Enhanced exon skipping and prolonged dystrophin restoration achieved by TfR1-targeted delivery of antisense oligonucleotide using FORCE conjugation in mdx mice.

Current therapies for Duchenne muscular dystrophy (DMD) use phosphorodiamidate morpholino oligomers (PMO) to induce exon skipping in the dystrophin pre-mRNA, enabling the translation of a shortened but functional dystrophin protein. This strategy has been hampered by insufficient delivery of PMO to cardiac and skeletal muscle. To overcome these limitations, we developed the FORCETM platform consisting of an antigen-binding fragment, which binds the transferrin receptor 1, conjugated to an oligonucleotide. We demonstrate that a single dose of the mouse-specific FORCE-M23D conjugate enhances muscle delivery of exon skipping PMO (M23D) in mdx mice, achieving dose-dependent and robust exon skipping and durable dystrophin restoration. FORCE-M23D-induced dystrophin expression reached peaks of 51%, 72%, 62%, 90% and 77%, of wild-type levels in quadriceps, tibialis anterior, gastrocnemius, diaphragm, and heart, respectively, with a single 30 mg/kg PMO-equivalent dose. The shortened dystrophin localized to the sarcolemma, indicating expression of a functional protein. Conversely, a single 30 mg/kg dose of unconjugated M23D displayed poor muscle delivery resulting in marginal levels of exon skipping and dystrophin expression. Importantly, FORCE-M23D treatment resulted in improved functional outcomes compared with administration of unconjugated M23D. Our results suggest that FORCE conjugates are a potentially effective approach for the treatment of DMD.

The biggest problem confronting oligonucleotide therapeutics is a lack of compounds capable of targeting compounds to diseased tissues. This paper reports a major advance targeting the transferrin receptor to increase the delivery of morpholine oligomers to muscle cells in vivo. This work suggests the possibility for improved treatments of muscular dystrophy and other diseases.

Virus-Like Particle-Drug Conjugates Induce Protective, Long-lasting Adaptive Antitumor Immunity in the Absence of Specifically Targeted Tumor Antigens.

This study examined the ability of a papillomavirus-like particle drug conjugate, belzupacap sarotalocan (AU-011), to eradicate subcutaneous tumors after intravenous injection and to subsequently elicit long-term antitumor immunity in the TC-1 syngeneic murine tumor model. Upon in vitro activation with near-infrared light (NIR), AU-011-mediated cell killing was proimmunogenic in nature, resulting in the release of damage-associated molecular patterns such as DNA, ATP, and HMGB-1, activation of caspase-1, and surface relocalization of calreticulin and HSP70 on killed tumor cells. A single in vivo administration of AU-011 followed by NIR caused rapid cell death, leading to long-term tumor regression in ∼50% of all animals. Within hours of treatment, calreticulin surface expression, caspase-1 activation, and depletion of immunosuppressive leukocytes were observed in tumors. Combination of AU-011 with immune-checkpoint inhibitor antibodies, anti-CTLA-4 or anti-PD-1, improved therapeutic efficacy, resulting in 70% to 100% complete response rate that was durable 100 days after treatment, with 50% to 80% of those animals displaying protection from secondary tumor rechallenge. Depletion of CD4+ or CD8+ T cells, either at the time of AU-011 treatment or secondary tumor rechallenge of tumor-free mice, indicated that both cell populations are vital to AU-011's ability to eradicate primary tumors and induce long-lasting antitumor protection. Tumor-specific CD8+ T-cell responses could be observed in circulating peripheral blood mononuclear cells within 3 weeks of AU-011 treatment. These data, taken together, support the conclusion that AU-011 has a direct cytotoxic effect on tumor cells and induces long-term antitumor immunity, and this activity is enhanced when combined with checkpoint inhibitor antibodies.

An Infrared Dye-Conjugated Virus-like Particle for the Treatment of Primary Uveal Melanoma.

The work outlined herein describes AU-011, a novel recombinant papillomavirus-like particle (VLP) drug conjugate and its initial evaluation as a potential treatment for primary uveal melanoma. The VLP is conjugated with a phthalocyanine photosensitizer, IRDye 700DX, that exerts its cytotoxic effect through photoactivation with a near-infrared laser. We assessed the anticancer properties of AU-011 in vitro utilizing a panel of human cancer cell lines and in vivo using murine subcutaneous and rabbit orthotopic xenograft models of uveal melanoma. The specificity of VLP binding (tumor targeting), mediated through cell surface heparan sulfate proteoglycans (HSPG), was assessed using HSPG-deficient cells and by inclusion of heparin in in vitro studies. Our results provide evidence of potent and selective anticancer activity, both in vitro and in vivo AU-011 activity was blocked by inhibiting its association with HSPG using heparin and using cells lacking surface HSPG, indicating that the tumor tropism of the VLP was not affected by dye conjugation and cell association is critical for AU-011-mediated cytotoxicity. Using the uveal melanoma xenograft models, we observed tumor uptake following intravenous (murine) and intravitreal (rabbit) administration and, after photoactivation, potent dose-dependent tumor responses. Furthermore, in the rabbit orthotopic model, which closely models uveal melanoma as it presents in the clinic, tumor treatment spared the retina and adjacent ocular structures. Our results support further clinical development of this novel therapeutic modality that might transform visual outcomes and provide a targeted therapy for the early-stage treatment of patients with this rare and life-threatening disease. Mol Cancer Ther; 17(2); 565-74. ©2017 AACR.

Mechanistic investigation of the inhibition of Abeta42 assembly and neurotoxicity by Abeta42 C-terminal fragments.

Oligomeric forms of amyloid beta-protein (Abeta) are key neurotoxins in Alzheimer's disease (AD). Previously, we found that C-terminal fragments (CTFs) of Abeta42 interfered with assembly of full-length Abeta42 and inhibited Abeta42-induced toxicity. To decipher the mechanism(s) by which CTFs affect Abeta42 assembly and neurotoxicity, here, we investigated the interaction between Abeta42 and CTFs using photoinduced cross-linking and dynamic light scattering. The results demonstrate that distinct parameters control CTF inhibition of Abeta42 assembly and Abeta42-induced toxicity. Inhibition of Abeta42-induced toxicity was found to correlate with stabilization of oligomers with a hydrodynamic radius (R(H)) of 8-12 nm and attenuation of formation of oligomers with an R(H) of 20-60 nm. In contrast, inhibition of Abeta42 paranucleus formation correlated with CTF solubility and the degree to which CTFs formed amyloid fibrils themselves but did not correlate with inhibition of Abeta42-induced toxicity. Our findings provide important insight into the mechanisms by which different CTFs inhibit the toxic effect of Abeta42 and suggest that stabilization of nontoxic Abeta42 oligomers is a promising strategy for designing inhibitors of Abeta42 neurotoxicity.

Amino acid position-specific contributions to amyloid beta-protein oligomerization.

Understanding the structural and assembly dynamics of the amyloid beta-protein (Abeta) has direct relevance to the development of therapeutic agents for Alzheimer disease. To elucidate these dynamics, we combined scanning amino acid substitution with a method for quantitative determination of the Abeta oligomer frequency distribution, photo-induced cross-linking of unmodified proteins (PICUP), to perform "scanning PICUP." Tyr, a reactive group in PICUP, was substituted at position 1, 10, 20, 30, or 40 (for Abeta40) or 42 (for Abeta42). The effects of these substitutions were probed using circular dichroism spectroscopy, thioflavin T binding, electron microscopy, PICUP, and mass spectrometry. All peptides displayed a random coil --> alpha/beta --> beta transition, but substitution-dependent alterations in assembly kinetics and conformer complexity were observed. Tyr(1)-substituted homologues of Abeta40 and Abeta42 assembled the slowest and yielded unusual patterns of oligomer bands in gel electrophoresis experiments, suggesting oligomer compaction had occurred. Consistent with this suggestion was the observation of relatively narrow [Tyr(1)]Abeta40 fibrils. Substitution of Abeta40 at the C terminus decreased the population conformational complexity and substantially extended the highest order of oligomers observed. This latter effect was observed in both Abeta40 and Abeta42 as the Tyr substitution position number increased. The ability of a single substitution (Tyr(1)) to alter Abeta assembly kinetics and the oligomer frequency distribution suggests that the N terminus is not a benign peptide segment, but rather that Abeta conformational dynamics and assembly are affected significantly by the competition between the N and C termini to form a stable complex with the central hydrophobic cluster.

C-terminal peptides coassemble into Abeta42 oligomers and protect neurons against Abeta42-induced neurotoxicity.

Alzheimer's disease (AD) is an age-related disorder that threatens to become an epidemic as the world population ages. Neurotoxic oligomers of Abeta42 are believed to be the main cause of AD; therefore, disruption of Abeta oligomerization is a promising approach for developing therapeutics for AD. Formation of Abeta42 oligomers is mediated by intermolecular interactions in which the C terminus plays a central role. We hypothesized that peptides derived from the C terminus of Abeta42 may get incorporated into oligomers of Abeta42, disrupt their structure, and thereby inhibit their toxicity. We tested this hypothesis using Abeta fragments with the general formula Abeta(x-42) (x = 28-39). A cell viability screen identified Abeta(31-42) as the most potent inhibitor. In addition, the shortest peptide, Abeta(39-42), also had high activity. Both Abeta(31-42) and Abeta(39-42) inhibited Abeta-induced cell death and rescued disruption of synaptic activity by Abeta42 oligomers at micromolar concentrations. Biophysical characterization indicated that the action of these peptides likely involved stabilization of Abeta42 in nontoxic oligomers. Computer simulations suggested a mechanism by which the fragments coassembled with Abeta42 to form heterooligomers. Thus, Abeta(31-42) and Abeta(39-42) are leads for obtaining mechanism-based drugs for treatment of AD using a systematic structure-activity approach.

Neurotoxic protein oligomers--what you see is not always what you get.

An increasing body of evidence suggests that soluble assemblies of amyloid proteins are the predominant neurotoxic species in many amyloid-related diseases. Consequently, the focus of research on pathologic mechanisms underlying amyloidoses has shifted from amyloid fibrils to oligomers. Biophysical characterization of oligomers is difficult due to their metastable nature. The most popular experimental method for detection of oligomers has been SDS-PAGE. However, we provide experimental evidence that SDS-PAGE is not a reliable method for characterization of amyloid protein oligomers and discuss alternative approaches. In addition, we discuss how inconsistent nomenclature has obfuscated our understanding of the process and products of protein assembly. The goals of this paper are to identify pitfalls associated with the methods and language used to study protein oligomers and to provide alternatives, thereby facilitating successful elucidation of the mechanisms controlling amyloid protein oligomer assembly and toxicity.