Gaining the hard yard: pre-clinical evaluation of lentiviral-mediated gene therapy for the treatment of β-thalassemia

Gene therapy is one potential novel therapeutic avenue for the treatment of inherited monogenic disorders. Diseases of the blood are frequent targets for gene therapy because it is relatively easy to harvest haematopoietic stem cells (HSCs) from the bone marrow, genetically modify the cells ex vivo, and then re-administer the corrected cells back into the patient via intra-venous injection. In this Closeup, Milsom and Williams discuss the work of Roselli et al, who describe the pre-clinical evaluation of the treatment for β-thalassemia in erythroid cells via the genetic correction of patient HSCs using a lentiviral vector

Induction of Histiocytic Sarcoma in Mouse Skeletal Muscle

Myeloid sarcomas are extramedullary accumulations of immature myeloid cells that may present with or without evidence of pathologic involvement of the bone marrow or peripheral blood, and often coincide with or precede a diagnosis of acute myeloid leukemia (AML). A dearth of experimental models has hampered the study of myeloid sarcomas and led us to establish a new system in which tumor induction can be evaluated in an easily accessible non-hematopoietic tissue compartment. Using ex-vivo transduction of oncogenic Kras(G12V) into p16/p19−/− bone marrow cells, we generated transplantable leukemia-initiating cells that rapidly induced tumor formation in the skeletal muscle of immunocompromised NOD.SCID mice. In this model, murine histiocytic sarcomas, equivalent to human myeloid sarcomas, emerged at the injection site 30–50 days after cell implantation and consisted of tightly packed monotypic cells that were CD48+, CD47+ and Mac1+, with low or absent expression of other hematopoietic lineage markers. Tumor cells also infiltrated the bone marrow, spleen and other non-hematopoietic organs of tumor-bearing animals, leading to systemic illness (leukemia) within two weeks of tumor detection. P16/p19−/−; Kras(G12V) myeloid sarcomas were multi-clonal, with dominant clones selected during secondary transplantation. The systemic leukemic phenotypes exhibited by histiocytic sarcoma-bearing mice were nearly identical to those of animals in which leukemia was introduced by intravenous transplantation of the same donor cells. Moreover, murine histiocytic sarcoma could be similarly induced by intramuscular injection of MLL-AF9 leukemia cells. This study establishes a novel, transplantable model of murine histiocytic/myeloid sarcoma that recapitulates the natural progression of these malignancies to systemic disease and indicates a cell autonomous leukemogenic mechanism.Stem Cell and Regenerative Biolog

Disrupted Signaling through the Fanconi Anemia Pathway Leads to Dysfunctional Hematopoietic Stem Cell Biology: Underlying Mechanisms and Potential Therapeutic Strategies

Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome. FA patients suffer to varying degrees from a heterogeneous range of developmental defects and, in addition, have an increased likelihood of developing cancer. Almost all FA patients develop a severe, progressive bone marrow failure syndrome, which impacts upon the production of all hematopoietic lineages and, hence, is thought to be driven by a defect at the level of the hematopoietic stem cell (HSC). This hypothesis would also correlate with the very high incidence of MDS and AML that is observed in FA patients. In this paper, we discuss the evidence that supports the role of dysfunctional HSC biology in driving the etiology of the disease. Furthermore, we consider the different model systems currently available to study the biology of cells defective in the FA signaling pathway and how they are informative in terms of identifying the physiologic mediators of HSC depletion and dissecting their putative mechanism of action. Finally, we ask whether the insights gained using such disease models can be translated into potential novel therapeutic strategies for the treatment of the hematologic disorders in FA patients

New insight into the causes, consequences, and correction of hematopoietic stem cell aging

Aging of hematopoietic stem cells (HSCs) is characterized by lineage bias, increased clonal expansion, and functional decrease. At the molecular level, aged HSCs typically display metabolic dysregulation, upregulation of inflammatory pathways, and downregulation of DNA repair pathways. Cellular aging of HSCs, driven by cell-intrinsic and cell-extrinsic factors, causes a predisposition to anemia, adaptive immune compromise, myelodys, plasia, and malignancy. Most hematologic diseases are strongly associated with age. But what is the biological foundation for decreased fitness with age? And are there therapeutic windows to resolve age-related hematopoietic decline? These questions were the focus of the International Society for Experimental Hematology (ISEH) New Investigator Committee Fall 2022 Webinar. This review touches on the latest insights from two leading laboratories into inflammatory- and niche-driven stem cell aging and includes speculation on strategies to prevent or correct age-related decline in HSC function

Effects of a weight loss plus exercise program on physical function in overweight, older women: a randomized controlled trial

BACKGROUND: Obesity and a sedentary lifestyle are associated with physical impairments and biologic changes in older adults. Weight loss combined with exercise may reduce inflammation and improve physical functioning in overweight, sedentary, older adults. This study tested whether a weight loss program combined with moderate exercise could improve physical function in obese, older adult women. METHODS: Participants (N = 34) were generally healthy, obese, older adult women (age range 55-79 years) with mild to moderate physical impairments (ie, functional limitations). Participants were randomly assigned to one of two groups for 24 weeks: (i) weight loss plus exercise (WL+E; n = 17; mean age = 63.7 years [4.5]) or (ii) educational control (n = 17; mean age = 63.7 [6.7]). In the WL+E group, participants attended a group-based weight management session plus three supervised exercise sessions within their community each week. During exercise sessions, participants engaged in brisk walking and lower-body resistance training of moderate intensity. Participants in the educational control group attended monthly health education lectures on topics relevant to older adults. Outcomes were: (i) body weight, (ii) walking speed (assessed by 400-meter walk test), (iii) the Short Physical Performance Battery (SPPB), and (iv) knee extension isokinetic strength. RESULTS: Participants randomized to the WL+E group lost significantly more weight than participants in the educational control group (5.95 [0.992] vs 0.23 [0.99] kg; P < 0.01). Additionally, the walking speed of participants in the WL+E group significantly increased compared with that of the control group (reduction in time on the 400-meter walk test = 44 seconds; P < 0.05). Scores on the SPPB improved in both the intervention and educational control groups from pre- to post-test (P < 0.05), with significant differences between groups (P = 0.02). Knee extension strength was maintained in both groups. CONCLUSION: Our findings suggest that a lifestyle-based weight loss program consisting of moderate caloric restriction plus moderate exercise can produce significant weight loss and improve physical function while maintaining muscle strength in obese, older adult women with mild to moderate physical impairments

Multicistronic lentiviral vectors containing the FMDV 2A cleavage factor demonstrate robust expression of encoded genes at limiting MOI

BACKGROUND: A number of gene therapy applications would benefit from vectors capable of expressing multiple genes. In this study we explored the feasibility and efficiency of expressing two or three transgenes in HIV-1 based lentiviral vector. Bicistronic and tricistronic self-inactivating lentiviral vectors were constructed employing the internal ribosomal entry site (IRES) sequence of encephalomyocarditis virus (EMCV) and/or foot-and-mouth disease virus (FMDV) cleavage factor 2A. We employed enhanced green fluorescent protein (eGFP), O(6)-methylguanine-DNA-methyltransferase (MGMT), and homeobox transcription factor HOXB4 as model genes and their expression was detected by appropriate methods including fluorescence microscopy, flow cytometry, immunocytochemistry, biochemical assay, and western blotting. RESULTS: All the multigene vectors produced high titer virus and were able to simultaneously express two or three transgenes in transduced cells. However, the level of expression of individual transgenes varied depending on: the transgene itself; its position within the construct; the total number of transgenes expressed; the strategy used for multigene expression and the average copy number of pro-viral insertions. Notably, at limiting MOI, the expression of eGFP in a bicistronic vector based on 2A was ~4 times greater than that of an IRES based vector. CONCLUSION: The small and efficient 2A sequence can be used alone or in combination with an IRES for the construction of multicistronic lentiviral vectors which can express encoded transgenes at functionally relevant levels in cells containing an average of one pro-viral insert

Eaten out of house and home:impacts of grazing on ground-dwelling reptiles in Australian grasslands and grassy woodlands

Large mammalian grazers can alter the biotic and abiotic features of their environment through their impacts on vegetation. Grazing at moderate intensity has been recommended for biodiversity conservation. Few studies, however, have empirically tested the benefits of moderate grazing intensity in systems dominated by native grazers. Here we investigated the relationship between (1) density of native eastern grey kangaroos, Macropus giganteus, and grass structure, and (2) grass structure and reptiles (i.e. abundance, richness, diversity and occurrence) across 18 grassland and grassy Eucalyptus woodland properties in south-eastern Australia. There was a strong negative relationship between kangaroo density and grass structure after controlling for tree canopy cover. We therefore used grass structure as a surrogate for grazing intensity. Changes in grazing intensity (i.e. grass structure) significantly affected reptile abundance, reptile species richness, reptile species diversity, and the occurrence of several ground-dwelling reptiles. Reptile abundance, species richness and diversity were highest where grazing intensity was low. Importantly, no species of reptile was more likely to occur at high grazing intensities. Legless lizards (Delma impar, D. inornata) were more likely to be detected in areas subject to moderate grazing intensity, whereas one species (Hemiergis talbingoensis) was less likely to be detected in areas subject to intense grazing and three species (Menetia greyii, Morethia boulengeri, and Lampropholis delicata) did not appear to be affected by grazing intensity. Our data indicate that to maximize reptile abundance, species richness, species diversity, and occurrence of several individual species of reptile, managers will need to subject different areas of the landscape to moderate and low grazing intensities and limit the occurrence and extent of high grazing

Predicting risk of pelvic floor disorders 12 and 20 years after delivery

Background: Little progress has been made in the prevention of pelvic floor disorders, despite their significant health and economic impact. The identification of women who are at risk remains a key element in targeting prevention and planning health resource allocation strategies. Although events around the time of childbirth are recognized clinically as important predictors, it is difficult to counsel women and to intervene around the time of childbirth because of an inability to convey a patient’s risk accurately in the presence of multiple risk factors and the long time lapse, which is often decades, between obstetric events and the onset of pelvic floor disorders later in life. Prediction models and scoring systems have been used in other areas of medicine to identify patients who are at risk for chronic diseases. Models have been developed for use before delivery that predict short-term risk of pelvic floor disorders after childbirth, but no models that predict long-term risk exist.Objective: The purpose of this study was to use variables that are known before and during childbirth to develop and validate prognostic models that will estimate the risks of these disorders 12 and 20 years after delivery.Study Design: Obstetric variables were collected from 2 cohorts: (1) women who gave birth in the United Kingdom and New Zealand (n=3763) and (2) women from the Swedish Medical Birth Register (n=4991). Pelvic floor disorders were self-reported 12 years after childbirth in the United Kingdom/New Zealand cohort and 20 years after childbirth in the Swedish Register. The cohorts were split so that data during the first half of the cohort’s time period were used to fit prediction models, and validation was performed from the second half (temporal validation). Because there is currently no consensus on how to best define pelvic floor disorders from a patient’s perspective, we chose to fit the data for each model using multiple outcome definitions for prolapse, urinary incontinence, fecal incontinence, ≥1 pelvic floor disorder, and ≥2 pelvic floor disorders. Model accuracy was measured in the following manner: (1) by ranking an individual’s risk among all subjects in the cohort (discrimination) with the use of a concordance index and (2) by observing whether the predicted probability was too high or low (calibration) at a range of predicted probabilities with the use of visual plots.Results: Models were able to discriminate between women who experienced bothersome symptoms or received treatment at 12 and 20 years, respectively, for pelvic organ prolapse (concordance indices, 0.570, 0.627), urinary incontinence (concordance indices, 0.653, 0.689), fecal incontinence (concordance indices, 0.618, 0.676), ≥1 pelvic floor disorders (concordance indices, 0.639, 0.675), and ≥2 pelvic floor disorders (concordance indices, 0.635, 0.619). Route of delivery and family history of each pelvic floor disorder were strong predictors in most models. Urinary incontinence before and during the index pregnancy was a strong predictor for the development of all pelvic floor disorders in most models 12 years after delivery. The 12- and 20-year bothersome symptoms or treatment for prolapse models were accurate when predictions were provided for risk from 0% to approximately 15%. The 12- and 20-year primiparous model began to over predict when risk rates reached 20%. When we predicted bothersome symptoms or treatment for urinary incontinence, the 12-year models were accurate when predictions ranged from approximately 5–60%; the 20-year primiparous models were accurate from 5% and 80%. For bothersome symptoms or treatment for fecal incontinence, the 12- and 20-year models were accurate from 1–15% risk and began to over predict at rates at &gt;15% and 20%, respectively.Conclusion: Models may provide an opportunity before birth to identify women who are at low risk of the development of pelvic floor disorders and may provide institute prevention strategies such as pelvic floor muscle training, weight control, or elective cesarean section for women who are at higher risk. Models are provided at http://riskcalc.org/UR_CHOICE/

Oncogene <i>EVI1 </i>drives acute myeloid leukemia via a targetable interaction with CTBP2

Acute myeloid leukemia (AML) driven by the activation of EVI1 due to chromosome 3q26/MECOM rearrangements is incurable. Because transcription factors such as EVI1 are notoriously hard to target, insight into the mechanism by which EVI1 drives myeloid transformation could provide alternative avenues for therapy. Applying protein folding predictions combined with proteomics technologies, we demonstrate that interaction of EVI1 with CTBP1 and CTBP2 via a single PLDLS motif is indispensable for leukemic transformation. A 4× PLDLS repeat construct outcompetes binding of EVI1 to CTBP1 and CTBP2 and inhibits proliferation of 3q26/MECOM rearranged AML in vitro and in xenotransplant models. This proof-of-concept study opens the possibility to target one of the most incurable forms of AML with specific EVI1-CTBP inhibitors. This has important implications for other tumor types with aberrant expression of EVI1 and for cancers transformed by different CTBP-dependent oncogenic transcription factors.</p

Oncogene <i>EVI1 </i>drives acute myeloid leukemia via a targetable interaction with CTBP2

Acute myeloid leukemia (AML) driven by the activation of EVI1 due to chromosome 3q26/MECOM rearrangements is incurable. Because transcription factors such as EVI1 are notoriously hard to target, insight into the mechanism by which EVI1 drives myeloid transformation could provide alternative avenues for therapy. Applying protein folding predictions combined with proteomics technologies, we demonstrate that interaction of EVI1 with CTBP1 and CTBP2 via a single PLDLS motif is indispensable for leukemic transformation. A 4× PLDLS repeat construct outcompetes binding of EVI1 to CTBP1 and CTBP2 and inhibits proliferation of 3q26/MECOM rearranged AML in vitro and in xenotransplant models. This proof-of-concept study opens the possibility to target one of the most incurable forms of AML with specific EVI1-CTBP inhibitors. This has important implications for other tumor types with aberrant expression of EVI1 and for cancers transformed by different CTBP-dependent oncogenic transcription factors.</p