Gammaretroviral Vectors Research Articles

Gene therapy using haematopoietic stem and progenitor cells.

Haematopoietic stem and progenitor cell (HSPC) gene therapy has emerged as an effective treatment modality for monogenic disorders of the blood system such as primary immunodeficiencies and β-thalassaemia. Medicinal products based on autologous HSPCs corrected using lentiviral and gammaretroviral vectors have now been approved for clinical use, and the site-specific genome modification of HSPCs using gene editing techniques such as CRISPR-Cas9 has shown great clinical promise. Preclinical studies have shown engineered HSPCs could also be used to cross-correct non-haematopoietic cells in neurodegenerative metabolic diseases. Here, we review the most recent advances in HSPC gene therapy and discuss emerging strategies for using HSPC gene therapy for a range of diseases.

Genome-wide integration site detection using Cas9 enriched amplification-free long-range sequencing.

The gene and cell therapy fields are advancing rapidly, with a potential to treat and cure a wide range of diseases, and lentivirus-based gene transfer agents are the vector of choice for many investigators. Early cases of insertional mutagenesis caused by gammaretroviral vectors highlighted that integration site (IS) analysis was a major safety and quality control checkpoint for lentiviral applications. The methods established to detect lentiviral integrations using next-generation sequencing (NGS) are limited by short read length, inadvertent PCR bias, low yield, or lengthy protocols. Here, we describe a new method to sequence IS using Amplification-free Integration Site sequencing (AFIS-Seq). AFIS-Seq is based on amplification-free, Cas9-mediated enrichment of high-molecular-weight chromosomal DNA suitable for long-range Nanopore MinION sequencing. This accessible and low-cost approach generates long reads enabling IS mapping with high certainty within a single day. We demonstrate proof-of-concept by mapping IS of lentiviral vectors in a variety of cell models and report up to 1600-fold enrichment of the signal. This method can be further extended to sequencing of Cas9-mediated integration of genes and to in vivo analysis of IS. AFIS-Seq uses long-read sequencing to facilitate safety evaluation of preclinical lentiviral vector gene therapies by providing IS analysis with improved confidence.

Targeted Integration of a CAR at a Novel Genomic Safe Harbor Directs Potent Therapeutic Outcomes

Chimeric receptor antigen (CAR)-T cell therapy using CARs specific for CD19 have been remarkably successful for treating chemo-refractory/relapsed B cell malignancies. These successes not withstanding, therapeutic outcomes between patients are variable and occasional cases of clonal expansion of the transduced T cells have been observed, albeit without leukemic transformation. These issues are cause for concern and need to be addressed to achieve better and safer therapeutic outcomes. Clonal expansion due to insertional mutagenesis and variegated transgene expression due to position effects are well established to be the consequence of the semi-random integration pattern afforded by gamma-retroviral and lentiviral vectors. We previously established that integration of a CAR cDNA in the TCR alpha locus (TRAC) provides consistent, regulated expression of CD19 CARs and superior CAR T cell efficacy in a mouse model of B-cell acute lymphoblastic leukemia (B-ALL). Here, we identify a novel extragenic site devoid of any known function and remote from endogenous genes, i.e. a 'genomic safe harbor' (GSH), that can be efficiently targeted in human T cells and drives potent CAR T cell therapy in the B-ALL mouse model. To identify GSHs that could be efficiently targeted in T cells by CRISPR-Cas9 and that could also support durable transgene expression, we screened for genomic regions meeting both, GSH criteria and high chromatin accessibility in T cells as measured by ATAC-seq. In human primary T cells, we identified 379 such sites. Ten of the highest accessible sites were investigated. All showed high (>90%) cleavage efficiency and allowed for CAR cDNA targeted integration and expression which also translated into effective cytolytic activity of the CARs within a few days after transduction. However, thereafter CAR expression diminished over the course of a week at most but not all of these sites. In order to prevent possible heterochromatinization, we incorporated chromatin insulator elements with barrier activity flanking the CAR transcription unit. Incorporation of the chromatin insulator element dramatically improved CAR expression and functionality at one site, whereas 3 other GSHs tested were not affected. One of the 10 GSHs maintained long-term CAR expression without requiring an insulator and directed potent anti-leukemic CAR T cell efficacy in a B-ALL 'CAR stress test' mouse model, matching the T cell potency afforded by integrating the CAR cDNA at the TRAC locus. This finding highlights the major effect of the integration site on transgene expression and ensuing therapeutic efficacy. We identified an extragenic GSH site that can be used for effective T cell engineering and sustained expression of a CAR. Through this study, we provide a platform for identifying GSHs that could be reliably targeted for safe and predictable expression of CARs or other immunomodulatory transgenes to potentiate adoptive immunotherapy. Disclosures Sadelain: Fate Therapeutics: Patents & Royalties, Research Funding; Mnemo: Patents & Royalties; Atara: Patents & Royalties, Research Funding; Takeda: Patents & Royalties, Research Funding; Minerva: Other: Biotechnologies, Patents & Royalties.

Bipotent Lymphoid Progenitors Independently Maintain Long-Term Genetically Engineered T and NK Cell Production in Humans

Historically, survival and activity of individual human hematopoietic progenitor subtypes have been studied exclusively via transplantation in permissive mouse models. Despite being a relevant investigational tool, such animal models do not recapitulate the human hematopoietic milieu. Specifically, the production of human T and NK cells in these mice is severely impaired therefore, as of today, it has been impossible to measure with enough confidence the in vivo dynamics of human T/NK lymphoid progenitors. Hematopoietic stem cell gene therapy (GT) using integrating viral vectors has opened a unique opportunity to trace the fate of transplanted cells for the first time directly in vivo in humans by means of integration sites (IS) clonal tracking. Our mathematical modelling of IS data in clinical GT supported the existence of a population of long-term lymphoid progenitors (LtLP) capable of surviving independently from hematopoietic stem cells (HSC). However, to date, no experimental setting has been available to validate such statistical prediction neither in the mouse nor in humans. We here report the first formal evidence, directly in vivo in humans, that a population of bipotent lymphoid progenitors is capable of surviving and maintaining de novo T/NK production for at least 15 years after loss of transplanted HSC. In 5 SCID-X1 patients treated with gammaretroviral vector HSC-GT we observed that genetically engineered myeloid and B cells have stayed constantly below detection limits starting from 6 months after GT. On the contrary T and NK cells remained vector positive for up to 19 years (average vector copies/cell equal 2.1 and 2.3 respectively). We initially thought that such data would be consistent with permanent loss of engineered HSC and survival of vector-marked long-living circulating mature lymphocytes. However, strikingly and unexpectedly, by a comprehensive immunophenotyping of T cells overtime, we detected vector-positive short-living CD45RA+CD62L+CD95- naïve T cells (Tn) in the circulation of these patients. We first obtained functional validation of the identity of such Tn cells by IFN-y productionassays and we confirmed positive thymic activity by measurement of TREC content. High throughput sequencing of 1,193 T-cell receptor (TCR) rearrangements in FACS-sorted T cell subtypes confirmed that these engineered Tn cells had normal TCR diversity and that new rearrangements were detectable overtime. Similarly, Vbeta repertoire measured by spectratyping displayed normal profiles in all patients. These results suggested that a de novo T-cells production is maintained in these patients by a putative LtLP population in absence of supply by gene-corrected HSC. Using Tn data as surrogate markers of LtLP clonal dynamics and composition, we collected and analysed 12,756 unique IS from 5 populations including Tn cells in a window of 10.1 to 14.9 years after the observed loss of transplanted HSC. Clonal diversity was stable in all T subtypes and IS sharing across subpopulations and timepoints was high and consistent with active in vivo output by LtLP and differentiation of Tn into memory/effector T cells. Analyzing nature and recapture probability of IS we observed that IS in LtLP are significantly enriched in genes involved in lymphocyte survival/activation and we could estimate that T cell production is currently sustained by 2,092-6,056 individual engineered LtLP clones. Moreover, by studying 651 IS collected from CD3-CD56+ purified NK cells we observed that up to 41.2% of them were shared with independently analyzed Tn suggesting that the LtLP active in these patients have a dual T/NK restricted potential. Lastly, we detected and tracked overtime 52 clones bearing IS in MECOM, CCND2 and LMO2 including the IS originally associated with vector-induced leukemia in one patient. Our data show absence of any sustained clonal expansions within the engineered LtLP population for up 19 years after GT, the longest follow up available to date in clinical HSC-GT. In conclusion, our data provide the first formal evidence in vivo in human that a de novo production of genetically engineered T and NK cells can be physiologically maintained by a population of LtLP surviving up to 15 years after loss of transplanted HSC (manuscript submitted). Identification and exploitation of such human LtLP population might be crucial for the development of next generation GT and cancer immunotherapy approaches. Disclosures Baricordi: AVROBIO Inc: Current equity holder in publicly-traded company, Other: Trainee. Thrasher:Orchard Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership; Generation bio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; 4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Alteration of gammaretroviral vector integration patterns by insertion of histone and leucine zipper into integrase.

Retroviral vectors show long-term gene expression in gene therapy through the integration of transgenes into the human cell genome. Murine leukemia virus (MLV), a well-studied gammaretrovirus, has been often used as a representative retroviral vector. However, frequent integrations of MLV-based vectors into transcriptional start sites (TSSs) could lead to the activation of oncogenes by enhancer effects of the genetic components within the vectors. Therefore, the MLV integration preference for TSSs limits its wider use in clinical applications. To reduce the integration preference of MLV-based vectors, we attempted to perturb the structure of the viral integrase that plays a key role in determining integration sites. For this goal, we inserted histones and leucine zippers, having DNA-binding property, into internal sites of MLV integrase. This integrase engineering yielded multiple mutant vectors that showed significantly different integration patterns compared with that of wild-type vector. Some mutant vectors did not prefer the key regulatory genomic domains of human cells, TSSs. Moreover, a couple of engineered vectors did not integrate into the genomic sites near the TSSs of oncogenes. Overall, this study suggests that structural perturbation of integrase is a simple way to develop safer MLV-based retroviral vectors for use in clinical applications.

Abstract 6590: Epigenetic suppression of transgenic T-cell receptor (TCR) expression in adoptive cell transfer (ACT) therapy

Abstract Background: ACT of TCR transgenic T cells recognizing shared tumor antigens is associated with robust initial response rates, but frequent disease relapse. This usually occurs in the setting of poor long-term persistence of cells expressing the transgenic TCR, generated most frequently using a murine stem cell virus (MSCV) gamma retroviral vector. Patients and Methods: Between 2009 and 2016, 24 patients were treated at UCLA using transgenic TCR ACT directed against the tumor antigens MART-1 or NY-ESO-1 for the treatment of melanoma or sarcoma, respectively. We analyzed 16 of these patients' cell therapy products for in vivo persistence and expression of the transgenic TCR at baseline and over time. Transgenic TCR DNA, RNA, and surface protein expression were assessed over time via TCR sequencing, qRT-PCR, and MHC dextramer analysis via FACS, respectively. DNA methylation status of the MSCV 5'LTR promoter region was determined via targeted bisulfite sequencing. Custom biotinylated RNA probes that target the entire MSCV vector were utilized to assess whole vector methylation over time, as well as integration sites within the host genome. Results: Despite overall strong persistence of the transgenic TCR at the DNA level in all patients over time, its expression was profoundly decreased over time at the RNA and protein levels. In a subset of these patients (N=6), the degree of TCR surface expression at day +70 was <0.5%, and was significantly lower than the other patients (p<0.0001). These patients displayed significant increases in DNA methylation over time within the MSCV 5'LTR promoter region, as determined via targeted bisulfite sequencing. These increases in MSCV promoter methylation were inversely correlated with the degree of transgenic TCR RNA and protein expression, and the patients with increases in the MSCV 5'LTR promoter region displayed significantly lower expression of the transgenic TCR at the protein and RNA levels (p<0.001 and p<0.01, respectively). Analysis of the entire MSCV vector revealed progressive increases in DNA methylation within the entire MSCV vector over time, which recapitulated the significantly higher degree of vector methylation in patients with the most profound decreases in transgenic TCR expression (p<0.0001). These increases in vector methylation occurred independently of its integration site within the host genome. Conclusions: Progressive acquisition of DNA methylation within the MSCV retroviral vector is associated with suppression of expression of the encoded transgenic TCR. These results have significant implications for the design of future transgenic cell therapies and their associated viral vectors. Citation Format: Theodore S. Nowicki, Colin Farrell, Marco Morselli, Liudmilla Rubbi, Katie Campbell, Beata Berent-Maoz, Begonya Comin-Anduix, Matteo Pellegrini, Antoni Ribas. Epigenetic suppression of transgenic T-cell receptor (TCR) expression in adoptive cell transfer (ACT) therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6590.

Abstract 2173: CRISPR-Cas9 gene editing of inhibitory receptor signaling pathways improves CAR T cell activity against glioblastoma

Abstract Early clinical trials have demonstrated the therapeutic potential of chimeric antigen receptor (CAR) T cells against glioblastoma (GBM), but also highlighted the obstacles that need to be addressed to improve their anti-tumor activity. GBM is highly immunosuppressive and thus necessitates endowing adoptively transferred CAR T cells with resistance mechanisms to achieve sustained treatment responses. This is, in part, due to the effect of immune-inhibitory receptors, including PD1, CTLA4, LAG3 and BTLA, that antagonize the tumor-directed T cells. A common mechanism of T cell inhibition involves the recruitment of Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2) to the immune synapse upon engagement of multiple co-inhibitory receptors. SHP2, in turn, de-phosphorylates key signaling proteins involved in T cell activation. We hypothesized that disrupting SHP2 expression will offset multiple co-inhibitory pathways, thereby improving the anti-tumor activity of CAR T cells. Using CRISPR/Cas9 gene editing, we disrupted the SHP2-encoding gene locus, PTPN11, in primary human T cells by generating and testing a library of single-guide RNA (sgRNA). We selected the top candidate that reproducibly reduced the SHP2 protein expression to <10%. A HER2-targeting second-generation CAR (HER2-CAR) was then expressed in SHP2 knock-out (SHP2KO) T cells via gamma retroviral vector transduction. SHP2KO CAR T cell manufacturing following a single round of stimulation was optimized to minimize ex vivo manipulation of the cells. The phenotypic profiles of SHP2KO and wild-type HER2-CAR T cells were comparable at baseline. Loss of SHP2 did not alter the CAR T cell viability, expansion or proliferative capacity in comparison to the wild-type CAR T cells. Mass cytometry (CyTOF)-based analysis of SHP2KO CAR T cells after encountering GBM cells demonstrated the preservation of the central memory (CCR7+CD45RA-) T cell subset. The majority of PD1+ SHP2KO CAR T cells co-expressed functional activation markers such as ICOS and CD107a. The PD1+ T cell subset was comprised of mostly T-betHi progenitor population and only a smaller percentage of EomesHi terminally exhausted population. Functionally, HER2-specific SHP2KO CAR T cells from different donors (n=4) exhibited improved anti-tumor activity that was sustained in long-term cytotoxicity assays. In an experimental orthotopic LN229-GBM mouse model, SHP2KO HER2-CAR T cells better controlled the growth of established xenografts and improved survival in comparison to wild-type HER2-CAR T cells. In conclusion, our results show that disrupting SHP2 expression using a CRISPR/Cas9 gene editing strategy could improve the CAR T cell function against GBM and warrants further clinical testing. Citation Format: Khaled Sanber, Zeid Nawas, Vita Salsman, Ahmed Gad, Pretty Rose Matthew, Daniel Landi, Ciaran Lee, Amel Sengal, Rikhia Chakraborty, Sujith Joseph, Ahmed Nabil, Meenakshi Hegde. CRISPR-Cas9 gene editing of inhibitory receptor signaling pathways improves CAR T cell activity against glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2173.

Overexpression of ER Protein Processing and Apoptosis Regulator Genes in Human Embryonic Kidney 293 Cells Improves Gene Therapy Vectors Production.

Gammaretroviral and lentiviral vectors (γ-RV and LV) are among the most used vectors in gene therapy. Currently, human embryonic kidney (HEK) 293 cells, the manufacture platform of choice for these vectors, provide low transducing particle yields, challenging their therapeutic applications and commercialization. This work studies metabolic pathways, focusing on endoplasmic reticulum (ER) protein processing and anti-apoptotic mechanisms, influencing vector productivity in HEK 293 cell substrates. To that end, four candidate genes-protein disulfide isomerase family A member 2 gene, heat shock protein family A (Hsp70) member 5 gene, X-box binding protein 1 gene (ER protein processing), and B-cell lymphoma 2 protein gene (anti-apoptotic)-are individually stably expressed in the cells. How their overexpression level influence vector yields is analyzed by establishing cell populations with incremental genomic copies of each. γ-RV volumetric productivity increases up to 97% when overexpressing ER protein processing genes. LV volumetric production increases 53% when overexpressing the anti-apoptotic gene. Improvements are associated with higher cell specific productivities and dependent on gene overexpression level, highlighting the importance of fine-tuning gene expression. Overall, this work discloses gene engineering targets enabling efficient gene therapy product manufacture showing that ER protein processing and anti-apoptotic pathways are pivotal to producer cell performance.

Progress in treating chronic granulomatous disease.

Chronic granulomatous disease is a primary immunodeficiency due to a defect in one of sixsubunits that make up the nicotinamide adenine dinucleotide phosphate oxidase complex. The most commonly defective protein, gp91phox , is inherited in an X-linked fashion; other defects have autosomal recessive inheritance. Bacterial and fungal infections are common presentations, although inflammatory complications are increasingly recognized as a significant cause of morbidity and are challenging to treat. Haematopoietic stem cell transplantation offers cure from the disease with improved quality of life; overall survival in the current era is around 85%, with most achieving long-term cure free of medication. More recently, gene therapy is emerging as an alternative approach. Results using gammaretroviral vectors were disappointing with genotoxicity and loss of efficacy, but preliminary results using lentiviral vectors are extremely encouraging.

Scaffold-Mediated Static Transduction of T Cells for CAR-T Cell Therapy.

Chimeric antigen receptor T (CAR-T) cell therapy has produced impressive clinical responses in patients with B-cell malignancies. Critical to the success of CAR-T cell therapies is the achievement of robust gene transfer into T cells mediated by viral vectors such as gamma-retroviral vectors. However, current methodologies of retroviral gene transfer rely on spinoculation and the use of retronectin, which may limit the implementation of cost-effective CAR-T cell therapies. Herein, a low-cost, tunable, macroporous, alginate scaffold that transduces T cells with retroviral vectors under static condition is described. CAR-T cells produced by macroporous scaffold-mediated viral transduction exhibit >60% CAR expression, retain effector phenotype, expand to clinically relevant cell numbers, and eradicate CD19+ lymphoma in vivo. Efficient transduction is dependent on scaffold macroporosity. Taken together, the data show that macroporous alginate scaffolds serve as an attractive alternative to current transduction protocols and have high potential for clinical translation to genetically modify T cells for adoptive cellular therapy.

Nanopore sequencing as a scalable, cost-effective platform for analyzing polyclonal vector integration sites following clinical T cell therapy

BackgroundAnalysis of vector integration sites in gene-modified cells can provide critical information on clonality and potential biological impact on nearby genes. Current short-read next-generation sequencing methods require specialized instruments and...

Genome-Wide profiling of viral vector integration in clinical CAR T products using comprehensive methodologies

Background & Aim Chimeric antigen receptor (CAR)-engineered T cells have become an important cancer immunotherapy and the clinical application of these cells is increasing rapidly. For clinical CAR T-cell therapies, autologous T-cells are genetically modified to express a specific/bispecific chimeric antigen receptor. These CAR T-cells have high specificity and affinity for antigens expressed on the surface of target cells. At present, T-cells are genetically modified by introduction of the receptor by gamma- and lentiviral vectors, which integrate into the cell genome and is subsequently expressed. However, as transduction efficiency and the number of integration events increases, the risk of insertional mutagenesis also increases. In order to maintain the safety and effectiveness of clinical CAR T-cells, it is essential to monitor the insertion efficiency, accurately assess insertion sites and determine the effects of vector instertion on the CAR T-cell transcriptome profile. Methods, Results & Conclusion We developed a comprehensive methodology for evaluating the safety and effectiveness of CAR T-cells, utilizing droplet digital PCR to measure vector copy number, DNA-seq to measure vector insertion sites and RNA-seq to assess gene expression profiles. These assays were applied to 38 clinical CAR T products (18 gamma- and 20 lentiviral vector). We verified that ddPCR can reliably measure the average number of vector copies integrated into the CAR T-cell genome. CAR T products with a detectable number of vector copies were subjected to DNA-seq. The results showed that DNA-seq accurately revealed integration site maps in the whole genome and found some loci that were integration hot spots. Moreover, we found gamma-retroviral vectors resulted in more integration events than lentiviral vectors. Combining the RNA-seq and DNA-seq data revealed that most integration events within coding genes did not result in a change in gene expression. We did noticed that some gene loci both have an integration event and changed gene expression. Further studies will focus on these finding and the impact on CAR T-cell clinical outcomes. In summary, we established a series of methods to detect vector copy number, insertion sites and gene expression profiles in clincial CAR T products. These methods will be useful for exploring the risk of insertional mutagenesis of viral vector in CAR T products, the signficance of specific integration sites, and facilates improved safety in development of CAR T cell therapies.

Vectofusin-1 Improves Transduction of Primary Human Cells with Diverse Retroviral and Lentiviral Pseudotypes, Enabling Robust, Automated Closed-System Manufacturing.

Cell and gene therapies are finally becoming viable patient treatment options, with both T cell- and hematopoietic stem cell (HSC)-based therapies being approved to market in Europe. However, these therapies, which involve the use of viral vector to modify the target cells, are expensive and there is an urgent need to reduce manufacturing costs. One major cost factor is the viral vector production itself, therefore improving the gene modification efficiency could significantly reduce the amount of vector required per patient. This study describes the use of a transduction enhancing peptide, Vectofusin-1®, to improve the transduction efficiency of primary target cells using lentiviral and gammaretroviral vectors (LV and RV) pseudotyped with a variety of envelope proteins. Using Vectofusin-1 in combination with LV pseudotyped with viral glycoproteins derived from baboon endogenous retrovirus, feline endogenous virus (RD114), and measles virus (MV), a strongly improved transduction of HSCs, B cells and T cells, even when cultivated under low stimulation conditions, could be observed. The formation of Vectofusin-1 complexes with MV-LV retargeted to CD20 did not alter the selectivity in mixed cell culture populations, emphasizing the precision of this targeting technology. Functional, ErbB2-specific chimeric antigen receptor-expressing T cells could be generated using a gibbon ape leukemia virus (GALV)-pseudotyped RV. Using a variety of viral vectors and target cells, Vectofusin-1 performed in a comparable manner to the traditionally used surface-bound recombinant fibronectin. As Vectofusin-1 is a soluble peptide, it was possible to easily transfer the T cell transduction method to an automated closed manufacturing platform, where proof of concept studies demonstrated efficient genetic modification of T cells with GALV-RV and RD114-RV and the subsequent expansion of mainly central memory T cells to a clinically relevant dose.

Generation of Chimeric Antigen Receptor T Cells Using Gammaretroviral Vectors.

Manufacturing chimeric antigen receptor (CAR)-modified T cells requires incorporation of the CAR transgene, for which viral vectors are most often used. Here, we describe the generation of CAR T cells using primary human T cells and a non-self-inactivating gammaretroviral vector encoding a CAR transgene. The gammaretroviral vector is produced by 293T cells transiently transfected with DNA plasmids encoding necessary components of the viral vector. The resulting viral particles efficiently infect activated T cells and integrate the CAR transgene into the genome of dividing cells for stable expression.

Advances in the gene therapy of monogenic blood cell diseases.

Hematopoietic gene therapy has markedly progressed during the last 15 years both in terms of safety and efficacy. While a number of serious adverse events (SAE) were initially generated as a consequence of genotoxic insertions of gamma-retroviral vectors in the cell genome, no SAEs and excellent outcomes have been reported in patients infused with autologous hematopoietic stem cells (HSCs) transduced with self-inactivated lentiviral and gammaretroviral vectors. Advances in the field of HSC gene therapy have extended the number of monogenic diseases that can be treated with these approaches. Nowadays, evidence of clinical efficacy has been shown not only in primary immunodeficiencies, but also in other hematopoietic diseases, including beta-thalassemia and sickle cell anemia. In addition to the rapid progression of non-targeted gene therapies in the clinic, new approaches based on gene editing have been developed thanks to the discovery of designed nucleases and improved non-integrative vectors, which have markedly increased the efficacy and specificity of gene targeting to levels compatible with its clinical application. Based on advances achieved in the field of gene therapy, it can be envisaged that these therapies will soon be part of the therapeutic approaches used to treat life-threatening diseases of the hematopoietic system.

Gene therapy for primary immunodeficiency.

Gene therapy is now being trialled as a therapeutic option for an expanding number of conditions, based primarily on the successful treatment over the past two decades of patients with specific primary immunodeficiencies (PIDs) including severe combined immunodeficiency and Wiskott-Aldrich syndrome and metabolic conditions such as leukodystrophy. The field has evolved from the use of gammaretroviral vectors to more sophisticated lentiviral platforms that offer an improved biosafety profile alongside greater efficiency for hematopoietic stem cells gene transfer. Here we review more recent developments including licensing of gene therapies, use of gene corrected autologous T cells as an alternative strategy for some PIDs and the potential of targeted gene correction using various gene editing platforms. Given the promising results of recent clinical trials, it is likely that autologous gene therapies will become standard of care for a number of devastating diseases in the coming decade.

Gene therapy targeting haematopoietic stem cells for inherited diseases: progress and challenges.

Pioneering gene therapy trials have shown that the genetic engineering of haematopoietic stem and progenitor cells can be an alternative to allogeneic transplantation inthe treatment of primary immunodeficiencies. Early trials also highlighted the risk of insertionalmutagenesis and oncogene transactivation associated with the first generation of gammaretroviral vectors. These events prompted the development of safer, self-inactivating lentiviral or gammaretroviral vectors. These lentiviral vectors have been successfully used to treat over 200 patients with 10 different haematological disorders (including primary immunodeficiencies, haemoglobinopathies and metabolic disorders) and for the generation ofchimeric antigen receptor-T cells for cancer therapy. However, several challenges, such as effective reconstitution during inflammation, remain if gene therapy is to be extended to more complex diseases in which haematopoietic stem and progenitor cells can be altered by the disease environment. We discuss the progress made and future challenges for gene therapy and contrast gene therapy with gene-editing strategies.

Sustained and regulated gene expression by Tet-inducible “all-in-one” retroviral vectors containing the HNRPA2B1-CBX3 UCOE®

Genetic modification of induced pluripotent stem (iPS) cells may be necessary for the generation of effector cells for cellular therapies. Hereby, it can be important to induce transgene expression at restricted and defined time windows, especially if it interferes with pluripotency or differentiation. To achieve this, inducible expression systems can be used such as the tetracycline-inducible retroviral vector system, however, retroviral expression can be subjected to epigenetic silencing or to position-effect variegation. One strategy to overcome this is the incorporation of ubiquitous chromatin opening elements (UCOE®'s) into retroviral vectors to maintain a transcriptionally permissive chromatin state at the integration site.In this study, we developed Tet-inducible all-in-one gammaretroviral vectors carrying different sized UCOE®'s derived from the A2UCOE. The ability to prevent vector silencing by preserving the Tet-regulatory potential was investigated in different cell lines, and in murine and human iPS cells. A 670-bp fragment spanning the CBX3 promoter region of A2UCOE (U670) was the most potent element in preventing silencing, and conferred the strongest expression from the vector in the induced state. While longer fragments of A2UCOEs also sustained expression, vector titers and induction efficiencies were impaired. Finally, we demonstrate that U670 can be used for constitutive expression of the transactivator in the all-in-one vector for faithful regulation of transgenes by doxycycline, including the thrombopoietin receptor Mpl conferring cytokine-dependent cell growth.

Shifting Retroviral Vector Integrations Away from Transcriptional Start Sites via DNA-Binding Protein Domain Insertion into Integrase

The unique ability of retroviruses to integrate genes into host genomes is of great value for long-term expression in gene therapy, but only when integrations occur at safe genomic sites. To reap the benefit of using retroviruses without severe detrimental effects, we developed several murine leukemia virus (MLV)-based gammaretroviral vectors with safer integration patterns by perturbing the structure of the integrase via insertion of DNA-binding zinc-finger domains (ZFDs) into an internal position of the enzyme. ZFD insertion significantly reduced the inherent, strong MLV integration preference for genomic regions near transcriptional start sites (TSSs), which are the most dangerous spots. The altered retroviral integration pattern was related to increased formation of residual primer-binding site sequences at the 3′ end of proviruses. Several ZFD insertion mutants showed lower frequencies of integrations into the TSS genome regions when having the residual primer-binding site sequences in the proviruses. Our findings not only can extend the use of retroviruses in biomedical applications, but also provide a glimpse into the mechanisms underlying retroviral integration.

A Nontoxic Transduction Enhancer Enables Highly Efficient Lentiviral Transduction of Primary Murine T Cells and Hematopoietic Stem Cells

Lentiviral vectors have emerged as an efficient, safe therapeutic tool for gene therapy based on hematopoietic stem cells (HSCs) or T cells. However, the monitoring of transduced cells in preclinical models remains challenging because of the inefficient transduction of murine primary T cells with lentiviral vectors, in contrast to gammaretroviral vectors. The use of this later in preclinical proof of concept is not considered as relevant when a lentiviral vector will be used in a clinical trial. Hence, there is an urgent need to develop an efficient transduction protocol for murine cells with lentiviral vectors. Here, we describe an optimized protocol in which a nontoxic transduction enhancer (Lentiboost) enables the efficient transduction of primary murine T cells with lentiviral vectors. The optimized protocol combines low toxicity and high transduction efficiency. We achieved a high-level transduction of murine CD4+ and CD8+ T cells with a VSV-G-pseudotyped lentiviral vector with no changes in the phenotypes of transduced T cells, which were stable and long-lived in culture. This enhancer also increased the transduction of murine HSCs. Hence, use of this new transduction enhancer overcomes the limitations of lentiviral vectors in preclinical experiments and should facilitate the translation of strategies based on lentiviral vectors from the bench to the clinic.