Safety and activity of anti-CD14 antibody IC14 (atibuclimab) in ALS: Experience with expanded access protocol.

INTRODUCTION/AIMS: IC14 (atibuclimab) is a monoclonal anti-CD14 antibody. A previous phase 1 trial of 10 participants with amyotrophic lateral sclerosis (ALS) demonstrated initial safety of IC14 in an acute treatment setting. We provided long-term treatment with IC14 to individuals with ALS via an expanded access protocol (EAP) and documented target engagement, biomarker, safety, and disease endpoints. METHODS: Participants received intravenous IC14 every 2 weeks. Consistent with United States Food and Drug Administration guidelines, participants were not eligible for clinical trials and the EAP was inclusive of a broad population. Whole blood and serum were collected to determine monocyte CD14 receptor occupancy (RO), IC14 levels, and antidrug antibodies. Ex vivo T-regulatory functional assays were performed in a subset of participants. RESULTS: Seventeen participants received IC14 for up to 103 weeks (average, 30.1 weeks; range, 1 to 103 weeks). Treatment-emergent adverse events (TEAEs) were uncommon, mild, and self-limiting. There were 18 serious adverse events (SAEs), which were related to disease progression and unrelated or likely unrelated to IC14. Three participants died due to disease progression. Monocyte CD14 RO increased for all participants after IC14 infusion. One individual required more frequent dosing (every 10 days) to achieve over 80% RO. Antidrug antibodies were detected in only one participant and were transient, low titer, and non-neutralizing. DISCUSSION: Administration of IC14 in ALS was safe and well-tolerated in this intermediate-size EAP. Measuring RO guided dosing frequency. Additional placebo-controlled trials are required to determine the efficacy of IC14 in ALS.

Improved specificity of gene silencing by siRNAs containing unlocked nucleobase analogs.

siRNAs confer sequence specific and robust silencing of mRNA. By virtue of these properties, siRNAs have become therapeutic candidates for disease intervention. However, their use as therapeutic agents can be hampered by unintended off-target effects by either or both strands of the siRNA duplex. We report here that unlocked nucleobase analogs (UNAs) confer desirable properties to siRNAs. Addition of a single UNA at the 5'-terminus of the passenger strand blocks participation of the passenger strand in RISC-mediated target down-regulation with a concomitant increase in guide strand activity. Placement of a UNA in the seed region of the guide strand prevents miRNA-like off-target silencing without compromising siRNA activity. Most significantly, combined substitution of UNA at the 3'-termini of both strands, the addition of a UNA at the 5'-terminus of the passenger strand, and a single UNA in the seed region of the guide strand, reduced the global off-target events by more than 10-fold compared to unmodified siRNA. The reduction in off-target events was specific to UNA placement in the siRNA, with no apparent new off-target events. Taken together, these results indicate that when strategically placed, UNA substitutions have important implications for the design of safe and effective siRNA-based therapeutics.

An amino acid-based amphoteric liposomal delivery system for systemic administration of siRNA.

We demonstrate a systematic and rational approach to create a library of natural and modified, dialkylated amino acids based upon arginine for development of an efficient small interfering RNA (siRNA) delivery system. These amino acids, designated DiLA2 compounds, in conjunction with other components, demonstrate unique properties for assembly into monodisperse, 100-nm small liposomal particles containing siRNA. We show that DiLA2-based liposomes undergo a pH-dependent phase transition to an inverted hexagonal phase facilitating efficient siRNA release from endosomes to the cytosol. Using an arginine-based DiLA2, cationic liposomes were prepared that provide high in vivo siRNA delivery efficiency and are well-tolerated in both cell and animal models. DiLA2-based liposomes demonstrate a linear dose-response with an ED50 of 0.1 mg/kg against liver-specific target genes in BALB/c mice.

RNAi-based therapeutics targeting survivin and PLK1 for treatment of bladder cancer.

Harnessing RNA interference (RNAi) to silence aberrant gene expression is an emerging approach in cancer therapy. Selective inhibition of an overexpressed gene via RNAi requires a highly efficacious, target-specific short interfering RNA (siRNA) and a safe and efficient delivery system. We have developed siRNA constructs (UsiRNA) that contain unlocked nucleobase analogs (UNA) targeting survivin and polo-like kinase-1 (PLK1) genes. UsiRNAs were encapsulated into dialkylated amino acid-based liposomes (DiLA(2)) containing a nor-arginine head group, cholesteryl hemisuccinate (CHEMS), cholesterol and 1, 2-dimyristoyl-phosphatidylethanolamine-polyethyleneglycol 2000 (DMPE-PEG2000). In an orthotopic bladder cancer mouse model, intravesical treatment with survivin or PLK1 UsiRNA in DiLA(2) liposomes at 1.0 and 0.5 mg/kg resulted in 90% and 70% inhibition of survivin or PLK1 mRNA, respectively. This correlated with a dose-dependent decrease in tumor volumes which was sustained over a 3-week period. Silencing of survivin and PLK1 mRNA was confirmed to be RNA-induced silencing complex mediated as specific cleavage products were detected in bladder tumors over the duration of the study. This report suggests that intravesical instillation of survivin or PLK1 UsiRNA can serve as a potential therapeutic modality for treatment of bladder cancer.

A 52-kb deletion in the SOST-MEOX1 intergenic region on 17q12-q21 is associated with van Buchem disease in the Dutch population.

Van Buchem disease is an autosomal recessive sclerosing bone dysplasia characterized by skeletal hyperostosis, overgrowth of the mandible, and a liability to entrapment of the seventh and eighth cranial nerves. The genetic determinant maps to chromosome 17q12-q21. We refined the critical interval to the < 1-Mb region between D17S2250 and D17S2253 in 15 affected individuals, all of whom shared a common disease haplotype. Furthermore, we report here the identification of a 52-kb deletion located within the interval and encompassing D17S1789 that is 100% concordant with the disorder. Although the deletion itself does not appear to disrupt the coding region of any known or novel gene(s), the closest flanking genes are MEOX1 on the proximal side, and SOST on the distal side of the deletion. MEOX1 is known to be important for the development of the axial skeleton, whereas the SOST gene is the determinant of sclerosteosis, a disorder that shares many features with van Buchem disease, thus raising the possibility that van Buchem disease results from dysregulation of the expression of one or both of these genes.

Genotyping and site-directed mutagenesis of a cytochrome P450 meander Pro-X-Arg motif critical to CYP4B1 catalysis.

CYP4B1 isoforms from rodents and other common laboratory animals are involved in the bioactivation of a range of protoxins, including 2-aminofluorene, 4-ipomeanol, and valproic acid. However, an earlier study provided evidence for a human allele encoding a nonfunctional CYP4B1 enzyme due to a Pro427Ser transversion in the meander region of the protein. In the present study, the CYP4B1 gene from several racial groups, Caucasians, African-Americans, and Hispanics, and from six nonhuman primate species was genotyped using a PCR-Hinf1 restriction enzyme fragment length polymorphism assay or by direct sequencing. All human populations examined were found to possess only the Ser allele at codon 427 ((1279)TCT) and all of the nonhuman primate species possessed only the Pro (CCT) allele. Therefore, an inactivating (1279)C-->T mutation in the human CYP4B1 gene likely arose following divergence of the Homo and Pan clades. Amino acid sequence alignments revealed further that this key Pro residue is located two amino acid residues N-terminal to the distal Arg of a Glu-Arg-Arg triad thought to participate in heme binding and/or redox partner interactions. Mutation of the corresponding Arg424 residue in rabbit CYP4B1 to Leu, but not His, resulted in a loss of lauric acid hydroxylase activity and ability to generate a reduced-CO binding spectrum. These data provide additional evidence for the importance of this meander region Pro-X-Arg motif in CYP4B1 heme binding and catalytic function.

Polymorphisms in the sclerosteosis/van Buchem disease gene (SOST) region are associated with bone-mineral density in elderly whites.

Osteoporosis has a strong genetic component, but the genes involved are poorly defined. We studied whether the sclerosteosis/van Buchem disease gene (SOST) is an osteoporosis-risk gene by examining its association with bone-mineral density (BMD). Mutations in SOST result in sclerosteosis, and alterations in the SOST gene expression may be causal in the closely related van Buchem disease. We used a set of eight polymorphisms from the SOST gene region to genotype 1,939 elderly men and women from a large population-based prospective-cohort study of Dutch whites. A 3-bp insertion (f=0.38) in the presumed SOST promoter region (SRP3) was associated with decreased BMD in women at the femoral neck (FN) (P=.05) and lumbar spine (LS) (P=.01), with evidence of an allele-dose effect in the oldest age group (P=.006). Similarly, a G variant (f=0.40) in the van Buchem deletion region (SRP9) was associated with increased BMD in men at the FN (P=.007) and LS (P=.02). In both cases, differences between extreme genotypes reached 0.2 SD. We observed no genotype effects on fracture risk, for the 234 osteoporotic fractures validated during 8.2 years of follow-up and for the 146 vertebral prevalent fractures analyzed. We did not find association between any of several frequent haplotypes across the SOST gene region and BMD. We did find evidence of additive effects of SRP3 with the COLIA1 Sp1 polymorphism but not with haplotypes of 3' polymorphisms in the vitamin-D receptor gene. The SOST-COLIA1 additive effect increased with age and reached 0.5 SD difference in BMD at LS in the oldest age group (P=.02). The molecular mechanism whereby these moderate SOST genotype effects are mediated remains to be elucidated, but it is likely to involve differences in regulation of SOST gene expression.

A novel mutation in CD83 results in the development of a unique population of CD4+ T cells.

Using a mouse mutagenesis screen, we have identified CD83 as being critical for the development of CD4(+) T cells and for their function postactivation. CD11c(+) dendritic cells develop and function normally in mice with a mutated CD83 gene but CD4(+) T cell development is substantially reduced. Additionally, we now show that those CD4(+) cells that develop in a CD83 mutant animal fail to respond normally following allogeneic stimulation. This is at least in part due to an altered cytokine expression pattern characterized by an increased production of IL-4 and IL-10 and diminished IL-2 production. Thus, in addition to its role in selection of CD4(+) T cells, absence of CD83 results in the generation of cells with an altered activation and cytokine profile.