Synthetic biology design as a paradigm shift toward manufacturing affordable adeno-associated virus gene therapies

Gene therapy has demonstrated enormous potential for changing how we combat disease. By directly engineering the genetic composition of cells, it provides a broad range of options for improving human health. Adeno-associated viruses (AAVs) represent a leading gene therapy vector and are expected to address a wide range of conditions in the coming decade. Three AAV therapies have already been approved by the FDA to treat Leber\u27s congenital amaurosis, spinal muscular atrophy, and hemophilia B. Yet these therapies cost around $850,000,$2,100,000, and $3,500,000, respectively. Such prices limit the broad applicability of AAV gene therapy and make it inaccessible to most patients. Much of this problem arises from the high manufacturing costs of AAVs. At the same time, the field of synthetic biology has grown rapidly and has displayed a special aptitude for addressing biomanufacturing problems. Here, we discuss emerging efforts to apply synthetic biology design to decrease the price of AAV production, and we propose that such efforts could play a major role in making gene therapy much more widely accessible

Genetic commonality of macrolide-resistant group A beta hemolytic streptococcus pharyngeal strains

BACKGROUND: Group A beta hemolytic streptococcus (GABHS) pharyngitis is a common childhood illness. Penicillin remains the gold standard therapy, but macrolides are indicated for the penicillin allergic patient, and are often used for convenience. METHODS: We conducted a surveillance study of children with pharyngitis and positive streptococcal rapid antigen testing from 10/05 to 10/06 at 2 sites (A & B). Demographics, treatment, and resistance data was collected and compared to previous data from 2002. Erythromycin (EM) resistance was determined by disk diffusion and E-test on 500 isolates. Pulse field gel electrophoresis (PFGE) was performed to measure genetic relatedness of isolates. StatXact version 8 software (Cytel Inc., Cambridge, MA) was utilized to perform Fisher's exact test and exact confidence interval (CI) analysis. RESULTS: There were no differences in resistance rates or demographic features, with the exception of race, between sites A & B. EM resistance was 0 in 2002, 3.5% in 2005-06 at site A, and 4.5% in 2005-06 at site B. 3/7 and 3/9 had inducible resistance at A and B respectively. 8 isolates had relatedness ≥80%, 5 of which were 88% homologous on PFGE. CONCLUSION: Community macrolide resistance has increased following increased macrolide use. These results may have treatment implications if use continues to be high

Anti-Amyloid-β Single-Chain Antibody Brain Delivery Via AAV Reduces Amyloid Load But May Increase Cerebral Hemorrhages in an Alzheimer\u27s Disease Mouse Model

Accumulation of amyloid-β protein (Aβ) in the brain is thought to be a causal event in Alzheimer\u27s disease (AD). Immunotherapy targeting Aβ holds great promise for reducing Aβ in the brain. Here, we evaluated the efficacy and safety of anti-Aβ single-chain antibody (scFv59) delivery via recombinant adeno-associated virus (rAAV) on reducing Aβ deposits in an AD mouse model (TgAβPPswe/PS1dE9). First, delivery of scFv59 to the brain was optimized by injecting rAAV serotypes 1, 2, and 5 into the right lateral ventricle. Symmetrical high expression of scFv59 was found throughout the hippocampus and partly in the neocortex in both hemispheres via rAAV1 or rAAV5, while scFv59 expression via rAAV2 was mostly limited to one hemisphere. rAAV1, however, induced apoptosis and microglial activation but rAAV5 did not. Therefore, rAAV5 was selected for therapeutic scFv59 delivery in TgAβPPswe/PS1dE9 mice. rAAV5 was similarly injected into the ventricle of 10-month-old TgAβPPswe/PS1dE9 mice and 5 months later its efficacy and safety were evaluated. Immunoreactive Aβ deposits reduced in the hippocampus. Aβ42 levels in cerebrospinal fluid (CSF) tended to increase and the Aβ40 : 42 ratio decreased in CSF, suggesting that Aβ42 was relocated from the parenchyma to CSF. Hemorrhages associated with a focal increase in blood vessel amyloid were found in the brain. While immunotherapy has great potential for clearing cerebral Aβ, caution for cerebrovascular effects should be exercised when rAAV-mediated anti-Aβ immunotherapy is applied

Antibiotic prophylaxis is associated with subsequent resistant infections in children with an initial extended-spectrum-cephalosporin-resistant Enterobacteriaceae infection

ABSTRACT The objective of this study was to assess the association between previous antibiotic use, particularly long-term prophylaxis, and the occurrence of subsequent resistant infections in children with index infections due to extended-spectrum-cephalosporin-resistant Enterobacteriaceae . We also investigated the concordance of the index and subsequent isolates. Extended-spectrum-cephalosporin-resistant Escherichia coli and Klebsiella spp. isolated from normally sterile sites of patients aged &lt;22 years were collected along with associated clinical data from four freestanding pediatric centers. Subsequent isolates were categorized as concordant if the species, resistance determinants, and fumC-fimH ( E. coli ) or tonB ( Klebsiella pneumoniae ) type were identical to those of the index isolate. In total, 323 patients had 396 resistant isolates; 45 (14%) patients had ≥1 subsequent resistant infection, totaling 73 subsequent resistant isolates. The median time between the index and first subsequent infections was 123 (interquartile range, 43 to 225) days. In multivariable Cox proportional hazards analyses, patients were 2.07 times as likely to have a subsequent resistant infection (95% confidence interval, 1.11 to 3.87) if they received prophylaxis in the 30 days prior to the index infection. In 26 (58%) patients, all subsequent isolates were concordant with their index isolate, and 7 (16%) additional patients had at least 1 concordant subsequent isolate. In 12 of 17 (71%) patients with E. coli sequence type 131 (ST131)-associated type 40-30, all subsequent isolates were concordant. Subsequent extended-spectrum-cephalosporin-resistant infections are relatively frequent and are most commonly due to bacterial strains concordant with the index isolate. Further study is needed to assess the role prophylaxis plays in these resistant infections. </jats:p

Mycobacterium triplex Pulmonary Disease in Immunocompetent Host

Mycobacterium triplex, a recently described, potentially pathogenic species, caused disease primarily in immunocompromised patients. We report a case of pulmonary infection due to this mycobacterium in an immunocompetent patient and review the characteristics of two other cases. In our experience, Mycobacterium triplex pulmonary infection is unresponsive to antimycobacterial chemotherapy

Invasive Disease Caused by Nontuberculous Mycobacteria, Tanzania

Data on nontuberculous mycobacterial (NTM) disease in sub-Saharan Africa are limited. During 2006–2008, we identified 3 HIV-infected patients in northern Tanzania who had invasive NTM; 2 were infected with “Mycobacterium sherrisii” and 1 with M. avium complex sequevar MAC-D. Invasive NTM disease is present in HIV-infected patients in sub-Saharan Africa

Enterovirus D68 outbreak detection through a syndromic disease epidemiology network

BACKGROUND: In 2014, enterovirus D68 (EV-D68) was responsible for an outbreak of severe respiratory illness in children, with 1,153 EV-D68 cases reported across 49 states. Despite this, there is no commercial assay for its detection in routine clinical care. BioFire® Syndromic Trends (Trend) is an epidemiological network that collects, in near real-time, deidentified. BioFire test results worldwide, including data from the BioFire® Respiratory Panel (RP). OBJECTIVES: Using the RP version 1.7 (which was not explicitly designed to differentiate EV-D68 from other picornaviruses), we formulate a model, Pathogen Extended Resolution (PER), to distinguish EV-D68 from other human rhinoviruses/enteroviruses (RV/EV) tested for in the panel. Using PER in conjunction with Trend, we survey for historical evidence of EVD68 positivity and demonstrate a method for prospective real-time outbreak monitoring within the network. STUDY DESIGN: PER incorporates real-time polymerase chain reaction metrics from the RPRV/EV assays. Six institutions in the United States and Europe contributed to the model creation, providing data from 1,619 samples spanning two years, confirmed by EV-D68 gold-standard molecular methods. We estimate outbreak periods by applying PER to over 600,000 historical Trend RP tests since 2014. Additionally, we used PER as a prospective monitoring tool during the 2018 outbreak. RESULTS: The final PER algorithm demonstrated an overall sensitivity and specificity of 87.1% and 86.1%, respectively, among the gold-standard dataset. During the 2018 outbreak monitoring period, PER alerted the research network of EV-D68 emergence in July. One of the first sites to experience a significant increase, Nationwide Children's Hospital, confirmed the outbreak and implemented EV-D68 testing at the institution in response. Applying PER to the historical Trend dataset to determine rates among RP tests, we find three potential outbreaks with predicted regional EV-D68 rates as high as 37% in 2014, 16% in 2016, and 29% in 2018. CONCLUSIONS: Using PER within the Trend network was shown to both accurately predict outbreaks of EV-D68 and to provide timely notifications of its circulation to participating clinical laboratories

Nosocomial Outbreak of Parechovirus 3 Infection among Newborns, Austria, 2014

In 2014, sepsis-like illness affected 9 full-term newborns in 1 hospital in Austria. Although results of initial microbiological testing were negative, electron microscopy identified picornavirus. Archived serum samples and feces obtained after discharge were positive by PCR for human parechovirus 3. This infection should be included in differential diagnoses of sepsis-like illness in newborns

New Strategy for Rapid Diagnosis and Characterization of Fungal Infections: The Example of Corneal Scrapings

PURPOSE: The prognosis of people infected with Fungi especially immunocompromised depends on rapid and accurate diagnosis to capitalize on time administration of specific treatments. However, cultures produce false negative results and nucleic-acid amplification techniques require complex post-amplification procedures to differentiate relevant fungal types. The objective of this work was to develop a new diagnostic strategy based on real-time polymerase-chain reaction high-resolution melting analysis (PCR-HRM) that a) detects yeasts and filamentous Fungi, b) differentiates yeasts from filamentous Fungi, and c) discriminates among relevant species of yeasts. METHODS: PCR-HRM detection limits and specificity were assessed with a) isolated strains; b) human blood samples experimentally infected with Fungi; c) blood experimentally infected with other infectious agents; d) corneal scrapings from patients with suspected fungal keratitis (culture positive and negative) and e) scrapings from patients with suspected bacterial, viral or Acanthamoeba infections. The DNAs were extracted and mixed with primers diluted in the MeltDoctor® HRM Master Mix in 2 tubes, the first for yeasts, containing the forward primer CandUn (5'CATGCCTGTTTGAGCGTC) and the reverse primer FungUn (5'TCCTCCGCTT ATTGATATGCT) and the second for filamentous Fungi, containing the forward primer FilamUn (5'TGCCTGTCCGAGCGTCAT) and FungUn. Molecular probes were not necessary. The yields of DNA extraction and the PCR inhibitors were systematically monitored. RESULTS: PCR-HRM detected 0.1 Colony Forming Units (CFU)/µl of yeasts and filamentous Fungi, differentiated filamentous Fungi from yeasts and discriminated among relevant species of yeasts. PCR-HRM performances were higher than haemoculture and sensitivity and specificity was 100% for culture positive samples, detecting and characterizing Fungi in 7 out 10 culture negative suspected fungal keratitis. CONCLUSIONS: PCR-HRM appears as a new, sensitive, specific and inexpensive test that detects Fungi and differentiates filamentous Fungi from yeasts. It allows direct fungal detection from clinical samples and experimentally infected blood in less than 2.30 h after DNA extraction