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

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

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

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

Stem Cell-Specific Mechanisms Ensure Genomic Fidelity within HSCs and upon Aging of HSCs

SummaryWhether aged hematopoietic stem and progenitor cells (HSPCs) have impaired DNA damage repair is controversial. Using a combination of DNA mutation indicator assays, we observe a 2- to 3-fold increase in the number of DNA mutations in the hematopoietic system upon aging. Young and aged hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) do not show an increase in mutation upon irradiation-induced DNA damage repair, and young and aged HSPCs respond very similarly to DNA damage with respect to cell-cycle checkpoint activation and apoptosis. Both young and aged HSPCs show impaired activation of the DNA-damage-induced G1-S checkpoint. Induction of chronic DNA double-strand breaks by zinc-finger nucleases suggests that HSPCs undergo apoptosis rather than faulty repair. These data reveal a protective mechanism in both the young and aged hematopoietic system against accumulation of mutations in response to DNA damage

Protocol for Northern Ireland Caries Prevention in Practice Trial (NIC-PIP) trial: a randomised controlled trial to measure the effects and costs of a dental caries prevention regime for young children attending primary care dental services

<p>Abstract</p> <p>Background</p> <p>Dental caries is a persistent public health problem with little change in the prevalence in young children over the last 20 years. Once a child contracts the disease it has a significant impact on their quality of life. There is good evidence from Cochrane reviews including trials that fluoride varnish and regular use of fluoride toothpaste can prevent caries.</p> <p>The Northern Ireland Caries Prevention in Practice Trial (NIC-PIP) trial will compare the costs and effects of a caries preventive package (fluoride varnish, toothpaste, toothbrush and standardised dental health education) with dental health education alone in young children.</p> <p>Methods/Design</p> <p>A randomised controlled trial on children initially aged 2 and 3 years old who are regular attenders at the primary dental care services in Northern Ireland. Children will be recruited and randomised in dental practices. Children will be randomised to the prevention package of both fluoride varnish (twice per year for three years), fluoride toothpaste (1,450 ppm F) (supplied twice per year), a toothbrush (supplied twice a year) or not; both test and control groups receive standardised dental health education delivered by the dentist twice per year. Randomisation will be conducted by the Belfast Trust Clinical Research Support Centre ([CRSC] a Clinical Trials Unit).</p> <p>1200 participants will be recruited from approximately 40 dental practices. Children will be examined for caries by independent dental examiners at baseline and will be excluded if they have caries. The independent dental examiners will examine the children again at 3 years blinded to study group.</p> <p>The primary end-point is whether the child develops caries (cavitation into dentine) or not over the three years. One secondary outcome is the number of carious surfaces in the primary dentition in children who experience caries. Other secondary outcomes are episodes of pain, extraction of primary teeth, other adverse events and costs which will be obtained from parental questionnaires.</p> <p>Discussion</p> <p>This is a pragmatic trial conducted in general dental practice. It tests a composite caries prevention intervention, which represents an evidence based approach advocated by current guidance from the English Department of Health which is feasible to deliver to all low risk (caries free) children in general dental practice. The trial will provide valuable information to policy makers and clinicians on the costs and effects of caries prevention delivered to young children in general dental practice.</p> <p>Trial registration</p> <p>EudraCT No: 2009 - 010725 - 39</p> <p>ISRCTN: <a href="http://www.controlled-trials.com/ISRCTN36180119">ISRCTN36180119</a></p> <p>Ethics Reference No: 09/H1008/93:</p

Identification of Regulatory Networks in HSCs and Their Immediate Progeny via Integrated Proteome, Transcriptome, and DNA Methylome Analysis

SummaryIn this study, we present integrated quantitative proteome, transcriptome, and methylome analyses of hematopoietic stem cells (HSCs) and four multipotent progenitor (MPP) populations. From the characterization of more than 6,000 proteins, 27,000 transcripts, and 15,000 differentially methylated regions (DMRs), we identified coordinated changes associated with early differentiation steps. DMRs show continuous gain or loss of methylation during differentiation, and the overall change in DNA methylation correlates inversely with gene expression at key loci. Our data reveal the differential expression landscape of 493 transcription factors and 682 lncRNAs and highlight specific expression clusters operating in HSCs. We also found an unexpectedly dynamic pattern of transcript isoform regulation, suggesting a critical regulatory role during HSC differentiation, and a cell cycle/DNA repair signature associated with multipotency in MPP2 cells. This study provides a comprehensive genome-wide resource for the functional exploration of molecular, cellular, and epigenetic regulation at the top of the hematopoietic hierarchy