Accurate prediction of busulfan exposure is critical to achieve target myeloablative exposure while minimizing toxicity in pediatric hematopoietic stem cell transplantation. Model-informed precision dosing is recommended for improved accuracy and flexibility; dose adjustments in clinical practice are frequently performed using linear proportionality between dose changes and area under the concentration–time curve (AUC), optionally accounting for decrease of busulfan clearance over time. We retrospectively analyzed 54 consecutive pediatric patients receiving once-daily intravenous busulfan with therapeutic drug monitoring to characterize the relationship between dose adjustments and AUC variations, and to explore determinants of acute toxicity. Correlation between dose adjustments and AUC variations was linear for dose changes up to 1.16-fold, but linearity underestimated AUC increases following larger dose escalations. A quadratic model better described the dose–AUC relationship, particularly for dose increases, and was supported by an inverse correlation between dose variation and clearance change. While cumulative AUC was consistently maintained within target ranges, higher peak single-dose AUCs were associated with increased acute toxicity, including longer duration of mucositis and greater parenteral nutrition requirements. Initial dosing at 80 mg/m2 was associated with lower peak AUCs and reduced need for subsequent dose corrections compared with 120 mg/m2, without differences in engraftment outcomes. These findings highlight the clinical relevance of non-linear dose–exposure relationships and peak exposure in busulfan-related toxicity.

This study evaluated the efficacy of atidarsagene autotemcel (arsa-cel) gene therapy in mitigating the severity and progression of peripheral neuropathy as assessed by nerve conduction velocity (NCV) in individuals affected by late-infantile metachromatic leukodystrophy (LI-MLD). A post hoc analysis was conducted on pre-symptomatic patients affected by LI-MLD treated with ex vivo autologous haematopoietic stem cell gene therapy (arsa-cel) in the context of prospective open-label, single-arm, interventional trials and expanded access programmes. All patients were followed longitudinally with nerve conduction studies (NCSs) of peripheral motor (ulnar, deep peroneal) and sensory (median, sural) nerves. These results were compared with those from a control group of untreated patients (NHx) studied with the same standardized protocol. We then analysed the effects of baseline characteristics (age at treatment, severity of neuropathy pre-treatment expressed as age-matched NCV Z-scores) and arylsulphatase A (ARSA) enzyme activity (measured in peripheral blood myeloid CD15+ cells post-treatment) on NCVs of treated patients. The primary end point of this post hoc analysis was NCV, reflecting the severity of demyelinating neuropathy. Changes in dermal nerve histopathology in skin biopsies were used as an exploratory outcome. Fifteen treated and 16 NHx patients were included in the analyses, with a median age (interquartile range) at treatment of 13 (9.1-14.5) months. At 36 months of age, treated patients showed higher estimated NCVs in all nerves compared with age-matched controls (∼15 m/s difference in motor nerves). Peripheral neuropathy was observed in the majority of treated patients at their pre-treatment examination (age range 7.3-17.4 months). Severity of pre-treatment neuropathy in treated patients did not have an effect on NCV values at 2 years post-gene therapy or on the rate of NCV-slowing afterwards. A younger age at treatment was associated with higher NCVs of motor ulnar nerve and sensory medial nerve 2 years post-gene therapy. Overall, ARSA levels in CD15+ cells correlated with NCVs of motor deep peroneal nerve at 2 years post-gene therapy, and ARSA levels were associated with a slower decrease or a slight increase in NCVs of the deep peroneal, ulnar and medial nerves afterwards. In summary, peripheral neuropathy assessed by NCV is significantly ameliorated in LI patients treated with arsa-cel compared with untreated patients of similar age. In addition to the potential role of early age at treatment in the preservation of myelin, supraphysiological ARSA levels may slow demyelination of the deep peroneal and other peripheral nerves. Arsa-cel may exert a stronger effect on NCV than allogeneic haematopoietic stem cell transplantation due to its greater ARSA expression.

BACKGROUND:Metachromatic leukodystrophy (MLD) is an ultrarare, severe lysosomal storage disorder caused by a deficiency of arylsulfatase A (ARSA). METHODS:We treated patients who had MLD with atidarsagene autotemcel (arsa-cel), a hematopoietic stem-cell-based gene therapy, in two prospective open-label clinical studies and expanded-access programs. We compared their outcomes with those of untreated patients (natural history cohort). The primary end point was survival free from severe motor impairment (the time from birth to the first occurrence of loss of locomotion and of sitting without support or death from any cause). RESULTS:A total of 39 treated patients and 49 untreated patients were included. The median follow-up was 6.76 years (range, 0.64 to 12.19). Arsa-cel resulted in a significantly lower risk of severe motor impairment or death than no treatment among patients with presymptomatic late-infantile MLD (P<0.001), those with presymptomatic early-juvenile MLD (P = 0.04), and those with early-symptomatic early-juvenile MLD (P<0.001). The estimated percentage of patients surviving without severe motor impairment at 6 years of age was 0% (95% confidence interval [CI], not evaluable) among untreated patients with late-infantile MLD and 100% (95% CI, 100 to 100) among treated patients with presymptomatic late-infantile MLD. The estimated percentage of patients surviving without severe motor impairment at 10 years of age was 11.2% (95% CI, 0.9 to 36.4) among untreated patients with early-juvenile MLD and 87.5% (95% CI, 38.7 to 98.1) and 80.0% (95% CI, 40.9 to 94.6) among treated patients with presymptomatic and early-symptomatic early-juvenile MLD, respectively. No evidence of insertional oncogenesis was found. The most common grade 3 or higher adverse event was febrile neutropenia. Anti-ARSA antibodies were detected transiently in 6 of 39 patients (15%). Three deaths occurred, all of which were considered by the investigators to be unrelated to arsa-cel. CONCLUSIONS:Among patients with presymptomatic late-infantile or early-juvenile MLD and those with early-symptomatic early-juvenile MLD, the risk of severe motor impairment or death was significantly lower among those who received treatment with arsa-cel than in a natural history cohort that did not receive treatment. (Funded by Orchard Therapeutics and others; ClinicalTrials.gov numbers, NCT01560182 and NCT03392987.).

Metachromatic leukodystrophy (MLD) is a rare lysosomal storage disorder caused by biallelic pathogenic variants in the arylsulfatase A (ARSA) gene that result in deficiency of the ARSA enzyme. ARSA deficiency leads to accumulation of the undegraded substrate (sulfatides) in the central and peripheral nervous systems, and subsequently progressive demyelination, neurodegeneration, loss of motor and cognitive skills, and early death. MLD is a devastating and debilitating disease with profound burden and poor quality of life (QoL) for affected patients and their caregivers.

Mucopolysaccharidosis type I Hurler (MPSIH) is characterized by severe and progressive skeletal dysplasia that is not fully addressed by allogeneic hematopoietic stem cell transplantation (HSCT). Autologous hematopoietic stem progenitor cell–gene therapy (HSPC-GT) provides superior metabolic correction in patients with MPSIH compared with HSCT; however, its ability to affect skeletal manifestations is unknown. Eight patients with MPSIH (mean age at treatment: 1.9 years) received lentiviral-based HSPC-GT in a phase 1/2 clinical trial (NCT03488394). Clinical (growth, measures of kyphosis and genu velgum), functional (motor function, joint range of motion), and radiological [acetabular index (AI), migration percentage (MP) in hip x-rays and MRIs and spine MRI score] parameters of skeletal dysplasia were evaluated at baseline and multiple time points up to 4 years after treatment. Specific skeletal measures were retrospectively compared with an external cohort of HSCT-treated patients. At a median follow-up of 3.78 years after HSPC-GT, all patients treated with HSPC-GT exhibited longitudinal growth within WHO reference ranges and a median height gain greater than that observed in patients treated with HSCT after 3-year follow-up. Patients receiving HSPC-GT experienced complete and earlier normalization of joint mobility compared with patients treated with HSCT. Mean AI and MP showed progressive decreases after HSPC-GT, suggesting a reduction in acetabular dysplasia. Typical spine alterations measured through a spine MRI score stabilized after HSPC-GT. Clinical, functional, and radiological measures suggested an early beneficial effect of HSPC-GT on MPSIH-typical skeletal features. Longer follow-up is needed to draw definitive conclusions on HSPC-GT’s impact on MPSIH skeletal dysplasia.

Severe, progressive skeletal dysplasia is a major symptom of multiple mucopolysaccharidoses (MPS) types. While a gene therapy approach initiated at birth has been shown to prevent the development of bone pathology in different animal models of MPS, the capacity to correct developed bone disease is unknown. In this study, ex vivo micro-computed tomography was used to demonstrate that bone mass and architecture of murine MPS VII L5 vertebrae were within the normal range at 1 month of age but by 2 months of age were significantly different to normal. The difference between normal and MPS VII BV/TV increased with age reaching a maximal difference at approximately 4 months of age. In mature MPS VII bone BV/TV is increased (51.5% versus 21.5% in normal mice) due to an increase in trabecular number (6.2 per mm versus 3.8 per mm in normal mice). The total number of osteoclasts in the metaphysis of MPS VII mice was decreased, as was the percentage of osteoclasts attached to bone. MPS VII osteoblasts produced significantly more osteoprotegerin (OPG) than normal osteoblasts and supported the production of fewer osteoclasts from spleen precursor cells than normal osteoblasts in a co-culture system. In contrast, the formation of osteoclasts from MPS VII spleen monocytes was similar to normal in vitro, when exogenous RANKL and m-CSF was added to the culture medium. Administration of murine β-glucuronidase to MPS VII mice at 4 months of age, when bone disease was fully manifested, using lentiviral gene delivery resulted in a doubling of osteoclast numbers and a significant increase in attachment capacity (68% versus 29.4% in untreated MPS VII animals). Bone mineral volume rapidly decreased by 39% after gene therapy and fell within the normal range by 6 months of age. Collectively, these results indicate that lentiviral-mediated gene therapy is effective in reversing established skeletal pathology in murine MPS VII.

BACKGROUND:Effective treatment for metachromatic leukodystrophy (MLD) remains a substantial unmet medical need. In this study we investigated the safety and efficacy of atidarsagene autotemcel (arsa-cel) in patients with MLD. METHODS:This study is an integrated analysis of results from a prospective, non-randomised, phase 1/2 clinical study and expanded-access frameworks. 29 paediatric patients with pre-symptomatic or early-symptomatic early-onset MLD with biochemical and molecular confirmation of diagnosis were treated with arsa-cel, a gene therapy containing an autologous haematopoietic stem and progenitor cell (HSPC) population transduced ex vivo with a lentiviral vector encoding human arylsulfatase A (ARSA) cDNA, and compared with an untreated natural history (NHx) cohort of 31 patients with early-onset MLD, matched by age and disease subtype. Patients were treated and followed up at Ospedale San Raffaele, Milan, Italy. The coprimary efficacy endpoints were an improvement of more than 10% in total gross motor function measure score at 2 years after treatment in treated patients compared with controls, and change from baseline of total peripheral blood mononuclear cell (PBMC) ARSA activity at 2 years after treatment compared with values before treatment. This phase 1/2 study is registered with ClinicalTrials.gov, NCT01560182. FINDINGS:At the time of analyses, 26 patients treated with arsa-cel were alive with median follow-up of 3·16 years (range 0·64-7·51). Two patients died due to disease progression and one due to a sudden event deemed unlikely to be related to treatment. After busulfan conditioning, all arsa-cel treated patients showed sustained multilineage engraftment of genetically modified HSPCs. ARSA activity in PBMCs was significantly increased above baseline 2 years after treatment by a mean 18·7-fold (95% CI 8·3-42·2; p<0·0001) in patients with the late-infantile variant and 5·7-fold (2·6-12·4; p<0·0001) in patients with the early-juvenile variant. Mean differences in total scores for gross motor function measure between treated patients and age-matched and disease subtype-matched NHx patients 2 years after treatment were significant for both patients with late-infantile MLD (66% [95% CI 48·9-82·3]) and early-juvenile MLD (42% [12·3-71·8]). Most treated patients progressively acquired motor skills within the predicted range of healthy children or had stabilised motor performance (maintaining the ability to walk). Further, most displayed normal cognitive development and prevention or delay of central and peripheral demyelination and brain atrophy throughout follow-up; treatment benefits were particularly apparent in patients treated before symptom onset. The infusion was well tolerated and there was no evidence of abnormal clonal proliferation or replication-competent lentivirus. All patients had at least one grade 3 or higher adverse event; most were related to conditioning or to background disease. The only adverse event related to arsa-cel was the transient development of anti-ARSA antibodies in four patients, which did not affect clinical outcomes. INTERPRETATION:Treatment with arsa-cel resulted in sustained, clinically relevant benefits in children with early-onset MLD by preserving cognitive function and motor development in most patients, and slowing demyelination and brain atrophy. FUNDING:Orchard Therapeutics, Fondazione Telethon, and GlaxoSmithKline.

BACKGROUND Allogeneic hematopoietic stem-cell transplantation is the standard of care for Hurler syndrome (mucopolysaccharidosis type I, Hurler variant [MPSIH]). However, this treatment is only partially curative and is associated with complications. METHODS We are conducting an ongoing study involving eight children with MPSIH. At enrollment, the children lacked a suitable allogeneic donor and had a Developmental Quotient or Intelligence Quotient score above 70 (i.e., none had moderate or severe cognitive impairment). The children received autologous hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with an α-L-iduronidase (IDUA)-encoding lentiviral vector after myeloablative conditioning. Safety and correction of blood IDUA activity up to supraphysiologic levels were the primary end points. Clearance of lysosomal storage material as well as skeletal and neurophysiological development were assessed as secondary and exploratory end points. The planned duration of the study is 5 years. RESULTS We now report interim results. The children's mean (±SD) age at the time of HSPC gene therapy was 1.9±0.5 years. At a median follow-up of 2.10 years, the procedure had a safety profile similar to that known for autologous hematopoietic stem-cell transplantation. All the patients showed prompt and sustained engraftment of gene-corrected cells and had supraphysiologic blood IDUA activity within a month, which was maintained up to the latest follow-up. Urinary glycosaminoglycan (GAG) excretion decreased steeply, reaching normal levels at 12 months in four of five patients who could be evaluated. Previously undetectable levels of IDUA activity in the cerebrospinal fluid became detectable after gene therapy and were associated with local clearance of GAGs. Patients showed stable cognitive performance, stable motor skills corresponding to continued motor development, improved or stable findings on magnetic resonance imaging of the brain and spine, reduced joint stiffness, and normal growth in line with World Health Organization growth charts. CONCLUSIONS The delivery of HSPC gene therapy in patients with MPSIH resulted in extensive metabolic correction in peripheral tissues and the central nervous system. (Funded by Fondazione Telethon and others; ClinicalTrials.gov number, NCT03488394; EudraCT number, 2017-002430-23.).

In this study, we characterize the natural course of metachromatic leukodystrophy (MLD), explore intra/inter group differences, and identify biomarkers to monitor disease progression. This is a longitudinal observational study. Genotype and characteristics at disease onset were recorded. Time-to-event analyses were performed to assess time to major disease-related milestones in different subgroups. Longitudinal trajectories of nerve conduction velocities (NCV), brain MRI score, and brainstem auditory evoked responses (BAERs) were described. We recruited 22 late-infantile, 14 early-juvenile, 5 late-juvenile, and 4 adult MLD patients. Thirty-four were prospectively evaluated (median FU time 43 months). In late-infantile patients, the attainment of independent walking was associated with a later age at dysphagia. In early-juvenile, the presence of isolated cognitive impairment at onset was not a favorable prognostic factor. Late-infantile and early-juvenile subjects showed similar rapid loss of ambulation and onset of seizures, but late-infantile displayed earlier loss of trunk control, dysphagia, and death. We found significant differences in all major disease-related milestones (except death) between early-juvenile and late-juvenile patients. Late-juvenile and adult patients both presented with a predominant cognitive impairment, mild/no peripheral neuropathy, lower brain MRI score at plateau compared to LI/EJ, and later cerebellar involvement. NCV and BAER were consistently severely abnormal in late-infantile but not in older subjects, in whom both NCV and BAER were variably affected, with no deterioration over time in some cases. This study clarifies intra/inter group differences between MLD subtypes and provides additional indications regarding reliable clinical and instrumental tools to monitor disease progression and to serve as areference to evaluate the efficacy of future therapeutic interventions inthe different MLD variants.

The natural history of cognitive growth in the neuronopathic form of Mucopolysaccharidosis type II (MPS II) is not well defined especially their patterns of development and decline. The ability to predict the developmental course of the neurologically impaired patient is necessary to assess treatment outcomes aimed at the brain. Thirteen intravenous enzyme replacement therapy-treated Japanese patients with neuronopathic MPSII who had mutation analysis were followed on one standard measure of cognitive development over time. Six children in Group MS had missense mutations and 7 children in Group NT had null type mutations such as deletions, recombination with the pseudogene, and nonsense mutations. The patients as a whole demonstrated cognitive growth until about 36–42 months of age, followed by a plateau in development. The mean age equivalent score at age 3 was similar to that at age 6. While the decline was slow for the entire group, the patients in Group NT showed a more rapid decline than those in Group MS. Two patients with deletions showed decline to a very low level by age 5. The long plateau in cognitive development in patents with MPS II was substantiated and was consistent with other studies. This is the first demonstration that different mutation types within the neuronopathic MPS II patients are associated with different rates of decline. We also were able to identify the chronological age before which a trial would need to start in order to maintain cognitive growth and a ceiling beyond which a relatively normal outcome would not be likely.

Background Wiskott-Aldrich syndrome is a rare, life-threatening, X-linked primary immunodeficiency characterised by microthrombocytopenia, infections, eczema, autoimmunity, and malignant disease. Lentiviral vector-mediated haemopoietic stem/progenitor cell (HSPC) gene therapy is a potentially curative treatment that represents an alternative to allogeneic HSPC transplantation. Here, we report safety and efficacy data from an interim analysis of patients with severe Wiskott-Aldrich syndrome who received lentiviral vector-derived gene therapy. Methods We did a non-randomised, open-label, phase 1/2 clinical study in paediatric patients with severe WiskottAldrich syndrome, defined by either WAS gene mutation or absent Wiskott-Aldrich syndrome protein (WASP) expression or a Zhu clinical score of 3 or higher. We included patients who had no HLA-identical sibling donor available or, for children younger than 5 years of age, no suitable 10/10 matched unrelated donor or 6/6 unrelated cord blood donor. After treatment with rituximab and a reduced-intensity conditioning regimen of busulfan and fludarabine, patients received one intravenous infusion of autologous CD34+ cells genetically modified with a lentiviral vector encoding for human WAS cDNA. The primary safety endpoints were safety of the conditioning regimen and safety of lentiviral gene transfer into HSPCs. The primary efficacy endpoints were overall survival, sustained engraftment of genetically corrected HSPCs, expression of vector-derived WASP, improved T-cell function, antigen-specific responses to vaccinations, and improved platelet count and mean platelet volume normalisation. This interim analysis was done when the first six patients treated had completed at least 3 years of follow-up. The planned analyses are presented for the intention-to-treat population. This trial is registered with ClinicalTrials. gov (number NCT01515462) and EudraCT (number 2009-017346-32). Findings Between April 20, 2010, and Feb 26, 2015, nine patients (all male) were enrolled of whom one was excluded after screening; the age range of the eight treated children was 1.1-12.4 years. At the time of the interim analysis (data cutoff April 29, 2016), median follow-up was 3.6 years (range 0.5-5.6). Overall survival was 100%. Engraftment of genetically corrected HSPCs was successful and sustained in all patients. The fraction of WASP-positive lymphocytes increased from a median of 3. 9% (range 1.8-35.6) before gene therapy to 66. 7% (55.7-98.6) at 12 months after gene therapy, whereas WASP-positive platelets increased from 19. 1% (range 4.1-31.0) to 76. 6% (53.1-98.4). Improvement of immune function was shown by normalisation of in-vitro T-cell function and successful discontinuation of immunoglobulin supplementation in seven patients with follow-up longer than 1 year, followed by positive antigen-specific response to vaccination. Severe infections fell from 2. 38 (95% CI 1.44-3.72) per patient-year of observation (PYO) in the year before gene therapy to 0. 31 (0.04-1.11) per PYO in the second year after gene therapy and 0. 17 (0.00-0.93) per PYO in the third year after gene therapy. Before gene therapy, platelet counts were lower than 20 x 10(9) per L in seven of eight patients. At the last follow-up visit, the platelet count had increased to 20-50 x 10(9) per L in one patient, 50-100 x 10(9) per L in five patients, and more than 100 x 10(9) per L in two patients, which resulted in independence from platelet transfusions and absence of severe bleeding events. 27 serious adverse events in six patients occurred after gene therapy, 23 (85%) of which were infectious (pyrexia [five events in three patients], device-related infections, including one case of sepsis [four events in three patients], and gastroenteritis, including one case due to rotavirus [three events in two patients]); these occurred mainly in the first 6 months of follow-up. No adverse reactions to the investigational drug product and no abnormal clonal proliferation or leukaemia were reported after gene therapy. Interpretation Data from this study show that gene therapy provides a valuable treatment option for patients with severe Wiskott-Aldrich syndrome, particularly for those who do not have a suitable HSPC donor available.

Collection of an adequate amount of autologous haematopoietic stem progenitor cells (HSPC) is required for ex vivo manipulation and successful engraftment for certain inherited disorders. Fifty-seven paediatric patients (age 0.5–11.4 years) underwent a bone marrow harvest for the purpose of HSPC gene therapy (GT), including adenosine deaminase-severe combined immunodeficiency (ADA-SCID), Wiskott–Aldrich syndrome (WAS) and metachromatic leukodystrophy (MLD) patients. Total nucleated cells and the percentage and absolute counts of CD34+ cells were calculated at defined steps of the procedure (harvest, CD34+ cell purification, transduction with the gene transfer vector and infusion of the medicinal product). A minimum CD34+ cell dose for infusion was 2 × 106/kg, with an optimal target at 5–10 × 106/kg. Median volume of bone marrow harvested was 34.2 ml/kg (range 14.2–56.6). The number of CD34+ cells collected correlated inversely with weight and age in all patients and particularly in the MLD children group. All patients reached the minimum target dose for infusion: median dose of CD34+ cells/kg infused was 10.3 × 106/kg (3.7–25.9), with no difference among the three groups. Bone marrow harvest of volumes > 30 ml/kg in infants and children with ADA-SCID, WAS and MLD is well tolerated and allows obtaining an adequate dose of a medicinal product for HSPC-GT.

Burkholderia cepacia complex (BCC) bacteria are a group of opportunistic pathogens that cause severe lung infections in cystic fibrosis (CF). Treatment of BCC infections is difficult, due to the inherent and acquired multidrug resistance of BCC. There is a pressing need to find new bacterial targets for antimicrobials. Here, we demonstrate that the novel compound Q22, which is related to the bacterial cytoskeleton destabilising compound A22, can reduce the growth rate and inhibit growth of BCC bacteria. We further analysed the phenotypic effects of Q22 treatment on BCC virulence traits, to assess its feasibility as an antimicrobial. BCC bacteria were grown in the presence of Q22 with a broad phenotypic analysis, including resistance to H2O2-induced oxidative stress, changes in the inflammatory potential of cell surface components, and in-vivo drug toxicity studies. The influence of the Q22 treatment on inflammatory potential was measured by monitoring the cytokine responses of BCC whole cell lysates, purified lipopolysaccharide, and purified peptidoglycan extracted from bacterial cultures grown in the presence or absence of Q22 in differentiated THP-1 cells. BCC bacteria grown in the presence of Q22 displayed varying levels of resistance to H2O2-induced oxidative stress, with some strains showing increased resistance after treatment. There was strain-to-strain variation in the pro-inflammatory ability of bacterial lysates to elicit TNFα and IL-1β from human myeloid cells. Despite minimal toxicity previously shown in vitro with primary CF cell lines, in-vivo studies demonstrated Q22 toxicity in both zebrafish and mouse infection models. In summary, destabilisation of the bacterial cytoskeleton in BCC, using compounds such as Q22, led to increased virulence-related traits in vitro. These changes appear to vary depending on strain and BCC species. Future development of antimicrobials targeting the BCC bacterial cytoskeleton may be hampered if such effects translate into the in-vivo environment of the CF infection.

The clinical development of antibiotics with a new mode of action combined with efficient pulmonary drug delivery is a priority against untreatable Pseudomonas aeruginosa lung infections. POL7001 is a macrocycle antibiotic belonging to the novel class of protein epitope mimetic (PEM) molecules with selective and potent activity against P. aeruginosa We investigated ventilator-associated pneumonia (VAP) and cystic fibrosis (CF) as indications of the clinical potential of POL7001 to treat P. aeruginosa pulmonary infections. MICs of POL7001 and comparators were measured for reference and clinical P. aeruginosa strains. The therapeutic efficacy of POL7001 given by pulmonary administration was evaluated in murine models of P. aeruginosa acute and chronic pneumonia. POL7001 showed potent in vitro activity against a large panel of P. aeruginosa isolates from CF patients, including multidrug-resistant (MDR) isolates with adaptive phenotypes such as mucoid or hypermutable phenotypes. The efficacy of POL7001 was demonstrated in both wild-type and CF mice. In addition to a reduced bacterial burden in the lung, POL7001-treated mice showed progressive body weight recovery and reduced levels of inflammatory markers, indicating an improvement in general condition. Pharmacokinetic studies indicated that POL7001 reached significant concentrations in the lung after pulmonary administration, with low systemic exposure. These results support the further evaluation of POL7001 as a novel therapeutic agent for the treatment of P. aeruginosa pulmonary infections.

ABSTRACT Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia and chronic lung infections in cystic fibrosis patients. Iron is essential for bacterial growth, and P. aeruginosa expresses multiple iron uptake systems, whose role in lung infection deserves further investigation. P. aeruginosa Fe3+ uptake systems include the pyoverdine and pyochelin siderophores and two systems for heme uptake, all of which are dependent on the TonB energy transducer. P. aeruginosa also has the FeoB transporter for Fe2+ acquisition. To assess the roles of individual iron uptake systems in P. aeruginosa lung infection, single and double deletion mutants were generated in P. aeruginosa PAO1 and characterized in vitro, using iron-poor media and human serum, and in vivo, using a mouse model of lung infection. The iron uptake-null mutant (tonB1 feoB) and the Fe3+ transport mutant (tonB1) did not grow aerobically under low-iron conditions and were avirulent in the mouse model. Conversely, the wild type and the feoB, hasR phuR (heme uptake), and pchD (pyochelin) mutants grew in vitro and caused 60 to 90% mortality in mice. The pyoverdine mutant (pvdA) and the siderophore-null mutant (pvdA pchD) grew aerobically in iron-poor media but not in human serum, and they caused low mortality in mice (10 to 20%). To differentiate the roles of pyoverdine in iron uptake and virulence regulation, a pvdA fpvR double mutant defective in pyoverdine production but expressing wild-type levels of pyoverdine-regulated virulence factors was generated. Deletion of fpvR in the pvdA background partially restored the lethal phenotype, indicating that pyoverdine contributes to the pathogenesis of P. aeruginosa lung infection by combining iron transport and virulence-inducing capabilities.