Hematopoietic stem cell gene therapy for inherited monogenic diseases and its implications for future gene therapy trials in Turkey

Stem cell therapy offers a great advantage for the development of new treatments in the field of regenerative and restorative medicine. However, the use of stem cell therapies and their clinical indications can even be further improved using genetic modification of the cells. Due to the high level of consanguineous marriages in Turkey, the country suffers from an increased frequency of inborn genetically inherited diseases. Treatment of these diseases is difficult, since 1) diagnosis is often delayed in rural areas, 2) distance to specialized centers may be considerable, 3) treatment may require frequent hospital visits and 4) treatment procedures are often both invasive and expensive. Here, we discuss the current status of gene therapy of hematopoietic stem cells (HSCs) for rare, inherited monogenic diseases and the advantages to use these cells as an alternative treatment option for patients in Turkey. We discuss results of clinical trials using retroviral and lentiviral gene therapy for the treatment of immune deficiencies, hemoglobinopathies and several enzyme deficiencies, new developments in the field of the HSC gene therapy to improve safety and efficacy and recommendations for the future.Kök hücre tedavisi rejeneratif tıp ve restoratif tıp alanlarında yeni tedavilerin geliştirilmesi için büyük avantaj sağlamaktadır. Böylelikle, kök hücre tedavilerinin kullanımı ve bunların klinik endikasyonları hücrelerin genetik modifikasyonu ile geliştirilebilmektedir. Türkiye’deki akraba evlilik oranının fazla olmasından dolayı, yenidoğan genetik kalıtsal hastalıkların insidası artmaktadır ve bu durum bir sorun teşkil etmektedir. Bu hastalıkların tedavi edilmesi; 1) kırsal bölgelerde hastalığın sıklıkla geç teşhisi, 2) Özel merkezlerin uzak olması, 3) Tedavilerin sık hastane kontrolü gerektirmesi, 4) Tedavi prosedürlerinin hem pahalı hem de invazif olması nedenleriyle zordur. Bu makalede nadir kalıtsal monogenik hastalıklar için hematopoetik kök hücre (HKH) gen tedavisinin güncel durumları ve Türkiye’deki hastalar için alternatif bir tedavi seçeneği olarak kullanımının avantajları tartışılacaktır. İmmun yetmezlikler, hemoglobinopatiler, birçok enzim eksikliklerinde retroviral ve lentiviral gen tedavi klinik çalışma sonuçları, HKH gen tedavisi alanındaki yeni gelişmeler, güvenlik ve etkinliğin artırılması ve gelecekteki öneriler tartışılacaktır

Immune modulation in gene therapy studies

Summary Host immune responses play a major role in clearance of viral infections from the body, and may limit long-term expression and clinical efficacy of viral vectors. Methods to prevent these immune responses may also increase the risk for infections, recombination with wild type virus and affect biodistribution, persistence, shedding and transmission. The study described in this report was initiated to assess possible environmental risks associated with the use of immune modulation in combination with gene therapy and set up as a literature study, by performing PubMed searches for certain keywords, by interviewing experts and by attending selected meetings. Lack of availability of clinical data combining gene therapy and immune modulation and limited animal data warranted additional exploration of relevant non-gene therapy studies from closely related fields such as stem cell and organ transplantation, and vaccination studies with live attenuated vaccines. ...... Finally, we propose the use of a checklist to assess current environmental risks in the use of immune modulation during gene therapy. This report is expected to provide guidance to risk assessors and regulatory officers as well as to applicants for a gene therapy licence

G-CSF Treatment of Healthy Pediatric Donors Affects Their Hematopoietic Microenvironment Through Changes in Bone Marrow Plasma Cytokines and Stromal Cells

Although G-CSF mobilized peripheral blood stem cell (PBSC) transplantation is commonly used in adults, bone marrow (BM) is still the preferred stem cell source in pediatric stem cell transplantation. Despite the fact that G- CSF is increasingly being used to enhance the hematopoietic stem/progenitor cell (HSPC) yield in BM trans-plantation (G-BM), the direct effects of G-CSF on the pediatric BM microenvironment have never been investi-gated. The BM hematopoietic niche provides the physical space where the HSPCs reside. This BM niche regulates HSPC quiescence and proliferation through direct interactions with other niche cells, including Mesenchymal Stromal Cells (MSCs). These cells have been shown to secrete a wide range of hematopoietic cytokines (CKs) and growth factors (GFs) involved in differentiation, retention and homing of hematopoietic cells. Here, we assessed changes in the BM microenvironment by measuring levels of 48 different CKs and GFs in G-BM and control BM (C-BM) plasma from pediatric donors. In addition, the effect of G-CSF on cell numbers and characteristics of HSPCs and MSCs was assessed. IL-16, SCGF-b, MIP-1b (all >1000 pg/mL) and RANTES (>10.000 pg/mL) were highly expressed in healthy donor pediatric BM plasma. Levels of IL-3, IL-18, GROa, MCP-3 (p<0.05) were increased in G-BM, whereas levels of RANTES (p<0.001) decreased after G-CSF treatment. We found a negative correlation with increasing age for IL2-Ra and LIF (p<0.05). In addition, a concomitant increase in the number of both hematopoietic and fibroblast colony forming units was observed, indicating that G-CSF affects both HSPC and MSC numbers. In conclusion, G-CSF treatment of healthy pediatric donors affects the hematopoietic BM microenvironment by expansion of HSPC and MSC numbers and modifying local CK and GF levels.Wo

Characterization Of Mesenchymal Stem Cells In Mucolipidosis Type Ii (I-Cell Disease)

Mucolipidosis type II (ML-II, I-cell disease) is a fatal inherited lysosomal storage disease caused by a deficiency of the enzyme N-acetylglucosamine-1-phosphotransferase. A characteristic skeletal phenotype is one of the many clinical manifestations of ML-II. Since the mechanisms underlying these skeletal defects in ML-II are not completely understood, we hypothesized that a defect in osteogenic differentiation of ML-II bone marrow mesenchymal stem cells (BM-MSCs) might be responsible for this skeletal phenotype. Here, we assessed and characterized the cellular phenotype of BM-MSCs from a ML-II patient before (BBMT) and after BM transplantation (ABMT), and we compared the results with BM-MSCs from a carrier and a healthy donor. Morphologically, we did not observe differences in ML-II BBMT and ABMT or carrier MSCs in terms of size or granularity. Osteogenic differentiation was not markedly affected by disease or carrier status. Adipogenic differentiation was increased in BBMT ML-II MSCs, but chondrogenic differentiation was decreased in both BBMT and ABMT ML-II MSCs. Immunophenotypically no significant differences were observed between the samples. Interestingly, the proliferative capacity of BBMT and ABMT ML-II MSCs was increased in comparison to MSCs from age-matched healthy donors. These data suggest that MSCs are not likely to cause the skeletal phenotype observed in ML-II, but they may contribute to the pathogenesis of ML-II as a result of lysosomal storage-induced pathology.PubMedWoSScopu

Hematopoetik Kök Hücre Hibernasyonunda Nöropeptid Y (NPY)´nin Rolünün İncelenmesi

Hematopoetik kök hücreler (HKH), kök hücre nişi olarak da adlandırılan özel bir mikroçevrede bulunur. Bu mikrocevrede mezenkimal kök hucreler (MKH), endotelyal hücreler (EH), retiküler hücreler bulunur. Perivasküler mikroortamda bulunan EH, MKH, makrofajlar ve HKH yaşamını ve kemik iliği rejenerasyonunu düzenler.Nörotransmitter nöropeptit Y (NPY), makrofajlardan ve osteoblastlardan salınır. NPY, immün ve kemik hücre homeostazını veya bu yapıların vasküler yeniden yapılanmasını, kemik iliği hücrelerinde ifade edilen Y reseptörüyle düzenlerler. Hibernasyonda HKH'ler sıklıkla trabeküler endosteuma yakın bulunurlar. İhtiyaç duyulduğunda bu hücreler çoğalır ve farklılaşır, stres ve yaralanmalarda salgılanan büyüme faktörleri ile hücre döngüsüne girerler. NPY eksikliğinde, HKH’lerin sayıları düşmekte ve kemik iliği rejenerasyonu bozulmaktadır. Önerilen projenin amacı, NPY ve NPY reseptör sinyal yolağı etkisiyle HKH'lerin uyku durumuna geri dönmesi ve HKH havuz boyutu ile kemik iliği fonksiyonunun korunmasında sessizliğin rolünün ortaya çıkmasına ilişkin anahtar bir mekanizmanın belirlenmesidir

Neuropeptide Y is involved in the regulation of quiescence of hematopoietic stem cells

Differentiation, self-renewal and quiescence of Hematopoietic stem cells (HSCs) is tightly regulated in order to protect the HSCs from the strain of constant cell division and depletion of the stem cell pool. The neurotransmitter Neuropeptide Y (NPY) is released from sympathetic nerves in the bone marrow and has been shown to indirectly affect HSC function through effects on bone marrow (BM) multipotent Mesenchymal Stromal Cells (MSCs), osteoblasts (OBs) and macrophages. Although the absence of NPY has been shown to be accompanied by severe BM impairment and delayed engraftment of HSCs, the direct effects of NPY on HSCs have never been assessed. Here, we aimed to explore the effect of NPY on the regulation of HSCs. All NPY receptors Y1, Y2, Y4 and Y5 were found to be highly expressed on most HSCs and mature hematopoietic cell subsets. In culture, in particularly expression of the Y1 receptor was shown to decrease in time. Doses of 300 nM NPY suppressed HSC proliferation in cell cultures, as confirmed by an increase of HSCs in G(0) phase and an increase in the gene expression levels of FOXO3, DICERI, SMARCA2 and PDK1, which all have been shown to play an important role in the regulation of cell quiescence. These data support the idea that NPY may have a direct effect on the regulation of HSC fate by modulating cell quiescence

Automated washing of long-term cryopreserved peripheral blood stem cells promotes cell viability and preserves CD34+ cell numbers

Peripheral blood stem cell (PBSC) transplantation has become an established treatment option for a range of malignant and inherited diseases. PBSCs are usually cryopreserved in the presence of dimethyl sulfoxide (DMSO) and stored in liquid nitrogen. However, cryopreservation and thawing of PBSCs may affect cell viability, resulting in delayed engraftment and other risks related to low stem cell numbers [1]. DMSO is commonly used at a concentration of 10% during freezing of PBSCs. However, DMSO itself is toxic and lowering doses of DMSO may have a favorable effect on hematopoietic recovery after transplantation [2, 3]. Generally, PBSCs are thawed and infused without removal of DMSO. This has been associated with a wide range of adverse effects (AEs), ranging from minor to severe life-threatening events [4, 5]. Although not all toxic events can be contributed to DMSO, grafts containing lower concentrations of DMSO typically display a reduced incidence of AEs [5, 6]. To decrease AEs and improve graft quality after thawing, it has been suggested to remove DMSO prior to transplantation [4, 7] using different washing systems [8, 9]. Here, we aimed to remove DMSO from long-term cryopreserved PBSCs using an automated, fully closed system and assessed effects on CD34+ stem cells, viability, and colony-forming capacity

Neurological Regulation of the Bone Marrow Niche

The bone marrow (BM) hematopoietic niche is the microenvironment where in the adult hematopoietic stem and progenitor cells (HSPCs) are maintained and regulated. This regulation is tightly controlled through direct cell-cell interactions with mesenchymal stromal stem (MSCs) and reticular cells, adipocytes, osteoblasts and endothelial cells, through binding to extracellular matrix molecules and through signaling by cytokines and hematopoietic growth factors. These interactions provide a healthy environment and secure the maintenance of the HSPC pool, their proliferation, differentiation and migration. Recent studies have shown that innervation of the BM and interactions with the peripheral sympathetic neural system are important for maintenance of the hematopoietic niche, through direct interactions with HSCPs or via interactions with other cells of the HSPC microenvironment. Signaling through adrenergic receptors (ARs), opioid receptors (ORs), endocannabinoid receptors (CRs) on HSPCs and MSCs has been shown to play an important role in HSPC homeostasis and mobilization. In addition, a wide range of neuropeptides and neurotransmitters, such as Neuropeptide Y (NPY), Substance P (SP) and Tachykinins, as well as neurotrophins and neuropoietic growth factors have been shown to be involved in regulation of the hematopoietic niche. Here, a comprehensive overview is given of their role and interactions with important cells in the hematopoietic niche, including HSPCs and MSCs, and their effect on HSPC maintenance, regulation and mobilization