Inorganic polyphosphates (polyPs) are linear polymers composed of repeated phosphate (PO43−) units linked together by multiple high-energy phosphoanhydride bonds. In addition to being a source of energy, polyPs have cytoprotective and antiviral activities. Here, we investigated the antiviral activities of long-chain polyPs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In molecular docking analyses, polyPs interacted with several conserved amino acid residues in angiotensin-converting enzyme 2 (ACE2), the host receptor that facilitates virus entry, and in viral RNA-dependent RNA polymerase (RdRp). ELISA and limited proteolysis assays using nano– LC-MS/MS mapped polyP120 binding to ACE2, and site-directed mutagenesis confirmed interactions between ACE2 and SARS-CoV-2 RdRp and identified the specific amino acid residues involved. PolyP120 enhanced the proteasomal degradation of both ACE2 and RdRp, thus impairing replication of the British B.1.1.7 SARS-CoV-2 variant. We thus tested polyPs for functional interactions with the virus in SARS-CoV-2–infected Vero E6 and Caco2 cells and in primary human nasal epithelial cells. Delivery of a nebulized form of polyP120 reduced the amounts of viral positive-sense genomic and subgenomic RNAs, of RNA transcripts encoding proinflammatory cytokines, and of viral structural proteins, thereby presenting SARS-CoV-2 infection in cells in vitro.

The COVID-19 pandemic has forced diagnostic laboratories to focus on the early diagnostics of SARS-CoV-2. The positivity of a molecular test cannot respond to the question regarding the viral capability to replicate, spread, and give different clinical effects. Despite the fact that some targets are covered by commercially-available assays, the identification of new biomarkers is desired in order to improve the quality of the information given by these assays. Therefore, since the subgenomic transcripts (sgN and sgE) are considered markers of viral activity, we evaluated these subgenomic transcripts in relation to the genomic amplification obtained using five different commercial CE-IVD tools. Methods: Five CE-IVD kits were compared in terms of their capability to detect both synthetic SARS-CoV-2 viral constructs (spiked in TMB or PBS medium) and targets (N, E, RdRp and Orf1ab genes) in twenty COVID-19–positive patients’ swabs. The sgN and sgE were assayed by real-time RT-qPCR and digital PCR. Results: None of the diagnostic kits missed the viral target genes when they were applied to targets spiked in TMB or PBS (at dilutions ranging from 100 pg to 0.1 pg). Nevertheless, once they were applied to RNA extracted from the patients’ swabs, the superimposability ranged from 50% to 100%, regardless of the extraction procedure. The sgN RNA transcript was detected only in samples with a higher viral load (Ct ≤ 22.5), while sgE was within all of the Ct ranges. Conclusions: The five kits show variable performances depending on the assay layout. It is worthy of note that the detection of the sgN transcript is associated with a higher viral load, thus representing a new marker of early and more severe infection.

BCR-ABL1 fusion transcript is the minimal residual disease marker in chronic myeloid leukemia; 2% of patients show unusual breakpoints generating atypical transcripts, not quantifiable by standardized real-time PCR (RT–PCR). Response monitoring is performed by non-quantitative NESTED PCR, useless for evaluating patients’ molecular remission, excluding them from treatment-free-remission protocols. Droplet digital PCR (ddPCR) is highly sensitive technology, allowing an absolute quantification independent of standard curves. Based on this, we have developed assays able to evaluate the molecular response in atypical patients. We designed new ddPCR-based molecular assays able to quantify atypical BCR-ABL1 transcripts, with a detection limit of 0.001%, validated in a cohort of 65 RNA from 11 patients. Fifty samples were identified congruently by ddPCR and NESTED PCR (40 positives and 10 negatives for atypical BCR–ABL1 transcript), while 11 positive samples were detected only by ddPCR. Our results highlight ddPCR usefulness, primarily when the BCR–ABL1/ABL1 level is less than 1.5% and NESTED PCR results are often inaccurate. Furthermore, we identified 3 patients who maintained a deep molecular response for at least one year, who could be considered good candidates for treatment-free remission approaches. Here, we describe a new promising molecular approach, highly sensitive, to monitor atypical BCR–ABL1 patients, paving the foundation to include them in treatment-free remission protocols.

We have previously demonstrated in CML patients enrolled to the Australian TIDEL trial, (600mg imatinib upfront in newly diagnosed patients) that patients with high OCT-1 activity, measured in patient blood mononuclear cells prior to imatinib start, achieve a superior molecular response, compared to those with low OCT-1 activity 1. Furthermore, the impact of low OCT-1 activity could be partially overcome with increased imatinib dose. We now prospectively test the predictive value of OCT-1 activity on the achievement of a major molecular response (<0.1 BCR-ABL IS) by 12 months, in CML patients enrolled to the TOPS trial (randomised 400 vs 800 mg imatinib). A subset of 131 TOPS2 patients had OCT-1 activity measured prior to the start of therapy, as part of the Global Novartis Correlative Science Studies. 41 had high OCT-1 activity (>7.2ng/200,000 cells) as defined in our original study. Patients with high OCT-1 activity had a markedly superior rate of MMR, on either standard or high dose imatinib (table 1). Significantly, a greater proportion of patients with low OCT-1 activity achieved MMR on the high dose arm compared to those on standard dose. This finding was not evident in the high OCT-1 activity group. The % of patients achieving MMR by 12 months (n) Total Low OCT-1 Activity High OCT-1 Activity p- value Total 48% (90) 90% (41) <0.001 400mg 59% (34) 24% (17) 94% (17) <0.001 800mg 62% (97) 53% (73) 87% (24) 0.044 p-value 0.27 0.012 0.64 Table 1: The % of patients achieving MMR based on OCT-1 activity and Randomised dose. The median OCT-1 activity for those patients achieving a MMR (n=80) was 6.05ng/200,000 cells compared to 3.9 for those patients failing to achieve MMR (n=51:p=0.003). Of the 131 patients, trough imatinib levels were available on 61. A greater proportion of patients with a trough imatinib plasma level of >1000ng/ml 3 (n=50) at 1 month achieved MMR (88%) compared to those with plasma levels of <1000ng/ml (n=11:45%: p=0.032). Importantly, OCT-1 activity is not significantly different comparing those patients with trough levels >1000ng/ml (5.6ng/200,000 cells) at 1 month to those with lower trough levels (7.3ng/200,000 cells: p=0.117). This indicates that OCT-1 activity is not providing a surrogate marker of imatinib PK. Dividing the imatinib PK data into quartiles, there is no significant difference in the % of patients achieving MMR based on trough imatinib levels, in patients with high OCT-1 activity. In contrast significantly fewer patients with low OCT-1 activity and low trough levels achieve MMR by 12 months. (Table 2) The % of patients achieving MMR by 12 months (n) Imatinib PK (ng/ml) Total Low OCT-1 Activity High OCT-1 Activity P value Quartile 1 <1600 47% (15) 12% (8) 86% (7) 0.013 Quartile 2 >1600 < 2500 80% (15) 67% (6) 89% (9) 0.469 Quartile 3 >2500 < 3500 80% (15) 77% (13) 100% (2) 0.654 Quartile 4 >3500 75% (16) 60% (10) 91% (6) 0.559 Table 2: The percentage of patients achieving MMR based on quartile analysis of imatinib PK at day 29 In the Australian cohort of 60 patients where detailed molecular response data is available the median molecular response in the 4 subgroups at 12 months shows a significant difference between the 400 mg group with low and high OCT-1 activity (Median BCR-ABL 0.2% IS v 0.02% IS p=0.03) but no difference in the 800 mg groups (low OCT-1 activity v high Median BCR-ABL 0.05% IS v 0.03% IS p=0.139). These analyses support our original proposal that OCT-1 activity defined at diagnosis has a major impact on molecular response and raises the possibility of patient-specific dosing. Patients with low OCT-1 activity are likely to achieve superior molecular responses if they receive imatinib at doses greater than 400 mg, whereas we could not find evidence of a molecular benefit to high dose imatinib for patients with high OCT-1 activity. The clinical value of monitoring trough imatinib drug levels remains to be clearly defined but it is likely to be greatly enhanced if it assessed in the context of the patient’s OCT-1 activity.

Anti-viral activities of long-chain inorganic polyphosphates (PolyPs) against severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infection were investigated. In molecular docking analyses, PolyPs interacted with several conserved angiotensin-converting enzyme (ACE)2 and RNA-dependent RNA polymerase (RdRp) amino acids. We thus tested PolyPs for functional interactions in vitro in SARS-CoV-2–infected Vero E6, Caco2 and human primary nasal epithelial cells. Immunofluorescence, qPCR, direct RNA sequencing, FISH and Immunoblotting were used to determine virus loads and transcription levels of genomic(g)RNAs and sub-genomic(sg)RNAs. We show that PolyP120 binds to ACE2 and enhances its proteasomal degradation. PolyP120 shows steric hindrance of the genomic Sars-CoV-2-RNA/RdRP complex, to impair synthesis of positive-sense gRNAs, viral subgenomic transcripts and structural proteins needed for viral replication. Thus, PolyP120 impairs infection and replication of Korean and European (containing non-synonymous variants) SARS-CoV-2 strains. As PolyPs have no toxic activities, we envision their use as a nebulised formula for oropharyngeal delivery to prevent infections of SARS-CoV-2 and during early phases of antiviral therapy.