Hematopoietic stem/progenitor cells (HSPCs) are capable of supporting the lifelong production of blood cells exerting a wide spectrum of functions. Lentiviral vector HSPC gene therapy generates a ...human hematopoietic system stably marked at the clonal level by vector integration sites (ISs). Using IS analysis, we longitudinally tracked >89,000 clones from 15 distinct bone marrow and peripheral blood lineages purified up to 4 years after transplant in four Wiskott-Aldrich syndrome patients treated with HSPC gene therapy. We measured at the clonal level repopulating waves, populations' sizes and dynamics, activity of distinct HSPC subtypes, contribution of various progenitor classes during the early and late post-transplant phases, and hierarchical relationships among lineages. We discovered that in-vitro-manipulated HSPCs retain the ability to return to latency after transplant and can be physiologically reactivated, sustaining a stable hematopoietic output. This study constitutes in vivo comprehensive tracking in humans of hematopoietic clonal dynamics during the early and late post-transplant phases.
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•Hematopoietic reconstitution occurs in two distinct clonal waves•A few thousand HSPC clones stably sustain multilineage blood cell production•Steady-state hematopoiesis after transplant is maintained by both HSCs and MPPs•Natural killer clones have closer relationships to myeloid cells than to lymphoid cells
Biasco et al. report a clonal tracking study on the dynamics and nature of hematopoietic reconstitution in humans after transplant. Using integration sites as molecular tags, they measured, in gene therapy patients, repopulating waves, population size and dynamics, activity of progenitor subtypes during the early and late post-transplant phases, and hierarchical relationships among lineages.
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.
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 tetrahydropyrazolo1,5-apyrimidine-3-carboxamide (THPP) and N-benzyl-6',7'-dihydrospiropiperidine-4,4'-thieno3,2-cpyran (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.
Hypersensitivity to abacavir affects about 4% of patients who receive the drug for HIV-1 infection. We did a retrospective, case-control study to identify multiple markers in the vicinity of
HLA-B ...associated with hypersensitivity reactions.
HLA-B57 was present in 39 (46%) of 84 patients versus four (4%) of 113 controls (p< 0·0001). However, because of low numbers of women and other ethnic groups enrolled, these findings relate largely to white men. The lower sensitivity of
HLA-B57 for predicting hypersensitivity to abacavir identified in this study compared with a previous report highlights that predictive values for markers will vary across populations. Clinical monitoring and management of hypersensitivity reactions among patients receiving abacavir must remain unchanged.
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.
Abstract 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.
Several lines of evidence support a role for CCL2 (monocyte chemotactic protein-1) and its receptor CCR2 in the development of atherosclerosis. The aim of the present study was to determine the ...association of the CCR2 Val64Ile polymorphism with the development of coronary artery disease in the WOSCOPS study sample set.
A total of 443 cases and 1003 controls from the West of Scotland Coronary Prevention Study (WOSCOPS) were genotyped for the Val64Ile polymorphism in the CCR2 gene. Genotype frequencies were compared between cases and controls. The CCR2 Val64Ile polymorphism was found not to be associated with coronary events in this study population (odds ratio 1.15, 95% CI 0.82-1.61, p = 0.41).
This case-control study does not support an association of the CCR2 Val64Ile polymorphism with coronary artery disease in the WOSCOPS sample set and does not confirm a possible protective role for CCR2 Val64Ile in the development of coronary artery disease.
There is a genetic component in the aetiology of Alzheimer disease (AD). Recently, Blacker et al. reported an odds ratio of 3.56 for being affected by AD in individuals carrying at least one copy of ...an alpha -2 macroglobulin gene (A2M) allele (A2M*2) corresponding to a deletion near the 5 theta splice site of exon 18 (ref. 2). Cases were aged 50 years and older and 22% had autopsy diagnoses. This effect was independent of APOE. We tried to replicate these findings using three different samples. Our first sample was composed of 125 autopsy-confirmed AD cases from Brain Banks in England and 218 elderly non-demented controls. Where age of onset was documented, 3 cases were younger than 50 years and 30 cases were 50-65 years. In cases where the age at onset was not available, 35 individuals died before age 66, including 2 before 50 years. We used non-demented elderly English individuals as controls: 59 from Ely (a town 14 miles from Cambridge) aged 70 years and older meeting neither DSM-III-R nor AGECAT dementia criteria; and 159 from Cambridge aged 84 years and older with Mini Mental State Examination (MMSE) scores of more than 23 (ref. 3). Our second sample was composed of 53 pairs of age-matched (within 5 years) demented cases and controls, aged 70 years and older, from the epidemiologically based Ely component of the Medical Research Council Cognitive Function and Ageing Study. Cases met both DSM-III-R and AGECAT criteria and controls met neither of these dementia criteria. The controls used in our first sample included those from the matched case-control series with six unmatched controls from the same cohort. Our third sample was composed of individuals from an epidemiologically based cohort from Cambridge aged 84 and older in which cognitive function was assessed using the MMSE 3. Of 229 individuals analysed, 47 had MMSE scores less than 22 and 159 had MMSE scores greater than 23 (used as controls in sample 1, above). The odds ratio reported previously for A2M (3.56; ref. 2) is detectable with 37 cases and 74 controls (80% power, 5% significance; ref. 5), thus our combined sample of 230 cases and 218 controls should detect this effect. Our autopsy-confirmed sample alone should detect an odds ratio of 2 (80% power, 5% significance).
Perfusion magnetic resonance imaging (MRI) studies quantify the microcirculatory status of liver parenchyma and liver lesions, and can be used for the detection of liver metastases, assessing the ...effectiveness of antiangiogenic therapy, evaluating tumor viability after anticancer therapy or ablation, and diagnosis of liver cirrhosis and its severity. In this review, we discuss the basic concepts of perfusion MRI using tracer kinetic modeling, the common kinetic models applied for analyses, the MR scanning t...
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Hematopoietic stem cells (HSC) are endowed with the unique role of generating an adequate and efficient pool of blood cells throughout human life. Data derived from clonal tracking of HSC activity ...and hematopoietic dynamics directly in vivo in humans would be of paramount importance for the design of therapies for hematological disorders and cancers. Our gene therapy (GT) clinical trials for adenosine deaminase (ADA) deficient-SCID and Wiskott-Aldrich Syndrome (WAS) based on the infusion of genetically engineered HSC, constitute unique clinical settings where each vector-marked progenitors and its blood cell progeny is traceable being univocally barcoded by a vector integration site (IS). To study early dynamics of hematopoietic reconstitution in humans, we collected by LAM-PCR + Illumina-Miseq sequencing 14.807.407 sequence reads corresponding to 71.981 IS tagging clones belonging to 13 different cell types purified from the bone marrow and the peripheral blood of 4 WAS patients up to 36 months after GT. We firstly identified and quantified identical IS shared among CD34+ progenitors, and mature Myeloid/Lymphoid cells as marker of the real-time clonal output of individual vector-marked HSC clones in vivo. We unraveled the timing of short, intermediate and long term HSC output showing that CD34+ clones active at 3-6 months after GT are not detectable at later follow up. By unsupervised clustering of IS similarities among lineages we unveiled diverse input of HSPC clonal differentiation towards lymphoid, myeloid and megakaryo-erythroid cells and found that NK cells have a distinct relationship with HSPC as compared to T and B cells. We also profiled the level of HSPC output overtime showing that early reconstitution is markedly skewed towards myeloid production. Importantly, clonogenic progenitors generated in vitro from ex vivo purified CD34+ patients’ cells, showed a IS profile coherent with that of freshly purified BM and PB cell types from the same time-point.
We also studied population clonal entropy through 7 different diversity indexes and uncovered that progenitor output occurs in distinct waves during the first 6-9 months after transplantation reaching a “homeostatic equilibrium” only by 12 months after GT. At steady state we estimated by mark-recapture mathematical approaches that 1900-7000 transduced HSC clones were stably contributing to the progenitors repertoire for up to 3 years after infusion of gene corrected CD34+ cells. To evaluate the long-term preservation of activity by transplanted HSC we exploited data derived from the IS-based tracking of 4.845 clones in ADA-SCID patients performed for up to 6 years after GT. We showed that identical IS are consistently detected at multiple lineages level even several years after GT. Strikingly, by semi-quantitative PCRs on specific vector-genome junctions we tracked a fluctuating but consistent output of marked HSC over a period of 5 years without the manifestation of clonal quiescence phases. Additionally, since the gamma-retroviral vector used in ADA-SCID HSC-GT trial is able to transduce only actively replicating cells, we provided the first evidence that in vitro activated HSC, “awaken” from dormancy, can still, once infused, retain in vivo long-term activity in humans. We exploited IS similarities among the lineages for both WAS and ADA-SCID datasets to reconstruct the hematopoietic hierarchy by combining conditional probability distributions and static/dynamic graphical models of dependencies. Notably, preliminary data unveiled a link between myeloid progenitors and mature lymphoid cells that supports the recently suggested model of hematopoiesis based on a delayed branching of myeloid and lymphoid lineages. Further mathematical models are being applied to specifically study population dynamics and single HSPC contribution to hematopoiesis including stochastic models of neutral clonal drift. More detailed analysis are also being performed on IS collected from 7 distinct CD34+ subtypes isolated from GT patients and FACS sorted according to the most recent markers of HSPC differentiation. Overall our work constitute the first molecular tracking of individual hematopoietic clones in humans providing an unprecedented detailed analysis of HSC activity and dynamics in vivo. The information gathered will be crucial for the design of therapeutic approaches for a broad spectrum of hematological diseases and tumors.
Neduva:GSK: Employment. Dow:GSK: Employment.
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