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Subcellular localization of LEDGF/p75-hybrids in interphase cells. LEDGF/p75 depleted cell lines were complemented with the respective LEDGF-fusions. Laser scanning confocal images of HelaP4 cells, stained using an Ab recognizing LEDGF480-530, are shown in green. Nuclei were stained using Dapi (shown in blue). A merge of green and blue fluorescence is shown. Data depicted are representative for the respective cell lines. PFV, Prototype foamy virus; LANA, Latency associated nuclear antigen; HPV, Human papilloma virus; LEDGF, Lens epithelium-derived growth factor; DAPI, 4',6-Diamidino-2-Phenylindole.
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The capacity to integrate transgenes into the host cell genome makes retroviral vectors an interesting tool for gene therapy. Although stable insertion resulted in successful correction of several monogenic disorders, it also accounts for insertional mutagenesis, a major setback in otherwise successful clinical gene therapy trials due to leukemia d...
Citations
... It is therefore reasonable to assume that this manufacturing process did not lead to a downregulation of this receptor either. This indicates that the integration of the transgene did not lead to any insertional mutagenesis capable of down-or upregulate genes responsible for hMSCs' immunophenotype (Vranckx et al. 2016;Milone and O'Doherty 2018). ...
Due to their immunomodulatory properties and in vitro differentiation ability, human mesenchymal stromal cells (hMSCs) have been investigated in more than 1000 clinical trials over the last decade. Multiple studies that have explored the development of gene-modified hMSC-based products are now reaching early stages of clinical trial programmes. From an engineering perspective, the challenge lies in developing manufacturing methods capable of producing sufficient doses of ex vivo gene-modified hMSCs for clinical applications. This work demonstrates, for the first time, a scalable manufacturing process using a microcarrier-bioreactor system for the expansion of gene-modified hMSCs. Upon isolation, umbilical cord tissue mesenchymal stromal cells (UCT-hMSCs) were transduced using a lentiviral vector (LV) with green fluorescent protein (GFP) or vascular endothelial growth factor (VEGF) transgenes. The cells were then seeded in 100 mL spinner flasks using Spherecol microcarriers and expanded for seven days. After six days in culture, both non-transduced and transduced cell populations attained comparable maximum cell concentrations (≈1.8 × 10⁵ cell/mL). Analysis of the culture supernatant identified that glucose was fully depleted after day five across the cell populations. Lactate concentrations observed throughout the culture reached a maximum of 7.5 mM on day seven. Immunophenotype analysis revealed that the transduction followed by an expansion step was not responsible for the downregulation of the cell surface receptors used to identify hMSCs. The levels of CD73, CD90, and CD105 expressing cells were above 90% for the non-transduced and transduced cells. In addition, the expression of negative markers (CD11b, CD19, CD34, CD45, and HLA-DR) was also shown to be below 5%, which is aligned with the criteria established for hMSCs by the International Society for Cell and Gene Therapy (ISCT). This work provides a foundation for the scalable manufacturing of gene-modified hMSCs which will overcome a significant translational and commercial bottleneck.
Key points
• hMSCs were successfully transduced by lentiviral vectors carrying two different transgenes: GFP and VEGF
• Transduced hMSCs were successfully expanded on microcarriers using spinner flasks during a period of 7 days
• The genetic modification step did not cause any detrimental impact on the hMSC immunophenotype characteristics
... on 17 December 2022). / Karyopherin involved in nuclear import [60] RanBP2 / Nuclear pore protein involved in Nuclear import [60] Nup153 / Karyopherin involved in nuclear import [61,62] CPSF6 / Nuclear import factor binding to HIV-1 capsid [63][64][65][66][67] LEDGF/p75 / IBD binds viral IN, PWWP domain binds H3K36me2/3 [22,23,[35][36][37][38][39][40][41][42]45,52,[68][69][70][71][72][73][74][75][76][77][78][79][80][81][82][83][84][85][86][87] HRP2 / IBD binds viral IN, PWWP domain binds H3K36me2/3 [74] / IN CCD and CTD bind target DNA [88][89][90][91][92][93][94][95][96][97][98] [4]. BET proteins consist of two bromodomains (BD), BDI and BDII, that bind acetylated lysines in the nucleosomes, and an extra-terminal (ET) domain that interacts with the CTD of the MLV IN [28][29][30][31][32][33][34]. ...
... Altering the integration of lentiviral vectors was also adopted as an approach to increase safety. Vranckx et al. generated LEDGF hybrids by replacing the PWWP domain of LEDGF with pan-chromatin binders, which resulted in more random integration within the host cell genome while maintaining stable transgene expression [79]. Lapaillerie [94]. ...
... A clinical application of this insight is the development of Bin MLV vectors, which have been used to reduce the risk of insertional mutagenesis [55][56][57]. Similarly, retargeting HIV-1 integration by intervening with LEDGF/p75 [79] or with the CTD of HIV-1 IN [93,94] also reduced the risk of insertional mutagenesis for HIV-1 viral vectors. Moreover, by inhibiting the interaction between HIV-1 IN and LEDGF/p75 with small molecules named LEDGINs, HIV-1 integration can be retargeted away from H3K36me2/3, the marker recognized by LEDGF/p75 [22,23,77]. ...
To complete their replication cycle, retroviruses need to integrate a DNA copy of their RNA genome into a host chromosome. Integration site selection is not random and is driven by multiple viral and cellular host factors specific to different classes of retroviruses. Today, overwhelming evidence from cell culture, animal experiments and clinical data suggests that integration sites are important for retroviral replication, oncogenesis and/or latency. In this review, we will summarize the increasing knowledge of the mechanisms underlying the integration site selection of the gammaretrovirus MLV and the lentivirus HIV-1. We will discuss how host factors of the integration site selection of retroviruses may steer the development of safer viral vectors for gene therapy. Next, we will discuss how altering the integration site preference of HIV-1 using small molecules could lead to a cure for HIV-1 infection.
... This work contributes to a growing body of evidence that LV vectors have a lower genotoxic risk when compared to gamma-retroviral vectors [75][76][77] . Although multiple strategies have and are being employed to increase the safety of lentiviral vectors in general 78,79 , patients participating in clinical trials involving lentiviral vectors will continue to require close long-term surveillance. It is worth noting that HT1 patients already require frequent cancer-related surveillance and HCC screening 5 , and this requirement would not be fundamentally altered after curative gene therapy to treat HT1. ...
Conventional therapy for hereditary tyrosinemia type-1 (HT1) with 2-(2-nitro-4-trifluoromethylbenzoyl)−1,3-cyclohexanedione (NTBC) delays and in some cases fails to prevent disease progression to liver fibrosis, liver failure, and activation of tumorigenic pathways. Here we demonstrate cure of HT1 by direct, in vivo administration of a therapeutic lentiviral vector targeting the expression of a human fumarylacetoacetate hydrolase ( FAH ) transgene in the porcine model of HT1. This therapy is well tolerated and provides stable long-term expression of FAH in pigs with HT1. Genomic integration displays a benign profile, with subsequent fibrosis and tumorigenicity gene expression patterns similar to wild-type animals as compared to NTBC-treated or diseased untreated animals. Indeed, the phenotypic and genomic data following in vivo lentiviral vector administration demonstrate comparative superiority over other therapies including ex vivo cell therapy and therefore support clinical application of this approach.
... Because lentiviral vectors are integrative vectors, they might be thought to induce mosaic expression if the expression levels across cells vary as a function of where exactly the vector is integrated in the genome. However, integration, although considered as random, is in fact preferentially observed in active regions of the chromatin [33]. In addition, transduced cells often contain multiple integration sites that usually normalize the expression level between transduced cells that are all different in terms of chromosomal integration. ...
Understanding the role of the noradrenergic nucleus locus coeruleus (LC) in cognition and behavior is critical: It is involved in several key behavioral functions such as stress and vigilance, as well as in cognitive processes such as attention and decision making. In recent years, the development of viral tools has provided a clear insight into numerous aspects of brain functions in rodents. However, given the specificity of primate brains and the key benefit of monkey research for translational applications, developing viral tools to study the LC in monkeys is essential for understanding its function and exploring potential clinical strategies. Here, we describe a pharmacogenetics approach that allows to selectively and reversibly inactivate LC neurons using Designer Receptors Exclusively Activated by Designer Drugs (DREADD). We show that the expression of the hM4Di DREADD can be restricted to noradrenergic LC neurons and that the amount of LC inhibition can be adjusted by adapting the dose of the specific DREADD activator deschloroclozapine (DCZ). Indeed, even if high doses (>0.3 mg/kg) induce a massive inhibition of LC neurons and a clear decrease in vigilance, smaller doses (<0.3 mg/kg) induce a more moderate decrease in LC activity, but it does not affect vigilance, which is more compatible with an assessment of subtle cognitive functions such as decision making and attention.
... Thus, the proportion of Scx + cells may decrease with expansion and some of the transduced cells might have lost Scx expression due to gene silencing. 64 Cell sorting could potentially be used to obtain a purer iMSC SCX+ population. Although, cell sorting was not necessary in this study, since . ...
Regenerative therapies for tendon are falling behind other tissues due to the lack of an appropriate and potent cell therapeutic candidate. This study aimed to induce cell tenogenesis using stable Scleraxis (Scx) overexpression in combination with uniaxial mechanical stretch of mesenchymal stromal cells (MSCs) of different origins. Scleraxis (Scx) is the single direct molecular regulator of tendon differentiation known so far. Mechanoregulation is known to be a central element guiding tendon development and healing. Cells explored were bone marrow-derived (BM-)MSCs as well as MSCs differentiated from induced pluripotent stem cells (iMSCs). Upregulation of early and late tendon markers, increased collagen deposition, and morphometric and cytoskeleton-related changes in mechanically stimulated Scx-overexpressing iMSCs compared to BM-MSCs and controls. Our findings suggest that these cells can be differentiated into tenocytes and may be a better candidate for tendon cell therapy applications than BM-MSCs.
... Insertion is catalysed by the viral integrase enzyme; however integration site choice is attributed to host cellular chromatin readers that are co-opted by the viral integrase. LV cDNA integration site is strongly biased towards actively transcribed genes (with a further preference towards insertion into introns) making the insertion site cell type dependent [24,25]. It is not necessary for AAVs to integrate for gene expression. ...
Spinal cord injury (SCI) is a devastating trauma that can cause permanent disability, life-long chronic issues for sufferers and is a big socioeconomic burden. Regenerative medicine aims to overcome injury caused deficits and restore function after SCI through gene therapy and tissue engineering approaches. SCI has a multifaceted pathophysiology. Due to this, producing therapies that target multiple different cellular and molecular mechanisms might prove to be a superior approach in attempts at regeneration. Both biomaterials and nucleic acid delivery via lentiviral vectors (LVs) have proven to promote repair and restoration of function post SCI in animal models. Studies indicate that a combination of biomaterials and LVs is more effective than either approach alone. This review presents studies supporting the use of LVs and LVs delivered with biomaterials in therapies for SCI and summarises methods to combine LVs with biomaterials for SCI treatment. By summarising this knowledge this review aims to demonstrate how LV delivery with biomaterials can augment/compliment both LV and biomaterial therapeutic effects in SCI.
... The second step is extracting the standardized coefficient from the model as a proxy to measure the single base DNA shape and DNA motif effect around retrovirus integration sites. [37][38][39] 。慢病毒载体的使用扩大了基因编辑的领域，并 被最近启动的体细胞基因组编辑联盟(SCGE)认为是广泛有前景的递送系统 [40] 。在基因治 疗方面，使用慢病毒载体最成功的案例是在嵌合抗原受体 T 细胞(chimeric antigen receptor T cells ，CAR-T)的癌症治疗 [37] 。这些慢病毒应用中的关键步骤是通过慢病毒载体将外部 基因传递到特定的宿主基因组位点，但慢病毒整合常常发生在错误的位置，并可能导致基 因突变，甚至进一步发展为癌症 [41,42] 。本研究表明，HIV-1 和 SIV 都受 DNA 基序和形状 的强烈调控，由此推测此调控模式适用于慢病毒种属。本研究提示在未来设计慢病毒载体 时应综合考虑包括 DNA 形状在内的影响因素，以更安全有效地用于基因编辑和基因疗法 [43,44] 。 最近有报道称 COVID-19 可以使用 HIV 逆转录酶逆转录其 RNA，并将该 cDNA 整合 到人类宿主基因组中 [45] ，但目前尚不清楚哪个整合酶负责其整合过程。对于 COVID-19 整 合位点附近 DNA 结构和序列特性的进一步研究可以有助于推测行驶功能的整合酶，并协 助设计干预策略以避免复发性感染。 本研究从 DNA 层面上探究了 DNA 基序和 DNA 形状对逆转录病毒整合的影响，有报 道显示宿主基因组的表观修改状态 [46] 和调控因子(如 LEDGF/p75 [20] ，ZAP [47] )可以影响 逆转录病毒的整合及转录，这表明逆转录病毒的活动周期受到多层级的精确调控。除逆转 录病毒外，其他种类的病毒如乙型肝炎病毒(Hepatitis B virus，HBV)，人类乳头瘤病毒 (Human papillomavirus，HPV)也被报道偏好性地整合到基因组特定区域 [48,49] ,虽然本文将 所研究的六种逆转录病毒分为三大类：即"强兼性偏好型"，"弱兼性偏好型"和"强形 状偏好型"，但是否所有种类的病毒均遵从类似的偏好性规则或是否存在"强基序偏好型" 病毒，仍需后续对于更多种类病毒的研究。基于此，本研究所使用的机器学习框架为未来 综合研究病毒的整合机制提供了新的策略，这不仅可以增进对病毒相关疾病的理解，还有 助于更准确地预测病毒的病原性整合热点以进行精准干预。 参考文献 ...
... Genomic integration of synthetic devices can introduce significant positional effects, where transcriptional activity in the local DNA environment, such as read-through from upstream transcription Loughran et al., 2014), DNA torsional effects (Sevier and Levine, 2018;Yeung et al., 2017), DNA looping (Hao et al., 2019), and the activity of enhancers and silencers (Liu et al., 2015) interfere with the prescribed function of the genetic device ( Figure 5C). Transposons and viral integration strategies generally lead to semi-random integration of exogenous genetic materials (Staunstrup et al., 2009;Vranckx et al., 2016;Yant et al., 2005), with little to no control over the genetic position of the device in the mammalian cell genome. In an effort to overcome positional effects, recent developments in genetic engineering technologies have enabled more predictable control over integration sites of exogenous genetic material, including the landing pad DNA integration platform (Duportet et al., 2014;Gaidukov et al., 2018) and targeted CRISPR-based approaches for insertion into ''safe harbor'' loci, which experience reduced epigenetic silencing and ensure the device does not interfere with core cell functions such as cellcycle regulation (Papapetrou and Schambach, 2016). ...
The rise of systems biology has ushered a new paradigm: the view of the cell as a system that processes environmental inputs to drive phenotypic outputs. Synthetic biology provides a complementary approach, allowing us to program cell behavior through the addition of synthetic genetic devices into the cellular processor. These devices, and the complex genetic circuits they compose, are engineered using a design-prototype-test cycle, allowing for predictable device performance to be achieved in a context-dependent manner. Within mammalian cells, context effects impact device performance at multiple scales, including the genetic, cellular, and extracellular levels. In order for synthetic genetic devices to achieve predictable behaviors, approaches to overcome context dependence are necessary. Here, we describe control systems approaches for achieving context-aware devices that are robust to context effects. We then consider cell fate programing as a case study to explore the potential impact of context-aware devices for regenerative medicine applications.
... Both γRV and LV -based vectors integrate semi -randomly into the host genome producing stable replication and passage of the inserted therapeutic gene upon cell division but the corollary is the inadvertent risk of rare insertional mutagenesis -induced malignancies. While LV preferably integrate into transcriptionally active chromatin, γRV predominantly integrate in the vicinity of gene regulatory regions (Vranckx et al., 2016). Particularly γRV show a long terminal repeat (LTR) -mediated propensity for recurrent insertion into 'hot spots' frequently near transcription start sites of protooncogenes explaining the heightened risk profile of early γRV vectors over LVs vectors for gene therapy applications (Cattoglio et al., 2007). ...
Central Nervous System (CNS) homeostasis and function rely on intercellular synchronization of metabolic pathways. Developmental and neurochemical imbalances arising from mutations are frequently associated with devastating and often intractable neurological dysfunction. In the absence of pharmacological treatment options, but with knowledge of the genetic cause underlying the pathophysiology, gene therapy holds promise for disease control. Consideration of leukodystrophies provide a case in point; we review cell type - specific expression pattern of the disease - causing genes and reflect on genetic and cellular treatment approaches including ex vivo hematopoietic stem cell gene therapies and in vivo approaches using Adeno-associated virus (AAV) vectors. We link recent advances in vectorology to glial targeting directed towards gene therapies for specific leukodystrophies and related developmental or neurometabolic disorders affecting the CNS white matter and frame strategies for therapy development in future.
... This work contributes to a growing body of evidence that LV vectors have a lower genotoxic risk when compared to gamma-retroviral vectors. [65][66][67] Although multiple strategies have and are being employed to increase the safety of lentiviral vectors in general, 68,69 patients participating in clinical trials involving lentiviral vectors will continue to require close long-term surveillance. It is worth noting that HT1 patients already require frequent cancerrelated surveillance and HCC screening, 5 and this requirement would not be fundamentally altered after curative gene therapy to treat HT1. ...
Conventional therapy for hereditary tyrosinemia type-1 (HT1) with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) delays but in some cases fails to prevent disease progression to liver fibrosis, liver failure, and activation of tumorigenic pathways. Here we demonstrate for the first time a complete cure of HT1 by direct, in vivo administration of a therapeutic lentiviral vector targeting the expression of a human fumarylacetoacetate hydrolase (FAH) transgene in the porcine model of HT1. This therapy was well tolerated and provided stable long-term expression of FAH in pigs with HT1. Genomic integration displayed a benign profile, with subsequent fibrosis and tumorigenicity gene expression patterns similar to wild-type animals as compared to NTBC-treated or diseased untreated animals. Indeed, the phenotypic and genomic data following in vivo lentiviral vector administration demonstrate comparative superiority over other therapies including ex vivo cell therapy and therefore support clinical application of this approach.
