Cytomegalovirus drug resistance mutations in transplant recipients with suspected resistance.

Resistant CMV infections are challenging complications after SOT and HSCT. Prompt recognition of ARMs is imperative for appropriate therapy. 108 plasma samples from 96 CMV + transplant recipients with suspected resistance were analysed in CNM in a retrospective nationwide study from January 2018 to July 2022 for resistance genotyping. ARMs in UL97 and UL54 were found in 26.87% (18/67) and 10.60% (7/66) of patients, respectively. Patients' ARM distribution in UL97 was as follows: L595S n = 3; L595S/M460I n = 1; L595S/N510S n = 1; L595W n = 1; C603W n = 4; A594V n = 3; A594E n = 1; C607Y n = 1; L397R/T409M/H411L/M460I n = 1; L397I n = 1; H520Q n = 1; four patients showed ARMs in UL54 as well (F412C n = 1; T503I n = 2; P522S n = 1), whereas three patients exhibited ARMs in UL54 only (L501I/T503I/L516R/A834P n = 1; A987G n = 2). L516R in UL54 and L397R/I and H411L in UL97 have been found for the first time in a clinical sample. L595S/W was the most prevalent ARM found to lend resistance to GCV. In UL54 all ARMs lent resistance to GCV and CDV. In addition, A834P, found in one patient, also lent resistance to FOS. CMV load did not differ significantly in patients with or without ARMs, and no differences were found either between patients with ARMs in UL97 or in UL97 and UL54. Despite extensive use of classical antivirals for the treatment of CMV infection after HSCT and SOT, ARMs occurred mainly in viral UL97 kinase, which suggests that CDV and mostly FOS continue to be useful alternatives to nucleoside analogues after genotypic detection of ARMs.

Clinical next-generation sequencing assay combining full-length gene amplification and shotgun sequencing for the detection of CMV drug resistance mutations.

Cytomegalovirus (CMV) causes severe systemic and tissue-invasive disease in immunocompromised patients, particularly solid organ and hematopoietic stem cell transplant recipients. While antiviral drugs offer promising efficacy, clinical management is complicated by the high frequency of drug resistance-associated mutations. The most commonly encountered mutations occur in the genes encoding for the drug targets: UL54 (DNA polymerase), UL56 (terminase complex), and UL97 (phosphotransferase), conferring resistance to ganciclovir/cidofovir/foscarnet, letermovir, and ganciclovir/maribavir, respectively. Currently, standard practice for detecting drug resistance is sequencing-based genotypic analysis by commercial reference laboratories with strictly prescribed sample requirements and reporting parameters that can often restrict testing in a highly vulnerable population. In order to circumvent these limitations, we developed a dual-step next-generation sequencing (NGS)-based clinical assay that utilizes full-length gene amplification by long-range PCR followed by shotgun sequencing for mutation analysis. This laboratory-developed test (LDT) achieved satisfactory performance with 96.4% accuracy, 100% precision, and an analytical sensitivity of 300IU/mL with 20% allele frequency. Highlighted by two clinical cases, our NGS LDT was able to provide critical results from patient specimens with viral loads <500IU/mL and volumes <0.5 mL - conditions otherwise unacceptable by reference laboratories. Here, we describe the development and implementation of a robust NGS LDT that offers greater testing flexibility and sensitivity to accommodate a more diverse patient population.

Phase I study of opaganib, an oral sphingosine kinase 2-specific inhibitor, in relapsed and/or refractory multiple myeloma.

Multiple myeloma (MM) remains an incurable disease and there is an unmet medical need for novel therapeutic drugs that do not share similar mechanisms of action with currently available agents. Sphingosine kinase 2 (SK2) is an innovative molecular target for anticancer therapy. We previously reported that treatment with SK2 inhibitor opaganib inhibited myeloma tumor growth in vitro and in vivo in a mouse xenograft model. In the current study, we performed a phase I study of opaganib in patients with relapsed/refractory multiple myeloma (RRMM). Thirteen patients with RRMM previously treated with immunomodulatory agents and proteasome inhibitors were enrolled and treated with single-agent opaganib at three oral dosing regimens (250 mg BID, 500 mg BID, or 750 mg BID, 28 days as a cycle). Safety and maximal tolerated dose (MTD) were determined. Pharmacokinetics, pharmacodynamics, and correlative studies were also performed. Opaganib was well tolerated up to a dose of 750 mg BID. The most common possibly related adverse event (AE) was decreased neutrophil counts. There were no serious AEs considered to be related to opaganib. MTD was determined as at least 750 mg BID. On an intent-to-treat basis, one patient (7.7%) in the 500 mg BID dose cohort showed a very good partial response, and one other patient (7.7%) achieved stable disease for 3 months. SK2 is an innovative molecular target for antimyeloma therapy. The first-in-class SK2 inhibitor opaganib is generally safe for administration to RRMM patients, and has potential therapeutic activity in these patients. Clinicaltrials.gov: NCT02757326.

Therapeutic approach with commercial supplements for pantothenate kinase-associated neurodegeneration with residual PANK2 expression levels.

BACKGROUND: Neurodegeneration with brain iron accumulation (NBIA) is a group of rare neurogenetic disorders frequently associated with iron accumulation in the basal nuclei of the brain characterized by progressive spasticity, dystonia, muscle rigidity, neuropsychiatric symptoms, and retinal degeneration or optic nerve atrophy. Pantothenate kinase-associated neurodegeneration (PKAN) is one of the most widespread NBIA subtypes. It is caused by mutations in the gene of pantothenate kinase 2 (PANK2) that result in dysfunction in PANK2 enzyme activity, with consequent deficiency of coenzyme A (CoA) biosynthesis, as well as low levels of essential metabolic intermediates such as 4'-phosphopantetheine, a necessary cofactor for essential cytosolic and mitochondrial proteins. METHODS: In this manuscript, we examined the therapeutic effectiveness of pantothenate, panthetine, antioxidants (vitamin E and omega 3) and mitochondrial function boosting supplements (L-carnitine and thiamine) in mutant PANK2 cells with residual expression levels. RESULTS: Commercial supplements, pantothenate, pantethine, vitamin E, omega 3, carnitine and thiamine were able to eliminate iron accumulation, increase PANK2, mtACP, and NFS1 expression levels and improve pathological alterations in mutant cells with residual PANK2 expression levels. CONCLUSION: Our results suggest that several commercial compounds are indeed able to significantly correct the mutant phenotype in cellular models of PKAN. These compounds alone or in combinations are of common use in clinical practice and may be useful for the treatment of PKAN patients with residual enzyme expression levels.

Cytomegalovirus UL97 ganciclovir resistance mutations in kidney transplant recipients.

OBJECTIVES: Widespread and prolonged therapy with ganciclovir (GCV) may result in the emergence of GCV-resistant mutations in human cytomegalovirus (HCMV) genome. The aim of this study was to detect the UL97 mutations associated with GCV resistance in kidney transplant recipients. METHODS: Forty-nine kidney recipients with positive HCMV DNAemia, who received GCV therapy were included in this study. A 707 bp fragment of UL97 gene spanning codons (436 to 655) was amplified by nested PCR and sequenced. RESULTS: Thirteen (26.5 %) out of 49 recipients contained mutations associated with amino acid changes. Two UL97 mutations related to GCV resistance were detected in 2 recipients (4 %), including alanine to valine (A594V) and proline to leucine (P521L). The D605E mutation was identified in 8 out of 49 (16.3 %) recipients. Silent mutations G598G, G503G, L553L, L634L, D456D and G579G were commonly observed. CONCLUSION: Our results indicate that mutations in the UL97 gene associated with GCV resistance may occur in 1 in 25 recipients treated with GCV. In addition, a higher mutation rate of D605E was detected in our recipients. This study provides the first evidence of the prevalence and pattern of GCV related mutations in Iranian Turkish recipients (Tab. 2, Fig. 1, Ref. 28).

Intra-host diversity of drug-resistant cytomegalovirus: A case report of cytomegalovirus infection after allogeneic hematopoietic cell transplantation.

Cytomegalovirus (CMV) is a major infectious agent causing severe complications in allogeneic hematopoietic cell transplantation (HCT) recipients, thereby warranting the need for aggressive preemptive or targeted antiviral therapy. However, prolonged or repeated use of antiviral agents, such as ganciclovir (GCV), foscarnet (FOS), and cidofovir (CDV), can result in drug-resistant CMV infection, posing challenges to successful outcomes. Here, we report a case of a patient with acute myeloid leukemia and drug-resistant CMV infection who presented with persistent CMV DNAemia, colitis, pneumonia, and encephalitis. An intra-host diversity of UL97 and UL54 mutations were detected through the genotypic resistance testing conducted on two blood samples (D+199 and D+224) and a cerebrospinal fluid (CSF) specimen (D+260) collected from the patient. UL97 L595W/L595F and L595W mutations were detected in the blood and CSF samples, respectively, that conferred GCV resistance. UL54 F412L mutation detected in all three samples conferred GCV/CDV resistance. However, the V787L mutation of UL54, conferring GCV/FOS resistance, was observed only in the D+224 blood sample. Despite combination therapy with FOS and high dose GCV and adjunctive therapy with leflunomide, the patient died from CMV infection and multiple organ failure on D+279. Further data on resistant mutations and intra-host diversity of CMV should be accumulated to elucidate the antiviral resistance and related outcomes.

Drug Resistance Mutations and Associated Phenotypes Detected in Clinical Trials of Maribavir for Treatment of Cytomegalovirus Infection.

BACKGROUND: In separate phase 2 trials, 120 patients received maribavir for cytomegalovirus (CMV) infection failing conventional therapy (trial 202) and 119 received maribavir for asymptomatic infection (trial 203). Overall, 172 cleared their CMV infection (CMV DNA <200 copies/mL) within 6 weeks. METHODS: Baseline and posttreatment plasma samples were tested for mutations in viral genes UL97, UL54, and/or UL27. Selected viral mutants were phenotyped for drug susceptibility. RESULTS: Baseline samples revealed UL54 mutations newly phenotyped as conferring resistance to standard DNA polymerase inhibitor(s), including K493N, P497S, K513T, L565V, V823A, A987V, and E989D. Of 29 patients (including 25 from trial 202) who cleared but later experienced recurrent CMV infection while on maribavir, 23 had available UL97 genotyping data; 17 had known resistance mutations (T409M or H411Y) and 5 additional had UL97 C480F alone. The newly phenotyped mutation C480F conferred high-grade maribavir resistance and low-grade ganciclovir resistance. Among 25 who did not respond to >14 days of therapy, 9 showed T409M or H411Y and 4 others showed C480F alone. CONCLUSIONS: After maribavir therapy (400-1200 mg twice daily), UL97 mutations T409M, H411Y, or C480F emerge to confer maribavir resistance in patients with recurrent CMV infection while on therapy or no response to therapy. CLINICAL TRIALS REGISTRATION: NCT01611974 and EudraCT 2010-024247-32.

A Potential Role for Fructosamine-3-Kinase in Cataract Treatment.

Cataracts are the major cause of blindness worldwide, largely resulting from aging and diabetes mellitus. Advanced glycation end products (AGEs) have been identified as major contributors in cataract formation because they alter lens protein structure and stability and induce covalent cross-linking, aggregation, and insolubilization of lens crystallins. We investigated the potential of the deglycating enzyme fructosamine-3-kinase (FN3K) in the disruption of AGEs in cataractous lenses. Macroscopic changes of equine lenses were evaluated after ex vivo intravitreal FN3K injection. The mechanical properties of an equine lens pair were evaluated after treatment with saline and FN3K. AGE-type autofluorescence (AF) was measured to assess the time-dependent effects of FN3K on glycolaldehyde-induced AGE-modified porcine lens fragments and to evaluate its actions on intact lenses after in vivo intravitreal FN3K injection of murine eyes. A potential immune response after injection was evaluated by analysis of IL-2, TNFα, and IFNγ using an ELISA kit. Dose- and time-dependent AF kinetics were analyzed on pooled human lens fragments. Furthermore, AF measurements and a time-lapse of macroscopic changes were performed on intact cataractous human eye lenses after incubation with an FN3K solution. At last, AF measurements were performed on cataractous human eyes after crossover topical treatment with either saline- or FN3K-containing drops. While the lenses of the equine FN3K-treated eyes appeared to be clear, the saline-treated lenses had a yellowish-brown color. Following FN3K treatment, color restoration could be observed within 30 min. The extension rate of the equine FN3K-treated lens was more than twice the extension rate of the saline-treated lens. FN3K treatment induced significant time-dependent decreases in AGE-related AF values in the AGE-modified porcine lens fragments. Furthermore, in vivo intravitreal FN3K injection of murine eyes significantly reduced AF values of the lenses. Treatment did not provoke a systemic immune response in mice. AF kinetics of FN3K-treated cataractous human lens suspensions revealed dose- and time-dependent decreases. Incubation of cataractous human eye lenses with FN3K resulted in a macroscopic lighter color of the cortex and a decrease in AF values. At last, crossover topical treatment of intact human eyes revealed a decrease in AF values during FN3K treatment, while showing no notable changes with saline. Our study suggests, for the first time, a potential additional role of FN3K as an alternative treatment for AGE-related cataracts.

Phenotype and Genotype Study of Novel C480F Maribavir-Ganciclovir Cross-Resistance Mutation Detected in Hematopoietic Stem Cell and Solid Organ Transplant Recipients.

Two transplant recipients (1 kidney and 1 hematopoietic stem cell) received maribavir (MBV) after cytomegalovirus (CMV) infection clinically resistant to standard therapy. Both patients achieved CMV DNA clearance within 30 and 18 days; however, the UL97 C480F variant emerged, causing recurrent CMV infection after a cumulative 2 months of MBV and 15 or 4 weeks of ganciclovir treatment, respectively. C480F was not detected under ganciclovir before MBV treatment. Recombinant phenotyping showed that C480F conferred the highest level of MBV resistance and ganciclovir cross-resistance, with impaired viral growth. Clinical follow-up and genotypic and phenotypic studies are essential for the assessment and optimization of patients with suspected MBV resistance.

Polyprenols mitigate cognitive dysfunction and neuropathology in the APP/PS1 mouse.

Alzheimer's disease (AD) is a very common neurodegenerative disorder in the elderly and brings considerable financial and social problems worldwide. In this study, polyprenols were firstly evaluated the effects on the cognitive deficits and neuropathology in APP/PS1 mice model of AD. At 3 months old, the APP/PS1 mice were divided into model group; polyprenols low, middle, and high dosage group; and positive drug group. Age-matched wild-type mice were chosen in control group. The administration by oral gavage lasted 6 months. Polyprenols treatment significantly improved cognitive impairment of double transgenic mice compared with vehicle control treatment in behavioral tests. In addition, immunohistochemistry and enzyme-linked immunosorbent assay showed that there were significantly reductions in neuritic plaques and the level of hyperphosphorylated tau in brain of polyprenols-treated mice. Furthermore, we found that polyprenols treatment reduced the apoptotic cells in brain sections of 9-month-old APP/PS1 mice. These results reveal that polyprenols exert neuroprotective effects in APP/PS1 mice and could represent an effective treatment for AD.

Implication of sphingosine-1-phosphate signaling in diseases: molecular mechanism and therapeutic strategies.

Sphingosine-1-phosphate signaling is emerging as a critical regulator of cellular processes that is initiated by the intracellular production of bioactive lipid molecule, sphingosine-1-phosphate. Binding of sphingosine-1-phosphate to its extracellular receptors activates diverse downstream signaling that play a critical role in governing physiological processes. Increasing evidence suggests that this signaling pathway often gets impaired during pathophysiological and diseased conditions and hence manipulation of this signaling pathway may be beneficial in providing treatment. In this review, we summarized the recent findings of S1P signaling pathway and the versatile role of the participating candidates in context with several disease conditions. Finally, we discussed its possible role as a novel drug target in different diseases.

Sphingosine kinase-1 protects differentiated N2a cells against beta-amyloid25-35-induced neurotoxicity via the mitochondrial pathway.

Although the etiology of Alzheimer's disease (AD) is not fully understood, multiple lines of evidence suggests the importance of amyloid-ß (Aß) in the initiation/progression of the disease. Aß has been shown to induce neuronal apoptosis via the sphingomyelin/ceramide pathway. This study was designed to elucidate whether the sphingosine kinase-1 (SPK1), a critical regulator of the ceramide/sphingosine 1-phosphate rheostat, plays a pivotal role in the regulation of death and survival of differentiated neuro-2a cells in response to beta-amyloid peptide fragment 25-35 (Aß25-35). These results show that the expression of SPK1 was markedly decreased in Aß25-35-induced neurotoxicity, as evidenced by the decreased cell viability and the increased apoptotic rate. Overexpression of SPK1 significantly attenuated Aß25-35-induced neurotoxicity, whereas silencing the expression of SPK1 exacerbated it. Moreover, overexpression of SPK1 can significantly attenuate Aß25-35-induced upregulation of Bax and rehabilitate the level of Bcl-2; concomitantly, it can ameliorate mitochondrial ultrastructure. These studies demonstrate that overexpression of SPK1 may moderate Aß25-35-induced neurotoxicity by regulating the Bcl-2/Bax ratio and improving mitochondrial ultrastructure. Based on these findings, SPK1 is a potential therapeutic target for AD.

The multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster as a therapeutic suicide gene of breast cancer cells.

BACKGROUND: The multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) was investigated for its broader substrate specificity and higher catalytic rate as a suicide gene in a combined gene/chemotherapy of cancer. METHODS: To evaluate the effects of nucleoside analog phosphorylation by Dm-dNK in vitro and in vivo, we generated a replication-deficient retroviral vector expressing Dm-dNK to transduce human breast cancer cells MCF7 (ER+) and MDA-MB-231 (ER-). We further determined the enzymatic activity and the sensitivity of the nontransduced and Dm-dNK-transduced 231/dNK and MCF7/dNK cells to the pyrimidine nucleoside analogs araC and araT. RESULTS: The data obtained show that Dm-dNK is enzymatically active and its overexpression in the nuclei of breast cancer cells results in an increased sensitivity to the nucleoside analogs araC and araT in vitro. Furthermore, subcutaneously transplanted 231/dNK cells were significantly inhibited after araC treatment, whereas nontransduced cancer cells continued to grow and develop in vivo. CONCLUSIONS: These results suggest that the Dm-dNK/nucleoside analog system could be a novel therapeutic strategy for treating breast cancer and improving anti-tumor efficacy, as well as for optimizing approaches for suicide gene therapy.

Adenoviral gene transfer of sphingosine kinase 1 protects heart against ischemia/reperfusion-induced injury and attenuates its postischemic failure.

Sphingosine kinase 1 (SPK1) has been identified as a central mediator of ischemia preconditioning and plays a protective role in ischemia/reperfusion (I/R)-induced cardiomyocyte death. In the present study, we investigated the protective effect of adenovirus-mediated SPK1 gene (Ad-SPK1) transfer on I/R-induced cardiac injury, and evaluated its therapeutic action on postinfarction heart failure. Cardiac SPK1 activity was increased about 5-fold by injection of Ad-SPK1, compared with injection of adenovirus carrying the green fluorescent protein gene (Ad-GFP). A more potent performance and a lower incidence of arrhythmia were observed in Ad-SPK1-injected hearts during the reperfusion period, compared with Ad-GFP-injected hearts. An enzymatic activity assay showed that creatine kinase release was also less in Ad-SPK1-injected hearts. To investigate the therapeutic action of the SPK1 gene on postischemic heart failure, the left anterior descending branch of the coronary artery in Wistar rats was ligated after direct intramyocardial injection of Ad-SPK1 or Ad-GFP as a control. Ad-SPK1 injection significantly preserved cardiac systolic and diastolic function, as evidenced by left ventricular (LV) systolic pressure, LV end-diastolic pressure, and peak velocity of contraction (dP/dt). The LV morphometric parameters of Ad-SPK1-treated animals were also preserved. In addition, SPK1 gene delivery significantly enhanced angiogenesis and reduced fibrosis. These results demonstrate that adenovirus-mediated SPK1 gene transfer could efficiently prevent I/R-induced myocardial injury and attenuate postischemic heart failure. Thus, SPK1 gene delivery would be a novel strategy for the treatment of coronary heart disease.

A novel adenoviral gutless vector encoding sphingosine kinase promotes arteriogenesis and improves perfusion in a rabbit hindlimb ischemia model.

OBJECTIVES: We previously demonstrated that sphingosine kinase (SPK) increases the level of extracellular sphingosine-1-phosphate and promotes neovascularization in a mouse matrigel model. In this study, we tested the hypothesis that SPK gene transfer using a novel adenoviral 'gutless' vector (AGV) can enhance arteriogenesis in a rabbit hindlimb ischemia model. METHODS: Thirty-five male New Zealand white rabbits were randomized to the AGV-SPK group (n=13), AGV-null group (n=13), and control group (n=9). On day 10, after the induction of unilateral hindlimb ischemia, gene vectors or buffer were introduced and the effect examined on day 30, using calf blood pressure, quantitative angiographic analysis, and histology. RESULTS: Calf systolic blood pressure ratios of the ischemic limb to the normal limb on day 30 were 0.77+/-0.13 in control groups, including the AGV-null group, and 0.91+/-0.14 in the AGV-SPK group (P<0.05). Angiographic vessel counts were significantly increased (8.0+/-2.1 at baseline and 11.8+/-3.2 on day 30, P<0.001) in the AGV-SPK group. Histologic analysis showed that microscopic total vessel counts on day 30 were 3.5+/-1.8/field in the control and AGV-null group and 5.4+/-1.0/field in the AGV-SPK group. Arterioles (AGV-SPK; 3.0+/-0.8 versus control and AGV-null; 2.1+/-1.1, P<0.05) were significantly increased in the AGV-SPK group. CONCLUSIONS: This study shows that SPK promotes arteriogenesis, as evidenced by the maximal improvement in the blood pressure restoration and collateral vessel counts. SPK may be an important angiogenic target to improve perfusion in ischemic tissues.