Nalini Mehta

Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disease caused by arylsulfatase A (ARSA) deficiency. Patients with MLD exhibit progressive motor and cognitive impairment and die within a few years of symptom onset. We used a lentiviral vector to transfer a functional ARSA gene into hematopoietic stem cells (HSCs) from three presymptomatic patients who showed genetic, biochemical, and neurophysiological evidence of late infantile MLD. After reinfusion of the gene-corrected HSCs, the patients showed extensive and stable ARSA gene replacement, which led to high enzyme expression throughout hematopoietic lineages and in cerebrospinal fluid. Analyses of vector integrations revealed no evidence of aberrant clonal behavior. The disease did not manifest or progress in the three patients 7 to 21 months beyond the predicted age of symptom onset. These findings indicate that extensive genetic engineering of human hematopoiesis can be achieved with lentiviral vectors and that this approach may offer therapeutic benefit for MLD patients.

Next-Generation Gene Therapy Few disciplines in contemporary clinical research have experienced the high expectations directed at the gene therapy field. However, gene therapy has been challenging to translate to the clinic, often because the therapeutic gene is expressed at insufficient levels in the patient or because the gene delivery vector integrates near protooncogenes, which can cause leukemia (see the Perspective by Verma ). Biffi et al. ( 1233158 , published online 11 July) and Aiuti et al. ( 1233151 ; published online 11 July) report progress on both fronts in gene therapy trials of three patients with metachromatic leukodystrophy (MLD), a neurodegenerative disorder, and three patients with Wiskott-Aldrich syndrome (WAS), an immunodeficiency disorder. Optimized lentiviral vectors were used to introduce functional MLD or WAS genes into the patients' hematopoietic stem cells (HSCs) ex vivo, and the transduced cells were then infused back into the patients, who were then monitored for up to 2 years. In both trials, the patients showed stable engraftment of the transduced HSC and high expression levels of functional MLD or WAS genes. Encouragingly, there was no evidence of lentiviral vector integration near proto-oncogenes, and the gene therapy treatment halted disease progression in most patients. A longer follow-up period will be needed to further validate efficacy and safety.

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. As part of our efforts towards the discovery of new anti-tubercular leads, a number of potent tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (THPP) and N-benzyl-6',7'-dihydrospiro[piperidine-4,4'-thieno[3,2-c]pyran] (Spiro) analogues were recently identified against Mycobacterium tuberculosis and Mycobacterium bovis BCG through a high-throughput whole-cell screening campaign. Herein, we describe the attractive in vitro and in vivo anti-tubercular profiles of both lead series. The generation of M. tuberculosis spontaneous mutants and subsequent whole genome sequencing of several resistant mutants identified single mutations in the essential mmpL3 gene. This 'genetic phenotype' was further confirmed by a 'chemical phenotype', whereby M. bovis BCG treated with both the THPP and Spiro series resulted in the accumulation of trehalose monomycolate. In vivo efficacy evaluation of two optimized THPP and Spiro leads showed how the compounds were able to reduce >2 logs bacterial cfu counts in the lungs of infected mice.

Chimeric antigen receptor (CAR) T cell therapy in glioblastoma faces many challenges including insufficient CAR T cell abundance and antigen-negative tumor cells evading targeting. Unfortunately, preclinical studies evaluating CAR T cells in glioblastoma focus on tumor models that express a single antigen, use immunocompromised animals, and/or pre-treat with lymphodepleting agents. While lymphodepletion enhances CAR T cell efficacy, it diminishes the endogenous immune system that has the potential for tumor eradication. Here, we engineered CAR T cells to express IL7 and/or Flt3L in 50% EGFRvIII-positive and -negative orthotopic tumors pre-conditioned with non-lymphodepleting irradiation. IL7 and IL7 Flt3L CAR T cells increased intratumoral CAR T cell abundance seven days after treatment. IL7 co-expression with Flt3L modestly increased conventional dendritic cells as well as the CD103+XCR1+ population known to have migratory and antigen cross-presenting capabilities. Treatment with IL7 or IL7 Flt3L CAR T cells improved overall survival to 67% and 50%, respectively, compared to 9% survival with conventional or Flt3L CAR T cells. We concluded that CAR T cells modified to express IL7 enhanced CAR T cell abundance and improved overall survival in EGFRvIII heterogeneous tumors pre-conditioned with non-lymphodepleting irradiation. Potentially IL7 or IL7 Flt3L CAR T cells can provide new opportunities to combine CAR T cells with other immunotherapies for the treatment of glioblastoma.

Traumatic brain injury (TBI) triggers multiple biochemical and cellular processes that exacerbate brain tissue damage through a secondary injury. Therapies that prevent or limit the evolution of secondary injury could significantly reduce the neurological deficits associated with TBI. Mesenchymal stem cell (MSC) transplantation after TBI can ameliorate neurological deficits by modulating inflammation and enhancing the expression of neurotrophic factors. However, transplanted MSCs can be actively rejected by host immune responses, such as those mediated by cytotoxic CD8+ T cells, thereby limiting their therapeutic efficacy. Here, we designed an agarose hydrogel that releases Fas ligand (FasL), a protein that can induce apoptosis of cytotoxic CD8+ T cells. We studied the immunosuppressive effect of this hydrogel near the allogeneic MSC transplantation site and its impact on the survival of transplanted MSCs in the injured brain. Agarose-FasL hydrogels locally reduced the host cytotoxic CD8+ T cell population and enhanced the survival of allogeneic MSCs transplanted near the injury site. Furthermore, the expression of crucial neurotrophic factors was elevated in the injury penumbra, suggesting an enhanced therapeutic effect of MSCs. These results suggest that the development of immunosuppressive hydrogels for stem cell delivery can enhance the benefits of stem cell therapy for TBI.

Next-generation sequencing (NGS) technologies represent a paradigm shift in sequencing capability. The technology has already been extensively applied to biological research, resulting in significant and remarkable insights into the molecular biology of cells. In this review, we focus on current and potential applications of the technology as applied to the drug discovery and development process. Early applications have focused on the oncology and infectious disease therapeutic areas, with emerging use in biopharmaceutical development and vaccine production in evidence. Although this technology has great potential, significant challenges remain, particularly around the storage, transfer and analysis of the substantial data sets generated.

We previously reported an association with a putative functional variant in the ADAMTSL3 gene, just below genome-wide significance in a genome-wide association study of schizophrenia. As variants impacting the function of ADAMTSL3 (a disintegrin-like and metalloprotease domain with thrombospondin type I motifs-like-3) could illuminate a novel disease mechanism and a potentially specific target, we have used complementary approaches to further evaluate the association. We imputed genotypes and performed high density association analysis using data from the HapMap and 1000 genomes projects. To review all variants that could potentially cause the association, and to identify additional possible pathogenic rare variants, we sequenced ADAMTSL3 in 92 schizophrenics. A total of 71 ADAMTSL3 variants were identified by sequencing, many were also seen in the 1000 genomes data, but 26 were novel. None of the variants identified by re-sequencing was in strong linkage disequilibrium (LD) with the associated markers. Imputation analysis refined association between ADAMTSL3 and schizophrenia, and highlighted additional common variants with similar levels of association. We evaluated the functional consequences of all variants identified by sequencing, or showing direct or imputed association. The strongest evidence for function remained with the originally associated variant, rs950169, suggesting that this variant may be causal of the association. Rare variants were also identified with possible functional impact. Our study confirms ADAMTSL3 as a candidate for further investigation in schizophrenia, using the variants identified here. The utility of imputation analysis is demonstrated, and we recommend wider use of this method to re-evaluate the existing canon of suggestive schizophrenia associations.

Treatment of aggressive glioblastoma brain tumors is challenging, largely due to diffusion barriers preventing efficient drug dosing to tumors. To overcome these barriers, bacterial carriers that are actively motile and programmed to migrate and localize to tumor zones were designed. These carriers can induce apoptosis via hypoxia-controlled expression of a tumor suppressor protein p53 and a pro-apoptotic drug, Azurin. In a xenograft model of human glioblastoma in rats, bacterial carrier therapy conferred a significant survival benefit with 19% overall long-term survival of >100 days in treated animals relative to a median survival of 26 days in control untreated animals. Histological and proteomic analyses were performed to elucidate the safety and efficacy of these carriers, showing an absence of systemic toxicity and a restored neural environment in treated responders. In the treated non-responders, proteomic analysis revealed competing mechanisms of pro-apoptotic and drug-resistant activity. This bacterial carrier opens a versatile avenue to overcome diffusion barriers in glioblastoma by virtue of its active motility in extracellular space and can lead to tailored therapies via tumor-specific expression of tumoricidal proteins.

Abstract Background A major hurdle to effectively treating glioblastoma (GBM) patients is the lack of longitudinal information about tumor progression, evolution, and treatment response. Methods In this study, we report the use of a neural tract-inspired conduit containing aligned polymeric nanofibers (i.e., an aligned nanofiber device) to enable on-demand access to GBM tumors in two rodent models. Depending on the experiment, a humanized U87MG xenograft tumor and/or F98-GFP+ syngeneic rat tumor models were chosen to test the safety and functionality of the device in providing continuous sampling access to the tumor and its microenvironment. Results The aligned nanofiber device was safe and provided a high quantity of quality genomic materials suitable for omics analyses and yielded a sufficient number of live cells for in vitro expansion and screening. Transcriptomic and genomic analyses demonstrated continuity between material extracted from the device and that of the primary, intracortical tumor (in the in vivo model). Conclusions The results establish the potential of this neural tract-inspired, aligned nanofiber device as an on-demand, safe, and minimally invasive access point which enables rapid, high-throughput, longitudinal assessment of tumor and its microenvironment, leading to more informed clinical treatment strategies.

Intravenous zanamivir (IVZ) is a neuraminidase (NA) inhibitor (NAI) under investigation for the treatment of subjects hospitalised with influenza. The study included 130 symptomatic, hospitalised adults with influenza. Subjects received IVZ for 5-10 days. Viruses were cultured and analysed for susceptibility to zanamivir. Mean IC50s (n = 50) (±SD) for influenza A/H1N1pdm09, A/H3N2 and influenza B were 0.20 ± 0.06, 0.26 ± 0.07 and 1.61 ± 0.35 nM, respectively, and are comparable to data observed for sensitive isolates. A total of 185 NA and 180 haemagglutinin (HA) sequences were obtained from 123 subjects; the majority did not contain resistance substitutions. Four influenza A/H1N1pdm09 viruses from four subjects harboured NA resistance substitutions: three, Y155H, D199G and S247N, were present at Day 1 before IVZ exposure and the fourth, E119D/E, was detected at Post Treatment +5 Days but was not present at 5 other timepoints. Five subjects harboured virus with treatment-emergent NA substitutions not associated with resistance; N63D, V83A, W190C, M269K (A/H1N1pdm09) and R210K (A/H3N2). Viruses from fifteen subjects harboured HA resistance substitutions, (A/H1N1pdm09) one emerged during treatment: S162N (Day 5). Five viruses harboured treatment-emergent HA substitutions (A/H1N1pdm09) not associated with resistance: E81K, V108L, S164D, D168N and S185N. 10/92 subjects with A/H1N1pdm09 harboured a D222 HA substitution, which has been associated with increased virulence. The emergent substitutions are not associated with resistance but may have arisen due to selection pressure during IVZ treatment or by chance. In this study, there was evidence for resistance selection in a post treatment sample but the resistant variant did not persist in later visit samples.

ABSTRACT A zanamivir postapproval efficacy study was conducted in children ( n = 279) in Japan during three influenza seasons. Pharyngeal swab specimens ( n = 714) were obtained for detailed resistance analysis. From 371 cultured viruses, 3 viruses (A/H1N1) from two subjects showed reduced susceptibility to zanamivir at day 1 (before treatment), 1 had an N74S amino acid substitution (fold shift, 46), and 2 (day 1 and day 2) had a Q136K amino acid substitution (fold shifts, 292 and 301). Q136K was detected only in cultured virus and not in the swab. From the remaining 118 cultured viruses obtained during or after treatment with zanamivir, no shifts in virus susceptibility were detected. Neuraminidase (NA) population sequencing showed that viruses from 12 subjects had emergent amino acid substitutions, but 3 with susceptibility data were not zanamivir resistant. The remainder may be natural variants. Further analysis is planned. Hemagglutinin (HA) sequencing showed that viruses from 20 subjects had 9 HA amino acid substitutions that were previously implicated in resistance to neuraminidase inhibitors in in vitro assays or that were close to the receptor binding site. Their role in in vivo resistance appears to be less important but is not well understood. NA clonal sequence analysis was undertaken to determine if minority species of resistant viruses were present. A total of 1,682 clones from 90 subjects were analyzed. Single clones from 12 subjects contained amino acid substitutions close to the NA active site. It is unclear whether these single amino acid substitutions could have been amplified after drug pressure or are just chance mutations introduced during PCR.

Abstract On 11th March 2020, the UK government announced plans for the scaling of COVID-19 testing, and on 27th March 2020 it was announced that a new alliance of private sector and academic collaborative laboratories were being created to generate the testing capacity required. The Cambridge COVID-19 Testing Centre (CCTC) was established during April 2020 through collaboration between AstraZeneca, GlaxoSmithKline, and the University of Cambridge, with Charles River Laboratories joining the collaboration at the end of July 2020. The CCTC lab operation focussed on the optimised use of automation, introduction of novel technologies and process modelling to enable a testing capacity of 22,000 tests per day. Here we describe the optimisation of the laboratory process through the continued exploitation of internal performance metrics, while introducing new technologies including the Heat Inactivation of clinical samples upon receipt into the laboratory and a Direct to PCR protocol that removed the requirement for the RNA extraction step. We anticipate that these methods will have value in driving continued efficiency and effectiveness within all large scale viral diagnostic testing laboratories.

Influenza infection causes substantial morbidity and mortality during seasonal epidemics and pandemics. Antivirals, including neuraminidase inhibitors, play an important role in the treatment of severely ill patients infected with influenza. Resistance is a key factor that can affect the efficacy of neuraminidase inhibitors (NAIs). It is a recommendation by regulatory authorities to monitor for resistance during the development of anti-influenza medications. An additional requirement by regulators is to examine amino acid sequences for minority species harbouring resistance substitutions. In a Phase III study of intravenous (IV) zanamivir respiratory samples were analysed for the presence of resistant quasi species using Next Generation Sequencing (NGS). In this study ten resistance substitutions, two of which were treatment emergent, were detected by NGS that otherwise would not have been detectable by Sanger sequencing. None of the substitutions were present at any other timepoints analysed. The effect these mutations have on clinical response is difficult to characterize; in fact, all patients from which these variants were isolated had a successful clinical outcome and the effect on clinical response was therefore likely minimal. Although NGS is becoming a routine method for nucleic acid sequencing and will detect substitutions previously undetected by Sanger sequencing, the value of this technique in identifying minority species with resistance substitutions that are clinically meaningful remains to be demonstrated, particularly with acute infections such as influenza.

Abstract On 11th March 2020, the UK government announced plans for the scaling of COVID-19 testing, and on 27th March 2020 it was announced that a new alliance of private sector and academic collaborative laboratories were being created to generate the testing capacity required. The Cambridge COVID-19 Testing Centre (CCTC) was established during April 2020 through collaboration between AstraZeneca, GlaxoSmithKline, and the University of Cambridge, with Charles River Laboratories joining the collaboration at the end of July 2020. The CCTC lab operation focussed on the optimised use of automation, introduction of novel technologies and process modelling to enable a testing capacity of 22,000 tests per day. Here we describe the optimisation of the laboratory process through the continued exploitation of internal performance metrics, while introducing new technologies including the Heat Inactivation of clinical samples upon receipt into the laboratory and a Direct to PCR protocol that removed the requirement for the RNA extraction step. We anticipate that these methods will have value in driving continued efficiency and effectiveness within all large scale viral diagnostic testing laboratories.

Primary squamous cell carcinoma of the thyroid represents less than 1% of all thyroid malignancies. Unlike other thyroid cancers, it is often fatal. We report a rare case of a male patient with primary squamous cell carcinoma of the thyroid. This paper emphasizes the importance of fine needle aspiration in diagnosis, the urgency of early diagnosis and the importance of multi-modality treatment.

Cytokine mobilized stem cells collected from peripheral blood are routinely used for allogeneic hematopoietic stem cell transplantation (alloHSCT). Advantages to PBSC use include better donor tolerance and more rapid engraftment than marrow source stem cells. Disadvantages of PBSC include the increased incidence and severity of chronic GVHD due to higher numbers of alloreactive T cells.1.2ObjectiveWe hypothesized that a short course of dexamethasone (Dex) given to donors prior to PBSC collection would reduce the T cell content of the graft resulting in decreased incidence and severity of GVHD without compromise to engraftment, infection rate, immune reconstitution or relapse rates.MethodsAt our institution, 122 consecutive patients treated with alloHSCT for various malignancies between 1998-2007 received PBSCs purged of T cells in vivo by oral administration of Dex (10mg/m2) to the donor on each of the last 3 days of G-CSF mobilization. The median patient age was 48 (range 19-68), all were conditioned with busulfan based regimens, 41 patients had low risk ASBMT disease status vs 53 with high risk disease. All received CSA+MTX GVH prophylaxis.ResultsAn earlier report of the first 98 patients vs 29 controls mobilized without Dex demonstrated a 2 log reduction in the CD3+graft content of the collected product, and a low incidence and severity of acute GVHD(19% grade II - IV at 100 days). We now report an updated retrospective multivariate analysis of the incidence of acute and chronic GVHD, time to engraftment, relapse rates, and 1 year overall survival of the entire cohort, with longer follow up (min = 1yr). Final analysis of all graft products contained CD3+ and CD34+contents of 3.46x108(sd = 2.32x108) and 7.85x106(sd = 5.02x106) cells/kg respectively vs. 1.09x1010 CD3+(sd 2.19x1010) and 7.84x106 CD 34+cells/kg (sd 5.34x106) in the 29 institutional controls. Patients in the G-Dex group experienced 26% grade II-IV acute and 9.3% extensive chronic GVHD, uniform engraftment success, normal pattern immune reconstitution, and an 18% malignant disease relapse rate at 1 year.ConclusionA short course of Dex given to donors prior to PBSC collection is a viable strategy for in vivo T cell depletion. Donors tolerated the steroid well, bone pain symptoms associated with GCSF mobilization were ameliorated, and there were no untoward effects of steroid use. Comparison of this patient population with 3:1 matched CIBMTR controls is approved and in progress. Cytokine mobilized stem cells collected from peripheral blood are routinely used for allogeneic hematopoietic stem cell transplantation (alloHSCT). Advantages to PBSC use include better donor tolerance and more rapid engraftment than marrow source stem cells. Disadvantages of PBSC include the increased incidence and severity of chronic GVHD due to higher numbers of alloreactive T cells.1.2 ObjectiveWe hypothesized that a short course of dexamethasone (Dex) given to donors prior to PBSC collection would reduce the T cell content of the graft resulting in decreased incidence and severity of GVHD without compromise to engraftment, infection rate, immune reconstitution or relapse rates. We hypothesized that a short course of dexamethasone (Dex) given to donors prior to PBSC collection would reduce the T cell content of the graft resulting in decreased incidence and severity of GVHD without compromise to engraftment, infection rate, immune reconstitution or relapse rates. MethodsAt our institution, 122 consecutive patients treated with alloHSCT for various malignancies between 1998-2007 received PBSCs purged of T cells in vivo by oral administration of Dex (10mg/m2) to the donor on each of the last 3 days of G-CSF mobilization. The median patient age was 48 (range 19-68), all were conditioned with busulfan based regimens, 41 patients had low risk ASBMT disease status vs 53 with high risk disease. All received CSA+MTX GVH prophylaxis. At our institution, 122 consecutive patients treated with alloHSCT for various malignancies between 1998-2007 received PBSCs purged of T cells in vivo by oral administration of Dex (10mg/m2) to the donor on each of the last 3 days of G-CSF mobilization. The median patient age was 48 (range 19-68), all were conditioned with busulfan based regimens, 41 patients had low risk ASBMT disease status vs 53 with high risk disease. All received CSA+MTX GVH prophylaxis. ResultsAn earlier report of the first 98 patients vs 29 controls mobilized without Dex demonstrated a 2 log reduction in the CD3+graft content of the collected product, and a low incidence and severity of acute GVHD(19% grade II - IV at 100 days). We now report an updated retrospective multivariate analysis of the incidence of acute and chronic GVHD, time to engraftment, relapse rates, and 1 year overall survival of the entire cohort, with longer follow up (min = 1yr). Final analysis of all graft products contained CD3+ and CD34+contents of 3.46x108(sd = 2.32x108) and 7.85x106(sd = 5.02x106) cells/kg respectively vs. 1.09x1010 CD3+(sd 2.19x1010) and 7.84x106 CD 34+cells/kg (sd 5.34x106) in the 29 institutional controls. Patients in the G-Dex group experienced 26% grade II-IV acute and 9.3% extensive chronic GVHD, uniform engraftment success, normal pattern immune reconstitution, and an 18% malignant disease relapse rate at 1 year. An earlier report of the first 98 patients vs 29 controls mobilized without Dex demonstrated a 2 log reduction in the CD3+graft content of the collected product, and a low incidence and severity of acute GVHD(19% grade II - IV at 100 days). We now report an updated retrospective multivariate analysis of the incidence of acute and chronic GVHD, time to engraftment, relapse rates, and 1 year overall survival of the entire cohort, with longer follow up (min = 1yr). Final analysis of all graft products contained CD3+ and CD34+contents of 3.46x108(sd = 2.32x108) and 7.85x106(sd = 5.02x106) cells/kg respectively vs. 1.09x1010 CD3+(sd 2.19x1010) and 7.84x106 CD 34+cells/kg (sd 5.34x106) in the 29 institutional controls. Patients in the G-Dex group experienced 26% grade II-IV acute and 9.3% extensive chronic GVHD, uniform engraftment success, normal pattern immune reconstitution, and an 18% malignant disease relapse rate at 1 year. ConclusionA short course of Dex given to donors prior to PBSC collection is a viable strategy for in vivo T cell depletion. Donors tolerated the steroid well, bone pain symptoms associated with GCSF mobilization were ameliorated, and there were no untoward effects of steroid use. Comparison of this patient population with 3:1 matched CIBMTR controls is approved and in progress. A short course of Dex given to donors prior to PBSC collection is a viable strategy for in vivo T cell depletion. Donors tolerated the steroid well, bone pain symptoms associated with GCSF mobilization were ameliorated, and there were no untoward effects of steroid use. Comparison of this patient population with 3:1 matched CIBMTR controls is approved and in progress.