Animal model or human cells: efficacy evidence for approved oligonucleotides

“Subcutaneous (SC) administration of vutrisiran to hTTR V30M transgenic mice and cynomolgus monkeys resulted in maximal reductions of ≥98% in serum TTR protein.”

“As the binding region of vutrisiran in the TTR mRNA is identical between humans and monkeys, but is not conserved in mice, rats and rabbits, non-clinical in vivo “proof-of-concept” investigations were restricted to monkeys and transgenic mice expressing the most prevalent human valine to methionine variant of TTR at amino acid 30 (V30M).”

“Efficacy studies for CYFENDUS were conducted in guinea pigs and cynomolgus macaques. Animals received two intramuscular vaccinations two weeks apart with various dilutions of CYFENDUS and were challenged with a lethal dose of aerosolized B. anthracis spores on Day 28 or 70.”

“The activity of the CpG 7909 adjuvant has been observed to be significantly weaker in rabbits; therefore, to provide a suitable additional animal to demonstrate the effectiveness of CYFENDUS, the applicant developed and used a guinea pig model of inhalational anthrax.”

“Four weekly administrations of the subcutaneous 3 mg/kg dose of givosiran prevented the production of ALA and PBG and a single subcutaneous dose (20 mg/kg in mice and 10 mg/kg in rats) of givosiran reduced ALA and PBG in both the mouse and rat models of AIP.”

“In addition to being pharmacologically active in humans, based on the nonclinical data described below, givosiran is also pharmacologically active in both rats and monkeys but is not pharmacologically active in rabbits.”

“In vivo, administration of pegaptanib sodium inhibited hypoxia-induced retinal neovascularization in a murine model of retinopathy of prematurity, VEGF-induced corneal angiogenesis in a rat corneal pocket model, and dermal vascular leakage in a dermal vascular permeability (modified Miles) assay in guinea pigs.”

“Pegaptanib also binds to VEGF188, the murine ortholog of human VEGF189, which is normally bound to the cell surface.”

“In a mouse disease model (the V30M/Hsf-1 KO mouse model), the tissue TTR protein suppression is 97-100% after 6 doses of ALN-TTR01 (LNP with same siRNA as patisiran-LNP) at 3mg/kg (bi-weekly; only one dose was tested).”

“Patisiran is a siRNA directed against human TTR mRNA and has cross-reactivity in the cynomolgus monkeys, but not in rat mouse or rabbit.”

“In vivo study in a genetic PH1 mouse model (AGXT-/-) (single dose): Single doses of 0.3, 1 and 3 mg/kg lumasiran resulted in a dose-dependent reduction of urinary oxalate and an increase in urinary glycolate. A single 3 mg/kg dose of lumasiran resulted in a reduction of urinary oxalate by approximately 50%.”

“The target site of lumasiran in the HAO1 mRNA is conserved across mammalian species and both rodent and monkey are pharmacologically relevant species.”

“In vivo studies conducted in transgenic mice and rats that express human mutant SOD1 (i.e., SOD1G93A), indicated reductions in SOD1 MRNA in tissue, and improved lifespan and rotorod performance in mouse.”

“This transgenic mouse model was chosen because the binding site of tofersen is not conserved in wild-type mouse or rats.”

“The HA and HB mouse proof of concept data were further corroborated by studies in a genetic dog model of HA and in an HA monkey model where hemophilia was induced using an anti-FVIII monoclonal antibody (MAb).”

“Fitusiran is pharmacologically active in mice, rats, dogs, and cynomolgus monkeys, as the AT mRNA target sequence is fully conserved (mice, rats, monkeys) or highly conserved (dogs) with the human sequence.”

“In vivo pharmacological characterization of plozasiran was conducted in a transgenic mouse expressing human ApoC-III (human ApoC-III mouse), healthy cynomolgus monkey, and dyslipidemic rhesus macaque monkeys.”

“The antisense strand of plozasiran is designed to be fully complementary to the monkey and human ApoC-III mRNA transcripts whereas seven sequence mismatches exist with the rat ApoC-III mRNA sequence.”

“In vivo, tumour growth inhibition and prolonged survival were demonstrated in xenograft mice models using cancer cell lines (ovarian cancer or AML cell lines) and patient-derived cancer cells (paediatric AML).”

“Due to the lack of MDS specific cell lines and mouse xenograft models and given the increased expression of telomerase across most cancer types, particularly myeloid malignancies that are closely related to MDS, nonclinical data were generated in models of other tumor types that are relevant to demonstrate imetelstat’s mechanism of action and antitumor activity.”

“In vivo studies (using intracerebroventricular and intrathecal [IT] administration) were conducted in transgenic mouse models of SMA because only humans are known to have the SMN2 gene.”

“Since humans are the only species known to have the SMN2 gene, in vivo pharmacology was examined in mouse models of SMA in which the endogenous mouse gene has been removed and the human SMN2 gene is expressed.”

“In vitro (human and monkey hepatocytes) and in vivo (monkey and transgenic mouse expressing the human TTR Ile84Ser mutation) studies documented decreases in TTR mRNA and protein levels following repeat-dose subcutaneous (SC) administration of inotersen.”

“The target sequence of inotersen is conserved between monkeys and humans, but contains 8 mismatches in rodents and 4 mismatches in rabbits.”

“Because the binding site for volanesorsen is 100% homologous between rhesus monkey and human apoC-III mRNAs and lipid metabolism in nonhuman primates resembles that observed in humans, the effects of volanesorsen in rhesus monkeys that were made hypertriglyceridemic via administration of a high-fructose supplement were evaluated.”

“Because of the divergence between the mouse, hamster and human apoC-III sequences, species-specific ASOs were employed in their respective in vivo evaluations.”

“Specifically, administration of zilganersen reduced GFAP mRNA and AxD-like features in transgenic mice overexpressing human GFAP; additionally, a species surrogate ASO reduced brain GFAP mRNA and decreased GFAP protein in the CSF of a rat model of AxD.”

“Given the lack of cross-reactivity to rodent or monkey GFAP mRNA, pharmacologic effects of zilganersen in vivo were evaluated in”

“The immunogenicity of HBsAg adjuvanted with 1018 ISS has been evaluated in mice, rat and baboons.”

“The immunogenicity studies also demonstrated that the anti-HBV antibody response and more importantly the frequency of animals responding with antibody levels ≥10 mIU/ml (the defined threshold for seroprotection in humans) is depending on the 1018 ISS dose.”

“Avacincaptad pegol has demonstrated complement inhibitory activity in several in vitro and ex vivo functional assays and in vivo in the monkey.”

“The monkey is the only species identified that could be considered relevant showing 1/10th of the ARC1905 in an in vitro activity assay against human C5. The rat has 1/1000th of the in vitro activity against human C5.”

“Pharmacodynamic activity, measured as reduced PCSK9 levels and/or LDL-C lowering, was demonstrated in transgenic mice expressing human PCSK9 (hPCSK9) and in healthy non-human primates (NHPs).”

“In response to a request, the applicant clarified that in vivo studies in animal disease models of hypercholesterolemia would be of minimal impact to the understanding of inclisiran’s biology and pharmacology in human. The argumentation included among others the facts that use of PCSK9 antibodies in clinical practice has validated PCSK9 as a pharmacologic target for reducing LDL-C and that there are species differences in cholesterol fractions.”

“Olezarsen was evaluated in vitro in primary hepatocytes, transgenic mice expressing human apoC-III, wild-type (normal) CD-1 mice and cynomolgus monkeys.”

“due to the divergence in sequence between mouse and human apoC-III mRNA”

“Primary: In vitro proof-of-concept studies, conducted primarily in DMD patient fibroblasts differentiated into myotubes, demonstrated viltolarsen-induced exon 53 skipping (EC50 values of 0.63-0.90 µM and 2.3 µM) and production of dystrophin protein in cells from DMD patients with deletion of exons 45-52 and exons 48-52, respectively.”

“In vivo assessment of exon 53 skipping was included in the 12- and 39-week toxicity studies in monkey; viltolarsen is not pharmacologically active in rodents.”

“The pharmacological in vivo activity of eplontersen was investigated in comparison to inotersen in transgenic mice overexpressing a mutant human TTR variant known to cause familial amyloid polyneuropathy in humans (hTTR I84S; Benson et al., 2006).”

“The target sequence of eplontersen and inotersen is 100 % complementary to human and Cynomolgus monkey TTR mRNA, whereas 8 mismatches in mice and rats preclude effective hybridisation and, hence, pharmacological activity in these animals.”

“Therefore, a mouse-specific surrogate ASO, ISIS 482584, was used in five in vivo studies to elucidate the role of PKK in vascular permeability.”

“Donidalorsen is not pharmacologically active in the mouse due to its lack of complementarity to the mouse PKK gene transcript.”

“The ability of eteplirsen to induce exon 51 skipping in human dystrophin mRNA has been demonstrated in the following: human muscle cell cultures; muscle explants from a DMD patient; gastrocnemius muscle of transgenic hDMD mice (after direct IM injection); quadriceps muscle, heart, and diaphragm of cynomolgus monkey (after 12 weeks of once weekly IV infusions); and EDB muscle of 7 DMD patients (after direct IM injection).”

“For mdx mouse, the pharmacological activity of the human product cannot be tested because of differences in the dystrophin gene mutation, requiring use of a mouse-specific PMO, exon 23 skipping AON and, therefore, resulting in a different truncated dystrophin.”

“Nonclinical pharmacology studies with these apoB antisense inhibitors have shown rapid reduction of hepatic apoB mRNA and protein, and reductions in serum apoB, LDL-C and total-C in mouse, hamster, rabbit and monkey models.”

“Species-specific differences in the nucleotide sequence of apoB mRNA caused a reduction (monkey) or complete loss (mouse, rat, dog and rabbit) of the intended pharmacological activity of mipomersen in these species.”

“In a mouse model of PH1 with knockout of the alanine-glyoxylate aminotransferase gene involved in limiting glyoxylate oxidation to oxalate, administration of the mouse active analog of nedosiran (DCR-m355) at doses of 0.5, 1, and 5 mg/kg caused dose-dependent suppression of hepatic LDH protein”

“The target sequence of nedosiran in LDHA mRNA is conserved in humans and nonhuman primates, but not in mice; thus, nedosiran is fully active in cynomolgus monkeys but inactive in mice.”

“The pharmacological activity of casimersen was tested in vitro using human rhabdomyosarcoma cells and normal primary myoblasts, as there are no relevant animal models due to the species- and mutation-specific nature of PMO ASOs.”

“The pharmacological activity of casimersen was tested in vitro using human rhabdomyosarcoma cells and normal primary myoblasts, as there are no relevant animal models due to the species- and mutation-specific nature of PMO ASOs.”

“Studies evaluating the pharmacological effects of defibrotide sodium on endothelial cells (ECs) were conducted primarily in the human microvascular endothelial cell line.”

“In order to evaluate the ability of fomivirsen to inhibit CMV replication in retinal cells, primary human retinal pigment epithelial (RPE) cells were used as indicator cells.”

“Due to lack of animal models for HCMV infection, the anti-CMV activity of fomivirsen has been demonstrated in in vitro tissue culture settings.”

“using a variety of cell types (rhabdomyosarcoma cells and immortalized and primary myoblast from DMD patients). All ASOs demonstrated activity, but golodirsen was selected for development based on “ease and cost of manufacture and IP status (freedom-to-operate) …” No in vivo nonclinical studies were conducted to assess the extent of exon 53 skipping by golodirsen.”

“Golodirsen is believed to be highly specific for the human dystrophin gene sequence and reportedly exhibits no hybridization affinity to any other sequence in the human genome that would present a safety concern for human use.”