A human genome editing-based MLL::AF4 ALL model recapitulates key cellular and molecular leukemogenic features.

Cellular ontogeny and MLL breakpoint site influence the capacity of MLL-edited CD34+ hematopoietic cells to initiate and recapitulate infant patients' features in pro-B-cell acute lymphoblastic leukemia (B-ALL). We provide key insights into the leukemogenic determinants of MLL-AF4+ infant B-ALL.

Specific correction of pyruvate kinase deficiency-causing point mutations by CRISPR/Cas9 and single-stranded oligodeoxynucleotides.

Pyruvate kinase deficiency (PKD) is an autosomal recessive disorder caused by mutations in the PKLR gene. PKD-erythroid cells suffer from an energy imbalance caused by a reduction of erythroid pyruvate kinase (RPK) enzyme activity. PKD is associated with reticulocytosis, splenomegaly and iron overload, and may be life-threatening in severely affected patients. More than 300 disease-causing mutations have been identified as causing PKD. Most mutations are missense mutations, commonly present as compound heterozygous. Therefore, specific correction of these point mutations might be a promising therapy for the treatment of PKD patients. We have explored the potential of precise gene editing for the correction of different PKD-causing mutations, using a combination of single-stranded oligodeoxynucleotides (ssODN) with the CRISPR/Cas9 system. We have designed guide RNAs (gRNAs) and single-strand donor templates to target four different PKD-causing mutations in immortalized patient-derived lymphoblastic cell lines, and we have detected the precise correction in three of these mutations. The frequency of the precise gene editing is variable, while the presence of additional insertions/deletions (InDels) has also been detected. Significantly, we have identified high mutation-specificity for two of the PKD-causing mutations. Our results demonstrate the feasibility of a highly personalized gene-editing therapy to treat point mutations in cells derived from PKD patients.

CRISPR/Cas9-mediated gene editing. A promising strategy in hematological disorders.

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has revolutionized the gene editing field, making it possible to interrupt, insert or replace a sequence of interest with high precision in the human genome. Its easy design and wide applicability open up a variety of therapeutic alternatives for the treatment of genetic diseases. Indeed, very promising approaches for the correction of hematological disorders have been developed in the recent years, based on the self-renewal and multipotent differentiation properties of hematopoietic stem and progenitor cells, which make this cell subset the ideal target for gene therapy purposes. This technology has been applied in different congenital blood disorders, such as primary immunodeficiencies, X-linked severe combined immunodeficiency, X-linked chronic granulomatous disease or Wiskott-Aldrich syndrome, and inherited bone marrow failure syndromes, such as Fanconi anemia, congenital amegakaryocytic thrombocytopenia or severe congenital neutropenia. Furthermore, CRISPR/Cas9-based gene editing has been implemented successfully as a novel therapy for cancer immunotherapy, by the development of promising strategies such as the use of oncolytic viruses or adoptive cellular therapy to the chimeric antigen receptor-T-cell therapy. Therefore, considering the variety of genes and mutations affected, we can take advantage of the different DNA repair mechanisms by CRISPR/Cas9 in different manners, from homology-directed repair to non-homologous-end-joining to the latest emerging technologies such as base and prime editing. Although the delivery systems into hematopoietic stem and progenitor cells are still the bottleneck of this technology, some of the advances in genome editing shown in this review have already reached a clinical stage and show very promising preliminary results.

Gene Editing for Inherited Red Blood Cell Diseases.

Today gene therapy is a real therapeutic option to address inherited hematological diseases that could be beneficial for thousands of patients worldwide. Currently, gene therapy is used to treat different monogenic hematological pathologies, including several red blood cell diseases such as ß-thalassemia, sickle cell disease and pyruvate kinase deficiency. This approach is based on addition gene therapy, which consists of the correction of hematopoietic stem cells (HSCs) using lentiviral vectors, which integrate a corrected version of the altered gene. Lentivirally-corrected HSCs generate healthy cells that compensate for the deficiency caused by genetic mutations. Despite its successful results, this approach lacks both control of the integration of the transgene into the genome and endogenous regulation of the therapeutic gene, both of which are important aspects that might be a cause for concern. To overcome these limitations, gene editing is able to correct the altered gene through more precise and safer approaches. Cheap and easy-to-design gene editing tools, such as the CRISPR/Cas9 system, allow the specific correction of the altered gene without affecting the rest of the genome. Inherited erythroid diseases, such as thalassemia, sickle cell disease and Pyruvate Kinase Deficiency, have been the test bed for these gene editing strategies, and promising results are currently being seen. CRISPR/Cas9 system has been successfully used to manipulate globin regulation to re-activate fetal globin chains in adult red blood cells and to compensate for hemoglobin defects. Knock-in at the mutated locus to express the therapeutic gene under the endogenous gene regulatory region has also been accomplished successfully. Thanks to the lessons learned from previous lentiviral gene therapy research and trials, gene editing for red blood cell diseases is rapidly moving from its proof-of-concept to its first exciting results in the clinic. Indeed, patients suffering from ß-thalassemia and sickle cell disease have already been successfully treated with gene editing, which will hopefully inspire the use of gene editing to cure erythroid disorders and many other inherited diseases in the near future.

Preclinical studies of efficacy thresholds and tolerability of a clinically ready lentiviral vector for pyruvate kinase deficiency treatment.

Pyruvate kinase deficiency (PKD) is a rare autosomal recessive disorder caused by mutations in the PKLR gene. PKD is characterized by non-spherocytic hemolytic anemia of variable severity and may be fatal in some cases during early childhood. Although not considered the standard of care, allogeneic stem cell transplantation has been shown as a potentially curative treatment, limited by donor availability, toxicity, and incomplete engraftment. Preclinical studies were conducted to define conditions to enable consistent therapeutic reversal, which were based on our previous data on lentiviral gene therapy for PKD. Improvement of erythroid parameters was identified by the presence of 20%-30% healthy donor cells. A minimum vector copy number (VCN) of 0.2-0.3 was required to correct PKD when corrected cells were transplanted in a mouse model for PKD. Biodistribution and pharmacokinetics studies, with the aim of conducting a global gene therapy clinical trial for PKD patients (RP-L301-0119), demonstrated that genetically corrected cells do not confer additional side effects. Moreover, a clinically compatible transduction protocol with mobilized peripheral blood CD34+ cells was optimized, thus facilitating the efficient transduction on human cells capable of repopulating the hematopoiesis of immunodeficient mice. We established conditions for a curative lentiviral vector gene therapy protocol for PKD.

Clinically relevant gene editing in hematopoietic stem cells for the treatment of pyruvate kinase deficiency.

Pyruvate kinase deficiency (PKD), an autosomal-recessive disorder, is the main cause of chronic non-spherocytic hemolytic anemia. PKD is caused by mutations in the pyruvate kinase, liver and red blood cell (P KLR) gene, which encodes for the erythroid pyruvate kinase protein (RPK). RPK is implicated in the last step of anaerobic glycolysis in red blood cells (RBCs), responsible for the maintenance of normal erythrocyte ATP levels. The only curative treatment for PKD is allogeneic hematopoietic stem and progenitor cell (HSPC) transplant, associated with a significant morbidity and mortality, especially relevant in PKD patients. Here, we address the correction of PKD through precise gene editing at the PKLR endogenous locus to keep the tight regulation of RPK enzyme during erythropoiesis. We combined CRISPR-Cas9 system and donor recombinant adeno-associated vector (rAAV) delivery to build an efficient, safe, and clinically applicable system to knock in therapeutic sequences at the translation start site of the RPK isoform in human hematopoietic progenitors. Edited human hematopoietic progenitors efficiently reconstituted human hematopoiesis in primary and secondary immunodeficient mice. Erythroid cells derived from edited PKD-HSPCs recovered normal ATP levels, demonstrating the restoration of RPK function in PKD erythropoiesis after gene editing. Our gene-editing strategy may represent a lifelong therapy to correct RPK functionality in RBCs for PKD patients.

The Current Status of Mesenchymal Stromal Cells: Controversies, Unresolved Issues and Some Promising Solutions to Improve Their Therapeutic Efficacy.

Mesenchymal stromal cells (MSCs) currently constitute the most frequently used cell type in advanced therapies with different purposes, most of which are related with inflammatory processes. Although the therapeutic efficacy of these cells has been clearly demonstrated in different disease animal models and in numerous human phase I/II clinical trials, only very few phase III trials using MSCs have demonstrated the expected potential therapeutic benefit. On the other hand, diverse controversial issues on the biology and clinical applications of MSCs, including their specific phenotype, the requirement of an inflammatory environment to induce immunosuppression, the relevance of the cell dose and their administration schedule, the cell delivery route (intravascular/systemic vs. local cell delivery), and the selected cell product (i.e., use of autologous vs. allogeneic MSCs, freshly cultured vs. frozen and thawed MSCs, MSCs vs. MSC-derived extracellular vesicles, etc.) persist. In the current review article, we have addressed these issues with special emphasis in the new approaches to improve the properties and functional capabilities of MSCs after distinct cell bioengineering strategies.

Enhanced anti-inflammatory effects of mesenchymal stromal cells mediated by the transient ectopic expression of CXCR4 and IL10.

BACKGROUND: Mesenchymal stromal cells (MSCs) constitute one of the cell types most frequently used in cell therapy. Although several studies have shown the efficacy of these cells to modulate inflammation in different animal models, the results obtained in human clinical trials have been more modest. Here, we aimed at improving the therapeutic properties of MSCs by inducing a transient expression of two molecules that could enhance two different properties of these cells. With the purpose of improving MSC migration towards inflamed sites, we induced a transient expression of the C-X-C chemokine receptor type 4 (CXCR4). Additionally, to augment the anti-inflammatory properties of MSCs, a transient expression of the anti-inflammatory cytokine, interleukin 10 (IL10), was also induced. METHODS: Human adipose tissue-derived MSCs were transfected with messenger RNAs carrying the codon-optimized versions of CXCR4 and/or IL10. mRNA-transfected MSCs were then studied, first to evaluate whether the characteristic phenotype of MSCs was modified. Additionally, in vitro and also in vivo studies in an LPS-induced inflamed pad model were conducted to evaluate the impact associated to the transient expression of CXCR4 and/or IL10 in MSCs. RESULTS: Transfection of MSCs with CXCR4 and/or IL10 mRNAs induced a transient expression of these molecules without modifying the characteristic phenotype of MSCs. In vitro studies then revealed that the ectopic expression of CXCR4 significantly enhanced the migration of MSCs towards SDF-1, while an increased immunosuppression was associated with the ectopic expression of IL10. Finally, in vivo experiments showed that the co-expression of CXCR4 and IL10 increased the homing of MSCs into inflamed pads and induced an enhanced anti-inflammatory effect, compared to wild-type MSCs. CONCLUSIONS: Our results demonstrate that the transient co-expression of CXCR4 and IL10 enhances the therapeutic potential of MSCs in a local inflammation mouse model, suggesting that these mRNA-modified cells may constitute a new step in the development of more efficient cell therapies for the treatment of inflammatory diseases.

Natural estrogens enhance the engraftment of human hematopoietic stem and progenitor cells in immunodeficient mice.

Hematopoietic Stem and Progenitor Cells are crucial in the maintenance of lifelong production of all blood cells. These Stem Cells are highly regulated to maintain homeostasis through a delicate balance between quiescence, self-renewal and differentiation. However, this balance is altered during the hematopoietic recovery after Hematopoietic Stem and Progenitor Cell Transplantation. Transplantation efficacy can be limited by inadequate Hematopoietic Stem Cells number, poor homing, low level of engraftment, or limited self-renewal. As recent evidences indicate that estrogens are involved in regulating the hematopoiesis, we sought to examine whether natural estrogens (estrone or E1, estradiol or E2, estriol or E3 and estetrol or E4) modulate human Hematopoietic Stem and Progenitor Cells. Our results show that human Hematopoietic Stem and Progenitor Cell subsets express estrogen receptors, and whose signaling is activated by E2 and E4 on these cells. Additionally, these natural estrogens cause different effects on human Progenitors in vitro. We found that both E2 and E4 expand human Hematopoietic Stem and Progenitor Cells. However, E4 was the best tolerated estrogen and promoted cell cycle of human Hematopoietic Progenitors. Furthermore, we identified that E2 and, more significantly, E4 doubled human hematopoietic engraftment in immunodeficient mice without altering other Hematopoietic Stem and Progenitor Cells properties. Finally, the impact of E4 on promoting human hematopoietic engraftment in immunodeficient mice might be mediated through the regulation of mesenchymal stromal cells in the bone marrow niche. Together, our data demonstrate that E4 is well tolerated and enhances human reconstitution in immunodeficient mice, directly by modulating human Hematopoietic Progenitor properties and indirectly by interacting with the bone marrow niche. This application might have particular relevance to ameliorate the hematopoietic recovery after myeloablative conditioning, especially when limiting numbers of Hematopoietic Stem and Progenitor Cells are available.

Targeted gene therapy into a safe harbor site in human hematopoietic progenitor cells.

Directed gene therapy mediated by nucleases has become a new alternative to lead targeted integration of therapeutic genes in specific regions in the genome. In this work, we have compared the efficiency of two nuclease types, TALEN and meganucleases (MN), to introduce an EGFP reporter gene in a specific site in a safe harbor locus on chromosome 21 in an intergenic region, named here SH6. The efficiency of targeted integration mediated by SH6v5-MN and SH6-TALEN in HEK-293H cells was up to 16.3 and 15.0%. A stable expression was observed both in the pool of transfected cells and in established pseudoclones, with no detection of off-target integrations by Southern blot. In human hematopoietic stem and progenitor CD34+ cells, the nucleofection process preserved the viability and clonogenic capacity of nucleofected cells, reaching up to 3.1% of specific integration of the transgene in colony forming cells when the SH6-TALEN was used, although no expression of the transgene could be found in these cells. Our results show the possibility to specifically integrate genes at the SH6 locus in CD34+ progenitor cells, although further improvements in the efficacy of the procedure are required before this approach could be used for the gene editing of hematopoietic stem cells in patients with hematopoietic diseases.

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 editing of PKLR gene in human hematopoietic progenitors through 5' and 3' UTR modified TALEN mRNA.

Pyruvate Kinase Deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene, which encodes the erythroid specific Pyruvate Kinase enzyme. Erythrocytes from PKD patients show an energetic imbalance and are susceptible to hemolysis. Gene editing of hematopoietic stem cells (HSCs) would provide a therapeutic benefit and improve safety of gene therapy approaches to treat PKD patients. In previous studies, we established a gene editing protocol that corrected the PKD phenotype of PKD-iPSC lines through a TALEN mediated homologous recombination strategy. With the goal of moving toward more clinically relevant stem cells, we aim at editing the PKLR gene in primary human hematopoietic progenitors and hematopoietic stem cells (HPSCs). After nucleofection of the gene editing tools and selection with puromycin, up to 96% colony forming units showed precise integration. However, a low yield of gene edited HPSCs was associated to the procedure. To reduce toxicity while increasing efficacy, we worked on i) optimizing gene editing tools and ii) defining optimal expansion and selection times. Different versions of specific nucleases (TALEN and CRISPR-Cas9) were compared. TALEN mRNAs with 5' and 3' added motifs to increase RNA stability were the most efficient nucleases to obtain high gene editing frequency and low toxicity. Shortening ex vivo manipulation did not reduce the efficiency of homologous recombination and preserved the hematopoietic progenitor potential of the nucleofected HPSCs. Lastly, a very low level of gene edited HPSCs were detected after engraftment in immunodeficient (NSG) mice. Overall, we showed that gene editing of the PKLR gene in HPSCs is feasible, although further improvements must to be done before the clinical use of the gene editing to correct PKD.

Highly efficient genome editing of human hematopoietic stem cells via a nano-silicon-blade delivery approach.

Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 bacterial immunity system has opened a promising avenue to treat genetic diseases that affect the human hematopoietic stem cells (HSCs). Therefore, finding a highly efficient delivery method capable of modifying the genome in the hard-to-transfect HSCs, combined with the advanced CRISPR-Cas9 system, may meet the challenges for dissecting the hematologic disease mechanisms and facilitate future clinical applications. Here, we developed an effective HSC-specified delivery microfluidic chip to disrupt the cell membrane transiently by inducing rapid mechanical deformation that allowed the delivery of biomaterials into the cytoplasm from the surrounding matrix. Compared with the previous designs, the new nano-silicon-blade structure was specifically optimized for HSCs. Using the silicon substrate, the sharpness and rigidity of the nano-blade constriction was largely enhanced to improve the biomaterials delivery efficiency. We achieved highly efficient delivery results by transporting various macro-molecules into the HSCs. Moreover, the treated HSCs possess high viability and maintain inherent pluripotency after the delivery via the Nano-Blade Chip (NB-Chip). Subsequently, we disrupted the p42 isoform in C/EBPα on the NB-Chip and induced HSCs into a myeloid proliferation behavior. In conclusion, the NB-Chip provides a harmless, rapid and high-throughput gene editing approach for the HSC study and therapeutics.

Generation of Patient-Specific induced Pluripotent Stem Cell from Peripheral Blood Mononuclear Cells by Sendai Reprogramming Vectors.

Induced pluripotent stem cells (iPSC) technology has changed preclinical research since their generation was described by Shinya Yamanaka in 2006. iPSCs are derived from somatic cells after being reprogrammed back to an embryonic state by specific combination of reprogramming factors. These reprogrammed cells resemble all the characteristic of embryonic stem cells (ESC). The reprogramming technology is even more valuable to research diseases biology and treatment by opening gene and cell therapies in own patient's iPSC. Patient-specific iPSC can be generated from a large variety of patient cells by any of the myriad of reprogramming platforms described. Here, we describe the generation of patient-specific iPSC from patient peripheral blood mononuclear cells by Sendai Reprogramming vectors.

Generation of a High Number of Healthy Erythroid Cells from Gene-Edited Pyruvate Kinase Deficiency Patient-Specific Induced Pluripotent Stem Cells.

Pyruvate kinase deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene. Erythrocytes from PKD patients show an energetic imbalance causing chronic non-spherocytic hemolytic anemia, as pyruvate kinase defects impair ATP production in erythrocytes. We generated PKD induced pluripotent stem cells (PKDiPSCs) from peripheral blood mononuclear cells (PB-MNCs) of PKD patients by non-integrative Sendai viral vectors. PKDiPSCs were gene edited to integrate a partial codon-optimized R-type pyruvate kinase cDNA in the second intron of the PKLR gene by TALEN-mediated homologous recombination (HR). Notably, we found allele specificity of HR led by the presence of a single-nucleotide polymorphism. High numbers of erythroid cells derived from gene-edited PKDiPSCs showed correction of the energetic imbalance, providing an approach to correct metabolic erythroid diseases and demonstrating the practicality of this approach to generate the large cell numbers required for comprehensive biochemical and metabolic erythroid analyses.

Targeted gene therapy and cell reprogramming in Fanconi anemia.

Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies.

New frontier in regenerative medicine: site-specific gene correction in patient-specific induced pluripotent stem cells.

Advances in cell and gene therapy are opening up new avenues for regenerative medicine. Because of their acquired pluripotency, human induced pluripotent stem cells (hiPSCs) are a promising source of autologous cells for regenerative medicine. They show unlimited self-renewal while retaining the ability, in principle, to differentiate into any cell type of the human body. Since Yamanaka and colleagues first reported the generation of hiPSCs in 2007, significant efforts have been made to understand the reprogramming process and to generate hiPSCs with potential for clinical use. On the other hand, the development of gene-editing platforms to increase homologous recombination efficiency, namely DNA nucleases (zinc finger nucleases, TAL effector nucleases, and meganucleases), is making the application of locus-specific gene therapy in human cells an achievable goal. The generation of patient-specific hiPSC, together with gene correction by homologous recombination, will potentially allow for their clinical application in the near future. In fact, reports have shown targeted gene correction through DNA-Nucleases in patient-specific hiPSCs. Various technologies have been described to reprogram patient cells and to correct these patient hiPSCs. However, no approach has been clearly more efficient and safer than the others. In addition, there are still significant challenges for the clinical application of these technologies, such as inefficient differentiation protocols, genetic instability resulting from the reprogramming process and hiPSC culture itself, the efficacy and specificity of the engineered DNA nucleases, and the overall homologous recombination efficiency. To summarize advances in the generation of gene corrected patient-specific hiPSCs, this review focuses on the available technological platforms, including their strengths and limitations regarding future therapeutic use of gene-corrected hiPSCs.

Cell fusion reprogramming leads to a specific hepatic expression pattern during mouse bone marrow derived hepatocyte formation in vivo.

The fusion of bone marrow (BM) hematopoietic cells with hepatocytes to generate BM derived hepatocytes (BMDH) is a natural process, which is enhanced in damaged tissues. However, the reprogramming needed to generate BMDH and the identity of the resultant cells is essentially unknown. In a mouse model of chronic liver damage, here we identify a modification in the chromatin structure of the hematopoietic nucleus during BMDH formation, accompanied by the loss of the key hematopoietic transcription factor PU.1/Sfpi1 (SFFV proviral integration 1) and gain of the key hepatic transcriptional regulator HNF-1A homeobox A (HNF-1A/Hnf1a). Through genome-wide expression analysis of laser captured BMDH, a differential gene expression pattern was detected and the chromatin changes observed were confirmed at the level of chromatin regulator genes. Similarly, Tranforming Growth Factor-ß1 (TGF-ß(1)) and neurotransmitter (e.g. Prostaglandin E Receptor 4 [Ptger4]) pathway genes were over-expressed. In summary, in vivo BMDH generation is a process in which the hematopoietic cell nucleus changes its identity and acquires hepatic features. These BMDHs have their own cell identity characterized by an expression pattern different from hematopoietic cells or hepatocytes. The role of these BMDHs in the liver requires further investigation.