong proteolytic stability, maintaining structure after exposure to multiple proteases and showing high stability in 10% and 25% fetal bovine serum, unlike the rapidly degraded HV3-TAT. Fluorogenic and liver microsome assays confirmed minimal degradation of P1, while HV3-TAT degraded rapidly. Moreover, HdhQ111 cells treated with enzyme or serum-pretreated P1 maintained viability, while HV3-TAT lost efficacy after pretreatment.

Hemocompatibility studies showed no significant impact on clotting for P1 at therapeutic levels, and P1 displayed negligible hemolytic activity and complement activation compared to controls. Pharmacokinetics of P1 demonstrated an initial 20-minute distribution half-life and a prolonged 152-hour elimination half-life, with primary localization in the liver and kidneys and low presence in the central nervous system (CNS).

Toxicity assays showed no significant pathology in wild-type mice across treatment groups. In the transgenic R6/2 model, P1 improved motor behavior, mitigated body weight loss, and extended survival, with more pronounced effects than HV3-TAT.

Neuropathological markers (dopamine- and cAMP-regulated phosphoprotein 32kDa, post-synaptic density protein, and brain-derived neurotrophic factor) were significantly elevated in P1-treated mice, with reduced mtHtt aggregation and VCP mitochondrial translocation, indicating neuroprotection and alignment with the hypothesized therapeutic mechanism.

Conclusion

In conclusion, the study demonstrated that peptide brush polymers, created through functional group tolerant polymerization, offer a promising therapeutic approach, showing stability, cell penetration, and effective disruption of disease-relevant protein interactions in the CNS. Unlike traditional polymers that serve as drug carriers, these polymers act as active therapeutics, potentially generalizable for targeting hard-to-drug protein interactions.

The platform's success in this model encourages further development, especially to optimize blood-brain barrier crossing, and suggests potential efficacy in human-derived HD neurons, supporting its translational therapeutic potential.

Journal reference:

• Proteomimetic polymer blocks mitochondrial damage, rescues Huntington's neurons, and slows onset of neuropathology in vivo. Wonmin Choi et al., Science Advances, 10:eado8307 (2024). d: 10.1126/sciadv.ado8307,  https://www.science.org/doi/10.1126/sciadv.ado8307