Stem Cells and Gene Therapy

Diseases affecting the central nervous system (CNS) pose a formidable obstacle to the delivery of effective therapeutics. A tight-knit collection of cells and macromolecules known as the blood- brain-barrier (BBB) prevents most substances from entering the brain. One intriguing approach to overcoming this obstacle involves transplanting neural stem cells (NSCs), the precursor cells to neurons and glia in the brain, as vehicles for the delivery of therapeutic proteins in their native environment. Notably, this strategy has already been successfully applied to several lysosomal storage diseases caused by genetic deficiencies in one of the many lysosomal hydrolases expressed throughout the body. A major drawback to this approach is that foreign NSCs, e.g. immortalized cell lines and primary fetal NSCs can be tumorigenic and immunogenic. Recently developed induced pluripotent stem cell (iPSC) technologies, combined with pluripotent stem cell differentiation techniques, have the potential to overcome these obstacles. This approach was evaluated using a comprehensive strategy targeting a prototypical lysosomal storage disease, Sly disease (MPS VII). MPS VII patient fibroblasts were transduced with retroviral vectors expressing the transcription factors Oct4, Sox2, Klf4, and c-Myc. Patient fibroblasts were reprogrammed into embryonic stem cell-like iPSCs that demonstrated hallmarks of pluripotency. Patient iPSCs, alongside iPSCs derived from an unaffected individual, were subjected to a stepwise

(aa253 – 364) is responsible for the translocation of the toxin across cellular membranes. The exact function of domainIb (aa365 – 404) has not been investigated yet, domainIII (aa405 – 613) with last 4 residues (aa400 – 404) of domainIb together form the catalytic subunit. 3,4 The natural killing ability of PE makes it an attractive candi- date for eradicating tumor cells. The mechanism of cell killing by PE is through ADP-ribosylation of eukaryotic elongation factor 2 (eEF-2) by transferring ADP-ribose from NAD + to diphthamide residue on eEF-2, and subse- quent inhibition of the protein synthesis, which leads to apoptosis of the host cells. 5–7 PE38 is a 38kDa truncated form of PE that contains extensive deletions in Domain Ia ( Δ 1 – 250) and Ib ( Δ 365 – 380). 7 Cell killing provoked by PE38 has successfully con fi rmed the cytotoxic potential of this toxin. To date, many PE38-based immunotoxins have been developed and they have been proved to ef fi ciently kill cancer cells both in vitro and in vivo. 8–12 There are also several reports on successful PE38-based cancer gene therapy. 13,14 Here, we employed BCSC cell-speci fi c expression of PE38 as a tool for breast cancer gene therapy.

Chronic obstructive pulmonary disease and emphysema are known to be associated with air pollution and smok- ing habit [36]. COPD and emphysema reduce the respi- ratory capability of the subject and induce lung injury which in several situations are life threatening [37]. Cur- rently several drugs such as inhaled bronchodilators, inhaled corticosteroids, phosphodiesterase-4 inhibitors and mucolytics are used as symptomatic therapy and blockage of inflammation [38-40]. Other drugs such as macrolides have been also used for control of COPD ex- acerbations by exploiting their anti-inflammatory pro- perties [41]. It has been observed that during COPD exacerbation the circulating endothelial stem cells are not decreased, however; their population is abnormally high, probably during an effort for tissue repair and in- flammation control [42,43]. Increased levels of vascular endothelial growth factor (VEGF) have been associated with increased levels of circulating endothelial stem cells [43]. It has been previously observed that mesenchymal stem cells protect cigarette smoke lung injury, by up-

The development of technology and a modern research approach in the 21st century has enabled access to new therapies in many fields of medicine and nanotechnology. Work on the use of cellular therapies is carried out in leading centers around the world. According to the "clinicaltraial.gov" service, it is estimated that there are about 5.5 thousand clinical trials using stem cells. The classification of stem cells is based on their potential to differentiate into other cells, tissues, organs or the whole organism. In cell therapy 3 groups of stem cells are used: Pluripotent Stem Cells (PSC), multipotent and unipotent, which have two common features: the ability to self-revalue, that is, to divide and to differentiate in many directions. To date, there are no objective, randomized clinical trials that would clearly determine the efficacy and safety of the cell therapy used in ophthalmic diseases. Hope is given by gene therapies such as the recently approved Luxturna™ gene therapy used in hereditary retinal degeneration caused by mutations in the RPE65 gene. Research is currently underway on experimental cell therapies to treat the following diseases of the optic system: glaucoma, retinopathy, age-related macular degeneration (AMD), optic nerve atrophy, retinal pigmentation (RP) and Stargardt's disease. Stem cell-based medical therapies are a promising and rapidly developing method of innovative treatment, especially for conditions that were previously considered incurable. The use of experimental cellular gene therapies in diseases of the visual organ gives hope to both patients and scientists, but the age of regenerative medicine has yet to come.

TRIMCyp without the potential pitfall of the host generating an immune response against xenoantigens. The TRIMCyp- based approach to lentiviral gene therapy offers advantages over other potential approaches to therapy; for example, by targeting viral entry, it denies the virus the opportunity to replicate, and thus the virus cannot generate escape mutants. As TRIMCyp does not target the function of an endogenously expressed molecule (recent targets for HIV gene therapy have included the coreceptor CCR5 [1, 13, 22]), it is unlikely to have side effects that are detrimental to the host. Treatment of the lentivirus-infected host using TRIMCyp fusions may be achieved by transduction of bone marrow-derived hema- topoietic progenitor stem cells and repopulation of the host immune system following bone marrow ablation. However, transduction of peripheral-blood-derived CD4 ⫹ T cells and ex vivo expansion of the transduced cells prior to repopulation of the host immune system may be sufficient to overcome the immunodeficiency associated with AIDS. Accordingly, the suc- cessful in vitro studies described here offer strong support for clinical trials of feTRIMCyp both as a therapy for FIV infec-

MSCs are a prototypical adult stem cell actively involved in tissue homeostasis, wound healing and repair processes. Allogeneic transplants of MSCs do not produce any adverse effect which may be due to their inherent immune-tolerance capacity. They release bio-molecules with anti- inflammatory, immunomodulating and anti- fibrogenic properties 62 . Recent studies have demonstrated that MSCs can be used to treat systemic diseases, local tissue defects or as a vehicle for genes in gene therapy protocols or to generate transplantable tissues and organs in tissue- engineering protocols. Aging is associated with a progressive failing of tissues and organs of the human body leading to a large number of age- related complications 63 . In order to treat degenerative or age-related diseases, infusion of stem cells into patients may lessen the complication of disease due to age.

Current conventional therapy, for deficient tumor selectivity, can lead to the destruction of healthy tissue and has serious side effects in the patient; beside this it is limited in effectiveness and the quantity of the used dose. Gene therapy presents an adequate alternative and it is very promising method for treating various types of disease, including cancer. Dendritic cells, as key elements of inducing anti-tumor immune response are promising means of gene therapy. There is an effort of combining immunotherapy with suicide gene therapy for the purpose of amplifying the effectiveness of suicide gene therapy. Tumor-suppressor genes avert the neoplastic behavior and induce tumor regression, however, the outcomes of clinical therapies are not satisfactory, mostly for the low effectiveness of transduction achieved by currently available distribution systems. The supplementation of genes coding antiangiogenic proteins is a promising procedure, avoiding the obstacles connected with systemic application of medicaments. The ability of mesenchymal stem cells to selectively proliferate in tumors designates them to become vectors in targeted gene therapy of cancer on the basis of stem cells. The introduction of transgenes to stem cells presents an attractive distribution strategy. Mesenchymal cells can be used in enzyme/prodrug conversion in targeted chemotherapy; they possess a specific resistance and migration capability into tumors and therefore should be considered as valuable stem cells for use in cancer therapy. Bacteria used as anti-cancer agents have the ability to infect and proliferate inside the tumor mass, enabling the use of bacteria as an alternative method of treating cancer.

Abstract: Allogeneic hematopoietic stem cell transplantation was until very recently, the only permanent curative option available for patients suffering from transfusion-dependent beta-thalassemia. Gene therapy, by autologous transplantation of genetically modi fi ed hema- topoietic stem cells, currently represents a novel therapeutic promise, after many years of extensive preclinical research for the optimization of gene transfer protocols. Nowadays, clinical trials being held on a worldwide setting, have demonstrated that, by re-establishing effective hemoglobin production, patients may be rendered transfusion- and chelation-inde- pendent and evade the immunological complications that normally accompany allogeneic hematopoietic stem cell transplantation. The present review will offer a retrospective scope of the long way paved towards successful implementation of gene therapy for beta-thalasse- mia, and will pinpoint the latest strategies employed to increase globin expression that extend beyond the classic transgene addition perspective. A thorough search was performed using Pubmed in order to identify studies that provide a proof of principle on the aforementioned topic at a preclinical and clinical level. Inclusion criteria also regarded gene transfer technologies of the past two decades, as well as publications outlining the pitfalls that precluded earlier successful implementation of gene therapy for beta-thalassemia. Overall, after decades of research, that included both successes and pitfalls, the path towards a permanent, donor-irrespective cure for beta-thalassemia patients is steadily becoming a realistic approach.

Future perspectives and concluding remarks In the present study, we propose several complementary methods for improving the therapeutic efficacy of MSCs. Each method targets different preparatory steps for a cell application; hence, these findings might contribute to es- tablishing comprehensive enhancement strategies by combinatorial use of each developed method. A combin- ation of 2D (e.g., priming) and 3D (e.g., spheroid culture) aids complements the therapeutic effects of MSCs. For example, MSCs modified to enhance proliferation and survival are inserted into the biocompatible scaffold and the complex implanted to the damaged joint with TNF- α inhibitor to treat degenerative arthritis. In addition, biomedical technologies at the cutting edge such as gene therapy or monoclonal antibody medicines are consid- ered for combinatorial treatment with MSCs. Indeed, Park et al. recently suggested a brand new function im- provement method called in vivo priming. In their study, the authors transduced BM-MSCs to consistently secrete HGF and the engineered cells were seeded on 3D patch mixing with naïve cells resulting in the improvement of therapeutic function compared to naive MSCs [75]. Therefore, optimization of the combinatorial use of each strategy could be envisioned to maximize the therapeutic outcome of MSC therapy.

Systematic delivery of drug or gene therapy has promising future but is currently limited by various factors such as immune detection, non-specific accumulation in normal tissues and poor permeation. The effects of many antican- cer agents are limited due to either their toxicities or their short half lives such as interferon β, which shows anti-pro- liferative and pro-apoptotic activities in vitro, but has shown restricted effects on human malignancies in vivo [66-68]. One proposed solution for these would be the cell-based carriers that may target the desired site. The recent concept of use of stem cells as delivery vehicles came from the fact that the tumours, similar to the wounds, send out chemo-attractants such as the vascular endothelial growth factor (VEGF) to recruit MSC to form the supporting stroma of the tumour, and pericytes for angiogenesis. MSC transduced with an adenoviral expres- sion vector carrying interferon-β gene has been demon-

Current treatment of hemophilia A (HemA) patients with repeated infusions of factor VIII (FVIII; abbreviated as F8 in constructs) is costly, inconvenient, and incompletely effective. In addition, approximately 25 % of treated patients develop anti-factor VIII immune responses. Gene therapy that can achieve long-term phenotypic correction without the complication of anti-factor VIII antibody formation is highly desired. Lentiviral vector (LV)-mediated gene transfer into hematopoietic stem cells (HSCs) results in stable integration of FVIII gene into the host genome, leading to persistent therapeutic effect. However, ex vivo HSC gene therapy requires pre-conditioning which is highly undesirable for hemophilia patients. The recently developed novel methodology of direct intraosseous (IO) delivery of LVs can efficiently transduce bone marrow cells, generating high levels of transgene expression in HSCs. IO delivery of E-F8-LV utilizing a ubiquitous EF1 α promoter generated initially therapeutic levels of FVIII, however, robust anti-FVIII antibody responses ensued neutralized functional FVIII activity in the circulation. In contrast, a single IO delivery of G-FVIII-LV utilizing a megakaryocytic-specific GP1b α promoter achieved platelet-specific FVIII expression, leading to persistent, partial correction of HemA in treated animals. Most interestingly, comparable therapeutic benefit with G-F8-LV was obtained in HemA mice with pre-existing anti-FVIII inhibitors. Platelets is an ideal IO delivery vehicle since FVIII stored in α -granules of platelets is protected from high-titer anti-FVIII antibodies; and that even relatively small numbers of activated platelets that locally excrete FVIII may be sufficient to promote efficient clot formation during bleeding. Additionally, combination of pharmacological agents improved transduction of LVs and persistence of transduced cells and transgene expression. Overall, a single IO infusion of G-F8-LV can generate long-term stable expression of hFVIII in platelets and correct hemophilia phenotype for long term. This approach has high potential to permanently treat FVIII deficiency with and without pre-existing anti-FVIII antibodies.

, p67 phox , and p40 phox . About 60% of CGD cases are X-linked (XL) and caused by mutations in the CYBB gene encoding for gp91 phox protein. The other form of disease is autosomal recessive (AR) CGD and most common form of AR-CGD is mutation in Neutrophil Cytosolic Factor 1 (NCF1) encoding p47- phox (about 20%) and another, mutations in NCF1 (6%) and CYBA (6%) genes encoding p67 phox and p22 phox respectively (Table 1) 6-8 . Mutation in NCF1 encoding p40-phox has been reported in one patient that has prevented activation of the NADPH oxidase at intracellular membrane without any effect on NADPH oxidase assembly. 9 Another study demonstrated a critical role of phosphatidylinositol 3-phosphate [PI(3)P] binding domain in p40 phox for the optimal activation of NADPH oxidase in macrophages. 10 Gene therapy of autologous hematopoietic stem cells (HSCs) or induced pluripotent stem cells (iPSCs) by delivery of a functional copy of mutated NADPH oxidase subunit is another promising approach to treat high-risk CGD patients without HLA-matched donor for HSCT. In this alternative approach, wild-type of the mutated gene transfers into autologous hematopoietic stem cells using retro or lentiviral vectors. Although, CGD patients were functionally corrected early after treatment, however, insertional activation of PRDM16 and EVI1 could lead to life-treating myelodysplasia. This demonstrates the potential of successful, however, the alternative strategies to eliminate the risk of insertional oncogenesis is needed. Genome editing technologies are powerful therapeutic strategies for gene correction at the precise location through introducing site-specific double-strand DNA break (DBS) by engineered endonucleases such as zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs) and the clustered regularly

All protocols were approved by the Animal Care and Use Committee of the Medical Research Institute at Yonsei Uni- versity College of Medicine. All experiments were conduct- ed in accordance with international guidelines on the ethical use of animals, and the number of animals used was mini- mized. For anesthesia, ICR mice (male, 6 weeks, 30 g, Ori- entBio, Seongnam, Gyeonggi-do, Korea) were intraperito- neally injected with ketamine (100 mg/kg, Yuhan, Seoul, Korea) and rompun (10 mg/kg, Bayer Korea, Seoul, Korea). Laminectomy was performed at thoracic level 11. SCI was carried out using self-closing forceps (compression injury for 10 sec). Animals were divided into two groups, those treat- ed with SV::Luci-NSCs and those treated with NSE::Luci- NSCs. NSCs (5×10 5 cells/2 μL) were directly injected into

In 2018, we wrote a paper on the drug treatment of Parkinson’s disease. In this article, we obtained that the wearing off was observed in 77%, but the incidence of dyskinesia was 37.7% for the Parkinson’s disease patients from the onset of the disease 16-20 years. In this review article, we will discuss some of the newer treatments of Parkinson’s disease first, i.e., transplantation with induced pluripotent stem cell-derived cells, gene therapy, deep brain stimulation, levodopa/ carbidopa intrajejunal gel infusion, MRI-supported focused ultrasound, and IPX066. Then, we will discuss our opinion on the mechanism of wearing off and dyskinesia, and modifications of levodopa treatment after the wearing off sets in.

Genetically engineered pacemakers could be a possible alternative to implantable electronic devices for the treatment of bradyarrhythmias. The strategies include upregulation of beta adrenergic receptors, conversion of myocytes into pacemaker cells and stem cell therapy. Pacemaker activity in adult ventricular myocytes is normally repressed by the inward rectifier potassium current (I K1 ). The I K1 current is encoded by the Kir2 gene family. Use of a negative construct that suppresses current when expressed with wild-type Kir2.1 is an experimental approach for genesis of genetic pacemaker. hyperpolarisation activated cyclic nucleotide gated (HCN) channels which generate If current, the pacemaker current of heart can be delivered to heart by using stem cell therapy approach and viral vectors. The unresolved issues include longevity and stability of pacemaker genes, limitations involved in adenoviral and stem cell therapy and creation of genetic pacemakers which can compete with the electronic units.

MiR-132 enhances HIV-1 replication, therefore, anti- miRNA oligonucleotide (AMO) would be applicable for therapy HIV-1 infection [119]. As concerning to longitu- dinal HIV infection, treatment for dual and single chronic HBV and/or HCV co-infection have to set up because of 3% - 5% of HIV-infected individuals [120]. MiR-122 is the most dominant miRNA gene (approximate 70% of the total miRNA) expressed in the liver. The miRNA gene can control lipid and cholesterol metabolism and maintain the levels of the plasma cholesterol [121]. Al- though lipid and cholesterol metabolism are regulated on day-light dependence, miR-122 targeted the circadian rhythm of the metabolic regulation in the liver [122]. Further, mouse miR-122 knockout (KO) in germline or in liver-specific resulted in hepatosteatosis, hepatitis, and the development of tumor, therefore, miR-122 functioned as tumor suppressor [123]. The expression of miR-122 in the liver permits HCV 1b replication [124]. And the HCV replication was repressed about 80% by anti-miR- 122. The miR-122 gene can bind to the 5’UTR of HCV genomic RNA [125] and stimulate HCV protein transla- tion [126] but contradictorily, miR-122 did not directly stimulate HCV replication [127,128]. Although treatment of anti-miR-122 LNA, miravirsen reduced HCV RNA in serum of HCV-infected individuals on Phase IIa trial [129], in the case of chronic hepatitis C, there is no cor- relation between miR-122 and HCV RNA levels of pa- tients [130]. The AMO LNA is clearly effective to HCV infection but information of miR-122 to HCV life cycle has not yet been ciphered. On the contrary, miR-122 has been reported to be suppressed in chronic HBV infected patient [131] and the reducing expression of miR-122 in chronic HBV was explained through expression inhibi- tion of HBV enhancer HNF3 and HNF4α because miR- 122 requires HNF for its own transcription [132]. The miR-122 mimic LNA may be considered as a candidate of HBV therapy. For HIV-1-infected individuals, miR- 122 was increased in HIV-1-infected T cells [133]. In a scenario, if HIV-1 replication could be augmented in CD4+ T cells and macrophages by miR-122, it is plausi- ble whether miravirsen can be examined for HIV-1 in- fection as well as dual HIV infection with HCV or not because the PBMCs pool of the liver. At a computation analysis, miR-3065-3p has recently been predicted as an candidate of antiviral therapeutic agent for HIV-1, HCV and HBV triple infection [134]. We have to challenge ahead to cure the mixed infection for HIV-1 eradication.

Although the genetically modified MSC strategy has shown promising results in preclinical animal models of ARDS, the application of genetically modified MSCs for translational purposes appears much more hypothetical and uncertain; there are still some concerns that need to be addressed in the near future. First, there is a concern that genetic manipulation may have an impact on the functional characteristics of MSCs, including their tri- lineage differentiation capability, immunosuppressive properties, and cell surface receptor phenotype. Second, the therapeutic use of genetically modified MSC therapy exposes the genetic instability that can be induced by the genetic manipulation of MSCs, and little information is available regarding the long-term behavior of genetic- ally modified cells in vivo after systematic or local ad- ministration. Additionally, it is important to note that diverse vectors have been applied to transfect MSCs, a variety of MSC sources have been used, various models of preclinical ARDS have been studied, and different methods have been used to evaluate the therapeutic effi- cacy in these studies, so caution should be used when interpreting these findings. Moreover, from the clinical application perspective, it may be difficult to rapidly gen- erate enough genetically modified MSCs within a few days to transplant following the onset of ARDS. In the vast majority of the published studies, researchers inves- tigated only the overexpression of a pre-selected gene of interest to improve MSC therapy; however, it is justifi- able to suppose that the expression of certain genes can blunt the therapeutic effects of MSCs; therefore, it might be interesting to knockdown/knock out genes in MSCs to enhance the therapeutic potency of MSCs in ARDS.

are still highly required. In recent years, incorporation of inorganic nanoparticles has emerged as an effective method to enhance the gene delivery efficiency of cationic carriers [28]. The enhancement can be attributed to the improved colloidal stability and cellular uptake of cationic polymers complexing with inorganic nanoparticles. Incorporation of inorganic metal nanoparticles also confers additional theranostic functions such as the photothermal effect [29-31], imaging contrast [32, 33], and fluorescence imaging [34]. In this study, we proposed a novel magnetic ternary nanohybrid (MTN) system for gene delivery into human MSCs. The nanohybrid can be rapidly fabricated by electrostatically assembling hyaluronic acid (HA)-decorated superparamagnetic iron oxide (SPIO) nanoparticles (HA-SPIONs) with common cationic polyplexes. A ligand exchange reaction was utilized to prepare HA-SPIO. HA is a biocompatible anionic polymer capable of binding to CD44 receptor-overexpressing MSCs [35, 36]. A combination of HA with SPIO is anticipated to facilitate stem cell uptake of cationic nanocomplexes through dual CD44- and magneto-targeting effects. Here, HA-SPIO was first synthesized and characterized to determine its material properties in detail. A series of MTNs were prepared by complexing HA-SPIO with various cationic materials and then were examined for estimating the in vitro transfection efficiency. The effects of magnetic and CD44 dual targeting on the transfection efficiency of MTN were investigated in vitro. Tumor tropism of MTN-constructed TRAIL-expressing hMSCs ( TRAIL hMSCs) was examined using a transwell model

Most myeloid leukaemias are thought to arise directly from haematopoietic stem cells. There is evidence, however, that the M3 acute promyelocytic subtype arises from a transit amplifying cell that has acquired the capacity for self-renewal as a consequence of expression of the PML/RAR α fusion protein [5]. When transduced with a leukaemogenic MLL fusion gene in vitro both haematopoietic stem cells and committed myeloid progenitors promptly immortalise, and trans- plantation leads to AML with apparently identical phenotypes in vivo [59]. This demonstrates not only that malignancy can arise from transit amplifying cells, but also that cancer stem cells do not necessarily correspond to tissue stem cells. Similar studies with mammary epithelium would be most informative, but unfortunately promotors selectively expressed in stem cells and committed progenitors have yet to be identified; in lieu of these, most investigators have employed the MMTV promotor, which is expressed in a diverse array of cell types. Nevertheless, there is preliminary evidence that different oncogenes target different levels of the differentiation hierarchy. Tumours induced by expression of Wnt (and downstream members of this pathway) express markers of both luminal and basal/myoepithelial lineages [60]. This is not seen with other oncogenes such as Neu and H-Ras, and suggests origin from a pluripotent precursor, presumably a mammary epithelial stem cell. In support of this hypothesis the number of CD24 + CD29 hi stem cell

Progress in the manipulation of genes and their characteristics has led to standardization and comparative evaluation of the performance of vectors in various experiments. The magnitude and specificity of gene modification in its original or natural position, the integration of vectors in genomic safe locations, and the exclusive silencing of alleles by synthetic nucleases and epigenetic modifications provide more opportunities for improving gene therapy strategies. A deeper understanding of the pathology of hereditary, multi-genic, or acquired diseases will lead to the development of new gene therapy strategies. Since gene transfer strategies use live biological agents that can induce long-term effects on patients and their sex cells, long-term care and preventive measures should be considered when using them. Furthermore, due to the limited understanding of stem cell configurations, tissue regeneration, and immune response inspections, there is increasing and undeniable concern about the undesirable effects of genetic manipulation. From a clinical point of view, the clinical delivery of genes and cell therapy requires multidisciplinary specialists and, in some cases, advanced cell processing in the clinical setting. Biological readouts should also be performed to monitor the safety and efficacy of the treatment. Since the first advancements, from registration to marketing, were made in gene therapy, pharmaceutical departments and regulatory agencies have provided quality standards for the production and distribution of these highly specialized medicines. In social terms, the complexity and cost of producing and delivering live biological medicine in conventional healthcare systems challenge the sustainability of these treatments and require the establishment of reimbursable policies so that all patients can benefit from it. Ultimately, whether medical science surrenders to technology laws or takes on more responsibility for these developments is an important ethical issue. Of course, the development of strategies to treat and reduce the suffering of patients justifies our efforts from an ethical point of view. 54