Wiskott-Aldrich Syndrome Locus Research Articles

569. Precision Editing of the WAS Locus via Homologous Recombination in Primary Human Hematopoietic Cells Mediated by Either TALEN or CRISPR/Cas Nucleases

Wiskott-Aldrich Syndrome (WAS) is an inherited primary immunodeficiency caused by mutations in the WAS gene which encodes a protein (WASp) that regulates the actin cytoskeleton in multiple hematopoietic cell lineages. Currently, allogeneic stem cell transplantation constitutes the only available cure for WAS, although phase I/II clinical trials using lentiviral gene therapy of autologous stem cells are currently underway and have shown improvement in immune system defects. Although effective, the gene therapy approach carries a risk of insertional mutagenesis, and unpredictable expression due to promoter choice as well as influences of the random integration site, including epigenetic status and the presence of neighboring transcription regulatory elements. As a refinement, our goal is to develop gene editing methodologies that would allow the specific targeting of a WASp cDNA to a position within the WAS locus allowing transcriptional regulation by the endogenous WAS promoter. To this end, we first designed and tested the cleavage efficiency of several guide RNAs (delivered as self-complementary AAV along with Cas9, delivered as mRNA), as well as candidate TALEN pairs (delivered as mRNAs), in primary human T cells. Using the T7 endonuclease assay, we identified candidate nucleases from both platforms (CRISPR/Cas9 and TALEN) that achieved a high Indel frequency: 73 and 85%, respectively. We next created a synthetic AAV6 donor template for homology-directed repair (HDR) that contained an MND promoter driven GFP cDNA with 1kb of WAS homology arms flanking it. When delivered with the TALEN or CRISPR/Cas9 nucleases, we observed stable integration of the GFP reporter within >25% of primary human T cells. Subsequently, we utilzed the identical reagents to target integration of the reporter cDNA into the WAS locus in adult human mobilized CD34+ cells, albeit at lower efficiencies. The off-target cleavage sites for TALENs identified using the Prognos software were amplified and sequenced, with no evidence of off-target cleavage observed at any of the predicted loci. Thus, we have generated genome editing tools that possess a high degree of specificity, providing the foundation for site-specific modification of the WAS locus as a therapeutic option.

Absence of WASp Enhances Hematopoietic and Megakaryocytic Differentiation in a Human Embryonic Stem Cell Model.

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency caused by mutations in the WAS gene and characterized by severe thrombocytopenia. Although the role of WASp in terminally differentiated lymphocytes and myeloid cells is well characterized, its role in early hematopoietic differentiation and in platelets (Plts) biology is poorly understood. In the present manuscript, we have used zinc finger nucleases targeted to the WAS locus for the development of two isogenic WAS knockout (WASKO) human embryonic stem cell lines (hESCs). Upon hematopoietic differentiation, hESCs-WASKO generated increased ratios of CD34+CD45+ progenitors with altered responses to stem cell factor compared to hESCs-WT. When differentiated toward the megakaryocytic linage, hESCs-WASKO produced increased numbers of CD34+CD41+ progenitors, megakaryocytes (MKs), and Plts. hESCs-WASKO-derived MKs and Plts showed altered phenotype as well as defective responses to agonist, mimicking WAS patients MKs and Plts defects. Interestingly, the defects were more evident in WASp-deficient MKs than in WASp-deficient Plts. Importantly, ectopic WAS expression using lentiviral vectors restored normal Plts development and MKs responses. These data validate the AND-1_WASKO cell lines as a human cellular model for basic research and for preclinical studies for WAS.

342. Gene Edition for Wiskott-Aldrich Syndrome Gene Therapy

Site Specific nucleases (SSN) are powerful tools for genome editing that are revolutionizing basic and applied science. However, for most therapeutic applications their efficiency and/or specificity must be still improved before translation into clinic. Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency (PID) caused by mutations in the WAS gene. An interesting characteristic of this disease is that, due the growth selective advantage of the WAS-expressing cells, a small number of corrected hematopoietic stem cells (HSCs) could be enough to achieve a complete cure of the patients. In this context, our final aim is to develop gene edition tools for WAS gene therapy with the idea to achieve safe genetic correction in a small numbers of patients’ HSCs. To study efficiency and safety of our tools, we first developed a WAS-specific GFP-reporter cellular model harboring GFP coding sequences disrupted by the intron 1 of the WAS gene. This reporter cell allowed us to have a direct measurement of the efficiency and specificity of WAS-specific nucleases to achieve genetic restoration. We constructed different CRISPR-based and ZFNs-based WAS-specific nucleases expressed by Lentiviral vectors (LV). With these set of plasmids we performed a side by side comparison of two different nucleases (ZFNs and CRISPR) and two different delivery systems (nucleofection versus Integrative deficient lentiviral vectors (IDLV)) to edit the WAS locus. We showed that CRISPR and ZFNs were equally efficient for WAS gene edition (over 60% gene disruption and 4% gene restoration) when delivered by plasmid nucleofection. However, IDLV-ZFNs were more efficient than IDLV-CRISPR, probably due to the low expression levels of Cas9 in the IDLV backbone. Our data indicates that CRISPR based systems can be as efficient as other SSN using standard delivery systems, however, their efficiency using IDLV is limited.

Wiskott-Aldrich Syndrome Protein (WASP) Is An Effector of Kit Signaling.

Wiskott-Aldrich Syndrome (WAS) is an X-linked recessive immunodeficiency disorder with eczema, thrombocytopenia and high susceptibility to opportunistic and pyogenic infections. The gene product of the WAS locus, WAS protein (WASP), is expressed in a hematopoietic-specific fashion and regulates cytoskeletal actin reorganization via Cdc42 and Arp2/3 interactions. Non-random inactivation of the X chromosome carrying the defective WASP gene in all peripheral blood cells from obligate female carriers of WAS suggests a selective advantage of hematopoietic stem cells or immature progenitor cells expressing the intact WASP gene. Because such progenitor cells are dependent on the Kit receptor/Kit ligand (KL) pathway, we tested whether or not WASP plays a role in signaling responses through Kit. WASP and interacting proteins WIP and Arp2/3 were strongly phosphorylated in response to KL stimulation of Mo7e cells. Time kinetics revealed onset of tyrosine phosphorylation of WASP as early as 1 min and a maximum at 5 min after KL stimulation. Although real-time KL-induced actin reorganization and KL-mediated spreading of bone marrow-derived mast cells (BMMC) on fibronectin-coated surfaces were grossly normal, KL-induced formation of filopodia was significantly decreased in number and size in the absence of WASP. In addition, KL-induced calcium-flux in BMMCs was aberrant in the absence of WASP suggesting that KL-dependent calcium signals and cytoskeletal rearrangement are linked through WASP. When BMMC cultures were established from WASP heterozygous female mice using KL as a growth factor, the cultures initially contained a mixture of WASP positive and negative populations. KL-driven differentiation into mature BMMCs eventually resulted in homogenous WASP positive cultures derived from the WASP positive progenitors. Thus, WASP expression conferred a selective advantage to the development of Kit-dependent hematopoiesis consistent with the selective advantage of WASP positive blood cells observed in WAS heterozygous female humans. Finally, KL-mediated gene expression in BMMCs derived from WASP negative mice or WT controls was compared and revealed in summary that at least 30% of all changes are WASP-dependent. The results indicate that WASP is downstream of Kit signaling and necessary for Kit-mediated filopodia formation, cell survival and gene expression.

Platelet serotonin (5-HT) is decreased not only in alcoholic but also in HCV cirrhosis

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency caused by mutations in the WAS gene and characterized by severe thrombocytopenia. Although the role of WASp in terminally differentiated lymphocytes and myeloid cells is well characterized, its role in early hematopoietic differentiation and in platelets (Plts) biology is poorly understood. In the present manuscript, we have used zinc finger nucleases targeted to the WAS locus for the development of two isogenic WAS knockout (WASKO) human embryonic stem cell lines (hESCs). Upon hematopoietic differentiation, hESCs-WASKO generated increased ratios of CD34+CD45+ progenitors with altered responses to stem cell factor compared to hESCs-WT. When differentiated toward the megakaryocytic linage, hESCs-WASKO produced increased numbers of CD34+CD41+ progenitors, megakaryocytes (MKs), and Plts. hESCs-WASKO-derived MKs and Plts showed altered phenotype as well as defective responses to agonist, mimicking WAS patients MKs and Plts defects. Interestingly, the defects were more evident in WASp-deficient MKs than in WASp-deficient Plts. Importantly, ectopic WAS expression using lentiviral vectors restored normal Plts development and MKs responses. These data validate the AND-1_WASKO cell lines as a human cellular model for basic research and for preclinical studies for WAS.

Identification of WASP mutations in patients with Wiskott-Aldrich syndrome and isolated thrombocytopenia reveals allelic heterogeneity at the WAS locus

Mutation in the gene encoding the recently isolated WASP protein has now been identified as the genetic defect responsible for the X-linked Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency disease associated with extensive phenotypic variability. To elucidate the range of WASP mutations responsible for WAS, we used PCR-SSCP analysis to screen for WASP gene mutation in 19 unrelated boys with the diagnosis of classical or attenuated WAS or isolated thrombocytopenia. All 19 patients had WASP mutations, each of which localized to the initial three or terminal three exons of the gene, and the majority of which were unique in each case. However, a missense mutation which results in substitution of the arginine at WAS codon 86 was identified in three boys with severe WAS as well as in one boy presenting with thrombocytopenia alone. While the three mutations found in the isolated thrombocytopenia patients leave the reading frame intact, about one-half of the gene alterations detected in both severe and attenuated WAS patients result in frameshifted transcript and premature translation termination. These findings therefore confirm the association of WAS with WASP mutation and identify WASP mutation as a cause for isolated congenital thrombocytopenia in males. While the WASP gene defects responsible for isolated thrombocytopenia and other mild presentations of WAS do not appear distinct from those resulting in severe WAS, these data indicate that analysis of WASP gene mutation provides a valuable tool for distinguishing the spectrum of WAS patients and the subset of males with isolated thrombocytopenia who represent mild cases of WAS.

The UDP-galactose translocator gene is mapped to band Xp11.23-p11.22 containing the Wiskott-Aldrich syndrome locus.

We have cloned a segment of the human gene encoding UDP-galactose translocator by genetic complementation of its defective mutant in mouse FM3A cells. Chromosome mapping using fluorescent in situ hybridization revealed that the cloned gene hybridized to the Xp11.23-11.23 region of the X chromosome. This region is shared by the locus of Wiskott-Aldrich syndrome, an X-linked recessive immunodeficiency disorder, characterized by defective sugar chains on cell surface components. Genetic and phenotypic similarities suggest a possible link between UDP-galactose translocator and the Wiskott-Aldrich syndrome (WAS).

Wiskott-Aldrich syndrome: New molecular and biochemical insights

The Wiskott-Aldrich syndrome is an uncommon X-linked recessive disease characterized by eczema, thrombocytopenia, and immunodeficiency. The clinical features begin early in life and include recurrent infections, bleeding, and severe eczema. Unless the condition is treated by bone marrow transplantation, the prognosis of Wiskott-Aldrich syndrome is grave, and premature death caused by sepsis, hemorrhage, or lymphoreticular malignancy is common. Although the biochemical defect responsible for the syndrome is not known, recent investigations with restriction fragment length polymorphisms have mapped the Wiskott-Aldrich syndrome locus to the proximal portion of the short arm of the human X chromosome (Xpl 1 ). The isolation of these DNA markers makes feasible both carrier detection and prenatal diagnosis of Wiskott-Aldrich syndrome and provides an important adjunct to the management of Wiskott-Aldrich syndrome for patients and their families. These genetic data, in conjunction with the recent identification of a specific O-glycosylation defect in lymphocytes from patients with Wiskott-Aldrich syndrome, present an opportunity for the eventual isolation of the Wiskott-Aldrich syndrome gene and identification of the underlying cellular defect. We review the clinical and laboratory features of this syndrome and summarize the new molecular and biochemical approaches that can be used in diagnosis, genetic counseling, and treatment.

X-linked thrombocytopenia and Wiskott-Aldrich syndrome: similar regional assignment but distinct X-inactivation pattern in carriers

While inherited X-linked (XL) isolated thrombocytopenia is a mild condition, the Wiskott-Aldrich syndrome (WAS) associates severe thrombocytopenia with an immunodeficiency component and has a poor prognosis. Whether these conditions correspond to separate genetic entities or to different mutations of the same gene(s) remains unresolved. The Wiskott-Aldrich syndrome locus has been assigned to Xp 11.2 by means of RFLP studies. The X-inactivation pattern in female carriers has been found to follow a skewed pattern in the hematopoietic cells, thus allowing carrier detection. We studied a family with four members affected by XL thrombocytopenia and report the results of genetic segregation analysis, together with the X-inactivation pattern of hematopoietic cells from an obligate female carrier. Although the affected locus mapped to the same region as that of WAS, lymphocytes presented a skewed pattern of X-inactivation, whereas polymorphonuclear lymphocytes (PMN) did not. These results provide further evidence that the Wiskott-Aldrich syndrome and XL thrombocytopenia are different expressions of mutations within a single locus and that the severity of the disease corresponds to distinct hematopoietic cell selections in obligate carriers.

Localization of the gene for the Wiskott-Aldrich syndrome between two flanking markers, TIMP and DXS255, on Xp11.22–Xp11.3

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive genetic disease in which the basic molecular defect is unknown. We previously located the WAS gene between two DNA markers, DXS7 (Xp11.3) and DXS14 (Xp11), and mapped it to the proximal short arm of the human X chromosome (Kwan et al., 1988, Genomics 3:39–43). In thisstudy, further mapping was performed on 17 WAS families with two additional RFLP markers, TIMP and DXS255. Our data suggest that DXS255 is closer to the WAS locus than any other markers that have been previously described, with a multipoint maximum lod score of Z = 8.59 at 1.2 cM distal to DXS255 and thus further refine the position of the WAS gene on the short arm of the X chromosome. Possible locations for the WAS gene are entirely confined between TIMP (Xp11.3) and DXS255 (Xp11.22). Use of these markers thus represents a major improvement in genetic prediction in WAS families.

Linkage studies of the Wiskott-Aldrich syndrome: polymorphisms at TIMP and the X chromosome centromere are informative markers for genetic prediction

Eleven families segregating for the X-linked recessive immune deficiency disorder, Wiskott-Aldrich syndrome (WAS), were studied by linkage analysis with an alpha satellite DNA probe, pBamX-7, which detects polymorphisms at the X chromosome centromere, locus DXZ1, as well as three other polymorphic markers defining loci on the proximal short arm of the X chromosome. Linkage has been established between WAS and DXZ1 (zeta (theta) = 7.08 at theta = 0.03) and WAS and the TIMP gene locus (zeta (theta) = 5.09 at theta = 0.0). We have also confirmed close linkage between DXZ1 and two marker loci, DXS14 and DXS7, previously shown to be linked to the WAS locus. The probe pBamX-7 detected allelic variation in all females tested, reflecting the high frequency of polymorphism at the centromere. One WAS carrier revealed a recombination between WAS and both marker loci DXZ1 and DXS14, indicating that WAS does not map between these loci. In conjunction with previous data from genetic mapping studies of WAS, these results confirm the pericentromeric Xp localization of WAS and demonstrate the usefulness of alpha satellite DNA probes as tools for genetic prediction in WAS as well as other pericentric X-linked diseases.

Linkage of the Wiskott-Aldrich syndrome with polymorphic DNA sequences from the human X chromosome.

The Wiskott-Aldrich syndrome (WAS) is one of several human immunodeficiency diseases inherited as an X-linked trait. The location of WAS on the X chromosome is unknown. We have studied 10 kindreds segregating for WAS for linkage with cloned, polymorphic DNA markers and have demonstrated significant linkage between WAS and two loci, DXS14 and DXS7, that map to the proximal short arm of the X chromosome. Maximal logarithm of odds (lod scores) for WAS-DXS14 and WAS-DXS7 were 4.29 (at theta = 0.03) and 4.12 (at theta = 0.00), respectively. Linkage data between WAS and six marker loci indicate the order of the loci to be (DXYS1-DXS1)-WAS-DXS14-DXS7-(DXS84-OTC). These results suggest that the WAS locus lies within the pericentric region of the X chromosome and provide an initial step toward identifying the WAS gene and improving the genetic counseling of WAS families.